11-cis-retinal and Retinitis-Pigmentosa

To discuss antisense oligonucleotide (ASON) therapy for autosomal dominant retinitis pigmentosa (adRP) caused by the proline-23-histidine (P23H) mutation in the rhodopsin gene.. Viral and nonviral therapies to treat adRP are currently under investigation. A promising therapeutic option is a nonviral approach using ASONs. This form of genetic therapy has demonstrated a dose-dependent and highly selective reduction of P23H mutant rhodopsin mRNA in animal models, and it is currently being investigated as a human phase 1/2 clinical trial.. There are promising new therapies to treat adRP. ASON has shown encouraging results in animal models and has undergone a phase 1 clinical trial. ASON does not use a viral vector, is delivered with standard intravitreal injection, and its effects are reversible.

Topics: Animals; Histidine; Humans; Mutation; Oligonucleotides, Antisense; Proline; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of hereditary degenerative diseases affecting 1 of 4000 people worldwide and being the most prevalent cause of visual handicap among working populations in developed countries. These disorders are mainly related to the abnormalities in the rod G protein-coupled receptor (GPCR), rhodopsin reflected in the dysregulated membrane trafficking, stability and phototransduction processes that lead to progressive loss of retina function and eventually blindness. Currently, there is no cure for RP, and the therapeutic options are limited. Targeting rhodopsin with small molecule chaperones to improve the folding and stability of the mutant receptor is one of the most promising pharmacological approaches to alleviate the pathology of RP. This review provides an update on the current knowledge regarding small molecule compounds that have been evaluated as rhodopsin modulators to be considered as leads for the development of novel therapies for RP.

Topics: Carrier Proteins; Humans; Molecular Chaperones; Mutation; Receptors, G-Protein-Coupled; Retinitis Pigmentosa; Rhodopsin

Vision in dim-light conditions is triggered by photoactivation of rhodopsin, the visual pigment of rod photoreceptor cells. Rhodopsin is made of a protein, the G protein coupled receptor (GPCR) opsin, and the chromophore 11-cis-retinal. Vertebrate rod opsin is the GPCR best characterized at the atomic level of detail. Since the release of the first crystal structure 20 years ago, a huge number of structures have been released that, in combination with valuable spectroscopic determinations, unveiled most aspects of the photobleaching process. A number of spontaneous mutations of rod opsin have been found linked to vision-impairing diseases like autosomal dominant or autosomal recessive retinitis pigmentosa (adRP or arRP, respectively) and autosomal congenital stationary night blindness (adCSNB). While adCSNB is mainly caused by constitutive activation of rod opsin, RP shows more variegate determinants affecting different aspects of rod opsin function. The vast majority of missense rod opsin mutations affects folding and trafficking and is linked to adRP, an incurable disease that awaits light on its molecular structure determinants. This review article summarizes all major structural information available on vertebrate rod opsin conformational states and the insights gained so far into the structural determinants of adCSNB and adRP linked to rod opsin mutations. Strategies to design small chaperones with therapeutic potential for selected adRP rod opsin mutants will be discussed as well.

Topics: Animals; Crystallography, X-Ray; Eye Diseases, Hereditary; Genetic Diseases, X-Linked; Humans; Myopia; Night Blindness; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a hereditary degenerative disorder in which mutations in the gene encoding RHO, the light-sensitive G protein-coupled receptor involved in phototransduction in rods, lead to progressive loss of rods and subsequently cones in the retina. Clinical phenotypes are diverse, ranging from mild night blindness to severe visual impairments. There is currently no cure for RHO-adRP. Although there have been significant advances in gene therapy for inherited retinal diseases, treating RHO-adRP presents a unique challenge since it is an autosomal dominant disease caused by more than 150 gain-of-function mutations in the RHO gene, rendering the established gene supplementation strategy inadequate. This review provides an update on RNA therapeutics and therapeutic editing genome surgery strategies and ongoing clinical trials for RHO-adRP, discussing mechanisms of action, preclinical data, current state of development, as well as risk and benefit considerations. Potential outcome measures useful for future clinical trials are also addressed.

Topics: Animals; Disease Management; Gene Editing; Genes, Dominant; Genetic Predisposition to Disease; Genetic Therapy; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin; Treatment Outcome

The aim of this study was to understand the relationship between the findings of spectral-domain optical coherence tomography (SD-OCT) of previously reported animal models of retinitis pigmentosa (RP) associated with known genetic mutations and their background structural and functional changes.. We reviewed previous publications reporting the SD-OCT findings of animal models of RP and summarized the characteristic findings of SD-OCT in nine different animal models (. Despite the various abnormal structural changes found in these different animal models, progressive thinning of the outer nuclear layer (ONL) and hyperreflective change in the inner and outer segment (IS-OS) layers of the photoreceptors were commonly observed on SD-OCT. In the rapidly progressive severe photoreceptor degeneration seen in rd10 and

Topics: Animals; Arrestins; cis-trans-Isomerases; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Humans; Mice; Mice, Knockout; Rats; Rats, Transgenic; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

This brief review summarizes the major proof-of-concept gene therapy studies for autosomal dominant retinitis pigmentosa (RP) caused by mutations in the rhodopsin gene (RHO-adRP) that have been conducted over the past 20 years in various animal models. We have listed in tabular form the various approaches, gene silencing reagents, gene delivery strategies, and salient results from these studies.

Topics: Animals; Gene Silencing; Genes, Dominant; Genetic Therapy; Mutation; Retinitis Pigmentosa; Rhodopsin

PRCD (progressive rod-cone degeneration) is a small ~6 kDa protein with unknown function that specifically resides in photoreceptor discs and interacts with rhodopsin. PRCD's discovery resulted from decades-long study of a canine retinal disease called progressive rod-cone degeneration which is one of the most frequent causes of blindness in dogs characterized by the slow, progressive death of rod photoreceptors followed by cones. A series of genetic studies eventually mapped the disease to a single point mutation in a novel gene which was then named Prcd. Highlighting the importance of this gene, this and several other mutations have been identified in human patients suffering from retinitis pigmentosa. In this review, we highlight what is currently known about PRCD protein, including the etiology and pathology of the retinal disease caused by its mutation, the protein's trafficking, localization, and biochemical characterization.

Topics: Animals; Carrier Proteins; Dogs; Eye Proteins; Humans; Mutation; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.

Topics: Apoptosis Regulatory Proteins; Cell Death; Cholagogues and Choleretics; Endoplasmic Reticulum; Histone Deacetylase Inhibitors; Humans; Molecular Chaperones; Mutation; Photoreceptor Cells; Protein Folding; Protein Processing, Post-Translational; Retinitis Pigmentosa; Rhodopsin

Mutations in rhodopsin are one of the most common causes of retinitis pigmentosa (RP). Misfolding of rhodopsin can result in disruptions in cellular protein homeostasis, or proteostasis. There is currently no available treatment for RP. In this review, we discuss the different approaches currently being investigated for treatment of rhodopsin RP, focusing on the potential of manipulation of the proteostasis network as a therapeutic approach to combat retinal degeneration.

Topics: Animals; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Molecular Targeted Therapy; Mutation; Proteostasis Deficiencies; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

The first autosomal dominant mutation identified to cause retinitis pigmentosa in the North American population was the substitution of proline to histidine at position 23 of the rhodopsin gene (P23H RHO). Many biochemical studies have demonstrated that P23H mutation induces rhodopsin (RHO) misfolding leading to endoplasmic reticulum stress. Herein, we review current thinking of this topic.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Genes, Dominant; Humans; Proteostasis Deficiencies; Retinitis Pigmentosa; Rhodopsin

Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells.

Topics: Acute-Phase Proteins; Animals; DNA; Genes, Dominant; Genetic Vectors; Humans; Mutation; Phenotype; Retinitis Pigmentosa; Rhodopsin

Understanding the relationship between the visual pigment rhodopsin and Zn2+ under normal conditions and in case of deficiency of the latter, as well as the realization of the role of Zn2+ in the development of the hereditary disease retinitis pigmentosa, have great theoretical and practical importance. In this mini-review, we briefly examine the basic experimental data on the role of Zn2+ in the retina and photoreceptors, binding of endogenous Zn2+ by zinc-binding sites of differing affinities in rhodopsin, the influence of the exogenous Zn2+ on various properties of rhodopsin, including its ability for phosphorylation and activation of transducin, as well as its thermal stability and regeneration. Conflicting results on the correlation between Zn2+ content in the blood serum and the development of retinitis pigmentosa in patients are demonstrated. The review also shows the success of the application of animal models of induced or hereditary retinal degeneration and discusses some of the methodological approaches and therapeutic techniques to relieve the manifestations of this disease.

Topics: Animals; Binding Sites; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Zinc

Rhodopsins (Rh) are G protein-coupled receptors that function as light-sensors in photoreceptors. In humans, Rh mutations cause retinitis pigmentosa (RP), a degenerative disease that ultimately results in blindness. Studies in Drosophila have provided many insights into basic Rh biology and have identified pathways that lead to retinal degeneration. It has been shown that, because Rh is very abundant in photoreceptors, its accumulation in numerous organelles induces severe stress and results in degeneration of these cells. Moreover, genetic lesions that affect proper activation of membrane-bound Rh lead to disruption in Ca(2+) homeostasis which also causes photoreceptor degeneration. We review here the molecular signals involved in Rh homeostasis and the mechanisms underlying retinal degeneration in flies, and discuss possible links to human diseases.

Topics: Animals; Drosophila melanogaster; Homeostasis; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

In light of the elucidation of the molecular pathogenesis of some dominantly inherited retinal degenerations over the past two decades, it is timely to explore possible means of therapeutic intervention for such diseases. However, the presence of significant levels of intergenic and intragenic genetic heterogeneity in this group of dominant conditions represents a barrier to the development of therapies focused on correcting the primary genetic defect. More than 60 genes have been implicated in dominant retinopathies and indeed over 150 different mutations in the rhodopsin gene alone have been identified in patients with autosomal dominant retinitis pigmentosa. Employing next-generation sequencing to characterise populations of retinal degeneration patients genetically over the coming years will beyond doubt serve to highlight further the immense genetic heterogeneity inherent in this group of disorders. Such diversity in genetic aetiologies has promoted the search for therapeutic solutions for dominantly inherited retinopathies that are independent of disease-causing mutations. The various approaches being considered to provide mutation-independent therapies for these dominant conditions will be discussed in the review, as will the preclinical data supporting the further development of such strategies.

Topics: Animals; Dependovirus; Gene Expression; Gene Silencing; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA Interference

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Retinitis pigmentosa is a retinal degeneration transmitted by varied modes of inheritance and affects approximately 1 in 4000 individuals. The photoreceptors of the outer retina, as well as the retinal pigmented epithelium which supports the outer retina metabolically and structurally, are the retinal regions most affected by the disorder. In several forms of retinitis pigmentosa, the mislocalization of the rod photoreceptor protein rhodopsin is thought to be a contributing factor underlying the pathophysiology seen in patients. The mutations causing this mislocalization often occur in genes coding proteins involved in ciliary formation, vesicular transport, rod outer segment disc formation, and stability, as well as the rhodopsin protein itself. Often, these mutations result in the most early-onset cases of both recessive and dominant retinitis pigmentosa, and the following presents a discussion of the proteins, their degenerative phenotypes, and possible treatments of the disease.

Topics: Animals; Humans; Models, Biological; Mutant Proteins; Ophthalmology; Protein Transport; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

With a worldwide prevalence of about 1 in 3500-5000 individuals, Retinitis Pigmentosa (RP) is the most common form of hereditary retinal degeneration. It is an extremely heterogeneous group of genetically determined retinal diseases leading to progressive loss of vision due to impairment of rod and cone photoreceptors. RP can be inherited as an autosomal-recessive, autosomal-dominant, or X-linked trait. Non-Mendelian inheritance patterns such as digenic, maternal (mitochondrial) or compound heterozygosity have also been reported. To date, more than 65 genes have been implicated in syndromic and non-syndromic forms of RP, which account for only about 60% of all RP cases. Due to this high heterogeneity and diversity of inheritance patterns, the molecular diagnosis of syndromic and non-syndromic RP is very challenging, and the heritability of 40% of total RP cases worldwide remains unknown. However new sequencing methodologies, boosted by the human genome project, have contributed to exponential plummeting in sequencing costs, thereby making it feasible to include molecular testing for RP patients in routine clinical practice within the coming years. Here, we summarize the most widely used state-of-the-art technologies currently applied for the molecular diagnosis of RP, and address their strengths and weaknesses for the molecular diagnosis of such a complex genetic disease.

Topics: Animals; Eye Proteins; Humans; Molecular Diagnostic Techniques; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA

Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success.

Topics: Adenoviridae; Animals; Disease Models, Animal; Gene Expression; Gene Transfer Techniques; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Mice; Mutation; Retinitis Pigmentosa; Rhodopsin; RNA Interference; Transcription, Genetic

Topics: Animals; c-Mer Tyrosine Kinase; Carrier Proteins; cis-trans-Isomerases; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Drug Design; Eye Proteins; Genetic Therapy; Humans; Intercellular Signaling Peptides and Proteins; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Targeted Therapy; Mutation; Nerve Tissue Proteins; Nifedipine; Peripherins; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Retinitis Pigmentosa; Rhodopsin; Vitamin A

The retinitis pigmentosa (RP)-causing mutant of rhodopsin, Pro23His (P23H) rhodopsin, is folding defective and unable to traffic beyond the endoplasmic reticulum (ER). This ER retention, and in some cases, aggregation are proposed to result in ER-stress and eventually cell death. The endogenous rhodopsin ligand 11-cis-retinal and its isomer 9-cis-retinal have been shown to act as pharmacological chaperones, promoting proper folding and trafficking of the P23H rhodopsin. In spite of this promising effect, the development of retinals and related polyenealdehydes as pharmacological agents has been hampered by their undesirable properties, which include chemical instability, photolability, and potential retinoidal actions. Here we report the design and synthesis of a class of more stable nonpolyene-type rhodopsin ligands, structurally distinct from, and with lower toxicity than, retinals. A structure-activity relationship study was conducted using cell-surface expression assay to quantify folding/trafficking efficiency of P23H rhodopsin.

Topics: Drug Design; Endoplasmic Reticulum; Humans; Ligands; Molecular Chaperones; Mutation; Protein Folding; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

Mutations that cause rhodopsin misfolding and retention within the endoplasmic reticulum (ER) are a prominent cause of retinitis pigmentosa. Here, we discuss the hypothesis that the failure of photoreceptor neurons to adapt to the stress caused by rhodopsin accumulation in the ER leads to a global collapse of homeostasis and to retinal degeneration. We review the molecular mechanisms underlying the activity of local ER conformational sensors and stress-relaying modules and consider how ER-derived stress signals are amplified and implemented to impact on downstream processes, including rhodopsin clearance and cell fate control. The emerging view is that alterations to the systems responsible for the detection, transduction and implementation of ER stress might be used therapeutically to treat retinitis pigmentosa.

Topics: Animals; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation; Humans; Molecular Targeted Therapy; Mutation; Proteostasis Deficiencies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Stress, Physiological

Mutations in rhodopsin cause autosomal dominant retinitis pigmentosa. The majority of these mutations (class II) lead to protein misfolding. The misfolded protein is retained in the ER then retrotranslocated into the cytoplasm for degradation by the proteasome. If degradation fails, the protein can aggregate to form intracellular inclusions. In addition, the mutant rod opsin exerts a dominant negative effect on the wild-type protein. Here, we review these pathways and how different drug treatments can affect mutant rod opsin. Interestingly, drugs targeted at general protein stability (kosmotropes) or improving the cellular folding and degradation machinery (molecular chaperone inducers and autophagy induction) reduced P23H rod opsin aggregation and inclusion formation together with associated caspase activation and cell death, but did not enhance mutant protein processing or reduce the dominant negative effects. In contrast, pharmacological chaperones (retinoids) enhanced P23H folding and reduced the dominant negative effects, as well as reducing the other gains of function. Therefore, targeting the toxic gain of function did not require improved folding, whereas reducing the dominant negative effects required improved folding. These studies suggest that some forms of rhodopsin retinitis pigmentosa could be treated by targeting protein folding and/or reducing protein aggregation.

Topics: Animals; Humans; Models, Biological; Mutant Proteins; Protein Folding; Proteostasis Deficiencies; Retinitis Pigmentosa; Rhodopsin

Topics: Adolescent; Adult; Animals; Carrier Proteins; cis-trans-Isomerases; Disease Models, Animal; Eye Proteins; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Leber Congenital Amaurosis; Macaca fascicularis; Nerve Growth Factors; Rats; Retinitis Pigmentosa; Rhodopsin; Serpins; Simian Immunodeficiency Virus; Young Adult

Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal structure was obtained. Several crystal structures have now been solved representing different activation states of the receptor. These structures, together with those from lower resolution techniques (e.g. electron microscopy), shed light on the stepwise process by which energy from an extracellular photon is transduced across the membrane to the intracellular compartment thereby activating signalling mechanisms responsible for very low-level light detection. Controversy remains in several areas including: (i) transmembrane helix movements responsible for the transduction process, (ii) the stoichiometry of coupling to G proteins and their mode of activation, (iii) the role, if any, of receptor oligomerisation and (iv) the suitability of using structures of this GPCR as templates for modelling the structures of other GPCRs, and their mechanisms of activation.

Topics: Amino Acid Sequence; Dimerization; Humans; Models, Molecular; Molecular Sequence Data; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Structure-Activity Relationship

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous group of diseases that cause blindness. Mutations within the rhodopsin gene account for approximately 25% of autosomal dominantly inherited RP cases. Therefore, understanding the mechanisms causing rhodopsin-mediated RP has a significant health impact. To date, results from multiple labs indicate that rhodopsin-mediated RP pathogenesis does not share a common mechanism of degeneration. There is strong evidence that multiple mechanisms are involved, including protein misfolding, mislocalization, release of toxic products, and aberrant signaling. Development of effective treatments requires investigation of the mechanism involved in the different rhodopsin mutations. This chapter focuses on the mechanisms by which rhodopsin mutations cause retinal degeneration, as well as potential therapeutic strategies to treat the disease.

Topics: Animals; Humans; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is an inherited retinal degeneration characterized by nyctalopia, ring scotoma, and bone-spicule pigmentation of the retina. So far, no effective therapy has been found for RP. As a possible molecular etiology of RP, retina-specific gene deficits are most likely involved, but little has been identified in terms of intracellular mechanisms leading to retinal photoreceptor cell death at post-translational levels. In order to find an effective therapy for RP, we must look for underlying common mechanisms that are responsible for the development of RP, instead of designing a specific therapy for each of the RP types with different causes. Therefore, in the present study, several animal models with different causes of RP were studied, including (1)Royal College of Surgeons (RCS) rats with a deficit of retinal pigment epithelium (RPE) function caused by rhodopsin mutation; (2) P23H rats, (3) S334ter rats, (4) photo stress rats, (5) retinal degeneration (rd) mice with a deficit of phosphodiesterase(PDE) function; and (6) cancer-associated retinopathy (CAR) model rats with a deficit of recoverin-dependent photoreceptor adaptation function. In each of these models, the following assessments were made in order to elucidate common pathological mechanisms among the models: (1) retinal function assessed by electroretinogram (ERG), (2) retinal morphology, (3) retinoid analysis, (4) rhodopsin regeneration, (5) rhodopsin phosphorylation and dephosphorylation, and (6) cytosolic cGMP levels. We found that unregulated photoreceptor adaptation processes caused by an imbalance of rhodopsin phosphorylation and dephosphorylation caused retinal dysfunction leading to photoreceptor cell death. As possible candidate drugs for normalizing these retinal dysfunctions and stopping further retinal degeneration, nilvadipine, a Ca channel blocker, retinoid derivatives, and anthocyanine were chosen and tested to determine their effect on the above animal models with retinal degeneration. Nilvadipine showed beneficial effects against retinal degeneration in all models tested, but retinoid derivatives and anthocyanine showed these beneficial effects in only some models. Thus our present data allowed us to test the effectiveness of nilvadipine in the treatment of human RP patients.

Topics: Animals; Calcium Channel Blockers; Dark Adaptation; Disease Models, Animal; Humans; Mice; Mutation; Nifedipine; Phosphorylation; Rats; Retinitis Pigmentosa; Rhodopsin

Since the discovery of the first rhodopsin mutation that causes retinitis pigmentosa in 1990, significant progresses have been made in elucidating the pathophysiology of diseases caused by inactivating mutations of G protein-coupled receptors (GPCRs). This review aims to compile the compelling evidence accumulated during the past 15 years demonstrating the etiologies of more than a dozen diseases caused by inactivating GPCR mutations. A generalized classification scheme, based on the life cycle of GPCRs, is proposed. Insights gained through detailed studies of these naturally occurring mutations into the structure-function relationship of these receptors are reviewed. Therapeutic approaches directed against the different classes of mutants are being developed. Since intracellular retention emerges as the most common defect, recent progresses aimed at correcting this defect through membrane permeable pharmacological chaperones are highlighted.

Topics: Animals; Diabetes Insipidus, Nephrogenic; Dwarfism; Humans; Hypogonadism; Mutation; Obesity; Receptor, Melanocortin, Type 1; Receptor, Melanocortin, Type 2; Receptor, Melanocortin, Type 3; Receptor, Parathyroid Hormone, Type 1; Receptors, Calcium-Sensing; Receptors, CCR5; Receptors, G-Protein-Coupled; Receptors, LHRH; Receptors, Vasopressin; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by the loss of photoreceptors and characterized by retinal pigment deposits visible on fundus examination. Prevalence of non syndromic RP is approximately 1/4,000. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. Some extreme cases may have a rapid evolution over two decades or a slow progression that never leads to blindness. In some cases, the clinical presentation is a cone-rod dystrophy, in which the decrease in visual acuity predominates over the visual field loss. RP is usually non syndromic but there are also many syndromic forms, the most frequent being Usher syndrome. To date, 45 causative genes/loci have been identified in non syndromic RP (for the autosomal dominant, autosomal recessive, X-linked, and digenic forms). Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs. Molecular diagnosis can be made for some genes, but is not usually performed due to the tremendous genetic heterogeneity of the disease. Genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, so the visual prognosis is poor. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitaminotherapy, treating the complications (cataract and macular edema), and helping patients to cope with the social and psychological impact of blindness. However, new therapeutic strategies are emerging from intensive research (gene therapy, neuroprotection, retinal prosthesis).

Topics: Adolescent; Child; Child, Preschool; Diagnosis, Differential; Female; Humans; Infant; Metabolic Diseases; Nervous System Diseases; Night Blindness; Pregnancy; Prenatal Diagnosis; Prognosis; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Sensation Disorders; Syndrome

Retinitis pigmentosa (RP) is one of the most genetically heterogeneous inherited disorders. Twelve genes have now been identified in the autosomal dominant form of the disease, including some recently characterized genes that show unprecedented and fascinating traits in both their function and in their expression profiles. These include many widely expressed genes encoding components of the spliceosome and a guanine nucleotide synthesis gene. Intriguingly, the most recently identified dominant gene does not appear to be expressed in the neuronal retina but is expressed in the capillaries of the choroid. In attempting to understand the effects of mutations in these genes, investigators are forced to re-evaluate their thinking on the molecular mechanisms of genetic blindness and to undertake an increasingly inter-disciplinary approach in their analysis of this disease. Recently, this has resulted in significant developments in the elucidation of the molecular pathogenesis of RP.

Topics: Animals; Genes, Dominant; Genetic Predisposition to Disease; Guanine; Guanosine Triphosphate; Humans; Light; Models, Biological; Mutation; Protein Folding; Retina; Retinitis Pigmentosa; Rhodopsin; Spliceosomes; Vision, Ocular

Retinitis pigmentosa (RP) is a group of retinal degenerative diseases that are characterised primarily by the loss of rod photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal-dominant RP (ADRP). Here, we propose a new classification for rhodopsin mutations based on their biochemical and cellular properties. Several different potential gain-of-function mechanisms for rhodopsin ADRP are described and discussed. Possible dominant-negative mechanisms, which affect the processing, translocation or degradation of wild-type rhodopsin, are also considered. Understanding the molecular and cellular consequences of rod-opsin mutations and the underlying disease mechanisms in ADRP are essential to develop future therapies for this class of retinal dystrophies.

Topics: Amino Acid Sequence; Cell Death; Humans; Models, Neurological; Molecular Sequence Data; Mutation; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa is a group of retinal degenerative diseases, within the broad family of hereditary retinopathies, for which there is no cure at present. Mutations in different genes coding for proteins related to the metabolism of photoreceptor cells, and to the visual phototransduction cascade, are the cause of this disease. Rhodopsin, the photoreceptor protein responsible for light absorption--and key in the first stages of vision--is one of the most studied molecules of the retina. Mutations in the opsin gene account for about 25% of all cases of autosomal dominant retinitis pigmentosa. Recent crystallization of this receptor in its inactive dark state has revealed new structural details yielding further insights into the intra and intermolecular mechanismsin which the protein is involved as a result of its activation.Furthermore, the in vitro study of recombinant rhodopsins carrying mutations previously found in retinitis pigmentosa patients (by means of spectroscopic and functional techniques) has shed new light on the structural requirements for its correct function, as well as the molecular defects underlying the mechanism of photoreceptor cell death. In this study, the main findings of the recent investigations carried out in this field are presented. The relevant information obtained at the molecular level is bound to facilitate our understandingof the molecular processes that will allow suitable therapiesfor different retinal degenerative diseases, particularly retinitis pigmentosa, to be proposed.

Topics: Humans; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

Biochemical data providing new insights into the packing of helices I and II in the transmembrane domain of rhodopsin reveals the existence of a specific set of size- and charge-sensitive interhelical interactions that influence protein tertiary structure. These findings have broad implications towards understanding the molecular consequences of naturally occurring mutations associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa.

Topics: Amino Acid Sequence; Cell Membrane; Genes, Dominant; Humans; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; Retinitis Pigmentosa; Rhodopsin

Mutations in the rhodopsin gene are the most common cause of retinitis pigmentosa (RP) among human patients. The nature of the rhodopsin mutations has critical implications for the design of strategies for gene therapy. Nearly all rhodopsin mutations are dominant. Although dominance does not arise because of haploinsufficiency, it is unclear whether it is caused by gain-of-function or dominant-negative mutations. Current strategies for gene therapy have been devised to deal with toxic, gain-of-function mutations. However, analysis of results of transgenic and targeted expression of various rhodopsin genes in mice suggests that dominance may arise as a result of dominant-negative mutations. This has important consequences for gene therapy. The effects of dominant-negative mutations can be alleviated, in principle, by supplementation with additional wild-type rhodopsin. If added wild-type rhodopsin could slow retinal degeneration in human patients, as it does in mice, it would represent a valuable new strategy for gene therapy of RP caused by dominant rhodopsin mutations.

Topics: Animals; Cell Death; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Humans; Mice; Mice, Transgenic; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Mice; Mice, Transgenic; Mutation; Night Blindness; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is primary, chronic, and hereditary chorioretinal degeneration characterized by photopsia, progressive visual loss with ring scotoma, and impairment of dark adaptation. Although modern molecular biology and molecular genetics have identified many causative genes, the molecular pathophysiology of RP is not fully understood, and no effective treatments have been found yet. In recent studies using animal models of RP, new treatments have been devised and their clinical use is being considered.. In terms of the molecular pathophysiology of RP, we summarized previous studies of genetic impairment of proteins involved in the phototransduction pathway and introduced new possible therapies for RP.. We found that most abnormalities of the genes related with the photoxcitation and its inhibition process in the photoreceptor cells caused a variety of clinical manifestations of RP.. So far, a variety of abnormalities of the genes causing RP have been identified. However, further studies of the relationship between the abnormalities and clinical expression are needed for better understanding of the pathophysiology of RP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Arrestin; Carrier Proteins; Eye Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Nerve Tissue Proteins; Peripherins; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is the membrane receptor responsible for photoreception in the vertebrate retina. Its characteristic seven-transmembrane helical structural motif is today widely recognised as a paradigm in signal transduction. Rhodopsin and the phototransduction system are frequently used as structural and mechanistic models for the G-protein coupled receptor superfamily. Recent advances in the activation mechanism (as derived from the structural available data) and the implications for normal and pathological - in retinal disorders - visual function will be reviewed.

Topics: Amino Acid Sequence; Animals; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Night Blindness; Protein Structure, Secondary; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Rhodopsin is the dim-light activated photoreceptor located in the rod cells of the eye. It belongs to the large superfamily of G-protein-coupled receptors (GPCRs). Many consider it the proto-typical GPCR as numerous studies since its cloning in 1983 (Nathans and Hogness 1983) have established many fundamental principles of seven transmembrane-spanning GPCRs. Abundant expression in the rod's outer segment, constituting about 90% of the total membrane protein in the discs, and the development of techniques to purify large quantities of functional protein has facilitated this process. Another distinct feature is rhodopsin's ligand, 11-cis-retinal, which is covalently bound via a Schiff base to transmembrane seven (TM VII), allowing extensive spectroscopic studies. Exciting recent developments include the discovery of naturally occurring mutations that lead to retinal degeneration, the determination of transmembrane movements using electron paramagnetic resonance (EPR) and biochemical techniques, and the discovery of its 3D X-ray crystal structure, the first among GPCRs. The impact of these major advances will be discussed in this review.

Topics: Amino Acid Sequence; Humans; Molecular Sequence Data; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP), the group of hereditary conditions involving death of retinal photoreceptors, represents the most prevalent cause of visual handicap among working populations in developed countries. Here we provide an overview of the molecular pathologies associated with such disorders, from which it becomes clearly apparent that RP is one of the most genetically heterogeneous of hereditary conditions for which molecular pathologies have so far been elucidated. While heterogeneity of such magnitude would appear to represent a major impediment to the development of therapeutics, mutation-independent approaches to therapy are being developed to effectively by-pass such diversity in genetic aetiology. The implications of such technologies in terms of therapeutic intervention in RP, and indeed other genetically heterogeneous conditions, will be addressed.

Topics: 3' Untranslated Regions; 5' Untranslated Regions; Animals; Apoptosis; Disease Progression; Eye Proteins; Genes, Dominant; Genes, Recessive; Genetic Heterogeneity; Genetic Linkage; Genetic Therapy; Humans; Mammals; Mice; Mice, Knockout; Models, Animal; Nerve Growth Factors; Optic Atrophy, Hereditary, Leber; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic; RNA, Messenger; Syndrome; Transcription Factors; X Chromosome

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.

Topics: Animals; Group II Chaperonins; Humans; Models, Molecular; Molecular Chaperones; Photoreceptor Cells; Protein Conformation; Protein Folding; Retina; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin

We review the sorting/targeting steps involved in the delivery of rhodopsin to the outer segment compartment of highly polarized photoreceptor cells. The transport of rhodopsin includes (1) the sorting/budding of rhodopsin-containing vesicles at the trans-Golgi network, (2) the directional translocation of rhodopsin-bearing vesicles through the inner segment, and (3) the delivery of rhodopsin across the connecting cilium to the outer segment. Several independent lines of evidence suggest that the carboxyl-terminal, cytoplasmic tail of rhodopsin is involved in the post-Golgi trafficking of rhodopsin. Inappropriate subcellular targeting of naturally occurring rhodopsin mutants in vivo leads to photoreceptor cell death. Thus, the genes encoding mutations in the cellular components involved in photoreceptor protein transport are likely candidate genes for retinal dystrophies.

Topics: Animals; Carrier Proteins; Cell Survival; Cytoskeleton; Molecular Motor Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

In this paper, an attempt is made to highlight some of the recent developments in genetics to understand the group of inherited eye disorders referred to as retinitis pigmentosa (RP). Of the seven genes identified, six are expressed specifically in the photoreceptor cells and four encode the enzymes involved in the phototransduction pathway. A short discussion is presented of the tremendous phenotypic heterogeneity. An understanding of RP requires knowledge of other genetic and environmental factors as well as tests to measure the status of the patient's photoreceptor cells in various disease stages.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic Nucleotide-Gated Cation Channels; Eye Proteins; Humans; Intermediate Filament Proteins; Ion Channels; Membrane Glycoproteins; Membrane Proteins; Nerve Tissue Proteins; Peripherins; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Tetraspanins; Transducin

Retinitis pigmentosa defines a genetically heterogenous group of disorders characterized by degenerations of photoreceptors and pigment epithelium. This article reviews our current knowledge of the genetical, clinical and pathophysiological aspects of this disease complex. Therapeutic concepts under current investigation are discussed as well. In recent years tremendous new insights have been made using molecular techniques for the investigation of retinal dystrophies. Ophthalmoscopically very similar patterns of photoreceptor dystrophies have been related to different gene mutations. In contrast, mutations in a single gene may cause different clinical patterns of photoreceptor dystrophies. Therefore, these recent results suggest that a reclassification of retinal dystrophies on the basis of their genetic origin may be favourable. In the future molecular genetics and the recent developments may play an increasing role for clinical classification and evaluation of photoreceptor dystrophies. The continued clinical and experimental research on hereditary disorders may help to elucidate further the wide disease spectrum and thereby developing new classifications and efficient therapeutic concepts.

Topics: Animals; Diagnosis, Differential; DNA Mutational Analysis; Eye Proteins; Genotype; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Nerve Tissue Proteins; Pedigree; Peripherins; Photoreceptor Cells; Pigment Epithelium of Eye; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Electroretinography; Humans; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutation; Protein Conformation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

To determine to what degree visual field size is correlated with electroretinogram (ERG) amplitude among patients with the common forms of retinitis pigmentosa (RP).. Visual field equivalent diameter to the V4e white test light of the Goldmann perimeter was correlated with log ERG amplitude elicited by 0.5 Hz or 30 Hz full-field flashes of white light. Primary analyses were conducted on data from 583 patients with the common forms of RP. Subset analyses were performed on data from patients with ERG responses with different ranges of amplitude to assess to what extent the correlation depends on ERG amplitude, as well as on data from patients of a given genetic type to determine whether the correlation depends on the mode of transmission. Data from patients with the rhodopsin, Pro23His mutation (n = 38) or with the rhodopsin, Pro347Leu mutation (n = 24) were analyzed to determine the correlation between visual field size and ERG amplitude for patients with the same mutation.. Visual field size was significantly correlated with ERG amplitude for every comparison (P < or = 0.0003). Correlations generally were higher for ERGs elicited by 30 Hz flashes (r = 0.62 for the entire sample) than they were for those elicited by 0.5 Hz flashes (r = 0.53 for the entire sample). They were lower for truncated ranges of ERG amplitude, higher for patients with dominant or recessive disease than for patients with x-linked disease or for patients of all genetic types combined, and strong for patients with the same rhodopsin mutation (reaching a value of 0.87).. Visual field size is significantly correlated with ERG amplitude for patients with RP. Correlation depends on the range of ERG amplitudes, the inheritance type, and, particularly, on whether the analysis is confined to a single gene mutation.

Topics: Adolescent; Adult; Aged; Electroretinography; Humans; Middle Aged; Point Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests; Visual Fields

Rhodopsin, the visual pigment of rod photoreceptors cells, is a member of the large family of G protein-coupled receptors. Rhodopsin is composed of two parts: a polypeptide chain called opsin and an 11-cis-retinal chromophore covalently bound to the protein by means of a protonated Schiff base linkage to Lys296 located in the seventh transmembrane segment of the protein. Several mutations have been described that constitutively activate the apoprotein opsin. These mutations appear to activate the protein by a common mechanism of action. They disrupt a salt-bridge between Lys296 and the couterion Glu113 that helps constrain the protein to an inactive conformation. Four of the mutations have been shown to cause two different diseases of the retina, retinitis pigmentosa and congenital night blindness. Recently, several other human diseases have been shown to be caused by constitutively activating mutations of G protein-coupled receptors.

Topics: GTP-Binding Proteins; Humans; Models, Molecular; Mutagenesis, Site-Directed; Night Blindness; Photochemistry; Protein Conformation; Retinitis Pigmentosa; Rhodopsin; Schiff Bases; Structure-Activity Relationship

Light signals are converted into electrical signals by vertebrate photoreceptor cells, and the generated electrical signals are then modulated within retinal neurons and brain. During the visual transduction precesses in the photoreceptor cells, there are basically three functions. That is, (1) photoexcitation, (2) quenching of the photoexcitation, and (3) adaptation. These functions are precisely regulated by enzymatic cascade reactions. Quite recently, it was shown that rhodopsin phosphorylation occurred at different sites with different kinetics, in vivo, and this may be a control mechanism for both quenching and adaptation. Furthermore, autosmal retinitis pigmentosa (RP) with rhodopsin mutation at 296 Lys, which in the binding site of 11-cis-retinal, showed constitutive activation of guanosine 5'-triphosphate (GTP) binding protein and no rhodopsin phosphorylation by rhodopsin kinase. These observations suggest that rhodopsin phosphorylation and dephosphorylation are critical for understanding visual transduction and pathophysiology of RP.

Topics: Adaptation, Ocular; Animals; Binding Sites; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; GTP-Binding Proteins; Mutation; Phosphorylation; Photic Stimulation; Photoreceptor Cells; Protein Kinases; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Topics: Amino Acid Sequence; Eye Diseases, Hereditary; Eye Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Sequence Data; Nerve Tissue Proteins; Peripherins; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Adult; Aged; Apoptosis; Chromosome Mapping; Environment; Female; Genes, Dominant; Humans; Male; Middle Aged; Phenotype; Retinal Diseases; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Hereditary degenerations and dysfunctions of the retina are an extremely heterogeneous group of diseases. This summary deals with recent advances in the molecular genetics of a subset of those disorders, namely, those encompassed under the diagnosis 'retinitis pigmentosa'. Over 20 loci where mutations cause retinitis pigmentosa have been mapped; the review focuses on the seven retinitis pigmentosa loci that have been identified.

Topics: Humans; Retinitis Pigmentosa; Rhodopsin

In recent years a rapid development in gene cloning techniques has been seen which has, in part, been responsible for the elucidation of the molecular mechanisms involved in many hereditary diseases. Most noteworthy is the rapid detection of mutations in various genes Most noteworthy is the rapid detection of mutations in various genes associated with various retinal degenerations and disorders. There is an increasing need for ophthalmologists to keep abreast with these new developments to better understand disease processes and recognize new opportunities for the diagnosis and treatment of these conditions. In addition, more recent discoveries in molecular genetics have revealed previously unrecognized associations between different clinical entities that share common gene mutations (gene sharing), as well as distinctly different molecular alterations within the spectrum of what traditionally was believed to be the same disease (locus heterogeneity). These findings will most likely, in the future, invoke the need to redefine disease categories at the molecular level. This article reviews basic molecular genetics useful to ophthalmologists and examines in detail these concepts with regard to one representative hereditary retinal disease--retinitis pigmentosa.

Topics: Amino Acid Sequence; Fundus Oculi; Humans; Molecular Biology; Molecular Sequence Data; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin

Molecular pathogenic cause of Retinitis pigmentosa leading from different mutations in rhodopsin gene and dependences between dominant or recessive mutations and clinical effects have been described. Importance of molecular analysis of rhodopsin in prognosis this disease have been emphasized.

Topics: Base Sequence; Humans; Molecular Sequence Data; Mutation; Phenotype; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa comprises a group of clinically variable and genetically heterogeneous inherited disorders of the retina. It is estimated that approximately 1.5 million people throughout the world are affected by this disease. It is a slowly progressive disorder and causes loss of night vision and peripheral visual field in adolescence. It can be inherited through an autosomal dominant, recessive, or X-linked mode; the autosomal dominant form is considered to be the mildest form. Molecular genetic studies on the autosomal dominant disorder have shown that, in some families, genes encoding the rhodopsin and peripherin/RDS map very close to the disease loci identified previously by the systematic linkage analyses. These results, together with the observation that a recessive nonsense mutation in the Drosophila opsin gene causes photoreceptor degeneration, prompted an extensive search for the alterations in the human rhodopsin and peripherin/RDS genes in families with autosomal dominant retinitis pigmentosa. As a result, several distinct rhodopsin and peripherin/RDS mutations have been found in approximately 30% of all autosomal dominant cases. A wide variety of clinical expression of the disorder even within a family with the same mutation, its late onset, slow progression, and cone degeneration clearly suggest that some other factors or genes in addition to rhodopsin are responsible for the phenotypic expression of the disorder. In this article, an attempt is made to highlight some of these recent developments and to correlate the various mutations and the phenotypes.

Topics: Eye Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Peripherins; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa was investigated with molecular genetic techniques, to identify gene abnormalities and to obtain a better understanding of the mechanism of retinal degeneration. First, a search for candidate genes was performed focusing on rhodopsin, peripherin/RDS, and phosducin genes, using non-radioisotopic SSCP and genomic DNA samples obtained from 387 Japanese patients with retinitis pigmentosa, including 56 families of the autosomal dominant type (ADRP). One ADRP family with rhodopsin Pro-347-Leu mutation and another with peripherin/RDS Asn-244-Lys mutation were identified. The genotype and phenotype correlation of each ADRP family was then analysed. Ocular findings associated with the rhodopsin Pro-347-Leu in the Japanese family were similar to those reported in Caucasian families, indicating that the same mutation can produce the common phenotype even among different ethnic populations. The phenotype associated with the peripherin/RDS Asn-244-Lys showed typical findings of retinitis pigmentosa associated with bull's-eye maculopathy. Finally, glutamate was immunohistochemically quantified in the photoreceptor inner segment of rds/rds mice using anti-Glu antibody. The results showed that glutamate was accumulated in the rds/rds mouse photoreceptor inner segment, suggesting that glutamate may play a role in the process of retinal degeneration caused by the peripherin/RDS gene abnormality, although the precise mechanism is currently unknown.

Topics: Animals; Glutamates; Glutamic Acid; Humans; Immunohistochemistry; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mutation; Nerve Tissue Proteins; Peripherins; Polymerase Chain Reaction; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Base Sequence; Humans; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa is an inherited progressive disease which is a major cause of blindness in western communities. It can be inherited as an autosomal dominant, autosomal recessive, or X-linked recessive disorder. In the autosomal dominant form (adRP), which comprises about 25% of total cases, approximately 30% of families have mutations in the gene encoding the rod photoreceptor-specific protein rhodopsin. This is the transmembrane protein which, when photoexcited, initiates the visual transduction cascade. So far, 41 single-base-pair (bp) substitutions, one two-bp substitution, and four deletions ranging from 3 to 42 bp have been identified in this gene. These mutations do not appear to be significantly clustered in a specific part of the protein, but occur in all three major domains, namely the intradiscal, transmembrane, and cytoplasmic regions. Different mutations appear to cause differences in the severity of the disease, though there is considerable variability in severity even within the same family, at least in certain of these mutations. Identification of all the mutations involved in rhodopsin-RP should allow accurate and early detection of affected individuals, informed genetic counselling, as well as furthering our knowledge of the disease process involved.

Topics: Amino Acid Sequence; Forecasting; Genes, Dominant; Humans; Molecular Sequence Data; Mutation; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Animals; Awards and Prizes; Base Sequence; Chromosome Aberrations; Chromosome Disorders; Disease Models, Animal; Electroretinography; Humans; Mice; Mice, Transgenic; Molecular Sequence Data; Mutation; Ophthalmology; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Societies, Medical; United States

Topics: Chromosome Mapping; Genes, Dominant; Humans; Mutation; Polymorphism, Restriction Fragment Length; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Color Vision Defects; Female; Humans; Male; Molecular Biology; Mutation; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

In recent years, research efforts in the basic and clinical sciences have yielded numerous new findings. The review given here outlines clinical findings, research results, and perspectives on the origin of hereditary retinal degeneration as far as molecular genetics, biochemistry, morphology, and clinical research are concerned: genotype-phenotype correlation, electroretinography, color perimetry, blue cone function, exogenous factors, refraction problems, fat metabolism, immunological aspects, retinal transplantation, and phenocopies of retinitis pigmentosa and related syndromes. The consequences for ophthalmological practice are pointed out and comprehensive, improved diagnostic procedures are recommended, using a checklist proposed here (see appendix).

Topics: Adult; Genes, Dominant; Genes, Recessive; Genetic Counseling; Genotype; Humans; Mutation; Phenotype; Retinitis Pigmentosa; Rhodopsin

The human retina carries specialized neurons, the rod and cone photoreceptors, which absorb and transduce light energy and transmit impulses through the optic nerve to the brain. The most prevalent group of inherited retinopathies, affecting approximately 1.5 million people, is collectively termed retinitis pigmentosa (RP). Mutations responsible for RP have now been found in two genes encoding transmembrane proteins of the rod photoreceptor outer segment disc, and a number of additional causative genes have been localized. It is likely that characterization of the majority of such genes over the next few years will lead to a substantial elucidation of the molecular pathology of this debilitating group of hereditary conditions.

Topics: Chromosome Mapping; Genetic Linkage; Humans; Models, Biological; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular; X Chromosome

Topics: Amino Acid Sequence; Animals; Humans; Molecular Sequence Data; Mutation; Photochemistry; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa is a model for the study of genetic diseases. Its genetic heterogeneity is reflected in the different forms of inheritance (autosomal dominant, autosomal recessive, or X-linked) and, in a few families, in the presence of mutations in the visual pigment rhodopsin. Clinical and molecular genetic studies of these disorders are discussed. Animal models of retinal degeneration have been investigated for many years with the hope of gaining insight into the cause of photoreceptor cell death. Recently, the genes responsible for two of these animal disorders, the rds and rd mouse genes, have been isolated and characterized. The retinal degeneration of the rd mouse is presented in detail. The possible involvement of human analogues of these mouse genes in human retinal diseases is being investigated.

Topics: Animals; Genes; Genetic Linkage; Humans; Mice; Molecular Biology; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

Ocular findings are presented in 17 unrelated patients with a form of autosomal dominant retinitis pigmentosa and the same C to A transversion in codon 23 of the rhodopsin gene. These patients (mean age, 36.6 years) had, on average, significantly better visual acuity and larger ERG amplitudes than 131 unrelated patients (mean age, 32.1 years) with autosomal dominant retinitis pigmentosa without this mutation. These 17 patients from separate families as well as 11 relatives with the mutation from 4 of these families showed interfamilial and intrafamilial variability with respect to severity of their ocular disease. This clinical heterogeneity among patients with the same mutation, with older patients sometimes showing less loss of visual function and less intraretinal bone spicule pigment than younger patients, suggests that some factor other than the gene defect itself is involved in the expression of this condition. This form of retinitis pigmentosa can now be detected by testing leukocyte DNA from peripheral blood. Patients so identified should have an ocular examination to determine the extent of their disease in view of the clinical heterogeneity that exists among patients with this mutation. Some mechanisms by which this mutation in the rhodopsin gene could lead to photoreceptor cell death are discussed. Opportunities for future clinical and laboratory research in search of possible treatments are considered.

Topics: Amino Acid Sequence; Base Sequence; DNA; Electroretinography; Fundus Oculi; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Pedigree; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

The diagnostic features and future research directions of retinitis pigmentosa were documented in this update and review of the subject. An extensive and current bibliography is provided.

Topics: Cell Survival; Electroretinography; Genetic Linkage; Humans; Night Blindness; Ophthalmoscopy; Retinitis Pigmentosa; Rhodopsin; Visual Fields; X Chromosome

Topics: Electrophysiology; Humans; Ophthalmoscopy; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Recent biochemical findings in the human disease, retinitis pigmentosa, and related retinal degenerative diseases in animals were reviewed and discussed. While the biochemical etiology of the human disease is not known, there are indications that retinal degeneration in the rd mouse and the Irish Setter dog are related to a deficiency in cGMP phosphodiesterase and the accumulation of cGMP in the photoreceptor outer segments. The biochemical basis of retinal degeneration in the Royal College of Surgeons (RCS) rat does not seem to be related to a defect in the metabolism of cGMP, but there are suggestions that a defect in retinoid metabolism may be involved. The possibility that the defect in RCS rats may involve receptors on the membranes of the cells of the retinal pigment epithelium or phagocytic markers on those of the rod outer segment disks was discussed.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Disease Models, Animal; Dogs; Eye Proteins; Guanylate Cyclase; Mice; Nucleotides, Cyclic; Rats; Retinitis Pigmentosa; Rhodopsin; Taurine; Vitamin A

Retinitis pigmentosa is a solitary manifestation of separate genetically determined disorders in which there is progressive loss of vision and the appearance of characteristic fundus abnormalities. It is likely that each disease contained within this family of disorders has a different aetiology, a consideration which is important to the clinician, the researcher and the therapist. To the clinician it is essential to be able to identify the inheritance of the disorder in order to give educated genetic advice. It is the responsibility of the clinician to sub-divide retinitis pigmentosa into purer samples of disease since without such a sub-division research is unlikely to be fruitful. It is unreasonable to expect a biochemist to define systemic biochemical abnormalities if blood is analysed from a series of patients, each of which has a different disorder. If the cause of retinal degeneration in an animal homologue of human retinitis pigmentosa is identified the question is then raised as to whether the abnormality is relevant to human disease and, if so, to which one. Moreover, if a metabolic abnormality is identified in one disease it will not necessarily to be found in others and similarly, if therapy is effective in one form of the disease it may not be effective for all patients. A sub-division of retinitis pigmentosa may be made on the basis of inheritance of the disorder, on the basis of morphologic changes in the fundus, and on the qualitative functional changes identified. Such observations may also give some clues as to the pathogenesis of the different forms of RP or at least indicate in which cell system the primary disorders lie and will also show in what way the defect interferes with cell function. In this paper it is hoped to show that studies undertaken by the various disciplines within ophthalmology have now made limited achievements towards the goals of subdividing retinitis pigmentosa (RP) into purer samples of disease and typifying the functional and morphological attributes of the constituent disorders.

Topics: Dark Adaptation; Electroretinography; Female; Humans; Male; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular

Topics: Chromosome Aberrations; Chromosome Disorders; Cyclic GMP; Electroretinography; Female; Humans; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin; Sex Chromosome Aberrations; Vision Disorders; Vitamin A Deficiency; X Chromosome

Topics: Animals; Cerebellum; Chimera; Circadian Rhythm; Conditioning, Classical; Drosophila; Flight, Animal; Humans; Locomotion; Membrane Potentials; Mice; Mosaicism; Muscular Dystrophy, Animal; Mutation; Nematoda; Nervous System Physiological Phenomena; Paramecium; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

There are no approved drug treatments for autosomal dominant retinitis pigmentosa, a relentlessly progressive cause of adult and childhood blindness.. To evaluate the potential efficacy and assess the safety of orally administered valproic acid (VPA) in the treatment of autosomal dominant retinitis pigmentosa.. Multicenter, phase 2, prospective, interventional, placebo-controlled, double-masked randomized clinical trial. The study took place in 6 US academic retinal degeneration centers. Individuals with genetically characterized autosomal dominant retinitis pigmentosa were randomly assigned to receive treatment or placebo for 12 months. Analyses were intention-to-treat.. Oral VPA 500 mg to 1000 mg daily for 12 months or placebo.. The primary outcome measure was determined prior to study initiation as the change in visual field area (assessed by the III4e isopter, semiautomated kinetic perimetry) between baseline and month 12.. The mean (SD) age of the 90 participants was 50.4 (11.6) years. Forty-four (48.9%) were women, 87 (96.7%) were white, and 79 (87.8%) were non-Hispanic. Seventy-nine participants (87.8%) completed the study (42 [95.5%] received placebo and 37 [80.4%] received VPA). Forty-two (46.7%) had a rhodopsin mutation. Most adverse events were mild, although 7 serious adverse events unrelated to VPA were reported. The difference between the VPA and placebo arms for mean change in the primary outcome was -150.43 degree2 (95% CI, -290.5 to -10.03; P = .035).. This negative value indicates that the VPA arm had worse outcomes than the placebo group. This study brings to light the key methodological considerations that should be applied to the rigorous evaluation of treatments for these conditions. This study does not provide support for the use of VPA in the treatment of autosomal dominant retinitis pigmentosa.. ClinicalTrials.gov Identifier: NCT01233609.

Topics: Administration, Oral; Adult; Aged; Anticonvulsants; Double-Blind Method; Electroretinography; Female; Humans; Male; Middle Aged; Mutation; Prospective Studies; Retina; Retinitis Pigmentosa; Rhodopsin; Valproic Acid; Vision Disorders; Visual Acuity; Visual Field Tests; Visual Fields

Retinal pigment epithelium (RPE) is a specialized pigmented monolayer dedicated to retinal support and protection. Given the fact that photoreceptor outer segments are the primary energy resource of RPE metabolism, it follows that, when photoreceptor function is compromised, RPE cells are impaired and vice versa. In retinitis pigmentosa (RP), genetic mutations lead to a massive degeneration of photoreceptors but only few studies have addressed systematically the consequences of rod and cone death on RPE cells, which, among others, undergo an abnormal organization of tight junctions (TJs) and a compromised barrier function. The biological mechanisms driving these barrier reorganizations are largely unknown. Studies aimed at addressing general and mutation-independent changes of the RPE in RP are relevant to reveal new pathogenic mechanisms of this heterogeneous family of diseases and prospectively develop effective therapeutic strategies. Here, we take advantage of a mouse model of RP in which retinal degeneration is spatially restricted to investigate a possible involvement of inflammatory responses in RPE remodeling. By immunostaining for Zona Occludens-1 (ZO-1), a structural and functional marker of TJs with pleiotropic functions, we found a partial rescue of TJs organization following local restoration of retinal organization, revealing that TJs structure can recover. Since lack of ZO-1 from TJs can alter cell density, we counted RPE cells without finding any differences between degenerated and controls animals, indicating preservation of RPE cells. However, we found an increased number of immune cells adhering to the RPE apical surface and a spatial correlation with areas of abnormal ZO-1 distribution. This suggests that inflammatory processes following photoreceptor degeneration can be responsible for TJs alterations during RP progression and deserve further investigation.

Topics: Animals; Epithelial Cells; Mice; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin

The pathogenic variant p.G90D in RHO is believed to be responsible for a spectrum of phenotypes, including congenital stationary blindness (for the purpose of this study termed night blindness without degeneration; NBWD), Sector RP, Pericentral RP, and Classic RP. We present a correlation between the serum concentration of vitamin A and disease severity in patients with this variant. This prospective study involved 30 patients from 7 families (17 male; median age 46 years, range 8−73). Full ophthalmological examination including visual acuity, Goldmann perimetry, slit-lamp exam, optical coherence tomography, fundus autofluorescence, and electrophysiology was performed to determine the presenting phenotype. The serum concentration of vitamin A was determined from a fasting blood sample taken on the day of the exam, where it was found that 23.3% (7/30) of patients had NBWD, 13.3% (4/30) had Sector RP, 3.3% (1/30) had Pericentral RP, and 60% (18/30) had Classic RP. Multiple logistic regression revealed a significantly higher probability of having a milder phenotype (NBWD or Sector RP) in association with younger age (p < 0.05) and a higher concentration of vitamin A (p < 0.05). We hypothesize that vitamin A in its 11-cis-retinal form plays a role in stabilizing the constitutively active p.G90D rhodopsin and its supplementation could be a potential treatment strategy for p.G90D RHO patients.

Topics: Electroretinography; Humans; Male; Mutation; Patient Acuity; Phenotype; Prospective Studies; Retinitis Pigmentosa; Rhodopsin; Vitamin A

To identify the genotypic and phenotypic characteristics of rhodopsin (RHO)-associated retinitis pigmentosa (RP) in the Japanese population.. Cross-sectional, single-center study METHODS: The medical records of 1336 patients with RP who underwent genetic testing at our clinic between November 2008 and September 2021 were reviewed, and patients with RHO variants were included. The patients were divided into class A and class B to assess genotype-phenotype correlations based on previous reports. The clinical findings, including best-corrected visual acuity (BCVA), OCT parameters (ellipsoid zone [EZ] width and central retinal thickness [CRT]), and presence of macular degeneration, of the 2 groups were compared.. The study included 28 patients diagnosed with RHO-associated RP (class A, 19; class B, 9). The BCVA was significantly worse in class A patients than in class B patients (P = 0.045). Superior EZ width was significantly shorter in class A than in class B patients (P = 0.016). Class A patients tended to have thinner CRT and shorter inferior EZ width than those of class B patients, although this difference was not significant. Macular degeneration was observed in 61.5% of class A and 12.5% of class B patients, demonstrating that macular degeneration can be a common complication in class A variants.. Patients with class A variants presented with a severer form of RP than that of patients with class B variants in the Japanese population. These results suggest that the phenotype of RHO-associated RP is linked to the location of the variants and that such a genotype-phenotype correlation is less affected by ethnicities with different genetic backgrounds.

Topics: Cross-Sectional Studies; East Asian People; Genotype; Humans; Macular Degeneration; Phenotype; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Topics: Animals; Disease Models, Animal; Dogs; Electroretinography; Humans; Mice; Mice, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering

Gene therapy for autosomal dominant retinitis pigmentosa (adRP) is challenged by the dominant inheritance of the mutant genes, which would seemingly require a combination of mutant suppression and wild-type replacement of the appropriate gene. We explore the possibility that delivery of a nanoparticle (NP)-mediated full-length mouse genomic rhodopsin (gRho) or human genomic rhodopsin (gRHO) locus can overcome the dominant negative effects of the mutant rhodopsin in the clinically relevant P23H

Topics: Animals; Disease Models, Animal; Genomics; Humans; Mice; MicroRNAs; Mutation; Nanoparticles; Retinitis Pigmentosa; Rhodopsin

To establish the proportion of patients with retinitis pigmentosa (RP) meeting the Australian fitness to drive (FTD) visual standards.. A prospective consecutive case series of patients with a clinical or genetic diagnosis of RP. Data on age at symptom onset, current driving status, inheritance pattern, better eye visual acuity (BEVA), binocular Esterman visual field (BEVF) parameters, genotype and ability to meet the driving standards based on BEVA and BEVF were collected. Outcome measures included the proportion of RP patients overall meeting the standards and clinical predictors for passing. A sub-analysis was performed on those RP patients who reported to drive. Change in BEVA and BEVF parameters across age in specific genotype groups was assessed.. Nearly 40% of RP patients met the driving standards. However, almost 50% of RP drivers were unaware of their failure to meet the current standards. BEVF testing is essential in the assessment of RP patients who are still driving. Phenotype and genotype predictors for passing the standards warrant further investigation.

Topics: Australia; Electroretinography; Eye Proteins; Frontotemporal Dementia; Humans; Mutation; Prospective Studies; Retinitis Pigmentosa; Rhodopsin

Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, we established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, we found that PERK signaling plays a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.

Topics: Animals; Autophagy; Drosophila; Drosophila Proteins; eIF-2 Kinase; Endoribonucleases; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Dogs; Electroretinography; Humans; Mice; Parvovirinae; Retina; Retinitis Pigmentosa; Rhodopsin

The unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration.. We used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H.. OCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation.. These data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.

Topics: Aging; Animals; Endoplasmic Reticulum Stress; Endoribonucleases; Mice; Protein Serine-Threonine Kinases; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Unfolded Protein Response

The need for robust and reliable animal models is a crucial step in studying any disease. This certainly applies to inherited retinal degenerative diseases, in which mutations of retinal specific genes result in photoreceptor cell death and subsequent visual loss. Animal models of retinal gene mutations have proven valuable to our understanding of disease mechanisms and as tools to evaluate therapeutic intervention strategies. Notable among these models are mice with a mutation of the rhodopsin gene at amino acid 23 in which proline is substituted for histidine (Rho-P23H). The RHO-P23H mutation is the most common cause of autosomal dominant retinitis pigmentosa. Here, we provide a brief review of the Rho-P23H mouse models currently available for research.

Topics: Animals; Disease Models, Animal; Mice; Mutation; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Most forms of outer retinal degenerative diseases involve the ectopic accumulation of microglia/macrophages in the subretinal space, including retinitis pigmentosa. However, their role in the loss of photoreceptor function during retinal degeneration remains unknown. Here, we examined the effect of conditional microglial depletion on photoreceptor numbers and visual function in mice with the rhodopsin P23H mutation, a dominant form of retinitis pigmentosa in humans. We found that microglial depletion led to an elevated level of rhodopsin and increased photoreceptor layer thickness. However, overall electrophysiological functions of the retina were reduced with microglial depletion. Therefore, these results identify an essential role of microglia specially in preserving visual function in outer retinal degeneration.

Topics: Animals; Disease Models, Animal; Humans; Mice; Microglia; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, we performed an unbiased in vivo RNAi screen with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1

Topics: Animals; Caspases; Drosophila; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Retinitis pigmentosa (RP) is a prevalent inherited retinal degenerative disease resulting from photoreceptor and pigment epithelial apoptosis. The

Topics: Apoptosis; Endoplasmic Reticulum Stress; Humans; Mutation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is a light-sensitive transmembrane receptor involved in the visual transduction cascade. Among the several rhodopsin mutations related to retinitis pigmentosa (RP), those affecting the C-terminal VAPA-COOH motif that is implicated in rhodopsin trafficking from the Golgi to the rod outer segment are notably associated with more aggressive RP forms. However, molecular reasons for defective rhodopsin signaling due to VAPA-COOH mutations, which might include steric hindrance, physicochemical features and structural determinants, are yet unknown, thus limiting further drug design approaches. In this work, clinically relevant rhodopsin mutations at the P347 site within the VAPA-COOH motif were investigated by molecular dynamics (MD) simulations and compared to the wild-type (WT) system. In agreement with experimental evidence, conformational fluctuations of the intrinsically disordered C-terminal tail of WT and mutant rhodopsin were found not to affect the overall structure of the transmembrane domain, including binding to the retinal cofactor. The WT VAPA-COOH motif adopts a unique conformation that is not found in pathological mutants, suggesting that structural features could better explain the pathogenicity of P347 rhodopsin mutants than physicochemical or steric determinants. These results were confirmed by MD simulations in both membrane-embedded full-length opsin and membrane-free C-terminal deca-peptides, these latter becoming very useful and small-size model systems for further investigations of rhodopsin C-terminal mutations. Structural details elucidated in this work might facilitate the understanding of the pathological mechanisms of this class of rhodopsin mutants, which will be instrumental to the development of new therapeutic strategies.

Topics: Humans; Molecular Conformation; Molecular Dynamics Simulation; Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Disease Models, Animal; Electroretinography; Humans; Mice; Mice, Transgenic; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Mutations in the photoreceptor protein rhodopsin are known as one of the leading causes of retinal degeneration in humans. Two rhodopsin mutations, Y102H and I307N, obtained in chemically mutagenized mice, are currently the subject of increased interest as relevant models for studying the process of retinal degeneration in humans. Here, we report on the biochemical and functional characterization of the structural and functional alterations of these two rhodopsin mutants and we compare them with the G90V mutant previously analyzed, as a basis for a better understanding of in vivo studies. This mechanistic knowledge is fundamental to use it for developing novel therapeutic approaches for the treatment of inherited retinal degeneration in retinitis pigmentosa. We find that Y102H and I307N mutations affect the inactive-active equilibrium of the receptor. In this regard, the mutations reduce the stability of the inactive conformation but increase the stability of the active conformation. Furthermore, the initial rate of the functional activation of transducin, by the I307N mutant is reduced, but its kinetic profile shows an unusual increase with time suggesting a profound effect on the signal transduction process. This latter effect can be associated with a change in the flexibility of helix 7 and an indirect effect of the mutation on helix 8 and the C-terminal tail of rhodopsin, whose potential role in the functional activation of the receptor has been usually underestimated. In the case of the Y102H mutant, the observed changes can be associated with conformational alterations affecting the folding of the rhodopsin intradiscal domain, and its presumed involvement in the retinal binding process by the receptor.

Topics: Animals; Cattle; Cell Line; Chlorocebus aethiops; COS Cells; HEK293 Cells; Humans; Mice; Models, Molecular; Molecular Dynamics Simulation; Mutation; Protein Folding; Protein Structure, Tertiary; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Arrestins; Disease Models, Animal; Electroretinography; Glucose; Lactic Acid; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The balanced homeostasis of the G protein-coupled receptor (GPCR), rhodopsin (Rho), is required for vision. Misfolding mutations in Rho cause photoreceptor death, leading to retinitis pigmentosa (RP) and consequently blindness. With no cure currently available, the development of efficient therapy for RP is an urgent need. Pharmacological supplementation with molecular chaperones, including flavonoids, improves stability, folding, and membrane targeting of the RP Rho mutants in vitro. Thus, we hypothesized that flavonoids by binding to P23H Rho and enhancing its conformational stability could mitigate detrimental effects of this mutation on retinal health. In this work, we evaluated the pharmacological potential of two model flavonoids, quercetin and myricetin, by using in silico, in vitro, and in vivo models of P23H Rho. Our computational analysis showed that quercetin could interact within the orthosteric binding pocket of P23H Rho and shift the conformation of its N-terminal loop toward the wild type (WT)-like state. Quercetin added to the NIH-3T3 cells stably expressing P23H Rho increased the stability of this receptor and improved its function. Systemic administration of quercetin to P23H Rho knock-in mice substantially improved retinal morphology and function, which was associated with an increase in levels of Rho and cone opsins. In addition, treatment with quercetin resulted in downregulation of the UPR signaling and oxidative stress-related markers. This study unravels the pharmacological potential of quercetin to slow down the progression of photoreceptor death in Rho-related RP and highlights its prospective as a lead compound to develop a novel therapeutic remedy to counter RP pathology.

Topics: Animals; Disease Models, Animal; Mice; Mutation; Prospective Studies; Quercetin; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Topics: Animals; Dietary Supplements; Mice; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Vitamin A

The P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal-dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has yet to be elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and wild-type (WT) rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin were reduced, but protein levels revealed selective degradation of the mutant protein. These results suggest that the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP. This article has an associated First Person interview with the first author of the paper.

Topics: Animals; Disease Models, Animal; Humans; Mice; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger

The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells.. Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression.. Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3.. Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

Topics: Cost-Benefit Analysis; DNA Mutational Analysis; Eye Proteins; Fibroblasts; Humans; Mutation; Pedigree; Phosphatidylinositol 3-Kinases; Retinitis Pigmentosa; Rhodopsin

Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.

Topics: Animals; Disease Models, Animal; Homeostasis; Mice; Molecular Chaperones; NIH 3T3 Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by gradual photoreceptor death, which lacks a definitive treatment. Here, we demonstrated the effect of 4-phenylbutyric acid (PBA), a chemical chaperon that can suppress endoplasmic reticulum (ER) stress, in P23H mutant rhodopsin knock-in RP models. In the RP models, constant PBA treatment led to the retention of a greater number of photoreceptors, preserving the inner segment (IS), a mitochondrial- and ER-rich part of the photoreceptors. Electroretinography showed that PBA treatment preserved photoreceptor function. At the early point, ER-associated degradation markers,

Topics: Epigenesis, Genetic; Humans; Mitochondria; Molecular Chaperones; Organelle Biogenesis; Retinitis Pigmentosa; Rhodopsin

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Michael Robichaux is first author on ' Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa', published in DMM. Michael conducted the research described in this article while a postdoctoral fellow in Ted Wensel's lab at Baylor College of Medicine, Houston, TX, USA. He is now an assistant professor at West Virginia University, Morgantown, WV, USA, investigating the intricate subcellular processes in photoreceptor neurons that enable vision.

Topics: Animals; Disease Models, Animal; Humans; Male; Mice; Retinitis Pigmentosa; Rhodopsin

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with a natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilize rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused by either bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.

Topics: Animals; Biological Products; Cattle; Ligands; Mice; Receptors, G-Protein-Coupled; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerations and pathogenic variants in the

Topics: DNA; Gene Editing; Genes, Dominant; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin; Staphylococcus aureus

Daily biological rhythms are fundamental to retinal physiology and visual function. They are generated by a local circadian clock composed of a network of cell type/layer-specific, coupled oscillators. Animal models of retinal degeneration have been instrumental in characterizing the anatomical organization of the retinal clock. However, it is still unclear, among the multiple cell-types composing the retina, which ones are essential for proper circadian function. In this study, we used a previously well-characterized mouse model for autosomal dominant retinitis pigmentosa to examine the relationship between rod degeneration and the retinal circadian clock. This model carries the P23H mutation in rhodopsin, which induces mild rod degeneration in heterozygous and rapid loss of photoreceptors in homozygous genotypes. By measuring PER2::LUC bioluminescence rhythms, we show that the retinal clock in

Topics: Animals; Circadian Clocks; Circadian Rhythm; Mice; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Both PRPF31 and PRPH2 are the causative genes for retinitis pigmentosa. And both of them are associated with the balance of rhodopsin. In this study, we aim to investigate the co-expression and interaction of PRPF31 and PRPH2. We used PRPF31-eGFP, PRPF31-3xFlag and PRPH2-mCherry vectors were transfected into HEK293T and APRE-19 cells. Immunoblotting and co-immunoprecipitation (Co-IP) were used for gene expression validation and protein interaction. Immunofluorescence staining assay was used to test the co-localization analysis of PRPF31 and PRPH2. Co-IP experiments showed that PRPF31 could be pulled down with an anti-PRPH2 antibody. There was co-localization between PRPF31 and PRPH2 in HEK293T, APRE-19 and mouse retina. The Co-IP and co-localization experiments suggest that PRPF31 interacted with PRPH2.

Topics: Animals; Eye Proteins; HEK293 Cells; Humans; Immunoprecipitation; Mice; Mutation; Pedigree; Peripherins; Retinitis Pigmentosa; Rhodopsin

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases.

Topics: Animals; Cysteine; Dogs; Eye Proteins; Humans; Membrane Proteins; Mice; Mutation; Protein Stability; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders characterized by photoreceptor degeneration. The rhodopsin gene (RHO) is the most frequent cause of autosomal dominant RP (ADRP), yet it remains unclear how RHO mutations cause heterogeneous phenotypes. Energy failure is a main cause of the secondary cone death during RP progression; however, its role in primary rod death induced by ADRP RHO mutants is unknown.. Three RHO missense mutations were chosen from different clinical classes. Wild-type (WT) RHO and its mutants, P23H (class B1), R135L (class A), and G188R (class B2), were overexpressed in 661w cells, a mouse photoreceptor cell line, and their effects on oxidative phosphorylation (OXPHOS) and aerobic glycolysis were compared separately.. Here, we report that energy failure is an early event in the cell death caused by overexpression of WT RHO and its mutants. RHO overexpression leads to OXPHOS deficiency, which might be a result of mitochondrial loss. Nonetheless, only in WT RHO and P23H groups, energy stress triggers AMP-activated protein kinase activation and metabolic reprogramming to increase glycolysis. Metabolic reprogramming impairment in R135L and G188R groups might be the reason why energy failure and cell injury are much more severe in those groups.. Our results imply that overexpression of RHO missense mutants have distinct impacts on the two energy metabolic pathways, which might be related to their heterogeneous phenotypes.

Topics: Animals; Energy Metabolism; Mice; Mutation; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Proteins are integral part of all living beings, which are building blocks of many amino acids. To be functionally active, amino acids chain folds up in a complex way to give each protein a unique 3D shape, where a minor error may cause misfolded structure. Genetic disorder diseases i.e. Alzheimer, Parkinson, etc. arise due to misfolding in protein sequences. Thus, identifying patterns of amino acids is important for inferring protein associated genetic diseases. Recent studies in predicting amino acids patterns focused on only simple protein misfolded disease i.e. Chromaffin Tumor, by association rule mining. However, more complex diseases are yet to be attempted. Moreover, association rules obtained by these studies were not verified by usefulness measuring tools.. In this work, we analyzed protein sequences associated with complex protein misfolded diseases (i.e. Sickle Cell Anemia, Breast Cancer, Cystic Fibrosis, Nephrogenic Diabetes Insipidus, and Retinitis Pigmentosa 4) by association rule mining technique and objective interestingness measuring tools. Experimental results show the effectiveness of our method.. Adopting quantitative experimental methods, this work can form more reliable, useful and strong association rules i. e. dominating patterns of amino acid of complex protein misfolded diseases. Thus, in addition to usual applications, the identified patterns can be more useful in discovering medicines for protein misfolded diseases and thereby may open up new opportunities in medical science to handle genetic disorder diseases.

Topics: Amino Acid Sequence; Amino Acids; Humans; Retinitis Pigmentosa; Rhodopsin

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.

Topics: Animals; Cone Dystrophy; Cone Opsins; Female; Genes, X-Linked; Humans; Male; Mice; Mutation, Missense; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Topics: Genetic Therapy; Humans; Retinitis Pigmentosa; Rhodopsin

Rhodopsin (RHO) is the most well-known genetic cause of autosomal dominant retinitis pigmentosa (adRP). This study aimed to investigate the genetic cause of a large Chinese adRP family and assess the pathogenicity of the detected RHO mutant.. Routine ocular examinations were conducted on all participants. Next-generation sequencing with targeted capture was performed to screen mutations in 179 genes associated with hereditary retinal diseases and 10 candidate genes. Variants detected by NGS were validated by Sanger sequencing and evaluated for pathogenicity. Fragments of mutant and wild-type RHO were cloned into the pEGFP-N1 vector and were transfected into different cell lines to observe the cellular localization of the Rhodopsin-GFP fusion protein and evaluate the expression of endoplasmic reticulum (ER) stress markers. RT-PCR analysis was used to detect transfected the splicing of X box-binding protein 1 (XBP1) mRNA, which is a critical factor affecting ER stress.. Genetic analysis identified a heterozygous missense variant, RHO, c.284 T > C (p.L95P) in this adRP family. Another RHO variant (p.P53R) that we reported previously was also included in further functional assessment. Both misfolded mutant proteins accumulated in the ER in a manner similar to that noted for the classic mutant P23H. Spliced XBP1 was observed in cells transfected with mutants, indicating an increase in ER stress.. Although the p.L95P variant is not a novel change, it was the first variant to be functionally evaluated and reported in Chinese RP patients. The results in our study provide significant evidence to classify the p.L95P mutation as a class II mutation.

Topics: Endoplasmic Reticulum Stress; High-Throughput Nucleotide Sequencing; Humans; Pedigree; Retinitis Pigmentosa; Rhodopsin

Rhodopsin (RHO) is a light-sensitive pigment in the retina and the main prototypical protein of the G-protein-coupled receptor (GCPR) family. After receiving a light stimulus, RHO and its cofactor retinylidene undergo a series of structural changes that initiate an intricate transduction mechanism. Along with RHO, other partner proteins play key roles in the signaling pathway. These include transducin, a GTPase, kinases that phosphorylate RHO, and arrestin (Arr), which ultimately stops the signaling process and promotes RHO regeneration. A large number of RHO genetic mutations may lead to very severe retinal dysfunction and eventually to impaired dark adaptation disease called autosomal dominant retinitis pigmentosa (adRP). In this study, we used molecular dynamics (MD) simulations to evaluate the different behaviors of the dimeric form of wild-type RHO (WT dRHO) and its mutant at position 135 of arginine to leucine (dR135L), both in the free (noncomplexed) and in complex with the transducin-like protein (Gtl). Gtl is a heterotrimeric model composed of a mixture of human and bovine G proteins. Our calculations allow us to explain how the mutation causes structural changes in the RHO dimer and how this can affect the signal that transducin generates when it is bound to RHO. Moreover, the structural modifications induced by the R135L mutation can also account for other misfunctions observed in the up- and downstream signaling pathways. The mechanism of these dysfunctions, together with the transducin activity reduction, provides structure-based explanations of the impairment of some key processes that lead to adRP.

Topics: Animals; Cattle; Humans; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Transducin

Sector and pericentral are two rare, regional forms of retinitis pigmentosa (RP). While usually defined as stable or only very slowly progressing, the available literature to support this claim is limited. Additionally, few studies have analyzed the spectrum of disease within a particular genotype. We identified all cases (9 patients) with an autosomal dominant Rhodopsin variant previously associated with sector RP (

Topics: Adolescent; Adult; Codon; Female; Fundus Oculi; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Phenotype; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Field Tests; Visual Fields

Inherited retinal degenerations (IRD) are a leading cause of visual impairment and can result from mutations in any one of a multitude of genes. Mutations in the light-sensing protein rhodopsin (RHO) is a leading cause of IRD with the most common of those being a missense mutation that results in substitution of proline-23 with histidine. This variant, also known as P23H-RHO, results in rhodopsin misfolding, initiation of endoplasmic reticulum stress, the unfolded protein response, and activation of cell death pathways. In this study, we investigate the effect of α-crystallins on photoreceptor survival in a mouse model of IRD secondary to P23H-RHO. We find that knockout of either αA- or αB-crystallin results in increased intraretinal inflammation, activation of apoptosis and necroptosis, and photoreceptor death. Our data suggest an important role for the ⍺-crystallins in regulating photoreceptor survival in the P23H-RHO mouse model of IRD.

Topics: Animals; Apoptosis; Cell Death; Crystallins; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Unfolded Protein Response

Mutations in RHO are the most common cause of autosomal dominant retinitis pigmentosa. However, the pathogenicity of many RHO variants is questionable. This study was designed to investigate the genotype-phenotype correlation for RHO variants. These RHO variants were collected from the in-house exome sequencing data of 7092 probands suffering from different types of eye conditions. The variants were classified using bioinformatics tools, family segregation, and clinical phenotypes. The RHO variants were assessed using multiple online tools and a genotype-phenotype analysis based on the data collected from of ours, gnomAD, and published literature. Totally, 52 heterozygous variants of RHO were detected in the 7092 probands. Of these 52, 17 were potentially pathogenic, were present in 35 families, and comprised 15 missense variants, one inframe deletion and one nonsense variant. All the 15 missense variants were predicted to be damaging by five different online tools. The analysis of the clinical data of the patients from the 35 families revealed certain common features, of an early damage to both the rods and the cones, relatively preserved visual acuity in adulthood, and mid-peripheral tapetoretinal degeneration with pigmentation or RPE atrophy. Our data, the data from gnomAD, and the systematic review of the 246 previously reported variants suggest that approximately two-thirds of the rare missense variants and most of the truncated variants involving upstream of K296 are likely benign. This study provides a brief summary of the characteristics of the pathogenic RHO variants. It emphasizes that the systematic evaluation of these variants at the individual-gene level is crucial in the current era of clinical genetic testing even for a well-known gene such as RHO.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Codon, Nonsense; Exome Sequencing; Female; Genetic Association Studies; Genetic Testing; Humans; Male; Middle Aged; Mutation Rate; Mutation, Missense; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion; Visual Acuity; Young Adult

We describe both phenotype and pathogenesis in two male siblings with typical retinitis pigmentosa (RP) and the potentially X-linked RP (XLRP) carrier phenotype in their mother. Two affected sons, two unaffected daughters, and their mother underwent detailed ophthalmological assessments including Goldmann perimetry, color vision testing, multimodal imaging and ISCEV-standard electroretinography. Genetic testing consisted of targeted next-generation sequencing (NGS) of known XLRP genes and whole exome sequencing (WES) of known inherited retinal disease genes (RetNet-WES). Variant validation and segregation analysis were performed by Sanger sequencing. The mutational load of the RHO variant in the mother was assessed in DNA from leucocytes, buccal cells and hair follicles using targeted NGS. Both affected sons showed signs of classical RP, while the mother displayed patches of hyperautofluorescence on blue light autofluorescence imaging and regional, intraretinal, spicular pigmentation, reminiscent of a carrier phenotype of XLRP. XLRP testing was negative. RetNet-WES testing revealed RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (21% of the reads) in the mother and in a heterozygous state in both sons. Targeted NGQSS of the RHO variant in different maternal tissues showed a mutation load between 25.06% and 41.72%. We report for the first time that somatic mosaicism of RHO variant c.404G > C p.(Arg135Pro) mimics the phenotype of a female carrier of XLRP, in combination with heterozygosity for the variant in the two affected sons.

Topics: Adult; Base Sequence; Female; Gene Dosage; Heterozygote; Humans; Male; Middle Aged; Mosaicism; Phenotype; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Young Adult

Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.

Topics: Alleles; Animals; Cell Line; CRISPR-Cas Systems; Dependovirus; Disease Models, Animal; Electroretinography; Gene Editing; Genetic Therapy; Humans; INDEL Mutation; Mice; Mice, Transgenic; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retina; Retinitis Pigmentosa; Rhodopsin

To describe clinical and genetic features in a series of Italian patients with sector retinitis pigmentosa (sector RP).. Fifteen patients with sector RP were selected from the database of Hereditary Retinal Degenerations Referring Center of Careggi Hospital (Florence, Italy). Eleven patients from five independent pedigrees underwent genetic analysis with next-generation sequencing (NGS) confirmed with Sanger sequencing. The diagnosis of sector RP was based on the detection of topographically limited retinal abnormalities consistent with corresponding sectorial visual field defects. Best-corrected visual acuity (BCVA), fundus color pictures as well as fundus autofluorescence (FAF), spectral domain-optical coherence tomography (SD-OCT), full-field electroretinography (ERG), and 30-2 Humphrey visual field (VF) data were retrospectively collected and analyzed.. For the 30 eyes, the mean BCVA was 0.05 ± 0.13 logMAR, and the mean refractive error was -0.52 ± 1.89 D. The inferior retina was the most affected sector (86.7%), and the VF defect corresponded to the affected sector. FAF showed a demarcation line of increased autofluorescence between the healthy and affected retina, corresponding on SD-OCT to an interruption of the ellipsoid zone (EZ) band in the diseased retina. Dark-adapted ERG amplitudes were decreased in comparison to normative values. In five unrelated families, the sector RP phenotype was associated with sequence variants in the. Typical sector RP is a mild form of RP characterized by preserved visual acuity with limited retinal involvement and, generally, a more favorable prognosis than other forms of RP.

Topics: Adult; Aged; Dark Adaptation; Electroretinography; Female; High-Throughput Nucleotide Sequencing; Humans; Italy; Male; Middle Aged; Pedigree; Phenotype; Refraction, Ocular; Retina; Retinitis Pigmentosa; Retrospective Studies; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; Visual Field Tests; Visual Fields; Young Adult

Mechanisms contributing to the progression of autosomal dominant retinitis pigmentosa (adRP) due to the P23H rhodopsin mutation are complex and diverse. Previous studies showed that mechanisms like endoplasmic reticulum (ER) stress, pyroptosis, and oxidative stress were involved in the pathogenesis of the disease. However, the roles and relationships of different mechanisms are not precisely known. In this study, we aimed to evaluate certain mechanisms and find novel genes involved in P23H-related adRP.. Total RNA extracted at postnatal day (PN) 14, PN21, and PN35 was used for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses were conducted for RNA-seq data. Additionally, data from the clustered regularly interspaced short palindromic repeats (CRISPR) screening library and the RNA-seq data of several mechanisms were used for generating custom gene sets for gene set enrichment analysis (GSEA). Next, we obtained the intersection of the aforementioned gene sets and our RNA-seq data to identify candidate genes, which were verified using real-time quantitative PCR (qPCR).. Functional enrichment analyses were consistent with disease phenotypes. All time points observed pyroptosis. In the results of GSEA, ER stress, pyroptosis, and oxidative stress were observed at PN14. ER stress and pyroptosis were shown on PN35. A total of 22 candidate genes were identified. The expression levels of selected genes verified by qPCR were concordant with the RNA-seq data.. In our study, we conclude that pyroptosis and ER stress might play a central role in RP progression. We also identified differentially expressed gene clusters related to ER stress and pyroptosis, which deserve further study. These findings provide a novel perspective for the investigation of P23H-related adRP.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Mice; Retinitis Pigmentosa; Rhodopsin; RNA-Seq

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

Topics: Dimerization; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humans; Micelles; Molecular Dynamics Simulation; Opsins; Retinitis Pigmentosa; Rhodopsin

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Topics: Animals; Dexamethasone; Disease Models, Animal; Evaluation Studies as Topic; Inflammation; Mice; Mice, Inbred C57BL; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Tight Junctions; Zonula Occludens-1 Protein

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients.

Topics: Animals; Humans; Mutation; Protein Conformation; Receptors, G-Protein-Coupled; Retinitis Pigmentosa; Rhodopsin; Valproic Acid

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Topics: Animals; Dependovirus; Disease Models, Animal; Female; Gene Expression; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Transcription Factors; Transcriptome; Zinc Fingers

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Topics: Animals; Animals, Genetically Modified; Behavior, Animal; Cell Line; Drug Evaluation, Preclinical; Genetic Diseases, Inborn; Humans; Mutation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transgenes; Vision, Ocular; Zebrafish

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

Topics: Animals; Chemokine CCL2; Female; Gene Expression Profiling; Gene Regulatory Networks; Glial Fibrillary Acidic Protein; GTP-Binding Proteins; Male; Mice; Mice, Transgenic; Neuroglia; Neuroprotection; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Environmental changes in the retina, including oxidative stress-induced cell death, influence photoreceptor degeneration in Retinitis Pigmentosa (RP). Previously, we tested and discovered that a cytoprotective chaperone protein, clusterin, produced robust preservation of rod photoreceptors of a rat autosomal dominant rhodopsin transgenic model of RP, S334ter-line3. To investigate the biochemical and molecular cytoprotective pathways of clusterin, we examined and compared a known source of cone cell death, nitric oxide (NO), observing nNOS expression using antibody against nNOS in RP retinas with intravitreal injections of saline, clusterin (10 μg/ml), or a non-isoform-selective NOS inhibitor (25 mM), L-NAME, or with an intraperitoneal injection (IP) of L-NAME (100 mg/kg). Rhodopsin-immunoreactive rod photoreceptor cells and nNOS-immunoreactive cells were quantified with immunohistochemistry in the presence or absence of L-NAME or clusterin, and the total nNOS retinal expression was determined by immunoblot analysis. In this study, the level of nNOS expression was significantly up-regulated postnatally (P) at P15 (P < 0.05), P30 (P < 0.001) and P60 (P < 0.0001) in RP retinas compared to normal controls. Clusterin treatment suppressed the up-regulated nNOS expression in RP retinas (P < 0.0001) and was enhanced in Type II amacrine cells. Additionally, IP injection of L-NAME at P15 prolonged rod survival in the later stage of RP retinas (P < 0.001). Conversely, rod survival in L-NAME-treated RP retinas was not equivalent to the rod survival number seen in clusterin-treated retinas, which suggests induction of nNOS expression in RP retinas and its reduction by clusterin is only partly responsible for the rescue observed through the reduction of nNOS expression in S334ter-line3 rat retinas.

Topics: Animals; Cell Survival; Clusterin; Disease Models, Animal; Gene Expression; Gene Expression Regulation; Male; Neurons; Nitric Oxide Synthase; Oxidative Stress; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa type 45 (RP45) is an autosomal-recessively inherited blinding disease caused by mutations in the cyclic nucleotide-gated channel subunit beta 1 (

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Dependovirus; Genetic Therapy; Mice; Mice, Knockout; Nerve Tissue Proteins; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho

Topics: Activating Transcription Factor 6; Animals; Disease Models, Animal; Female; Homeostasis; Male; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a Rho

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Knockdown Techniques; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Mice; MicroRNAs; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA; RNA Interference; RNA Splicing

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Topics: Amino Acid Substitution; Animals; Antirheumatic Agents; Disease Models, Animal; Endothelium, Vascular; Gene Expression; Gene Knock-In Techniques; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinases; Leukemia Inhibitory Factor; Mice; Mice, Transgenic; Microglia; Mutation; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; STAT Transcription Factors; Transgenes; Tumor Necrosis Factor-alpha

Nerve growth factor (NGF) is a neuroprotective molecule performing not only on central and peripheral neurons but also on cells of the visual system. Human retinitis pigmentosa (RP) is a major cause of blindness worldwide, and a resolute therapy is still lacking. Recent studies have shown that ocular NGF administration exerts a protective action on damaged retinal cells of mammalians, including human beings, although whether NGF also protects photoreceptors is not clear.We used the Royal College of Surgeons (RCS) strain in this study. The RCS is a rodent affected by inherited retinitis pigmentosa (RP) during postnatal life. For this study, we investigated whether ocular NGF treatment reduces/stops the progression of photoreceptor degeneration of rats with RP.This study was carried out in vitro on isolated photoreceptors to further investigate the action on these cells and whether the action is direct or mediated.The results indicate that ocular NGF administration can protect photoreceptors from degeneration into a model developing inherited RP and that the NGF action is direct. In this regard, we observed that binding of NGF to its receptor modulates expression of rhodopsin, a specific biological marker for photoreceptor survival and functionality.Part of the data reported in this chapter has been published in a previous study.

Topics: Animals; Disease Models, Animal; Nerve Growth Factor; Photoreceptor Cells; Rats; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration.

Topics: Animals; Disease Models, Animal; Mice; Mice, Mutant Strains; Mutation; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin

The c.620 T > G mutation in rhodopsin found in the first mapped autosomal dominant retinitis pigmentosa (adRP) locus is associated with severe, early-onset RP. Intriguingly, another mutation affecting the same nucleotide (c.620 T > A) is related to a mild, late-onset RP. Assuming that both mutations are missense mutations (Met207Arg and Met207Lys) hampering the ligand-binding pocket, previous work addressed how they might differentially impair rhodopsin function. Here, we investigated the impact of both mutations at the mRNA and protein level in HEK293 cells and in the mouse retina. We show that, in contrast to c.620 T > A, c.620 T > G is a splicing mutation, which generates an exceptionally strong splice acceptor site (SAS) resulting in a 90 bp in-frame deletion and protein mislocalization in vitro and in vivo. Moreover, we identified the core element underlying the c.620 T > G SAS strength. Finally, we demonstrate that the c.620 T > G SAS is very flexible in branch point choice, which might explain its remarkable performance. Based on these results, we suggest that (i) point mutations should be routinely tested for mRNA splicing to avoid dispensable analysis of mutations on protein level, which do not naturally exist. (ii) Puzzling disease courses of mutations in other genes might also correlate with their effects on mRNA splicing. (iii) Flexibility in branch point choice might be another factor influencing the SAS strength. (iv) The core splice element identified in this study could be useful for biotechnological applications requiring effective SAS.

Topics: Animals; HEK293 Cells; Humans; Mice; Mutation, Missense; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin; RNA Splice Sites; RNA Splicing

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Expression; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Injections, Intraocular; Mice; Mice, Knockout; Mice, Transgenic; Ophthalmoscopy; Parvovirinae; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Staining and Labeling; Tomography, Optical Coherence

The use of gene therapy may allow replacement of the defective gene. Minigenes, such as cDNAs, are often used. However, these may not express normal physiological genetic profiles due to lack of crucial endogenous regulatory elements. We constructed DNA nanoparticles (NPs) that contain either the mouse or human full-length rhodopsin genomic locus, including endogenous promoters, all introns, and flanking regulatory sequences of the 15-16 kb genomic rhodopsin DNA inserts. We transduced the NPs into primary retinal cell cultures from the rhodopsin knockout (RKO) mouse in vitro and into the RKO mouse in vivo and compared the effects on different functions to plasmid cDNA NP counterparts that were driven by ubiquitous promoters. Our results demonstrate that genomic DNA vectors resulted in long-term high levels of physiological transgene expression over a period of 5 months. In contrast, the cDNA counterparts exhibited low levels of expression with sensitivity to the endoplasmic reticulum (ER) stress mechanism using the same transgene copy number both in vitro and in vivo. This study demonstrates for the first time the transducing of the rhodopsin genomic locus using compacted DNA NPs.

Topics: Animals; Disease Models, Animal; DNA; Endoplasmic Reticulum Stress; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Humans; Mice; Mice, Knockout; Nanoparticles; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transgenes

Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αβγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.

Topics: Animals; Cell Death; cis-trans-Isomerases; Cyclic GMP; Disease Models, Animal; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transducin

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease.

Topics: Adolescent; Adult; Aged; Animals; Autophagy; Disease Models, Animal; Electroretinography; Extracellular Matrix Proteins; Female; Genetic Association Studies; Genotype; Hearing Loss, Sensorineural; Humans; Male; Middle Aged; Mutation; Opsins; Retina; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Usher Syndromes; Visual Acuity; Young Adult; Zebrafish

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

Topics: Animals; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum; Gene Knock-In Techniques; HSP40 Heat-Shock Proteins; Mice; Mice, Knockout; Molecular Chaperones; Mutation; Photoreceptor Cells, Vertebrate; Rats; Retina; Retinitis Pigmentosa; Rhodopsin; Transfection

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of

Topics: Animals; Armadillo Domain Proteins; Cytoskeletal Proteins; Disease Models, Animal; Electroretinography; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NAD; Photoreceptor Cells; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

To study cone structure and function in patients with retinitis pigmentosa (RP) owing to mutations in rhodopsin (RHO), expressed in rod outer segments, and mutations in the RP-GTPase regulator (RPGR) gene, expressed in the connecting cilium of rods and cones.. Four eyes of 4 patients with RHO mutations, 5 eyes of 5 patients with RPGR mutations, and 4 eyes of 4 normal subjects were studied. Cone structure was studied with confocal and split-detector adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral-domain optical coherence tomography. Retinal function was measured using a 543-nm AOSLO-mediated adaptive optics microperimetry (AOMP) stimulus. The ratio of sensitivity to cone density was compared between groups using the Wilcoxon rank-sum test.. AOMP sensitivity/cone density in patients with RPGR mutations was significantly lower than normal (P < 0.001) and lower than patients with RHO mutations (P < 0.015), whereas patients with RHO mutations were similar to normal (P > 0.9).. Retinal sensitivity/cone density was lower in patients with RPGR mutations than normal and lower than patients with RHO mutations, perhaps because cones express RPGR and degenerate primarily, whereas cones in eyes with RHO mutations die secondary to rod degeneration. High-resolution microperimetry can reveal differences in cone degeneration in patients with different forms of RP.

Topics: Adult; Age Factors; Case-Control Studies; Electroretinography; Eye Proteins; Female; Gene Expression Regulation; Humans; Male; Mutation; Ophthalmoscopy; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Risk Assessment; Scanning Laser Polarimetry; Sex Factors; Statistics, Nonparametric; Tomography, Optical Coherence; Young Adult

Retinitis pigmentosa is a retinal degenerative disease that leads to blindness through photoreceptor loss. Rhodopsin is the most frequently mutated protein in this disease. While many rhodopsin mutations have well-understood consequences that lead to cell death, the disease association of several rhodopsin mutations identified in retinitis pigmentosa patients, including F220C and F45L, has been disputed. In this study, we generated two knockin mouse lines bearing each of these mutations. We did not observe any photoreceptor degeneration in either heterozygous or homozygous animals of either line. F220C mice exhibited minor disruptions of photoreceptor outer segment dimensions without any mislocalization of outer segment proteins, whereas photoreceptors of F45L mice were normal. Suction electrode recordings from individual photoreceptors of both mutant lines showed normal flash sensitivity and photoresponse kinetics. Taken together, these data suggest that neither the F220C nor F45L mutation has pathological consequences in mice and, therefore, may not be causative of retinitis pigmentosa in humans.

Topics: Animals; Electrodes; Kinetics; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Adult; DNA Mutational Analysis; Electroretinography; Female; Fluorescein Angiography; Fundus Oculi; Humans; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin

Rhodopsin mutation and misfolding is a common cause of autosomal dominant retinitis pigmentosa (RP). Using a luciferase reporter assay, we undertook a small-molecule high-throughput screening (HTS) of 68, 979 compounds and identified nine compounds that selectively reduced the misfolded P23H rhodopsin without an effect on the wild type (WT) rhodopsin protein. Further, we found five of these compounds, including methotrexate (MTX), promoted P23H rhodopsin degradation that also cleared out other misfolded rhodopsin mutant proteins. We showed MTX increased P23H rhodopsin degradation via the lysosomal but not the proteasomal pathway. Importantly, one intravitreal injection (IVI) of 25 pmol MTX increased electroretinogram (ERG) response and rhodopsin level in the retinae of Rho

Topics: Animals; Cell Line; Electroretinography; Female; HEK293 Cells; Humans; Male; Mice; Mutant Proteins; Mutation; NIH 3T3 Cells; Photoreceptor Cells; Proteasome Endopeptidase Complex; Protein Folding; Retina; Retinitis Pigmentosa; Rhodopsin

A common inherited retinal disease is caused by mutations in RHO expressed in rod photoreceptors that provide vision in dim ambient light. Approximately half of all RHO mutations result in a Class B phenotype where mutant rods are retained in some retinal regions but show severe degeneration in other regions. We determined the natural history of dysfunction and degeneration of retained rods by serially evaluating patients. Even when followed for more than 20 years, rod function and structure at some retinal locations could remain unchanged. Other locations showed loss of both vision and photoreceptors but the rate of rod vision loss was greater than the rate of photoreceptor degeneration. This unexpected divergence in rates with disease progression implied the development of a rod function deficit beyond loss of cells. The divergence of progression rates was also detectable over a short interval of 2 years near the health-disease transition in the superior retina. A model of structure-function relationship supported the existence of a large rod function deficit which was also most prominent near regions of health-disease transition. Our studies support the realistic therapeutic goal of improved night vision for retinal regions specifically preselected for rod function deficit in patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Female; Humans; Male; Middle Aged; Mutation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular; Young Adult

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in

Topics: Animals; Cell Line; Disease Models, Animal; HEK293 Cells; Humans; Light Signal Transduction; Mice; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cells expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed this issue in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network, reminiscent of the neurodevelopmental state, and potentiation of rod - rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. These results suggest retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

Topics: Animals; Cells, Cultured; Female; Male; Mice; Mice, Inbred C57BL; Mutation; Neuronal Plasticity; Night Vision; Retina; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; Transcriptome

Retinitis pigmentosa (RP) is a group of blinding disorders caused by diverse mutations, including in rhodopsin (RHO). Effective therapies have yet to be discovered. The I307N Rho mouse is a light-inducible model of autosomal dominant RP. Our purpose was to describe the glial response in this mouse model to educate future experimentation. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes to induce retinal degeneration. Immunofluorescence staining of cross-sections and flat-mounts was performed to visualize the response of microglia and Müller glia. Histology was correlated with spectral-domain optical coherence tomography imaging (SD-OCT). Microglia dendrites extended between photoreceptors within two hours of induction, withdrew their dendrites between twelve hours and one day, appeared ameboid by three days, and assumed a ramified morphology by one month. Glial activation was more robust in the inferior retina and modulated across the boundary of light damage. SD-OCT hyper-reflectivity overlapped with activated microglia. Finally, microglia transiently adhered to the RPE before which RPE cells appeared dysmorphic. Our data demonstrate the spatial and temporal pattern of glial activation in the I307N Rho mouse, and correlate these patterns with SD-OCT images, assisting in interpretation of SD-OCT images in preclinical models and in human RP.

Topics: Animals; Dendrites; Disease Models, Animal; Genes, Dominant; Humans; Mice; Microglia; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

Retinitis pigmentosa (RP) is a collection of heterogenous genetic retinal disorders resulting in cumulative retinal deterioration involving progressive loss of photoreceptors and eventually in total blindness. Oxidative stress plays a central role in this photoreceptor loss. Gypenosides (Gyp) are the main functional component isolated from the climbing vine Gynostemma pentaphyllum and have been shown to defend cells against the effects of oxidative stress and inflammation, providing protection in experimentally-induced optic neuritis. The zebrafish model has been used to investigate a range of human diseases. Previously we reported early retinal degeneration in a mutant zebrafish line carrying a point-nonsense mutation in the retinitis pigmentosa GTPase regulator interacting protein 1 (rpgrip1) gene that is mutated in RP patients. The current study investigated the potential protective effects of Gyp against photoreceptor degeneration in the Rpgrip1 deleted zebrafish. Rpgrip1 mutant zebrafish were treated with 5 μg/ml of Gyp in E3 medium from 6 h post fertilization (hpf) till 1 month post fertilization (mpf). Rpgrip1 mutant zebrafish treated with 5 μg/ml of Gyp showed a significant decrease by 68.41% (p = 0.0002) in photoreceptor cell death compared to that of untreated mutant zebrafish. Expression of antioxidant genes catalase, sod1, sod2, gpx1, gclm, nqo-1 and nrf-2 was significantly decreased in rpgrip1 mutant zebrafish eyes by 61.51%, 77.40%, 60.11%, 81.17%, 72.07%, 78.95% and 85.42% (all p < 0.0001), respectively, when compared to that of wildtype zebrafish; superoxide dismutase and catalase activities, and glutathione levels in rpgrip1 mutant zebrafish eyes were significantly decreased by 87.21%, 21.55% and 96.51% (all p < 0.0001), respectively. There were marked increases in the production of reactive oxygen species (ROS) and malondialdehyde (MDA) by 2738.73% and 510.69% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes; expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α was also significantly increased by 150.11%, 267.79% and 190.72% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes, compared to that of wildtype zebrafish. Treatment with Gyp significantly counteracted these effects. This study indicates that Gyp has a potential role in the treatment of RP.

Topics: Animals; Gynostemma; Immunohistochemistry; Oxidative Stress; Photoreceptor Cells, Invertebrate; Plant Extracts; Reactive Oxygen Species; Retina; Retinitis Pigmentosa; Rhodopsin; Zebrafish

Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.

Topics: Animals; Disease Models, Animal; Eye Proteins; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Atomic Force; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Mutations in the gene rhodopsin are one of the major causes of autosomal dominant retinitis pigmentosa (adRP). Mutant forms of Rhodopsin frequently accumulate in the endoplasmic reticulum (ER), cause ER stress, and trigger photoreceptor cell degeneration. Here, we performed a genome-wide screen to identify suppressors of retinal degeneration in a Drosophila model of adRP, carrying a point mutation in the major rhodopsin, Rh1 (Rh1G69D). We identified two novel E3 ubiquitin ligases SORDD1 and SORDD2 that effectively suppressed Rh1G69D-induced photoreceptor dysfunction and retinal degeneration. SORDD1/2 promoted the ubiquitination and degradation of Rh1G69D through VCP (valosin containing protein) and independent of processes reliant on the HRD1 (HMG-CoA reductase degradation protein 1)/HRD3 complex. We further demonstrate that SORDD1/2 and HRD1 function in parallel and in a redundant fashion to maintain rhodopsin homeostasis and integrity of photoreceptor cells. These findings identify a new ER-associated protein degradation (ERAD) pathway and suggest that facilitating SORDD1/2 function may be a therapeutic strategy to treat adRP.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Electroretinography; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Gene Knockout Techniques; Humans; Larva; Microscopy, Electron, Scanning; Point Mutation; Proteasome Endopeptidase Complex; Proteolysis; Retina; Retinitis Pigmentosa; Rhodopsin; Ubiquitin-Protein Ligases; Ubiquitination; Valosin Containing Protein

Topics: Adult; Child; Female; Follow-Up Studies; Humans; Infant; Male; Mutation; Myopia; Pedigree; Prognosis; Retinitis Pigmentosa; Retrospective Studies; Rhodopsin; Scleral Diseases; Severity of Illness Index

We previously reported that modest running exercise protects photoreceptors in mice undergoing light-induced retinal degeneration and in the rd10 mouse model of autosomal recessive retinitis pigmentosa (arRP). We hypothesized that exercise would protect against other types of retinal degeneration, specifically, in autosomal dominant inherited disease. We tested whether voluntary running wheel exercise is protective in a retinal degeneration mouse model of class B1 autosomal dominant RP (adRP).. C57BL/6J mice heterozygous for the mutation in I307N rhodopsin (. In vivo measures revealed that induction of the I307N. Voluntary wheel running partially protected against retinal degeneration and inflammation, and RPE disruption in a model of inducible adRP. This is the first report of exercise protection in an adult adRP animal model. It is also the first report of an RPE phenotype in the I307N

Topics: Animals; Disease Models, Animal; Genes, Dominant; Inflammation; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Physical Conditioning, Animal; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-

Topics: Drug Discovery; Drug Evaluation, Preclinical; Gene Expression; Gene Expression Regulation; Genes, Reporter; Humans; Mutation; Receptors, G-Protein-Coupled; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Small Molecule Libraries; Yeasts

Gene therapy for adRP due to

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Child; Cross-Sectional Studies; Female; Genetic Predisposition to Disease; Humans; Longitudinal Studies; Male; Middle Aged; Mutation; Retinitis Pigmentosa; Retrospective Studies; Rhodopsin; Tomography, Optical Coherence; Young Adult

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.

Topics: Animals; Calpain; Cell Death; Disease Models, Animal; Endoplasmic Reticulum Stress; Gene Knock-In Techniques; Mice; Mice, Transgenic; Protein Folding; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

Topics: Animals; Cell Death; Disease Models, Animal; Genotype; Rats; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.

Topics: Cell Line, Tumor; Genes, Mating Type, Fungal; Humans; Mutation, Missense; Retinitis Pigmentosa; Rhodopsin; Saccharomyces cerevisiae; Signal Transduction

To evaluate the progression of retinitis pigmentosa (RP) due to mutations in rhodopsin (RHO) by measuring the short-wavelength autofluorescence (SW-AF) increased autofluorescence ring and ellipsoid zone (EZ)-line width.. Fundus autofluorescence (FAF) and spectral domain optical coherence tomography (SD-OCT) images were obtained from 10 patients with autosomal dominant RP due to mutations in the RHO gene. Measurements of ring area on FAF images, as well as the EZ line width on SD-OCT images and horizontal, vertical diameter, were performed by two independent masked graders.. The ring area decreased by a rate of 0.6 ± 0.2 mm. We observed SW-AF ring constriction and a progressive loss of EZ line width over time.

Topics: Adolescent; Adult; Aged; Child; Disease Progression; Female; Fluorescein Angiography; Fundus Oculi; Humans; Male; Middle Aged; Mutation; Optical Imaging; Retina; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

Rhodopsin mutations are associated with the autosomal-dominant form of retinitis pigmentosa (RP). Here we report simultaneous occurrence of RP associated with bilateral nanophthalmos and acute angle-closure glaucoma in patient with a new mutation in rhodopsin (R135W). ARPE-19 cells were transfected with myc-tagged wild-type (WT) and R135W rhodopsin constructs. The half-life of WT and R135W rhodopsin was analyzed via cycloheximide chase analysis. We found that R135W rhodopsin was accumulated in the endoplasmic reticulum (ER) and induced unfolded protein response (UPR) and apoptosis. Moreover, chaperone HSP70 alleviated ER stress and prevented apoptosis induced by R135W rhodopsin by attenuating UPR signaling. These findings reveal the novel pathogenic mechanism of RP and suggest that chaperone HSP70 has potential therapeutic significance for RP.

Topics: Adult; Apoptosis; Cell Line; Child; Child, Preschool; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Epithelial Cells; Exome Sequencing; Female; Glaucoma, Angle-Closure; HSP72 Heat-Shock Proteins; Humans; Hyperopia; Male; Microphthalmos; Middle Aged; Molecular Chaperones; Mutation; Pedigree; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Unfolded Protein Response

The rhodopsin mutation P23H is responsible for a significant portion of autosomal-dominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration.. We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure.. Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rod-saturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations.. Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

Topics: Animals; Animals, Genetically Modified; Dark Adaptation; Disease Models, Animal; Electrophysiological Phenomena; Electroretinography; Female; Male; Microscopy, Confocal; Photic Stimulation; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular; Xenopus laevis

Rhodopsin (Rho) is a visual G protein-coupled receptor expressed in the rod photoreceptors of the eye, where it mediates transmission of a light signal into a cell and converts this signal into a nerve impulse. More than 100 mutations in Rho are linked to various ocular impairments, including retinitis pigmentosa (RP). Accordingly, much effort has been directed toward developing ligands that target Rho and improve its folding and stability. Natural compounds may provide another viable approach to such drug discovery efforts. The dietary polyphenol compounds, ubiquitously present in fruits and vegetables, have beneficial effects in several eye diseases. However, the underlying mechanism of their activity is not fully understood. In this study, we used a combination of computational methods, biochemical and biophysical approaches, including bioluminescence resonance energy transfer, and mammalian cell expression systems to clarify the effects of four common bioactive flavonoids (quercetin, myricetin, and their mono-glycosylated forms quercetin-3-rhamnoside and myricetrin) on rod opsin stability, function, and membrane organization. We observed that by directly interacting with ligand-free opsin, flavonoids modulate its conformation, thereby causing faster entry of the retinal chromophore into its binding pocket. Moreover, flavonoids significantly increased opsin stability, most likely by introducing structural rigidity and promoting receptor self-association within the biological membranes. Of note, the binding of flavonoids to an RP-linked P23H opsin variant partially restored its normal cellular trafficking. Together, our results suggest that flavonoids could be utilized as lead compounds in the development of effective nonretinoid therapeutics for managing RP-related retinopathies.

Topics: Animals; Binding Sites; Cattle; Cell Line; Cell Membrane; Flavonoids; Protein Folding; Protein Stability; Protein Transport; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is the most common hereditary retinal degeneration and an important cause of visual disability worldwide. Rhodopsin gene is one of the most important genes implicated in autosomal dominant RP (ADRP). In this study, we investigated rhodopsin gene mutations in Iranian patients with ADRP.. Twenty-one patients from 21 unrelated families with a total of 51 affected members were enrolled in this study. After complete history taking, ophthalmic examination and genetic counseling, peripheral blood samples were obtained. Following genomic DNA extraction, all five exons and intron-exon boundaries of RHO gene were sequenced using Sanger method. Interpretation of detected variants was carried out using appropriate databases and bioinformatic tools. Novel variants were screened in 150 unrelated healthy subjects.. Results of direct sequencing revealed that five of 21 patients (23.8%) had mutation in the rhodopsin gene. Two of them had previously identified p.P347L mutation, and three had novel variants including p.L95P, p.R177K and p.N310K. None of these novel variants were detected in healthy controls. The p.L95P variant was associated with predominantly inferior retinal involvement.. Our study showed that mutations of the rhodopsin gene are relatively frequent in Iranian patients with ADRP and could be considered in further researches in the future. The novel p.L95P variant may be associated with a specific pattern of retinal degeneration in this population.

Topics: Adolescent; Adult; Child; Cross-Sectional Studies; DNA; DNA Mutational Analysis; Female; Humans; Incidence; Iran; Male; Middle Aged; Mutation; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Young Adult

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Electroretinography; Gene Knock-In Techniques; Glycosylation; Mice; Mice, Inbred C57BL; Mutation, Missense; Protein Structure, Tertiary; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Sequence Alignment

Long living animal models of retinitis pigmentosa (RP) can provide important information on the retinal changes that occur at the late stages of photoreceptor degeneration. The rhodopsin Pro347Leu transgenic rabbit (P347L Tg) is a model of RP, and it has been used to analyze the functional and morphological changes in the retina following the degeneration of the photoreceptors. They have also been used to test newly-developed therapies to treat eyes with photoreceptor degeneration. However, assessments of the retinal changes in P347L Tg rabbits older than 1-year have not been reported even though the data are important for research on developing new therapies to restore vision at the end stages of RP. The purpose of this study was to determine the time course of the loss of photoreceptor function and the changes in the morphology of the retina of P347L Tg rabbits. The experiments were performed on 26 older P347L Tg rabbits. The results showed that the amplitudes of the ERGs of the P347L Tg rabbits gradually decreased and reached <10 μV between 30- and 54-months-of-age. Histological analysis at these later stages showed a loss of the photoreceptor layer, and OCT analysis showed absence of the layering of the retina. However, the thickness between the inner limiting membrane and the outer plexiform layer was about 1.7 times thicker than the corresponding thickness of WT rabbits in the OCT images. This thickening was caused by a marked gliosis of the entire retina which was confirmed by light and transmission electron microscopy. In addition, immunohistochemical analysis showed there was excessive staining of the glial fibrillary acid protein in the older P347L Tg rabbits although the rod ON bipolar cells and horizontal cells were still present in the inner nuclear layer. Our results indicate that the P347L Tg rabbit progressed to complete photoreceptor loss within 30- and 54-months-of-age and severe gliosis altered the morphology of the retina.

Topics: Animals; Animals, Genetically Modified; Ependymoglial Cells; Gliosis; Photoreceptor Cells, Vertebrate; Rabbits; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Autophagosomes; Cell Membrane; Female; Homeostasis; Luminescent Proteins; Lysosomes; Male; Recombinant Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Sodium-Potassium-Exchanging ATPase; Xenopus laevis

Mutations in the RHO gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two RHO mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The in vitro biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.

Topics: Amino Acid Sequence; Animals; Chlorocebus aethiops; Circular Dichroism; COS Cells; Glycosylation; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Protein Folding; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Retinitis Pigmentosa; Rhodopsin; Spectroscopy, Fourier Transform Infrared

To determine the relationship between the amplitudes of the electrically evoked potentials (EEPs) and the number of optic nerve axons at a late stage of retinal degeneration in rhodopsin P347L transgenic (Tg) rabbits, a model of retinitis pigmentosa.. Six eyes of six wild-type (WT) (43.8 ± 7.5 months of age) and six eyes of six Tg (40.3 ± 2.6 months of age) rabbits were studied. The EEPs were elicited by 1 to 5 mA of transcorneal electrical stimulation. The first positive wave, the P1 component, was analyzed. After euthanasia, the number of axons in the optic nerve was counted.. The threshold current to elicit a P1 was significantly higher in Tg rabbits than WT rabbits. The amplitude of P1 elicited by 5 mA in Tg rabbits was about 24% of that in WT rabbits (P < 0.01). The number of axons in the optic nerve of Tg rabbits was reduced to about 59% of that of WT rabbits (P < 0.01). The correlation between the axon number and the amplitude of the P1 in Tg and WT rabbits was not significant. The mean ratio of the P1 amplitude/axon in Tg rabbits was decreased to 53% of that in WT rabbits (P < 0.05).. The degree of reduction in the EEP in Tg rabbits is more severe than the reduction in the number of optic nerve axons. The use of transcorneal electrical stimulation to determine the suitable candidates for prosthesis at the end-stage of retinitis pigmentosa may underestimate the condition of the optic nerves.

Topics: Animals; Animals, Genetically Modified; Axons; Cell Count; Disease Models, Animal; Electric Stimulation; Evoked Potentials, Visual; Optic Nerve; Photoreceptor Cells, Vertebrate; Rabbits; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP), the most common type of inherited retinal degeneration causing blindness, initially manifests as severely impaired rod function followed by deteriorating cone function. Mutations in the rhodopsin gene (

Topics: Adult; Asian People; Exome Sequencing; Female; Genetic Association Studies; Genetic Predisposition to Disease; Heterozygote; Humans; Male; Middle Aged; Mutation, Missense; Pedigree; Refractive Errors; Retinitis Pigmentosa; Rhodopsin

The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit.. Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts.. Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group.. Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

Topics: Animals; Animals, Genetically Modified; cis-trans-Isomerases; Disease Models, Animal; Electroretinography; Filtration; Genotyping Techniques; Immunohistochemistry; Light; Mice; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Phototherapy; Polymorphism, Single Nucleotide; Radio Waves; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal diseases with more than 80 identified causative genes to date. Mutations in the RHO (rhodopsin, OMIM, 180380) are the most common cause of autosomal dominant RP (adRP) worldwide. RHO is also one of the few RP genes that can cause autosomal recessive RP (arRP). To explore the frequency of RP mutations in Chinese populations, panel-based NGS (next-generation sequencing) screening and Sanger sequencing validation were performed for RP patients from 72 unrelated Chinese families. Here we reported the identified mutations only in the RHO gene. Our results showed that 4 mutations in RHO were detected in 5 (6.94%) of the 72 RP families, including two known missense mutations, c.158C > G (p.P53R) and c.551A > C (p.Q184P), and two novel mutations, c.34delC (p.P12NA) and c.82C > T (p.Q28X). The c.34delC (p.P12NA) mutation was detected in heterozygous state in one patient with intermediate RP phenotype. The c.82C > T (p.Q28X) mutation was found in a homozygous state in one proband with advanced RP phenotype at the age of 32. Clinical examination of the heterozygous carriers of c.82C > T (p.Q28X) in that family showed that the father at the age of 60s experienced no symptoms of RP and normal fundus examinations but displayed reduced electroretinography (ERG) and abnormal visual field. The sister and brother at the age of 30s showed no typical aspects of RP phenotypes. Our results not only expand the mutation spectrum of the RHO gene, but also suggest that the 2 null mutations might play minor dominant effects, leading to less severe and slower retinal degeneration in heterozygous state and more severe phenotype in homozygous state.

Topics: Adult; Asian People; China; Codon, Nonsense; DNA Mutational Analysis; Electroretinography; Female; Frameshift Mutation; High-Throughput Nucleotide Sequencing; Humans; Male; Microscopy, Fluorescence; Middle Aged; Mutation; Mutation, Missense; Pedigree; Retina; Retinitis Pigmentosa; Rhodopsin; Vision Disorders; Visual Fields; Young Adult

The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23H mice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23H mice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23H mice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.

Topics: Animals; Autophagy; Cell Death; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Homeostasis; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenylbutyrates; Phosphodiesterase 4 Inhibitors; Proteasome Endopeptidase Complex; Protein Folding; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7

Topics: Animals; Cytoskeletal Proteins; Disease Models, Animal; DNA-Binding Proteins; Electroretinography; Mice; Mice, Knockout; Proteins; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model. We have shown improved gene expression and fewer epigenetic modifications compared with the use of a Rho cDNA expression construct. In the current study, we altered our strategy by delivering a codon-optimized genomic form of Rho (co-sgRho) (for gene replacement) in combination with an RNAi-based inactivation of endogenous Rho alleles (gene suppression of both mutant Rho alleles, but mismatched with the co-sgRho) into a homozygous Rho

Topics: Animals; Disease Models, Animal; DNA; Gene Knock-In Techniques; Genes, Dominant; Genetic Therapy; Mice; Mice, Inbred C57BL; Nanoparticles; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Epigenetic and transcriptional mechanisms have been shown to contribute to long-lasting functional changes in adult neurons. The purpose of this study was to identify any such modifications in diseased retinal tissues from a mouse model of rhodopsin mutation-associated autosomal dominant retinitis pigmentosa (ADRP), Q344X, relative to age-matched wild-type (WT) controls.. We performed RNA sequencing (RNA-seq) at poly(A) selected RNA to profile the transcriptional patterns in 3-week-old ADRP mouse model rhodopsin Q344X compared to WT controls. Differentially expressed genes were determined by DESeq2 using the Benjamini & Hochberg p value adjustment and an absolute log. We observed significant differential expression in 2151 genes in the retina of Q344X mice compared to WT controls, including downregulation in the potassium channel gene,. These findings provide evidence that transcriptomic alterations occur in the ADRP mouse model rhodopsin Q344X retina and that these processes may contribute to the dysfunction and neurodegeneration seen in this animal model.

Topics: Amino Acid Substitution; Animals; Chromatin; Chromosome Disorders; Female; Gene Expression Profiling; Gene Knock-In Techniques; Genes, Dominant; Histone Deacetylases; Histones; Humans; Male; Mice; Mice, Transgenic; Mutation; Potassium Channels, Voltage-Gated; Protein Isoforms; Repressor Proteins; Retinitis Pigmentosa; Rhodopsin; Transcription, Genetic

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.

Topics: Diterpenes; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humans; Molecular Chaperones; Mutation; Protein Aggregation, Pathological; Protein Folding; Recombinant Proteins; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) comprises a group of inherited retinal degenerative conditions characterized by primary degeneration of the rod photoreceptors. Increased oxidative damage is observed in the retina, aqueous humor, and plasma of RP animal models and patients. The hepatic oxidative status may also be affected in RP due to oxidative damage influencing soluble macromolecules exiting the retina or to alterations in the melanopsin system resulting in chronic circadian desynchronization that negatively alters the oxidative stress defense system. P23H rats were crossed with pigmented Long Evans rats to produce offspring exhibiting the clinical conditions of RP. We measured hepatic malondialdehyde and 4-hydroxyalkenal concentrations as oxidative stress markers; nitrite level as a total nitrosative damage marker; total antioxidant capacity; and the activities of catalase, superoxide dismutase (SOD), and glutathione S-transferase. Retinal visual function was assessed based on optomotor and electroretinogram responses. P23H transgenic rats exhibited diminished visual acuity, contrast sensitivity, and electroretinographic responses according to the level of retinal degeneration. P23H rats at 30 days of age already demonstrated only 47% of the hepatic total antioxidant capacity of wild-type animals. Hepatic catalase and SOD activities were also reduced in P23H rats after 120 days, but we detected no difference in glutathione S-transferase activity. P23H rats had increased hepatic oxidative and nitrosative damage markers. GSH/GSSG ratio showed a significant diminution in P23H rats at P120 compared to WT. We conclude that the liver is under increased oxidative stress in P23H rats. Further studies are required, however, to clarify the contribution of systemic oxidative damage to the pathogenesis of RP.

Topics: Animals; Antioxidants; Free Radicals; Liver; Oxidative Stress; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

Topics: Animals; CRISPR-Cas Systems; DNA Mutational Analysis; Electroretinography; Eye Proteins; Genes, Recessive; Genotype; Humans; Larva; Mutation; Protein Domains; Retina; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA; Transducin; Vision, Ocular; Zebrafish; Zebrafish Proteins

G protein-coupled receptors can exist as dimers and higher-order oligomers in biological membranes. The specific oligomeric assembly of these receptors is believed to play a major role in their function, and the disruption of native oligomers has been implicated in specific human pathologies. Computational predictions and biochemical analyses suggest that two molecules of rhodopsin (Rho) associate through the interactions involving its fifth transmembrane helix (TM5). Interestingly, there are several pathogenic loss-of-function mutations within TM5 that face the lipid bilayer in a manner that could potentially influence the dimerization of Rho. Though several of these mutations are known to induce misfolding, the pathogenic defects associated with V209M and F220C Rho remain unclear. In this work, we utilized a variety of biochemical and biophysical approaches to elucidate the effects of these mutations on the dimerization, folding, trafficking, and function of Rho in relation to other pathogenic TM5 variants. Chemical cross-linking, bioluminescence energy transfer, and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy experiments revealed that each of these mutants exhibits a wild type-like propensity to self-associate within the plasma membrane. However, V209M and F220C each exhibit subtle defects in cellular trafficking. Together, our results suggest that the RP pathology associated with the expression of the V209M and F220C mutants could arise from defects in folding and cellular trafficking rather than the disruption of dimerization, as has been previously proposed.

Topics: Amino Acid Sequence; Cell Membrane; HEK293 Cells; Humans; Mutation; Protein Conformation; Protein Multimerization; Protein Transport; Retinitis Pigmentosa; Rhodopsin; Sequence Homology

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (

Topics: Animals; Dependovirus; Dogs; Gene Knock-In Techniques; Gene Knockdown Techniques; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic

Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases.. Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing.. Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively.. Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.

Topics: Aged; Arrestin; Basic Helix-Loop-Helix Transcription Factors; Case-Control Studies; Cell Differentiation; Cyclic Nucleotide Phosphodiesterases, Type 6; Eye Proteins; Female; Fibroblasts; Gene Expression Regulation; Heterozygote; Homeodomain Proteins; Homozygote; Humans; Male; Middle Aged; Mutation; Nerve Tissue Proteins; Otx Transcription Factors; Photoreceptor Cells, Vertebrate; Recoverin; Retinitis Pigmentosa; Rhodopsin; RNA Stability; RNA, Messenger; Rod Opsins; Trans-Activators; Transcription Factors

The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT).. SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels.. Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage.. Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.

Topics: Animals; Disease Models, Animal; Genes, Dominant; Immunohistochemistry; Mice; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Ganglion Cells; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Synaptophysin; Tomography, Optical Coherence; Zonula Occludens-1 Protein

Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.

Topics: Amino Acid Substitution; Animals; Cattle; Genetic Predisposition to Disease; HEK293 Cells; Hot Temperature; Humans; Light; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Phylogeny; Protein Interaction Domains and Motifs; Protein Stability; Recombinant Fusion Proteins; Recombinant Proteins; Retina; Retinitis Pigmentosa; Rhodopsin; Schiff Bases; Solubility; Spectrophotometry

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease, in which the death of mutant rod photoreceptors leads secondarily to the non-cell autonomous death of cone photoreceptors. Gene therapy is a promising treatment strategy. Unfortunately, current methods of gene delivery treat only a fraction of diseased cells, yielding retinas that are a mosaic of treated and untreated rods, as well as cones. In this study, we created two RP mouse models to test whether dying, untreated rods negatively impact treated, rescued rods. In one model, treated and untreated rods were segregated. In the second model, treated and untreated rods were diffusely intermixed, and their ratio was controlled to achieve low-, medium-, or high-efficiency rescue. Analysis of these mosaic retinas demonstrated that rescued rods (and cones) survive, even when they are greatly outnumbered by dying photoreceptors. On the other hand, the rescued photoreceptors did exhibit long-term defects in their outer segments (OSs), which were less severe when more photoreceptors were treated. In summary, our study suggests that even low-efficiency gene therapy may achieve stable survival of rescued photoreceptors in RP patients, albeit with OS dysgenesis.

Topics: Animals; Cell Death; Disease Models, Animal; Genetic Therapy; Mice; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in

Topics: Animals; Autophagosomes; Histone Deacetylase Inhibitors; Histones; Humans; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Valproic Acid; Xenopus laevis

Increasing evidence suggests that nerve growth factor (NGF) exerts protective effects against retinal degeneration in animal models of retinitis pigmentosa (RP). This study aims at investigating the effects of intravitreal injection of recombinant human NGF (rhNGF) on retinal photoreceptors apoptosis in an animal model of RP, the Royal College of Surgeons (RCS) rats.. Thirty-six RCS rats were treated with intravitreal injection of rhNGF or murine NGF (mNGF) or vehicle at 20 postnatal days (pd) and sacrificed at 40 pd. The eyes were enucleated and evaluated by histology, flow cytometric analysis for rhodopsin expression, Western blot for TrkA and activated (phosphorylated) TrkA (pTrkA) levels, and TUNEL assay for apoptosis' detection.. RCS rats showed a significant retinal degeneration associated with cell apoptosis at 40 pd when compared to wild-type animals. Histology showed that rhNGF intravitreal treatment significantly increased retinal thickness when compared to untreated eyes. Photoreceptors' number evaluated by flow cytometry was significantly increased in both intravitreal rhNGF- and mNGF-treated groups when compared to untreated eyes. This protective effect was associated with an increase in TrkA and activated pTrkA levels and an inhibition of apoptosis. Intravitreal NGF injection was well tolerated and did not show clinical and histological signs of adverse effects.. Intravitreal rhNGF injection proved safe and effective in favoring retinal cell survival in RCS rats. This is the first report showing that the novel rhNGF already proved safe in a phase I study exerts a biologic effect similar to the well-characterized mNGF-induced retinal protection. These results may trigger further studies to investigate rhNGF administration for the treatment of progressive degenerative retinal disorders such as retinitis pigmentosa.

Topics: Animals; Apoptosis; Blotting, Western; Cell Survival; Disease Models, Animal; Flow Cytometry; Humans; In Situ Nick-End Labeling; Intravitreal Injections; Male; Mice; Nerve Growth Factor; Photoreceptor Cells, Vertebrate; Rats; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin

The mutation-independent strategy for hammerhead ribozyme (hhRz) or RNA interference (RNAi)-based gene therapeutics to treat autosomal dominant diseases is predicated on the hypothesis that a single therapeutic would equivalently suppress all/most of the diverse mutant mRNAs in patients with the disease phenotype. However, the hypothesis has not been formally tested. We address this through a comprehensive bioinformatics study of how mutations affect target mRNA structure accessibility for a single lead hhRz therapeutic (725GUC↓), designed against human rod rhodopsin mRNA (hRHO), for patients with hRHO mutations that cause autosomal dominant retinitis pigmentosa.. A total of 199 in silico coding region mutations (missense, nonsense, insert, deletion, indel) were made in hRHO mRNA based on Human Gene Mutation Database and Database of Single Nucleotide Polymorphisms. Each mRNA was folded with MFold, SFold, and OligoWalk algorithms and subjected to a bioinformatics model called multiparameter prediction of RNA accessibility. Predicted accessibility of each mutant over both a broad local region and the explicit lead ribozyme annealing site were compared quantitatively to wild-type hRHO mRNA.. Accessibility of the 725GUC↓ site is sensitive to some mutations. For single nucleotide missense mutations, proximity of the mutation to the hhRz annealing site increases the impact on predicted accessibility, but some distant mutations also influence accessibility.. A mutation-independent strategy appears viable in this specific context but certain mutations could significantly influence ribozyme or RNAi efficacy through impact on accessibility at the target annealing site/region. This possibility must be considered in applications of this gene therapy strategy.

Topics: Codon, Nonsense; Computational Biology; DNA Mutational Analysis; Humans; Mutation, Missense; Polymorphism, Single Nucleotide; Retinitis Pigmentosa; Rhodopsin; RNA Interference; RNA, Catalytic; RNA, Messenger

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Disease Models, Animal; Female; Gene Editing; Genes, Dominant; Genes, Recessive; Green Fluorescent Proteins; Humans; Male; Phenotype; Point Mutation; Retinitis Pigmentosa; Rhodopsin; RNA, Guide, Kinetoplastida; Sequence Deletion; Xenopus laevis; Xenopus Proteins

Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the photoreceptor connecting cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the connecting cilium, thus facilitating rhodopsin transport to photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the photoreceptor connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP.Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in photoreceptors via the actin-severing protein, gelsolin.

Topics: Actins; Animals; Carrier Proteins; Cilia; Disease Models, Animal; Eye Proteins; Gelsolin; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Protein Transport; Retinitis Pigmentosa; Rhodopsin

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

Topics: Allosteric Regulation; Animals; Cattle; Flavonoids; Molecular Dynamics Simulation; Mutant Proteins; Protein Binding; Protein Conformation; Quercetin; Retinitis Pigmentosa; Rhodopsin

In some diseases the TrkC.T1 isoform is upregulated in glia, associated with glial TNF-α production and neuronal death. What remains unknown are the activating signals in glia, and how paracrine signals may be selective for a targeted neuron while sparing other proximate neurons. We studied these questions in the retina, where Müller glia contacts photoreceptors on one side and retinal ganglion cells on the other. In a mutant Rhodopsin mouse model of retinitis pigmentosa (RP) causing progressive photoreceptor death-but sparing retinal ganglion cells-TrkC.T1 and NT-3 ligand are upregulated in Müller glia. TrkC.T1 activity generates p-Erk, which causes increased TNF-α. These sequential events take place predominantly in Müller fibers contacting stressed photoreceptors, and culminate in selective death. Each event and photoreceptor death can be prevented by reduction of TrkC.T1 expression, by pharmacological antagonism of TrkC or by pharmacological inhibition Erk. Unmasking the sequence of non-cell autologous events and mechanisms causing selective neuronal death may help rationalize therapies.

Topics: Animals; Cell Death; Disease Models, Animal; Ependymoglial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; HEK293 Cells; Heterozygote; Humans; Mice; Mice, Knockout; Mutation; Nerve Growth Factors; Neuroglia; Neurotrophin 3; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-akt; Rats; Receptor, trkC; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 (KLF15) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin (RHO) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO-expressing cells). Adeno-associated virus (AAV) vector-mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene-targeted manipulation.

Topics: Animals; Dependovirus; Ectopic Gene Expression; Female; Gene Silencing; Gene Targeting; Genetic Therapy; Genetic Vectors; Kruppel-Like Transcription Factors; Mice, Transgenic; Mutation; Nuclear Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Topics: AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Humans; Metformin; Mice; Mutant Proteins; Photoreceptor Cells; Protein Folding; Proteostasis Deficiencies; Rats; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Transcriptional Activation

To characterize a light damage paradigm and establish structural and immunocytochemical measures of acute and protracted light-induced retinal degeneration in the rhodopsin (RHO) T4R dog model of RHO-autosomal dominant retinitis pigmentosa (ADRP).. Retinal light damage was induced in mutant dogs with a 1-minute exposure to various light intensities (0.1-1.0 mW/cm2) delivered with a Ganzfeld stimulator, or by fundus photography. Photoreceptor cell death was assessed by TUNEL assay, and alterations in retinal layers were examined by histology and immunohistochemistry 24 hours and 2 weeks after light exposure. Detailed topographic maps were made to document changes in the outer retinal layers of all four retinal quadrants 2 weeks post exposure.. Twenty-four hours post light exposure, the severity of photoreceptor cell death was dose dependent. Immunohistochemical analysis revealed disruption of rod outer segments, focal loss of the RPE integrity, and an increase in expression of endothelin receptor B in Müller cells with the two highest doses of light and fundus photography. Two weeks after light exposure, persistence of photoreceptor death, thinning of the outer nuclear layer, and induction of Müller cell gliosis occurred with the highest doses of light.. We have characterized outcome measures of acute and continuing retinal degeneration in the RHO T4R dog following light exposure. These will be used to assess the molecular mechanisms of light-induced damage and rescue strategies in this large animal model of RHO-ADRP.

Topics: Animals; Animals, Genetically Modified; Cell Death; DNA; DNA Mutational Analysis; Dogs; Immunohistochemistry; In Situ Nick-End Labeling; Light; Mutation; Photoreceptor Cells, Vertebrate; Radiation Injuries, Experimental; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa.

Topics: Animals; Chlorocebus aethiops; COS Cells; Docosahexaenoic Acids; Liposomes; Mutation; Phosphorylcholine; Protein Conformation; Protein Stability; Retinitis Pigmentosa; Rhodopsin; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes-presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene.

Topics: Androstadienes; Codon, Nonsense; HEK293 Cells; Humans; Nonsense Mediated mRNA Decay; Phenotype; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Wortmannin

Rhodopsin Pro347Leu transgenic rabbits were previously generated as models of retinitis pigmentosa (RP). While the mechanism underlying the retinal deterioration in these rabbits remains unresolved, it is likely that oxidative stress is one of the factors triggering cellular loss. We have recently succeeded in obtaining a novel activator (RS9) of nuclear factor erythroid 2-related factor (Nrf2, also known as NFE2L2), which regulates antioxidant transcriptional factors. The purpose of this study was to investigate whether RS9 delays progressive retinal degeneration in the transgenic rabbits.. RS9 microspheres (3 mM, 50 µL) were injected into the vitreous of rhodopsin Pro347Leu transgenic rabbits at 6 weeks, after which outer nuclear layer (ONL) thickness was measured by optical coherence tomography. Rabbits were sacrificed at 15 weeks.. After intravitreal injection of RS9 microspheres, the concentration of RS9 in the vitreous was maintained at 1 nM for 2 weeks. At a concentration of 0.3 mM and 50 µL, RS9 significantly inhibited thinning of the ONL in transgenic rabbits compared to vehicle-injected transgenic rabbits. In RS9-injected transgenic rabbits, Nrf2-targeted genes had increased significantly, and levels of interleukin-6 mRNA decreased.. Activation of Nrf2 signaling has potential as a novel approach for the prevention and treatment of RP, not only by driving intrinsic antioxidant enzymes, but also by inhibiting inflammatory responses. Although microspheres were employed in this study, small implants that release more compounds might be a realistic method for clinical trials.

Topics: Animals; Animals, Genetically Modified; Electroretinography; Intravitreal Injections; NF-E2-Related Factor 2; Rabbits; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence; Triterpenes

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications.

Topics: Animals; Disease Models, Animal; DNA; DNA Methylation; DNA, Complementary; Gene Expression; Gene Silencing; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Introns; Mice; Mice, Inbred C57BL; Mice, Knockout; Nanoparticles; Plasmids; Promoter Regions, Genetic; Retinitis Pigmentosa; Rhodopsin; Transgenes

Individuals carrying the same pathogenic mutation can present with a broad range of disease outcomes. While some of this variation arises from environmental factors, it is increasingly recognized that the background genetic variation of each individual can have a profound effect on the expressivity of a pathogenic mutation. In order to understand this background effect on disease-causing mutations, studies need to be performed across a wide range of backgrounds. Recent advancements in model organism biology allow us to test mutations across genetically diverse backgrounds and identify the genes that influence the expressivity of a mutation. In this study, we used the Drosophila Genetic Reference Panel, a collection of ∼200 wild-derived strains, to test the variability of the retinal phenotype of the Rh1(G69D) Drosophila model of retinitis pigmentosa (RP). We found that the Rh1(G69D) retinal phenotype is quite a variable quantitative phenotype. To identify the genes driving this extensive phenotypic variation, we performed a genome-wide association study. We identified 106 candidate genes, including 14 high-priority candidates. Functional testing by RNAi indicates that 10/13 top candidates tested influence the expressivity of Rh1(G69D). The human orthologs of the candidate genes have not previously been implicated as RP modifiers and their functions are diverse, including roles in endoplasmic reticulum stress, apoptosis and retinal degeneration and development. This study demonstrates the utility of studying a pathogenic mutation across a wide range of genetic backgrounds. These candidate modifiers provide new avenues of inquiry that may reveal new RP disease mechanisms and therapies.

Topics: Animals; Drosophila; Female; Genetic Association Studies; Genetic Variation; Genome-Wide Association Study; Genotype; Male; Models, Animal; Mutation; Pedigree; Phenotype; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Topics: Animals; cis-trans-Isomerases; Databases, Genetic; Evolution, Molecular; Mammals; Phylogeny; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis; Vertebrates

Retinitis pigmentosa (RP) is a genetically highly heterogeneous retinal disease and one of the leading causes of blindness in the world. Next-generation sequencing technology has enormous potential for determining the genetic etiology of RP. We sought to identify the underlying genetic defect in a 35-year-old male from an autosomal-dominant RP family with 14 affected individuals. By capturing next-generation sequencing (CNGS) of 144 genes associated with retinal diseases, we identified eight novel DNA variants; however, none of them cosegregated for all the members of the family. Further analysis of the CNGS data led to identification of a recurrent missense mutation (c.403C > T, p.R135W) in the rhodopsin (RHO) gene, which cosegregated with all affected individuals in the family and was not observed in any of the unaffected family members. The p.R135W mutation has a reference single nucleotide polymorphism (SNP) ID (rs104893775), and it appears to be responsible for the disease in this large family. This study highlights the importance of examining NGS data with reference SNP IDs. Thus, our study is important for data analysis of NGS-based clinical genetic diagnoses.

Topics: Adolescent; Adult; Amino Acid Sequence; Base Sequence; Child; DNA Mutational Analysis; Family; Female; Genes, Dominant; High-Throughput Nucleotide Sequencing; Humans; Male; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

The aim of this study was to unravel the molecular pathogenesis of an unusual retinitis pigmentosa (RP) phenotype observed in a Turkish consanguineous family. Homozygosity mapping revealed two candidate genes, SAMD7 and RHO. A homozygous RHO mutation c.448G > A, p.E150K was found in two affected siblings, while no coding SAMD7 mutations were identified. Interestingly, four non-coding homozygous variants were found in two SAMD7 genomic regions relevant for binding of the retinal transcription factor CRX (CRX-bound regions, CBRs) in these affected siblings. Three variants are located in a promoter CBR termed CBR1, while the fourth is located more downstream in CBR2. Transcriptional activity of these variants was assessed by luciferase assays and electroporation of mouse retinal explants with reporter constructs of wild-type and variant SAMD7 CBRs. The combined CBR2/CBR1 variant construct showed significantly decreased SAMD7 reporter activity compared to the wild-type sequence, suggesting a cis-regulatory effect on SAMD7 expression. As Samd7 is a recently identified Crx-regulated transcriptional repressor in retina, we hypothesize that these SAMD7 variants might contribute to the retinal phenotype observed here, characterized by unusual, recognizable pigment deposits, differing from the classic spicular intraretinal pigmentation observed in other individuals homozygous for p.E150K, and typically associated with RP in general.

Topics: Amino Acid Substitution; Animals; Female; Genetic Diseases, Inborn; Homeodomain Proteins; Humans; Male; Mice; Mutation, Missense; Pregnancy; Protein Domains; Response Elements; Retinitis Pigmentosa; Rhodopsin; Trans-Activators; Turkey

Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous hereditary retinal diseases that result in blindness due to photoreceptor degeneration. Mutations in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP) and are responsible for 16% to 35% of adRP cases in the Western population. Our purpose was to investigate the contribution of RHO to adRP in the Israeli and Palestinian populations.. Thirty-two adRP families participated in the study. Mutation detection was performed by whole exome sequencing (WES) and Sanger sequencing of RHO exons. Fluorescence PCR reactions of serially diluted samples were used to predict the percentage of mosaic cells in blood samples.. Eight RHO disease-causing mutations were identified in nine families, with only one novel mutation, c.548-638dup91bp, identified in a family where WES failed to detect any causal variant. Segregation analysis revealed that the origin of the mutation is in a mosaic healthy individual carrying the mutation in approximately 13% of blood cells.. This is the first report of the mutation spectrum of a known adRP gene in the Israeli and Palestinian populations, leading to the identification of seven previously reported mutations and one novel mutation. Our study shows that RHO mutations are a major cause of adRP in this cohort and are responsible for 28% of adRP families. The novel mutation exhibits a unique phenomenon in which an unaffected individual is mosaic for an adRP-causing mutation.

Topics: Adolescent; Adult; Child; DNA; DNA Mutational Analysis; Ethnicity; Exons; Family; Female; Genes, Dominant; Genetic Testing; Humans; Israel; Male; Mosaicism; Mutation; Pedigree; Polymerase Chain Reaction; Prevalence; Reference Values; Retinitis Pigmentosa; Rhodopsin; Young Adult

Most retinitis pigmentosa (RP) mutations arise in rod photoreceptor genes, leading to diminished peripheral and nighttime vision. Using a pig model of autosomal-dominant RP, we show glucose becomes sequestered in the retinal pigment epithelium (RPE) and, thus, is not transported to photoreceptors. The resulting starvation for glucose metabolites impairs synthesis of cone visual pigment-rich outer segments (OSs), and then their mitochondrial-rich inner segments dissociate. Loss of these functional structures diminishes cone-dependent high-resolution central vision, which is utilized for most daily tasks. By transplanting wild-type rods, to restore glucose transport, or directly replacing glucose in the subretinal space, to bypass its retention in the RPE, we can regenerate cone functional structures, reactivating the dormant cells. Beyond providing metabolic building blocks for cone functional structures, we show glucose induces thioredoxin-interacting protein (Txnip) to regulate Akt signaling, thereby shunting metabolites toward aerobic glucose metabolism and regenerating cone OS synthesis.

Topics: Animals; Disease Models, Animal; Fatty Acids; Glucose; Green Fluorescent Proteins; MicroRNAs; Retinal Cone Photoreceptor Cells; Retinal Photoreceptor Cell Inner Segment; Retinal Photoreceptor Cell Outer Segment; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Sus scrofa; Thioredoxins

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing ce

Topics: Animals; Disease Models, Animal; Dogs; Light; Photophobia; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Visual Perception

Topics: Amino Acid Substitution; Cell Membrane; Fluorescence Resonance Energy Transfer; Genes, Dominant; HEK293 Cells; Humans; Luminescent Proteins; Membrane Proteins; Mutation, Missense; Point Mutation; Protein Aggregates; Protein Folding; Protein Interaction Mapping; Protein Multimerization; Protein Transport; Proteostasis Deficiencies; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

T17M rhodopsin expression in rod photoreceptors leads to severe retinal degeneration and is associated with the activation of ER stress related Unfolded Protein Response (UPR) signaling. Here, we show a novel role of a UPR transcription factor, ATF4, in photoreceptor cellular pathology. We demonstrated a pro-death role for ATF4 overexpression during autosomal dominant retinitis pigmentosa (ADRP). Based on our results in ATF4 knockout mice and adeno-associated viral (AAV) delivery of ATF4 to the retina, we validated a novel therapeutic approach targeting ATF4 over the course of retinal degeneration. In T17M rhodopsin retinas, we observed ATF4 overexpression concomitantly with reduction of p62 and elevation of p53 levels. These molecular alterations, together with increased CHOP and caspase-3/7 activity, possibly contributed to the mechanism of photoreceptor cell loss. Conversely, ATF4 knockdown retarded retinal degeneration in 1-month-old T17M Rhodopsin mice and promoted photoreceptor survival, as measured by scotopic and photopic ERGs and photoreceptor nuclei row counts. Similarly, ATF4 knockdown also markedly delayed retinal degeneration in 3-month-old ADRP animals. This delay was accompanied by a dramatic decrease in UPR signaling, the launching of anti-oxidant defense, initiation of autophagy, and improvement of rhodopsin biosynthesis which together perhaps combat the cellular stress associated with T17M rhodopsin. Our data indicate that augmented ATF4 signals during retinal degeneration plays a cytotoxic role by triggering photoreceptor cell death. Future ADRP therapy regulating ATF4 expression can be developed to treat retinal degenerative disorders associated with activated UPR.

Topics: Activating Transcription Factor 4; Animals; Autophagy; Caspase 3; Caspase 7; Disease Models, Animal; Endoplasmic Reticulum; Mice; Mice, Inbred C57BL; Mice, Knockout; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Stress, Physiological; Transcription Factors; Unfolded Protein Response

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Mice; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

To study the phenotype in two families with genetically identified autosomal dominant retinitis pigmentosa (adRP) focusing on macular structure and function.. Clinical data were collected at the Department of Ophthalmology, Lund University, Sweden, for affected and unaffected family members from two pedigrees with adRP. Examinations included optical coherence tomography (OCT), full-field electroretinography (ffERG), and multifocal electroretinography (mfERG). Molecular genetic screening was performed for known mutations associated with adRP.. The mode of inheritance was autosomal dominant in both families. The members of the family with a mutation in the PRPF31 (p.IVS6+1G>T) gene had clinical features characteristic of RP, with severely reduced retinal rod and cone function. The degree of deterioration correlated well with increasing age. The mfERG showed only centrally preserved macular function that correlated well with retinal thinning on OCT. The family with a mutation in the RHO (p.R135W) gene had an extreme intrafamilial variability of the phenotype, with more severe disease in the younger generations. OCT showed pathology, but the degree of morphological changes was not correlated with age or with the mfERG results. The mother, with a de novo mutation in the RHO (p.R135W) gene, had a normal ffERG, and her retinal degeneration was detected merely with the reduced mfERG.. These two families demonstrate the extreme inter- and intrafamilial variability in the clinical phenotype of adRP. This is the first Swedish report of the clinical phenotype associated with a mutation in the PRPF31 (p.IVS6+1G>T) gene. Our results indicate that methods for assessment of the central retinal structure and function may improve the detection and characterization of the RP phenotype.

Topics: Adolescent; Aged; Aged, 80 and over; Child; Child, Preschool; DNA Mutational Analysis; Electroretinography; Eye Proteins; Female; Genes, Dominant; Humans; Infant; Macula Lutea; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sweden; Tomography, Optical Coherence; Visual Acuity; Young Adult

The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration.

Topics: Action Potentials; Afferent Pathways; Animals; Animals, Genetically Modified; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Form Perception; Rats; Rats, Long-Evans; Reaction Time; Retina; Retinal Degeneration; Retinal Neurons; Retinitis Pigmentosa; Rhodopsin; Visual Cortex; Visual Fields

Topics: Child; Humans; Magnetic Resonance Imaging; Male; Mutation; Optic Chiasm; Optic Nerve Glioma; Optic Nerve Neoplasms; Pedigree; Retinitis Pigmentosa; Rhodopsin; Vision Disorders; Visual Field Tests; Visual Fields

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations-F45L, V209M and F220C-yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Topics: Animals; Cattle; Chlorocebus aethiops; COS Cells; GTP-Binding Proteins; HEK293 Cells; Humans; Liposomes; Mice, Knockout; Mutation; Phospholipid Transfer Proteins; Point Mutation; Protein Multimerization; Retina; Retinitis Pigmentosa; Rhodopsin; Transducin

The diagnoses of retinitis pigmentosa (RP) and stationary night blindness (CSNB) are two distinct clinical entities belonging to a group of clinically and genetically heterogeneous retinal diseases. The current study focused on the identification of causative mutations in the RP-affected index patient and in several members of the same family that reported a phenotype resembling CSNB. Ophthalmological examinations of the index patient confirmed a typical form of RP. In contrast, clinical characterizations and ERGs of another affected family member showed the Riggs-type CSNB lacking signs of RP. Applying whole exome sequencing we detected the non-synonymous substitution c.337G > A, p.E113 K in the rhodopsin (RHO) gene. The mutation co-segregated with the diseases. The identification of the pathogenic variant p.E113 K is the first description of a naturally-occurring mutation in the Schiff base counterion of RHO in human patients. The heterozygous mutation c.337G > A in exon 1 was confirmed in the index patient as well as in five CSNB-affected relatives. This pathogenic sequence change was excluded in a healthy family member and in 199 ethnically matched controls. Our findings suggest that a mutation in the biochemically well-characterized counterion p.E113 in RHO can be associated with RP or Riggs-type CSNB, even within the same family.

Topics: Adult; Aged, 80 and over; Amino Acid Sequence; Amino Acid Substitution; Case-Control Studies; DNA Mutational Analysis; Female; Heterozygote; Humans; Male; Middle Aged; Mutation, Missense; Night Blindness; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Schiff Bases; Sequence Analysis, DNA

The effect of disease-causing missense mutations on protein folding is difficult to evaluate. To understand this relationship, we developed the unfolding mutation screen (UMS) for in silico evaluation of the severity of genetic perturbations at the atomic level of protein structure. The program takes into account the protein-unfolding curve and generates propensities using calculated free energy changes for every possible missense mutation at once. These results are presented in a series of unfolding heat maps and a colored protein 3D structure to show the residues critical to the protein folding and are available for quick reference. UMS was tested with 16 crystal structures to evaluate the unfolding for 1391 mutations from the ProTherm database. Our results showed that the computational accuracy of the unfolding calculations was similar to the accuracy of previously published free energy changes but provided a better scale. Our residue identity control helps to improve protein homology models. The unfolding predictions for proteins involved in age-related macular degeneration, retinitis pigmentosa, and Leber's congenital amaurosis matched well with data from previous studies. These results suggest that UMS could be a useful tool in the analysis of genotype-to-phenotype associations and next-generation sequencing data for inherited diseases.

Topics: Algorithms; cis-trans-Isomerases; Computer Simulation; Humans; Leber Congenital Amaurosis; Macular Degeneration; Mutation, Missense; Protein Conformation; Protein Unfolding; Retinitis Pigmentosa; Rhodopsin; Workflow

To determine the role of rhodopsin (RHO) gene mutations in patients with sector retinitis pigmentosa (RP) from Northern Ireland.. A case series of sector RP in a tertiary ocular genetics clinic.. Four patients with sector RP were recruited from the Royal Victoria Hospital (Belfast, Northern Ireland) and Altnagelvin Hospital (Londonderry, Northern Ireland) following informed consent.. The diagnosis of sector RP was based on clinical examination, International Society for Clinical Electrophysiology of Vision (ISCEV) standard electrophysiology, and visual field analysis. DNA was extracted from peripheral blood leucocytes and the coding regions and adjacent flanking intronic sequences of the RHO gene were polymerase chain reaction (PCR) amplified and cycle sequenced.. Rhodopsin mutational status.. A heterozygous missense mutation in RHO (c.173C > T) resulting in a non-conservative substitution of threonine to methionine (p. Thr58Met) was identified in one patient and was absent from 360 control individuals. This non-conservative substitution (p.Thr58Met) replaces a highly evolutionary conserved polar hydrophilic threonine residue with a non-polar hydrophobic methionine residue at position 58 near the cytoplasmic border of helix A of RHO.. The study identified a RHO gene mutation (p.Thr58Met) not previously reported in RP in a patient with sector RP. These findings outline the phenotypic variability associated with RHO mutations. It has been proposed that the regional effects of RHO mutations are likely to result from interplay between mutant alleles and other genetic, epigenetic and environmental factors.

Topics: DNA Mutational Analysis; Electrooculography; Electrophysiology; Electroretinography; Gene Amplification; Humans; Mutation, Missense; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests; Visual Fields

We aimed to determine the prevalence of mutations in the RHO gene in Spanish families with autosomal dominant Retinitis Pigmentosa (adRP), to assess genotype-phenotype correlations and to establish an accurate diagnostic algorithm after 23 years of data collection.. Two hundred patients were analysed through a combination of denaturing gradient gel electrophoresis, single-strand conformation polymorphism, genotyping microarray and Sanger sequencing of the RHO gene.. Overall, 42 of 200 Spanish adRP families were mutated for RHO (21.0%). Twenty-seven different RHO mutations were detected; seven of them were novel. A genotype-phenotype correlation was established with clinical data from 107 patients. The most prevalent p.Pro347Leu mutation, responsible for 4.5% (9/200) of all mutated adRP families, was associated with a phenotype of early onset and severe course diffuse RP.. This retrospective study provides a wide spectrum of mutations in the RHO gene in Spanish patients with adRP. Also, the prevalence of mutations is similar to that reported in European population. Genotyping microarray followed by RHO sequencing is proposed as a first step in molecular diagnosis of adRP Spanish families. An increasing understanding of causal RHO alleles in adRP facilitates disease diagnosis and prognosis, especially for the prevalent p.Pro347Leu mutation.

Topics: Adult; DNA Mutational Analysis; Female; Genetic Association Studies; Genotyping Techniques; Humans; Male; Middle Aged; Mutation; Oligonucleotide Array Sequence Analysis; Polymorphism, Single-Stranded Conformational; Prevalence; Retinitis Pigmentosa; Retrospective Studies; Rhodopsin; Spain

Genetic mapping was recently used to identify the underlying cause for a previously uncharacterized cohort of autosomal recessive retinitis pigmentosa cases. Genetic mapping of affected individuals resulted in the identification of an uncharacterized gene, C2Orf71, as the causative locus. However, initial homology searches failed to reveal similarities to any previously characterized protein or domain. To address this issue, we characterized the mouse homolog, BC027072. Immunohistochemistry with a custom polyclonal antibody showed staining localized to the inner segments (IS) of photoreceptor cells, as well as the outer segments (OS) of cone cells. A knockout mouse line (BC(-/-)) was generated and demonstrated that loss of this gene results in a severe, early-onset retinal degeneration. Histology and electron microscopy (EM) revealed disorganized OS as early as 3 weeks with complete loss by 24 weeks of age. EM micrographs displayed packets of cellular material containing OS discs or IS organelles in the OS region and abnormal retinal pigmented epithelium cells. Analyses of retinoids and rhodopsin levels showed <20% in BC(-/-) versus wild-type mice early in development. Electroretinograms demonstrated that affected mice were virtually non-responsive to light by 8 weeks of age. Lastly, RNAseq analysis of ocular gene expression in BC(-/-) mice revealed clues to the causes of the progressive retinal degenerations. Although its function remains unknown, this protein appears essential for normal OS development/maintenance and vision in humans and mice. RNAseq data are available in the GEO database under accession: GSE63810.

Topics: Animals; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Expression; Gene Expression Profiling; Gene Order; Gene Targeting; Genes, Recessive; Genetic Association Studies; Homozygote; Mice; Mice, Knockout; Protein Biosynthesis; Quantitative Trait Loci; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

Topics: Animals; Binding Sites; Cell Line, Transformed; Gene Expression; Genes, Dominant; Genetic Therapy; Genetic Vectors; Humans; Introns; Mice; Mice, Transgenic; Phenotype; Photoreceptor Cells; Protein Interaction Domains and Motifs; Protein Transport; Retinitis Pigmentosa; Rhodopsin; RNA Precursors; RNA Splicing; RNA, Messenger; Trans-Splicing; Transduction, Genetic

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation.

Topics: Animals; Disease Models, Animal; Gene Expression; Glycosylation; Humans; Mice; Mice, Transgenic; Microscopy, Electron; Mutation, Missense; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Ubiquitination

To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHO dog).. The left eyes of T4R RHO dogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure.. A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3.. The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.

Topics: Animals; Cell Death; Dogs; In Situ Nick-End Labeling; Light; Retina; Retinitis Pigmentosa; Rhodopsin; Unfolded Protein Response

High-throughput screening (HTS) is one of the major techniques for discovering promising molecules for drug development. Rhodopsin mutations cause the most common autosomal dominant form of retinitis pigmentosa, an inherited retinal degenerative disease that currently has no effective treatment. To find an optimal pharmacological treatment for rhodopsin-associated retinitis pigmentosa, we performed two cell-based HTSs with mammalian cells expressing the P23H rod opsin mutant and identified two sets of novel compounds for further validation and characterization. The first HTS screen identified pharmacological chaperones of P23H opsin that increased its translocation from the endoplasmic reticulum to the plasma membrane. The second HTS screen selected small molecules that enhanced the clearance of the mutant opsin while vision could be sustained by the healthy gene allele expressing wild-type rhodopsin. Here we describe the methodology of these two HTS assays in detail.

Topics: Cell Line, Tumor; High-Throughput Screening Assays; Humans; Mutation; Opsins; Retinitis Pigmentosa; Rhodopsin

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.

Topics: Animals; Blotting, Western; Disease Models, Animal; DNA; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Humans; Introns; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron, Transmission; Nanoparticles; Phenotype; Reproducibility of Results; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding.. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses.. Two stabilization assays of P23H mutant opsin were developed and validated, one based on β-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin.. Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Immunoblotting; Mice; Mutant Proteins; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.

Topics: Animals; Animals, Newborn; Cell Survival; Disease Models, Animal; Gene Expression; Mice; Nerve Growth Factor; Photoreceptor Cells, Vertebrate; Primary Cell Culture; Rats; Recombinant Proteins; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin

Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3' UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.

Topics: Amino Acid Sequence; Animals; Base Sequence; Color Vision; Disease Models, Animal; Electroretinography; Gene Dosage; Hemizygote; Molecular Sequence Data; Mutation; Phenotype; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Tomography, Optical Coherence; Transgenes

Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

Topics: Amino Acid Substitution; Animals; Cattle; Chlorocebus aethiops; COS Cells; Dimyristoylphosphatidylcholine; Lipid Bilayers; Mutation, Missense; Phospholipid Ethers; Protein Stability; Retinitis Pigmentosa; Rhodopsin

To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations.. Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells.. DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea.. Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina.

Topics: Aged; Aged, 80 and over; Arrestin; Electroretinography; Female; Humans; Immunohistochemistry; Male; Ophthalmoscopy; Pedigree; Point Mutation; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Tissue Donors; Tomography, Optical Coherence

To investigate the process of retinal degeneration by analyzing the functional and morphological findings in transgenic rabbits with a Pro347Leu rhodopsin mutation.. Wild-type (WT) and transgenic (Tg) rabbits at ages 4, 8 and 12 months were used. We conducted functional evaluation by recording the changes in the pupil response to red and blue light stimulation and the amplitude of the electroretinography (ERG). Morphologically, rod and cone distribution was examined using light and electron microscopy. Immunostaining for the identification of retinal ganglion cells (RGCs) was also confirmed by injecting a TUJ-1 monoclonal antibody.. Pupil constriction for infrared pupillography and the a- and b-waves for ERG in Tg rabbits decreased with increasing age; the differences were compared to the age-matched WT rabbits. The subnormal ERG in the Tg rabbits, especially the a-wave decrease and pupil constriction with a long latency time, was induced only during exposure to blue light stimulation at 12 months. Light and electron microscopic findings showed a progressive loss of photoreceptor cells over time manifesting by 8 months in the peripheral retina. Moreover, pyknotic nuclei of the outer nuclear layer in the center of the visual streak were observed. At 12 months, there was disappearance of the rods and ballooning degeneration of the cones. Some remaining RGCs had large cell bodies with long branching dendrites.. The changes in the pupil light response and amplitude of the ERG could be used to predict the state of retinal degeneration in the Tg rabbit.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Light; Photoreceptor Cells, Vertebrate; Point Mutation; Pupil; Rabbits; Reflex, Pupillary; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin; Tubulin

This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP.. Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.. We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR).. The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

Topics: 3' Untranslated Regions; Collagen Type VI; DNA Breaks; DNA Mutational Analysis; Exome; Exons; Female; Genes, Dominant; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Introns; Male; Multiplex Polymerase Chain Reaction; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.

Topics: Animals; Endoplasmic Reticulum; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humans; Luminescent Proteins; Mice; Microscopy, Confocal; Mutation; Opsins; Protein Folding; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry; Spectroscopy, Fourier Transform Infrared

Retinal prosthetic implants are the only approved treatment for retinitis pigmentosa, a disease of the eye that causes blindness through gradual degeneration of photoreceptors. An array of microelectrodes triggered by input from a camera stimulates surviving retinal neurons, with each electrode acting as a pixel. Unintended stimulation of retinal ganglion cell axons causes patients to see large oblong shapes of light, rather than focal spots, making it difficult to perceive forms. To address this problem, we performed calcium imaging in isolated retinas and mapped the patterns of cells activated by different electrical stimulation protocols. We found that pulse durations two orders of magnitude longer than those typically used in existing implants stimulated inner retinal neurons while avoiding activation of ganglion cell axons, thus confining retinal responses to the site of the electrode. Multielectrode stimulation with 25-ms pulses can pattern letters on the retina corresponding to a Snellen acuity of 20/312. We validated our findings in a patient with an implanted epiretinal prosthesis by demonstrating that 25-ms pulses evoke focal spots of light.

Topics: Animals; Biosensing Techniques; Disease Models, Animal; Electric Stimulation; Evoked Potentials, Visual; Female; Genetic Predisposition to Disease; Humans; In Vitro Techniques; Microelectrodes; Miniaturization; Mutation; Phenotype; Phosphenes; Prosthesis Design; Rats, Long-Evans; Retina; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin; Synaptic Potentials; Synaptic Transmission; Time Factors; Transfection; Visual Pathways; Visual Perception; Visual Prosthesis

We previously reported a transgenic Xenopus laevis model of retinitis pigmentosa in which tadpoles express the bovine form of P23H rhodopsin (bP23H) in rod photoreceptors. In this model, retinal degeneration was dependent on light exposure. Here, we investigated ultrastructural changes that occurred in the rod photoreceptors of these retinas when exposed to light.. Tadpoles expressing bP23H in rods were transferred from constant darkness to a 12-hour light:12-hour dark (12L:12D) regimen. For comparison, transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9) were reared in a 12L:12D regimen, and retinal degeneration was induced by administration of the drug AP20187. Tadpoles were euthanized at various time points, and eyes were processed for confocal light and transmission electron microscopy.. We observed defects in outer and inner segments of rods expressing bP23H that were aggravated by light exposure. Rod outer segments exhibited vesiculations throughout and were rapidly phagocytosed by the retinal pigment epithelium. In rod inner segments, we observed autophagic compartments adjacent to the endoplasmic reticulum and extensive vesiculation at later time points. These defects were not found in rods expressing iCasp9, which completely degenerated within 36 hours after drug administration.. Our results indicate that ultrastructural defects in outer and inner segment membranes of bP23H expressing rods differ from those observed in drug-induced apoptosis. We suggest that light-induced retinal degeneration caused by P23H rhodopsin occurs via cell death with autophagy, which may represent an attempt to eliminate the mutant rhodopsin and/or damaged cellular compartments from the secretory pathway.

Topics: Animals; Animals, Genetically Modified; Autophagy; Caspase 9; Disease Models, Animal; Light; Photoperiod; Radiation Injuries, Experimental; Retinal Photoreceptor Cell Inner Segment; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Tacrolimus; Xenopus laevis

Retinal rod photoreceptor cells have double membrane discs located in their outer segments (ROS) that are continuously formed proximally from connecting cilia (CC) and phagocytized distally by the retinal pigmented epithelium. The major component of these rod discs, the light-sensitive visual pigment rhodopsin (Rho), consists of an opsin protein linked to 11-cis-retinal. The P23H mutation of rod opsin (P23H opsin) is the most common cause of human blinding autosomal dominant retinitis pigmentosa (adRP). A mouse model of adRP with this mutation (Rho(P23H/+)) shows low levels of P23H opsin protein, partial misalignment of discs and progressive retinal degeneration. However, the impact of mutant P23H opsin on the formation of abnormal discs is unclear and it is still unknown whether this mutant pigment can mediate phototransduction. Using transretinal ERG recordings, we demonstrate that P23H mutant Rho can trigger phototransduction but Rho(P23H/P23H) rods are ∼17 000-fold less sensitive to light than Rho(+/+) rods and produce abnormally fast photo-responses. By analyzing homozygous Rho(P23H/P23H) knock-in mice, we show that P23H opsin is transported to ciliary protrusions where it forms sagittally elongated discs. Transmission electron microscopy of postnatal day (PND) 14 Rho(P23H/+) mouse retina revealed disordered sagittally oriented discs before the onset of retinal degeneration. Surprisingly, we also observed smaller, immature sagittally oriented discs in PND14 Rho(+/)(-) and Rho(+/+) mice that were not seen in older animals. These findings provide fundamental insights into the pathogenesis of the P23H mutant opsin and reveal a novel early sagittally aligned disc formation step in normal ROS disc expansion.

Topics: Animals; Disease Models, Animal; Gene Knock-In Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morphogenesis; Mutation; Retinal Degeneration; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Vision, Ocular

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

Topics: Animals; Blotting, Western; Cells, Cultured; Electroretinography; Female; G-Protein-Coupled Receptor Kinase 1; Genes, Dominant; Genetic Predisposition to Disease; HSP90 Heat-Shock Proteins; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Mutation; Pyridones; Pyrimidines; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Real-Time Polymerase Chain Reaction; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Tomography, Optical Coherence; Vision, Ocular

We previously reported activation of the unfolded protein response (UPR) in P23H rhodopsin (RHO) retinas with autosomal dominant retinitis pigmentosa (ADRP). Knowing that the UPR can trigger Ca(2+) release from the endoplasmic reticulum and regulate cellular signaling we examined the level of Ca(2+)-regulated proteins. We also looked for changes in the expression of Bcl2 family proteins, autophagy proteins and the mTOR/AKT pathways, as well as for the induction of mitochondria-associated apoptosis in the P23H RHO retina. Our data demonstrated that the elevation of calpain and caspase-12 activity was concomitantly observed with a decrease in the BCL2-XL/BAX ratio and an increase in mTor levels in the P23H-3 RHO retina suggesting a vulnerability of P23H RHO photoreceptors to apoptosis. The translocation of BAX to the mitochondria, as well as the release of cytochrome C and AIF into the cytosol supports this conclusion and indicates the involvement of mitochondria-induced apoptosis in the progression of ADRP. The level of autophagy proteins in general was found to be decreased in the P21-P30 P23H RHO retina. Injections of rapamycin, however, protected the P23H RHO rod photoreceptors from experiencing physiological decline. Despite this fact, the downregulation of mTOR did not alter the level of autophagy proteins. Our results imply that in addition to activation of the UPR during ADRP progression, photoreceptors also experience alterations in major proapoptotic pathways.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 12; Cytochromes c; Humans; Mitochondria; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation

Role of intravitreal injection of dexamethasone implant (Ozurdex) for refractory macular thickening.. A case report of a 13-year-old boy with Rhodopsin-positive, CRB1-negative retinitis pigmentosa presenting with Coat's-like exudative vitreoretinopathy and treatment-resistant cystoid macular oedema.. A reduction in the macular thickening following a single injection of Ozurdex.. We present our experience in successful treatment of refractory macular oedema with intravitreal injection of dexamethasone implant resulting in clinically significant resolution of macular thickening.

Topics: Adolescent; Anti-Inflammatory Agents; Dexamethasone; Drug Implants; Exudates and Transudates; Humans; Intravitreal Injections; Macular Edema; Male; Retinitis Pigmentosa; Rhodopsin; Treatment Outcome; Vitreoretinopathy, Proliferative

Following a previous study that demonstrated a correlation between rhodopsin stability and the severity of retinitis pigmentosa (RP), we investigated whether predictions of severity can be improved with a regional analysis of this correlation. The association between changes to the stability of the protein and the relative amount of rhodopsin reaching the plasma membrane was assessed.. Crystallography-based estimations of mutant rhodopsin stability were compared with descriptions in the scientific literature of the visual function of mutation carriers to determine the extent of associations between rhodopsin stability and clinical phenotype. To test the findings of this analysis, three residues of a green fluorescent protein (GFP) tagged rhodopsin plasmid were targeted with site-directed random mutagenesis to generate mutant variants with a range of stability changes. These plasmids were transfected into HEK-293 cells, and then flow cytometry was used to measure rhodopsin on the cells' plasma membrane. The GFP signal was used to measure the ratio between this membrane-bound rhodopsin and total cellular rhodopsin. FoldX stability predictions were then compared with the surface staining data and clinical data from the database to characterize the relationship between rhodopsin stability, the severity of RP, and the expression of rhodopsin at the cell surface.. There was a strong linear correlation between the scale of the destabilization of mutant variants and the severity of retinal disease. A correlation was also seen in vitro between stability and the amount of rhodopsin at the plasma membrane. Rhodopsin is drastically reduced on the surface of cells transfected with variants that differ in their inherent stability from the wild-type by more than 2 kcal/mol. Below this threshold, surface levels are closer to those of the wild-type.. There is a correlation between the stability of rhodopsin mutations and disease severity and levels of membrane-bound rhodopsin. Measuring membrane-bound rhodopsin with flow cytometry could improve prognoses for poorly characterized mutations and could provide a platform for measuring the effectiveness of treatments.

Topics: Animals; Base Sequence; Cattle; Cell Membrane; Flow Cytometry; Green Fluorescent Proteins; HEK293 Cells; Humans; Molecular Sequence Data; Mutagenesis; Mutant Proteins; Mutation; Phenotype; Protein Stability; Retinitis Pigmentosa; Rhodopsin; Statistics as Topic; Transfection; Visual Acuity

The P23H mutation in rhodopsin (Rho(P23H)) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho(P23H) rhodopsin transgene (GHL(+)). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for GHL(+), GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-) animals between 4-28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of Chop and Ask1 did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of GHL(+)/Chop(-/-) and GHL(+)/Ask1(-/-), a region that was severely degenerated in GHL(+) mice. Our results show that in the presence of the Rho(P23H) transgene, the rate of decline in retinal sensitivity is similar in Chop or Ask1 ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in GHL(+) mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.

Topics: Animals; Apoptosis; Disease Models, Animal; Electrophysiology; Electroretinography; Genotype; MAP Kinase Kinase Kinase 5; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Photophobia; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transcription Factor CHOP; Vision, Ocular

Retinitis pigmentosa (RP) is a group of inherited diseases that primarily affect light‑sensitive rods and cones in the retina. Rhodopsin mutations, including the T17M mutation, are associated with the autosomal dominant form of retinitis pigmentosa (ADRP) and have been linked to abnormal protein folding. However, the molecular mechanisms underlying T17M rhodopsin‑induced retinal degeneration are yet to be elucidated. In the present study, Human embryonic kidney (HEK) 293 and ARPE‑19 cells were transfected with myc‑tagged wild‑type (WT) and T17M rhodopsin constructs. Cells were fixed and stained with anti‑myc antibodies and the localization of WT and T17M rhodopsin was visualized using immunofluorescence microscopy. Turnover rates of WT and T17M rhodopsin were measured using western blot analysis. In addition, endoplasmic reticulum (ER) stress‑induced cell death was analyzed in WT and T17M rhodopsin‑transfected cells using nuclear staining. Misfolded T17M rhodopsin was observed to be abnormally localized in the ER, while WT rhodopsin was predominantly found at the plasma membrane. Protein turnover analysis revealed that T17M rhodopsin was more rapidly degraded by proteasomes than WT rhodopsin. Furthermore, overexpression of T17M rhodopsin was observed to induce cell death and increase cytotoxicity; predisposing cells to ER stress‑induced cell death. These findings show novel insight into the properties of T17M rhodopsin and highlight the role of ER stress in T17M‑associated RP.

Topics: Cell Death; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Free Radical Scavengers; Gene Expression; Humans; Mutation; Proteasome Endopeptidase Complex; Protein Stability; Protein Transport; Proteolysis; Retinitis Pigmentosa; Rhodopsin

Functional studies have detected deficits in retinal signaling in asymptomatic children from families with inherited autosomal dominant retinitis pigmentosa (RP). Whether retinal abnormalities are present earlier during gestation or shortly after birth in a subset of children with autosomal dominant RP is unknown and no appropriate animal RP model possessing visual function at birth has been available to examine this possibility. In a recently developed transgenic P23H (TgP23H) rhodopsin swine model of RP, we tracked changes in pre- and early postnatal retinal morphology, as well as early postnatal retinal function.. Domestic swine inseminated with semen from a TgP23H miniswine founder produced TgP23H hybrid and wild type (Wt) littermates. Outer retinal morphology was assessed at light and electron microscopic levels between embryonic (E) and postnatal (P) day E85 to P3. Retinal function was evaluated using the full field electroretinogram at P3.. Embryonic TgP23H rod photoreceptors are malformed and their rhodopsin expression pattern is abnormal. Consistent with morphological abnormalities, rod-driven function is absent at P3. In contrast, TgP23H and Wt cone photoreceptor morphology (E85-P3) and cone-driven retinal function (P3) are similar.. Prenatal expression of mutant rhodopsin alters the normal morphological and functional development of rod photoreceptors in TgP23H swine embryos. Despite this significant change, cone photoreceptors are unaffected. Human infants with similarly aggressive RP might never have rod vision, although cone vision would be unaffected. Such aggressive forms of RP in preverbal children would require early intervention to delay or prevent functional blindness.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; DNA; DNA Mutational Analysis; Electroretinography; Genotype; Humans; Immunohistochemistry; Microscopy, Electron, Transmission; Mutation; Polymerase Chain Reaction; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Swine, Miniature

Human and swine retinas have morphological and functional similarities. In the absence of primate models, the swine is an attractive model to study retinal function and disease, with its cone-rich visual streak, our ability to manipulate their genome, and the differences in susceptibility of rod and cone photoreceptors to disease. We characterized the normal development of cone function and its subsequent decline in a P23H rhodopsin transgenic (TgP23H) miniswine model of autosomal dominant RP.. Semen from TgP23H miniswine 53-1 inseminated domestic swine and produced TgP23H and Wt hybrid littermates. Retinal function was evaluated using ERGs between postnatal days (P) 14 and 120. Retinal ganglion cell (RGC) responses were recorded to full-field stimuli at several intensities. Retinal morphology was assessed using light and electron microscopy.. Scotopic retinal function matures in Wt pigs up to P60, but never develops in TgP23H pigs. Wt and TgP23H photopic vision matures similarly up to P30 and diverges at P60 where TgP23H cone vision declines. There are fewer TgP23H RGCs with visually evoked responses at all ages and their response to light is compromised. Photoreceptor morphological changes mirror these functional changes.. Lack of early scotopic function in TgP23H swine suggests it as a model of an aggressive form of RP. In this mammalian model of RP, normal cone function develops independent of rod function. Therefore, its retina represents a system in which therapies to rescue cones can be developed to prolong photopic visual function in RP patients.

Topics: Animals; Animals, Genetically Modified; Cell Count; Disease Models, Animal; Electroretinography; Microscopy, Electron, Transmission; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Swine, Miniature

The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect.

Topics: Animals; Apoptosis; Caspase 7; Disease Models, Animal; Electroretinography; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transcription Factor CHOP; Unfolded Protein Response

Many mutations in rhodopsin gene linked to retinitis pigmentosa (RP) cause rhodopsin misfolding. Rod photoreceptor cells expressing misfolded rhodopsin eventually die. Identifying mechanisms to prevent rhodopsin misfolding or to remove irreparably misfolded rhodopsin could provide new therapeutic strategies. IRE1, ATF6, and PERK signaling pathways, collectively called the unfolded protein response (UPR), regulate the functions of endoplasmic reticulum, responsible for accurate folding of membrane proteins such as rhodopsin. We used chemical and genetic approaches to selectively activate IRE1, ATF6, or PERK signaling pathways one at a time and analyzed their effects on mutant rhodopsin linked to RP. We found that both artificial IRE1 and ATF6 signaling promoted the degradation of mutant rhodopsin with lesser effects on wild-type rhodopsin. Furthermore, IRE1 and ATF6 signaling preferentially reduced levels of aggregated rhodopsins. By contrast, PERK signaling reduced levels of wild-type and mutant rhodopsin. These studies indicate that activation of either IRE1, ATF6, or PERK prevents mutant rhodopsin from accumulating in the cells. In addition, activation of IRE1 or ATF6 can selectively remove aggregated or mutant rhodopsin from the cells and may be useful in treating RP associated with rhodopsin protein misfolding.

Topics: Activating Transcription Factor 6; eIF-2 Kinase; Endoribonucleases; Humans; Protein Serine-Threonine Kinases; Proteostasis Deficiencies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.

Topics: Animals; Cell Line; Cell Survival; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Electroretinography; Heat-Shock Response; Humans; Hydroxylamines; Mutation; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Time Factors; Transfection; Unfolded Protein Response; Vision, Ocular

Identification and classification of all retinitis pigmentosa (RP) causing mutations contribute to a better understanding of disease variants. In this report we describe a New Zealand family, of European heritage, affected by a sectoral type RP phenotype in association with a novel rhodopsin mutation (proline-170-histidine) in a highly conserved site.

Topics: Adult; Electrophysiology; Histidine; Humans; Male; Pedigree; Point Mutation; Proline; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields; White People

Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds.. We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient's iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient's iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes.. The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.

Topics: Apoptosis; Autophagy; Base Sequence; Biomarkers; Cell Differentiation; Cell Line; Drug Evaluation, Preclinical; Endoplasmic Reticulum Stress; Female; Gene Targeting; Humans; Induced Pluripotent Stem Cells; Middle Aged; Molecular Sequence Data; Mutant Proteins; Mutation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Outer segments (OSs) of rod photoreceptors are cellular compartments specialized in the conversion of light into electrical signals. This process relies on the light-triggered change in the intracellular levels of cyclic guanosine monophosphate, which in turn controls the activity of cyclic nucleotide-gated (CNG) channels in the rod OS plasma membrane. The rod CNG channel is a macromolecular complex that in its core harbors the ion-conducting CNGA1 and CNGB1a subunits. To identify additional proteins of the complex that interact with the CNGB1a core subunit, we applied affinity purification of mouse retinal proteins followed by mass spectrometry. In combination with in vitro and in vivo co-immunoprecipitation and fluorescence resonance energy transfer (FRET), we found that the tetraspanin peripherin-2 links CNGB1a to the light-detector rhodopsin. Using immunoelectron microscopy, we found that this peripherin-2/rhodopsin/CNG channel complex localizes to the contact region between the disk rims and the plasma membrane. FRET measurements revealed that the fourth transmembrane domain (TM4) of peripherin-2 is required for the interaction with rhodopsin. Quantitatively, the binding affinity of the peripherin-2/rhodopsin interaction was in a similar range as that observed for rhodopsin dimers. Finally, we demonstrate that the p.G266D retinitis pigmentosa mutation found within TM4 selectively abolishes the binding of peripherin-2 to rhodopsin. This finding suggests that the specific disruption of the rhodopsin/peripherin-2 interaction in the p.G266D mutant might contribute to the pathophysiology in affected persons.

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Humans; Mice; Nerve Tissue Proteins; Peripherins; Protein Binding; Protein Structure, Tertiary; Retina; Retinal Photoreceptor Cell Outer Segment; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Next-generation sequencing has become more widely used to reveal genetic defect in monogenic disorders. Retinitis pigmentosa (RP), the leading cause of hereditary blindness worldwide, has been attributed to more than 67 disease-causing genes. Due to the extreme genetic heterogeneity, using general molecular screening alone is inadequate for identifying genetic predispositions in susceptible individuals. In order to identify underlying mutation rapidly, we utilized next-generation sequencing in a four-generation Chinese family with RP. Two affected patients and an unaffected sibling were subjected to whole exome sequencing. Through bioinformatics analysis and direct sequencing confirmation, we identified p.R135W transition in the rhodopsin gene. The mutation was subsequently confirmed to cosegregate with the disease in the family. In this study, our results suggest that whole exome sequencing is a robust method in diagnosing familial hereditary disease.

Topics: Adult; Asian People; Exome; Female; Genetic Heterogeneity; Genetic Predisposition to Disease; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Mutation; Pedigree; Polymorphism, Single Nucleotide; Retinitis Pigmentosa; Rhodopsin

This study was to analyze the spectrum and frequency of rhodopsin gene (RHO) mutations in Chinese patients with retinitis pigmentosa (RP).. Patients were given physical examinations, and blood samples were collected for DNA extraction. The RHO mutations were screened with direct sequencing.. Eight heterozygous nucleotide changes were detected in eight of 300 probands with RP, including six novel mutations and two known mutations. p.R21C, p.C110S, p.G182V, p.C187G, c.409-426delGTGGTGGTGTGTAAGCCC, and p.P347L were found in six autosomal dominant families. p.T92I and p.Y178C were found in two isolated cases.. The results reveal the spectrum and frequency of RHO mutations in Chinese patients with different forms of RP and demonstrate that RHO mutations account for a high proportion of autosomal dominant RP (adRP) cases.

Topics: Adult; Asian People; Base Sequence; China; DNA Mutational Analysis; Female; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Young Adult

For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X), cDNA encoding the enhanced green fluorescent protein (EGFP) at its 3' end, and a modified 5' untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP), which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear.

Topics: Aging; Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation, Developmental; Gene Knock-In Techniques; Green Fluorescent Proteins; Humans; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Mutation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transcriptional Activation

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

Topics: Analysis of Variance; Animals; Animals, Genetically Modified; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Humans; Light; Microscopy, Confocal; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Statistics, Nonparametric; Transfection; Vitamin A; Wheat Germ Agglutinins; Xenopus laevis

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.

Topics: Adult; Animals; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Female; Humans; Kinetics; Middle Aged; Mutation, Missense; Protein Stability; Protein Transport; Retinitis Pigmentosa; Rhodopsin

Mutations in more than 60 different genes have been associated with non-syndromic and syndromic retinitis pigmentosa (RP), a heterogeneous group of inherited retinal dystrophies. To increase the understanding of the molecular epidemiology of the disease in Italy, we analyzed 56 patients with syndromic and non-syndromic forms of RP attending the Retinitis Pigmentosa Center of San Paolo Hospital (Milan, Italy). Patients underwent detailed clinical examination. Genomic DNA isolated from peripheral blood samples was screened for mutations in different genes according to RP form by direct sequencing analysis. The impact of novel missense mutations on protein functions was predicted by in silico analysis and protein sequence alignment. Cosegregation analysis was performed between available family members. Forty-one of the 56 probands analyzed had non-syndromic and 15 had syndromic RP forms. Putative disease-causing mutations were identified in 19 of 56 unrelated RP probands. Mutation screening identified a total of 22 different heterozygous variants. Notably, 12 of these putative pathogenic mutations have not been previously reported. New variants were found to be located on the USH2A, RPGR, EYS, and RHO genes. All 3 new variants detected in X-linked RP probands were confirmed in other affected family members. We found a positivity rate of 24.4% and 60% for probands with non-syndromic and syndromic RP, respectively. This is the first report of RPGR X-linked RP proband-ORF15 mutations in Italian patients with X-linked (XL)-RP. In addition, this is the first report of data regarding the association between EYS mutations and non-syndromic RP forms in the Italian population.

Topics: Adult; Aged; Amino Acid Sequence; Base Sequence; DNA Mutational Analysis; Extracellular Matrix Proteins; Eye Proteins; Family Health; Female; Genetic Predisposition to Disease; Humans; Italy; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Amino Acid; Syndrome; Young Adult

The purpose of this study was to determine the molecular basis of retinitis pigmentosa (RP) in a 4 affected sib-family segregating this retinal phenotype.. Affected sibs underwent complete ophthalmologic examination including funduscopic inspection, electroretinogram, fluorescein angiography, visual field measurement, and optical coherence tomography. Both parents were deceased after their sixties and were reported with no visual handicap. Molecular analysis included direct nucleotide sequencing of the rhodopsin gene (RHO), at chromosome 3q21-q24, in DNA from a total of 4 affected sibs. A total of 200 ethnically matched alleles were included as mutation controls.. Sector RP was clinically documented in this family. Wide phenotypic variability was observed with visual acuities ranging from 20/20 to 20/200 and variable funduscopic appearance. Molecular analysis disclosed a c.233A>T mutation at RHO exon 1, predicting a missense p.N78I substitution.. Even though RP can be caused by mutations in a variety of genes, the RHO gene was chosen to be investigated in this RP family since it has been previously associated to sector disease. This case exemplifies the value of guiding RP molecular analysis based on funduscopic features.

Topics: Aged; DNA Mutational Analysis; Electroretinography; Exons; Female; Fluorescein Angiography; Genes, Dominant; Genetic Heterogeneity; Genotype; Humans; Male; Middle Aged; Mutation, Missense; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Stress, Physiological; Visual Acuity; Visual Fields

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.

Topics: Animals; Apoptosis; Caspase 7; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; JNK Mitogen-Activated Protein Kinases; Light; Mice; Mice, Transgenic; Poly(ADP-ribose) Polymerases; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA Interference; RNA, Small Interfering; TNF Receptor-Associated Factor 2; TOR Serine-Threonine Kinases; Unfolded Protein Response

The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.

Topics: Adaptation, Ocular; Age Factors; Animals; Disease Models, Animal; Electroretinography; Eye Proteins; Gene Expression Regulation; Humans; Nerve Tissue Proteins; Neuroglia; Neurons; Neurotransmitter Agents; Rats; Rats, Long-Evans; Rats, Transgenic; Receptors, Glutamate; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1-2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor's active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients.

Topics: Catalytic Domain; Cell Membrane; Half-Life; HEK293 Cells; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrolysis; Isomerism; Kinetics; Mutagenesis, Site-Directed; Mutation, Missense; Protein Denaturation; Protein Stability; Protein Transport; Retinitis Pigmentosa; Rhodopsin; Schiff Bases; Spectrophotometry, Ultraviolet

Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype.

Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Cattle; Cell Death; Cells, Cultured; Chlorocebus aethiops; COS Cells; Genes, Dominant; GTP-Binding Proteins; Humans; Ligands; Mice; Molecular Sequence Data; Mutant Proteins; Mutation; Protein Folding; Protein Transport; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.

Topics: Animals; Apoptosis Inducing Factor; Calpain; Cell Death; Cell Nucleus; Disease Models, Animal; Electroretinography; Gene Expression Regulation; In Situ Nick-End Labeling; Intravitreal Injections; Mitochondria; Mitochondrial Proteins; Mutation; Ophthalmic Solutions; Peptides; Photoreceptor Cells, Vertebrate; Protein Transport; Rats; Rats, Transgenic; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

A rhodopsin P347L transgenic (Tg) rabbit, a model of retinitis pigmentosa, has been generated in our laboratory. The purpose of this study was to determine the properties of focal areas of the retina in this rabbit model during the course of retinal degeneration. To accomplish this, we recorded focal ERGs from wild-type (WT) and Tg rabbits at ages 3, 6, and 12 months. A 15° stimulus spot was used to elicit the focal ERGs from the center of the visual streak and from four surrounding areas. We found that the amplitudes of the focal cone ERG b-waves and oscillatory potentials (OPs) of the Tg rabbits in the five areas decreased progressively with increasing age and became almost non-recordable at 12 months. There were no significant regional differences in the b-waves of Tg rabbits recorded from the 5 areas. The amplitudes of the OPs were better preserved than the b-waves and the OPs/b-wave ratio was higher than that in WT rabbits at every recording area. The summed OPs amplitudes, which most likely originate from the amacrine and/or ganglion cells, recorded from the area superior to the optic disc was significantly larger than that from other areas at 3- and 6-months-old. This indicated that the inner retinal neurons were not altered equally after photoreceptor degeneration in this rabbit model.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Rabbits; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.

Topics: Amino Acid Substitution; Animals; Cattle; HEK293 Cells; Humans; Mutation, Missense; Protein Folding; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho-Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho-Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho-NLS-cre) containing human rhodopsin promoter was cloned. The Rho-Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho-Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho-Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.

Topics: Animals; Cell Line; DNA Repair; Gene Knockout Techniques; Humans; Integrases; Mice; Mice, Transgenic; Organ Specificity; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger

Retinitis Pigmentosa (RP) is the leading cause of inherited blindness in the developed world, affecting approximately 1 in 3000 individuals. Although there is currently no cure for RP, the genetic pathology has been well established. In this study, we developed a novel mouse model of RP (huRhoP347S) expressing a pathogenic human rhodopsin gene with a Pro347Ser (P347S) mutation on a rhodopsin knockout background. These mice undergo severe retinal degeneration at 1 month of age. In contrast to prior studies, this model was administered a gene therapy treatment at 19 days postnata. We evaluated several self-complementary adeno-associated virus (AAV) serotypes for photoreceptor tropism, including scAAV2/2, scAAV2/5, scAAV2/6.2 and scAAV2/9, and found that scAAV2/9 transduced photoreceptors with greater efficiency and expression than other vectors. We engineered an scAAV2/9 vector to contain a microRNA sequence specifically targeting the human rhodopsin gene and demonstrated its ability to silence rhodopsin by 60.2±8.2% in vitro. In addition, we constructed an scAAV2/9 vector to contain a replacement 'codon-modified' rhodopsin transgene (RhoR2) that was resistant to degradation by the microRNA. We found that delivery of the RhoR2 by scAAV2/9 is capable of restoring vision to rhodopsin knockout mice, and rescuing our novel transgenic huRhoP347S mouse model of dominant RP. Average a-wave responses of RhoR2-injected eyes were 1.8-fold higher than those of control-injected eyes. We found that delivery of the microRNA and replacement rhodopsin in a 1:2 ratio produced an average electroretinography (ERG) a-wave response of 17.4±2.9 compared to 6.5±2.8 μV for eyes injected with negative control virus.

Topics: Animals; Dependovirus; Disease Models, Animal; Gene Silencing; Genetic Therapy; Mice; MicroRNAs; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Transgenes

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.

Topics: Age of Onset; Humans; Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA

Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6α(nmf363) mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6α(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6β deficiency, is also likely to be treatable by gene therapy.

Topics: Animals; Cyclic Nucleotide Phosphodiesterases, Type 6; Dependovirus; Disease Models, Animal; Electroretinography; Genetic Therapy; Genetic Vectors; Immunoblotting; Mice; Mice, Inbred C57BL; Mice, Transgenic; Promoter Regions, Genetic; Retina; Retinitis Pigmentosa; Rhodopsin; Transduction, Genetic

We have recently described the surviving cones and Müller-glia process remodeling in retinitis pigmentosa (RP) and shown that rod degeneration triggers the reorganization of the cone mosaic into an orderly array of rings. Within these rings, remodeled Müller-glia processes envelope cones. Here, we report the spatiotemporal pattern of healthy rods, their relationship with dying rods and the way that rod death stimulates the modification of cone spatial-distribution patterns and Müller-glia processes in the S334ter-line-3 rat, a transgenic model expressing a rhodopsin mutation that causes RP. The spatial patterns of rods, cones, microglial and Müller cells were labeled by immunocytochemistry with cell-type-specific markers at various stages of deveopment in rat whole-mount retinas. Spatial patterns of dying cells were examined by TUNEL staining. The S334ter rod mosaic began to develop small holes around postnatal day 10. These hot-spots of cell death progressively increased in size, leaving larger rod-less holes behind. The holes were temporarily occupied by active microglial cells, before being replaced by remodeled Müller-cell processes. Our data suggest that the hot spots of rod death create holes in the rod mosaic early in retinal degeneration and that the resulting pattern triggers the modification of the spatial-distribution patterns of cones and glia cells.

Topics: Animals; Cell Death; Disease Models, Animal; Disease Progression; Microglia; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Time Factors

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin's stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.

Topics: Amino Acid Substitution; Animals; Exons; GTP-Binding Protein alpha Subunits; Humans; Mice; Mice, Mutant Strains; Mutation, Missense; Phagocytosis; Protein Transport; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transducin

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Topics: Animals; Basic-Leucine Zipper Transcription Factors; CpG Islands; DNA Methylation; Electroretinography; Eye Proteins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

Mutations in the gene encoding the transcription factor neural retina leucine zipper (NRL) are known to cause autosomal dominant (adRP) or recessive (arRP) retinitis pigmentosa (RP). In an adRP Spanish family, we detected a novel sequence variation (c.287T>C) in the NRL gene that results in the p.M96T protein change. A functional test of the ability of NRL, in conjunction with cone-rod homeobox (CRX), to transactivate a human rhodopsin (RHO) promoter was used to evaluate the pathogenic mechanisms of NRL. We found upregulation of the RHO promoter by p.M96T protein similar to that shown by other missense NRL mutations that cause adRP. Affected RP patients of the family carry the nucleotide change, although two other family members that also carry the c.287T>C variation remain asymptomatic. This result complicates the genetic counselling of the family. The pathogenic mechanisms associated with adRP NRL mutations appear to be caused by a gain of function. To suppress the negative effect of an NRL mutant, the suppression and replacement strategy seems to be the most suitable therapeutic approach capable of overcoming the mutational heterogeneity associated with NRL-linked adRP. Thus, we evaluated this methodology in the NRL gene for the first time.

Topics: Adult; Aged; Amino Acid Sequence; Animals; Basic-Leucine Zipper Transcription Factors; Chlorocebus aethiops; COS Cells; Eye Proteins; Genes, Dominant; Genetic Heterogeneity; Genetic Variation; Homeodomain Proteins; Humans; Middle Aged; Molecular Sequence Data; Mutation, Missense; Pedigree; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Trans-Activators; Transcriptional Activation; Up-Regulation

In retinitis pigmentosa (RP), the death of cones normally follows some time after the degeneration of rods. Recently, surviving cones in RP have been studied and reported in detail. These cones undergo extensive remodeling in their morphology. Here we report an extension of the remodeling study to consider possible modifications of spatial-distribution patterns. For this purpose we used S334ter-line-3 transgenic rats, a transgenic model developed to express a rhodopsin mutation causing RP. In this study, retinas were collected at postnatal (P) days P5-30, 90, 180, and P600. We then immunostained the retinas to examine the morphology and distribution of cones and to quantify the total cone numbers. Our results indicate that cones undergo extensive changes in their spatial distribution to give rise to a mosaic comprising an orderly array of rings. These rings first begin to appear at P15 at random regions of the retina and become ubiquitous throughout the entire tissue by P90. Such distribution pattern loses its clarity by P180 and mostly disappears at P600, at which time the cones are almost all dead. In contrast, the numbers of cones in RP and normal conditions do not show significant differences at stages as late as P180. Therefore, rings do not form by cell death at their centers, but by cone migration. We discuss its possible mechanisms and suggest a role for hot spots of rod death and the remodeling of Müller cell process into zones of low density of photoreceptors.

Topics: Aging; Animals; Antibodies; Cell Death; Cell Line; Coloring Agents; Disease Progression; Hematoxylin; Immunohistochemistry; In Situ Nick-End Labeling; Opsins; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a pathological condition associated with blindness due to progressive retinal degeneration. RP-linked mutations lead to changes at the retinal binding pocket and in the absorption spectra. Here, we evaluate the geometries, electronic effects, and vertical excitation energies in the dark state of mutated human rhodopsins carrying the abnormal substitutions M207R or S186W at the retinal binding pocket. Two models are used, the solvated protein and the protein in a solvated POPC lipid bilayer. We apply homology modeling, classical molecular dynamics simulations, density functional theory (DFT), and quantum mechanical/molecular mechanical (QM/MM) methods. Our results for the wild type bovine and human rhodopsins, used as a reference, are in good agreement with experiment. For the mutants, we find less twisted QM/MM ground-state chromophore geometries around the C(11)-C(12) double bond and substantial blue shifts in the lowest vertical DFT excitation energies. An analysis of the QM energies shows that the chromophore-counterion region is less stable in the mutants compared to the wild type, consistent with recent protein folding studies. The influence of the mutations near the chromophore is discussed in detail to gain more insight into the properties of these mutants. The spectral tuning is mainly associated with counterion effects and structural features of the retinal chain in the case of the hM207R mutant, and with the presence of a neutral chromophore with deprotonated Lys296 in the case of the hS186W mutant.

Topics: Animals; Binding Sites; Cattle; Crystallography, X-Ray; Humans; Lipid Bilayers; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Molecular Structure; Mutation; Protein Conformation; Quantum Theory; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Dependovirus; Disease Models, Animal; Electroretinography; Gene Transfer Techniques; Genes, Dominant; Genetic Therapy; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Dependovirus; DNA-Binding Proteins; Electroretinography; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Transfer Techniques; Genetic Therapy; Heat Shock Transcription Factors; Heat-Shock Proteins; HeLa Cells; Humans; Photoreceptor Cells, Vertebrate; Protein Transport; Proteostasis Deficiencies; Rats; Rats, Transgenic; Retinitis Pigmentosa; Rhodopsin; RNA, Small Interfering; Transcription Factors

Topics: Autophagy; Cells, Cultured; Gene Expression; Genes, Dominant; Humans; In Vitro Techniques; Protein Transport; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Unfolded Protein Response

To describe early-onset cystoid macular edema (CME) in a family with retinitis pigmentosa (RP) due to the p.P347L in the rhodopsin gene (RHO).. All affected family members, including a 44-year-old mother and four children in their teens (two daughters, 17 and 15 years old, and two sons, 13 and 11 years old), have a mutation of p.P347L in RHO. Funduscopy, Goldmann perimetry, spectral domain optical coherence tomography (SD-OCT) and electroretinogram (ERG) were performed in all affected members to assess the retinal anatomy and function.. The mother had very poor visual acuity of light perception in both eyes, and marked foveal atrophy was observed via SD-OCT. Although the macular appearance in the funduscopy looked unremarkable in the four children, SD-OCT revealed bilateral CME in all the children. The rod response in ERG was extinguished and the cone response was decreased in all children.. The results present the possibility that CME in RP patients may be associated with a specific genotype such as the p.P347L in RHO. We speculate that the severe visual prognosis of this mutation may be related to early-onset CME, as shown in this family. However, further investigation in more RP patients with this mutation and CME will be needed.

Topics: Adolescent; Adult; Child; DNA Mutational Analysis; Electroretinography; Female; Genotype; Humans; Macular Edema; Male; Mutation; Pedigree; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; Visual Field Tests

The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene.. Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured.. A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H.. Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Topics: Animals; Animals, Genetically Modified; Blotting, Southern; Cell Line; Disease Models, Animal; Electroretinography; Female; Follow-Up Studies; Gene Expression Regulation; Genotype; Humans; In Situ Hybridization, Fluorescence; Male; Mutation; Nuclear Transfer Techniques; Retina; Retinitis Pigmentosa; Rhodopsin; RNA; Swine; Swine, Miniature

Rhodopsin is the G protein-coupled receptor in charge of initiating signal transduction in rod photoreceptor cells upon the arrival of the photon. D190N (Rho(D190n)), a missense mutation in rhodopsin, causes autosomal-dominant retinitis pigmentosa (adRP) in humans. Affected patients present hyperfluorescent retinal rings and progressive rod photoreceptor degeneration. Studies in humans cannot reveal the molecular processes causing the earliest stages of the condition, thus necessitating the creation of an appropriate animal model. A knock-in mouse model with the D190N mutation was engineered to study the pathogenesis of the disease. Electrophysiological and histological findings in the mouse were similar to those observed in human patients, and the hyperfluorescence pattern was analogous to that seen in humans, confirming that the D190N mouse is an accurate model for the study of adRP.

Topics: Animals; Disease Models, Animal; Gene Order; Gene Targeting; Genes, Dominant; Humans; Mice; Mice, Transgenic; Mutation; Protein Transport; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.

Topics: Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Mice; Mice, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; RNA

This study evaluated the capacity of Xenopus laevis retina to regenerate photoreceptor cells after cyclic light-mediated acute rod photoreceptor degeneration in a transgenic P23H mutant rhodopsin model of retinits pigmentosa. After discontinuation of cyclic light exposure, we monitored histologic progression of retinal regeneration over a 3 week recovery period. To assess their metabolomic states, contralateral eyes were processed for computational molecular phenotyping. We found that retinal degeneration in the P23H rhodopsin mutation could be partially reversed, with regeneration of rod photoreceptors recovering normal morphology (including full-length rod outer segments) by the end of the 3 week recovery period. In contrast, retinal degeneration mediated by directly induced apoptosis did not recover in the 3 week recovery period. Dystrophic rod photoreceptors with truncated rod outer segments were identified as the likely source of rod photoreceptor regeneration in the P23H retinas. These dystrophic photoreceptors remain metabolically active despite having lost most of their outer segments.

Topics: Amino Acid Substitution; Animals; Animals, Genetically Modified; Disease Models, Animal; Histidine; Mutation; Nerve Regeneration; Proline; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis

Topics: Animals; Female; Gene Expression Regulation; Humans; Male; Nuclear Transfer Techniques; Retina; Retinitis Pigmentosa; Rhodopsin; RNA; Swine, Miniature

Chronic stress in the endoplasmic reticulum (ER) underlies many degenerative and metabolic diseases involving apoptosis of vital cells. A well-established example is autosomal dominant retinitis pigmentosa (ADRP), an age-related retinal degenerative disease caused by mutant rhodopsins. Similar mutant alleles of Drosophila Rhodopsin-1 also impose stress on the ER and cause age-related retinal degeneration in that organism. Well-characterized signalling responses to ER stress, referred to as the unfolded protein response (UPR), induce various ER quality control genes that can suppress such retinal degeneration. However, how cells activate cell death programs after chronic ER stress remains poorly understood. Here, we report the identification of a signalling pathway mediated by cdk5 and mekk1 required for ER-stress-induced apoptosis. Inactivation of these genes specifically suppressed apoptosis, without affecting other protective branches of the UPR. CDK5 phosphorylates MEKK1, and together, they activate the JNK pathway for apoptosis. Moreover, disruption of this pathway can delay the course of age-related retinal degeneration in a Drosophila model of ADRP. These findings establish a previously unrecognized branch of ER-stress response signalling involved in degenerative diseases.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line; Cells, Cultured; Chromosome Aberrations; Cyclin-Dependent Kinase 5; Disease Models, Animal; Drosophila; Drosophila Proteins; Endoplasmic Reticulum Stress; Genes, Dominant; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Molecular Sequence Data; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Time Factors

To identify and functionally characterize the mutation responsible for autosomal dominant retinitis pigmentosa (adRP) in a large, six-generation French family.. Twenty individuals from this family participated in the genetic investigation. Six affected and 14 unaffected individuals from three-generations were available for linkage analysis using microsatellite markers flanking the rhodopsin (RHO) gene. A two-point logarithm of odds (LOD) score calculation was undertaken using GENEMARKER and MLINK software. Sanger sequencing of RHO was performed. Cellular localization of the mutant protein was performed by transforming SK-N-SH cells with pEGFP-N1-Rho, pEGFP-N1-Rho(P23H), and pEGFP-N1-Rho(c.614-622del).. The proband had nyctalopia, visual field constriction, peripheral bone spicule pigmentation of the fundus, central acuity (6/24 RE; 6/12 LE) at 55 years of age. Linkage analysis of this family suggested RHO as a possible candidate since the flanking marker D3S1292 yielded a LOD score of 2.43 at θ=0. Cloning of an exon 3 PCR product and direct sequencing of single clones identified a novel deletion in the third exon of RHO, c.614-622del (p.Y206-F208del). The deleted mutant protein localized to the endoplasmic reticulum and formed inclusion bodies.. This novel deletion in exon 3 of the RHO gene, c.614-622del results in a classical form of adRP in a multi-generation French family. Protein expression analyses confirmed that the deletion led to protein misfolding and suggest this is a class II mutation, similar to P23H, the most common class II mutation seen in North America.

Topics: Adolescent; Base Sequence; Case-Control Studies; Cell Line; Exons; Female; France; Genes, Dominant; Genetic Linkage; Humans; Microsatellite Repeats; Middle Aged; Molecular Sequence Data; Pedigree; Plasmids; Proteostasis Deficiencies; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Sequence Deletion; Transformation, Genetic; White People

Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Amino Acid Substitution; Animals; Animals, Genetically Modified; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 6; Enzyme Inhibitors; Fish Proteins; Gene Knockdown Techniques; Humans; Light; Mice; Mice, Inbred Strains; Protein Transport; Recombinant Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Second Messenger Systems; Transducin; Vision, Ocular; Zebrafish

Topics: Animals; Dependovirus; Dextrans; Eye Proteins; Genetic Therapy; Glycerides; Humans; Membrane Proteins; Mutation; Nanoparticles; Protamines; Retina; Retinitis Pigmentosa; Retinoschisis; Rhodopsin; RNA

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Disease Models, Animal; Electroretinography; Exons; Eye Proteins; Female; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Mutant Strains; Molecular Sequence Data; Mutation; Retina; Retinal Degeneration; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid

Retinitis pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. In this article, we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knockout (Rhod-ko) models. The RNA repertoire of single MGCs was comprehensively profiled, and a comparison was made between MGCs from wild-type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death.. Retinas were dissociated, and single MGCs were chosen under a dissecting microscope using a micropipette. Single cell cDNAs were generated and genome-wide profiles were obtained by hybridization to Affymetrix arrays. A comparison was made among all samples to discover the changes in gene expression during the periods of rod and cone photoreceptor death.. MGCs respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of glial fibrillary acidic protein (Gfap). Many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune-related response.. A high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells and/or inherent heterogeneity among MGCs.

Topics: Animals; Cell Death; Gene Expression; Gene Expression Profiling; Gene Knockout Techniques; Glial Fibrillary Acidic Protein; Mice; Nerve Tissue Proteins; Neuroglia; Oligonucleotide Array Sequence Analysis; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Single-Cell Analysis; Up-Regulation

Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients.. We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray.. The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%.. The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP.

Topics: Artifacts; Eye Proteins; Genes, Dominant; Genetic Testing; Genotype; Humans; Mutation; Oligonucleotide Array Sequence Analysis; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sensitivity and Specificity; Spain; White People

The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa.. Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance.. Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic.. Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.

Topics: Adult; Alleles; Amino Acid Sequence; Amino Acid Substitution; Biological Transport; Child, Preschool; Computational Biology; DNA Copy Number Variations; Endoplasmic Reticulum; Female; Genetic Predisposition to Disease; Genetic Testing; Genome, Human; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Reproducibility of Results; Retinitis Pigmentosa; Rhodopsin; Sensitivity and Specificity; Sequence Analysis, DNA

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Topics: Animals; Cyclic Nucleotide-Gated Cation Channels; Dependovirus; Disease Models, Animal; DNA, Complementary; Electroretinography; Genetic Therapy; Genetic Vectors; Mice; Mice, Knockout; Nerve Tissue Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding.. To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins.. ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell.. This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

Topics: Activating Transcription Factor 6; Cell Membrane; eIF-2 Kinase; Endoplasmic Reticulum Stress; Endoribonucleases; HEK293 Cells; Homeostasis; Humans; Membrane Proteins; Mutagenesis; Protein Serine-Threonine Kinases; Proteostasis Deficiencies; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Unfolded Protein Response

To evaluate microarray-based genotyping technology for the detection of mutations responsible for retinitis pigmentosa (RP) and to perform phenotypic characterization of patients with pathogenic mutations.. DNA from 336 patients with RP and 360 controls was analyzed using the GoldenGate assay with microbeads containing 95 previously reported disease-associated mutations from 28 RP genes. Mutations identified by microarray-based genotyping were confirmed by direct sequencing. Segregation analysis and phenotypic characterization were performed in patients with mutations. The disease severity was assessed by visual acuity, electroretinography, optical coherence tomography, and kinetic perimetry.. Ten RP-related mutations of five RP genes (PRP3 pre-mRNA processing factor 3 homolog [PRPF3], rhodopsin [RHO], phosphodiesterase 6B [PDE6B], peripherin 2 [PRPH2], and retinitis pigmentosa 1 [RP1]) were identified in 26 of the 336 patients (7.7%) and in six of the 360 controls (1.7%). The p.H557Y mutation in PDE6B, which was homozygous in four patients and heterozygous in nine patients, was the most frequent mutation (2.5%). Mutation segregation was assessed in four families. Among the patients with missense mutations, the most severe phenotype occurred in patients with p.D984G in RP1; less severe phenotypes occurred in patients with p.R135W in RHO; a relatively moderate phenotype occurred in patients with p.T494M in PRPF3, p.H557Y in PDE6B, or p.W316G in PRPH2; and a mild phenotype was seen in a patient with p.D190N in RHO.. The results reveal that the GoldenGate assay may not be an efficient method for molecular diagnosis in RP patients with rare mutations, although it has proven to be reliable and efficient for high-throughput genotyping of single-nucleotide polymorphisms. The clinical features varied according to the mutations. Continuous effort to identify novel RP genes and mutations in a population is needed to improve the efficiency and accuracy of the genetic diagnosis of RP.

Topics: Adult; Asian People; Cyclic Nucleotide Phosphodiesterases, Type 6; Eye Proteins; Female; Genotype; Heterozygote; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Microtubule-Associated Proteins; Middle Aged; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Pedigree; Peripherins; Retinitis Pigmentosa; Rhodopsin; Ribonucleoprotein, U4-U6 Small Nuclear; Sequence Analysis, DNA; Severity of Illness Index; Visual Acuity

Topics: Activating Transcription Factor 6; eIF-2 Kinase; Endoplasmic Reticulum Stress; Humans; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.

Topics: Alleles; Animals; Gene Expression; Genes, Dominant; Genetic Therapy; Mice; Mutation; Nerve Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

We investigated retinitis pigmentosa (RP) caused by a mutation in the gene rhodopsin (RHO) with a patient-specific rod cell model generated from induced pluripotent stem cells (iPSCs) derived from an RP patient. To generate the iPSCs and to avoid the unpredictable side effects associated with retrovirus integration at random loci in the host genome, a nonintegrating Sendai-virus vector was installed with four key reprogramming gene factors (POU5F1, SOX2, KLF4, and c-MYC) in skin cells from an RP patient. Subsequent selection of the iPSC lines was on the basis of karyotype analysis as well as in vitro and in vivo pluripotency tests. Using a serum-free, chemically defined, and stepwise differentiation method, the expressions of specific markers were sequentially induced in a neural retinal progenitor, a retinal pigment epithelial (RPE) progenitor, a photoreceptor precursor, RPE cells, and photoreceptor cells. In the differentiated rod cells, diffused distribution of RHO protein in cytoplasm and expressions of endoplasmic reticulum (ER) stress markers strongly indicated the involvement of ER stress. Furthermore, the rod cell numbers decreased significantly after successive culture, suggesting an in vitro model of rod degeneration. Thus, from integration-free patient-specific iPSCs, RP patient-specific rod cells were generated in vitro that recapitulated the disease feature and revealed evidence of ER stress in this patient, demonstrating its utility for disease modeling in vitro.

Topics: Biomarkers; Cell Count; Cell Culture Techniques; Cell Differentiation; Culture Media, Serum-Free; Endoplasmic Reticulum Stress; Fibroblasts; Genetic Vectors; Genome, Human; Humans; Induced Pluripotent Stem Cells; Karyotyping; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Models, Biological; Mutation; Octamer Transcription Factor-3; Proto-Oncogene Proteins c-myc; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Retroviridae; Rhodopsin; Sendai virus; SOXB1 Transcription Factors; Virus Integration

To describe the clinical and genetic findings in two Chinese families with retinitis pigmentosa (RP).. Two unrelated families were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Genotyping and haplotyping analysis was performed on the known genetic loci for autosomal dominant retinitis pigmentosa (adRP) with a panel of polymorphic markers in the two families, and then mutation screening of all coding exons of the RHO gene was performed by direct sequencing of PCR-amplified DNA fragments. Whenever substitutions were identified in a patient, restriction fragment length polymorphism analysis was performed on all available family members and on 100 normal controls.. Clinical examination and pedigree analysis revealed two four-generation families (83 and 112) with adRP. A significant two-point linkage odd disequilibrium (LOD) score was generated at marker D3S1292 (Zmax=1.90, θ=0) for family 83 and (Zmax=2.77, θ=0) for family 112, respectively, and further linkage and haplotype studies confined the disease locus to 3q21-22 where the RHO gene is located. Mutation screening of the RHO gene in the two families revealed a G→C transversion at position 505 (p.A169P) of the cDNA sequence in family 83 and a C→A transversion at position 1040 (p.P347Q) of the cDNA in family 112. The novel p.A169P and recurrent p.P347Q mutations cosegregated with the phenotypes of the two families. Secondary structure prediction suggested that the mutant rhodopsin 169P led to significant secondary structure changes between residues 165 and 169, which may interfere with the correct folding of the transmembrane domain.. Two mutations of the RHO gene were identified in two Chinese families with adRP. Our findings further suggest codon 347 is the mutation hotspot of the RHO.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Asian People; Codon; Exons; Female; Genetic Association Studies; Genetic Loci; Genetic Markers; Haplotypes; Humans; Linkage Disequilibrium; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Phenotype; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin

We had previously reported that transduction of the channelrhodopsin-2 (ChR2) gene into retinal ganglion cells restores visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats. In this study, we attempted to reveal the safety and influence of exogenous ChR2 gene expression. Adeno-associated virus (AAV) type 2 encoding ChR2 fused to Venus (rAAV-ChR2V) was administered by intra-vitreous injection to dystrophic RCS rats. However, rAAV-ChR2 gene expression was detected in non-target organs (intestine, lung and heart) in some cases. ChR2 function, monitored by recording visually evoked potentials, was stable across the observation period (64 weeks). No change in retinal histology and no inflammatory marker of leucocyte adhesion in the retinal vasculature were observed. Although antibodies to rAAV (0.01-12.21 μg ml(-1)) and ChR2 (0-4.77 μg ml(-1)) were detected, their levels were too low for rejection. T-lymphocyte analysis revealed recognition by T cells and a transient inflammation-like immune reaction only until 1 month after the rAAV-ChR2V injection. In conclusion, ChR2, which originates from Chlamydomonas reinhardtii, can be expressed without immunologically harmful reactions in vivo. These findings will help studies of ChR2 gene transfer to restore vision in progressed retinitis pigmentosa.

Topics: Animals; Antibodies, Viral; Dependovirus; DNA Primers; Enzyme-Linked Immunosorbent Assay; Evoked Potentials, Visual; Genetic Therapy; Immunohistochemistry; Microscopy, Fluorescence; Rats; Retinitis Pigmentosa; Rhodopsin; T-Lymphocytes; Transduction, Genetic

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein-lipid interphase, we used the von Heijne water-membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (>1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (<1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Child; Child, Preschool; Humans; Infant; Infant, Newborn; Middle Aged; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation, Missense; Night Blindness; Protein Folding; Retinitis Pigmentosa; Rhodopsin; Young Adult

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

Topics: Animals; Cloning, Molecular; Dependovirus; DNA Primers; DNA, Complementary; Electroretinography; Genes, Dominant; Genetic Therapy; Genetic Vectors; Immunoblotting; Mice; Mice, Transgenic; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

To report a novel, identical nonsense mutation in the rhodopsin (RHO) gene in two Indonesian families with autosomal recessive retinitis pigmentosa (arRP).. Mutation screening for the RHO gene was performed in 38 unrelated patients with retinitis pigmentosa (RP) by direct sequencing. Clinical features were also characterized, through complete ophthalmologic examination. Family members of RP patients testing positive for the RHO gene were subjected to genetic and clinical examination. To assess the founder effect in the two families, haplotype analysis also was performed.. A novel homozygous nonsense mutation was detected in two patients by a G to A transition at nucleotide position 482 in exon 2 of the RHO gene, resulting in substitution of a tryptophan-to-stop at codon 161 (c.482G>A, p.W161X). Examination of family members of these 2 patients showed that the affected members were homozygous and unaffected carriers were heterozygous for the p.W161X mutation. Haplotype analysis revealed that members of the two families carried the same disease-associated variants in markers (IVS1 RHO and D3S2322). No p.W161X mutations were detected in 45 normal Indonesian subjects, nor were any mutations detected in exons 1-5 of the RHO gene in the remaining 36 RP patients.. We detected a novel, recessive nonsense mutation (p.W161X) in the RHO gene of two families through mutation screening of RHO in 38 Indonesian RP patients. Haplotype analysis suggested that p.W161X was the founder mutation.

Topics: Adolescent; Adult; Asian People; Base Sequence; Child; Codon, Nonsense; DNA Mutational Analysis; DNA Primers; Female; Founder Effect; Genes, Recessive; Haplotypes; Humans; Indonesia; Male; Microsatellite Repeats; Middle Aged; Molecular Sequence Data; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

One of the characteristic signs of retinitis pigmentosa (RP) is the progressive loss of night vision. We have previously shown that the gain of rod photoreceptor activation is moderately reduced in some patients with RP, but this decrease in activation kinetics is not sufficient to account for the night blindness. Recently, single rod recording from animal models of RP showed rods under degeneration remain saturated for shorter periods than normal rods; i.e. are less able to sustain the rod photoresponse. Using paired-flash ERG, here we determine whether rod phototransduction inactivation parameters might also be abnormal in patients with RP. Inactivation parameters were derived from 13 subjects with normal vision, 16 patients with adRP, and 16 patients with autosomal recessive/isolate (rec/iso) RP. The adRP cases included 9 patients with rhodopsin mutations and 7 patients with peripherin/RDS mutations. The inactivation phase was derived using a double-flash paradigm, with a test flash of 2.7 log scot td-s followed at varying intervals by a 4.2 log scot td-s probe flash. Derived rod photoresponses to this just-saturating test flash in normal subjects exhibit a critical time to the initiation of recovery (T(sat)) of 525 ± 90 (SD) ms. The values of T(sat) were 336 ± 104 (SD) ms in patients with adRP (P < 0.001) and 271 ± 45 (SD) ms (P < 0.001) in patients with rec/iso RP. When T(sat) values were categorized by mutations, the values were 294 ± 91 (SD) ms (P < 0.001) for rhodopsin mutations, and 389 ± 100 (SD) ms (p = 0.01) for peripherin/RDS mutations. Overall, T(sat) in patients with RP was significantly correlated with the amplitude of ISCEV standard rod response (r = 0.56; P < 0.001) and the gain of the activation phase of phototransduction (r = 0.6, P < 0.001). T(sat) may be a useful marker for therapeutic efficacy in future clinical trials in RP.

Topics: Adult; Aged; Electroretinography; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Peripherins; Photic Stimulation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a 'repression-replacement' strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.

Topics: Animals; Disease Models, Animal; Down-Regulation; Gene Knockdown Techniques; Genetic Therapy; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ophthalmoscopy; Repressor Proteins; Retinitis Pigmentosa; Rhodopsin; Transcription, Genetic

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.

Topics: Alkyl and Aryl Transferases; Animals; Dolichols; Exons; Female; Genes, Dominant; Genetic Variation; Glycosylation; Humans; Male; Mutation; Pedigree; Phenotype; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Zebrafish; Zebrafish Proteins

The visual photoreceptor rhodopsin undergoes a series of conformational changes upon light activation, eventually leading to the active metarhodopsin II conformation, which is able to bind and activate the G-protein, transducin. We have previously shown that mutant rhodopsins G51V and G89D, associated with retinitis pigmentosa, present photobleaching patterns characterized by the formation of altered photointermediates whose nature remained obscure. Our current detailed UV-visible spectroscopic analysis, together with functional characterization, indicate that these mutations influence the relative stability of the different metarhodopsin photointermediates by altering their equilibria and maintaining the receptor in a nonfunctional light-induced conformation that may be toxic to photoreceptor cells. We propose that G51V and G89D shift the equilibrium from metarhodopsin I towards an intermediate, recently named as metarhodopsin Ib, proposed to interact with transducin without activating it. This may be one of the causes contributing to the molecular mechanisms underlying cell death associated with some retinitis pigmentosa mutations.

Topics: Humans; Light; Models, Molecular; Mutation; Protein Conformation; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry, Ultraviolet

To investigate the cellular expression of cis-acting splicing mutations in the rhodopsin gene (RHO) that lead to autosomal dominant or recessive retinitis pigmentosa (adRP/arRP) and the role of nonsense-mediated mRNA decay (NMD) in its pathogenic mechanism. To design a potential therapeutic RNAi-based suppression strategy for cis-acting adRP splicing mutants.. Cells were transfected with genomic constructs encoding the human wild-type (WT) and c.531-2A>G, c.936+1G>T, c.937-1G>T and c.745G>T RHO mutants. Total RNA was quantified by RT-PCR and protein was analyzed by immunocytochemistry. Three small interfering (si)RNAs directed against adRP mutant transcripts were designed and assayed in COS7 cells.. The RHO cis-acting splicing mutations causing adRP, c.531-2A>G and c.937-1G>T, induce cryptic splicing. In contrast, the c.936+1G>T mutation, which causes arRP, results in exon skipping. Although the c.531-2A>G and c.745G>T RHO sequence predicted a premature termination codon (PTC) that should be a target for NMD, these mutant proteins were detected in transfected cells. The siRNAs designed to interfere with adRP mutants silenced the corresponding mRNA with varying efficiency.. Although two RHO mutations that cause different RP phenotypes were the target for the NMD mechanism, a fraction of mutant RNA transcript may circumvent the NMD mechanism and be translated into protein. Thus, different levels of mutant protein may be necessary to trigger the RP phenotype. The findings demonstrate the potential use of siRNA to interfere with cis-acting splicing RHO transcripts. However, limitations in the mutation sequence and incomplete mutant transcript elimination should be considered in a therapeutic approach for adRP.

Topics: Alternative Splicing; Animals; Blotting, Western; Cells, Cultured; Chlorocebus aethiops; COS Cells; Genes, Dominant; HeLa Cells; Humans; Immunohistochemistry; Mutagenesis, Site-Directed; Mutant Proteins; Mutation; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA Interference; RNA Splicing; RNA, Messenger; RNA, Small Interfering; Transfection

Retinitis pigmentosa (RP) is a type of inherited retinal degenerative disease, which leads to blindness. The primary pathological event of this disease is the death of rods because of genetic mutations. The S334ter-line-3 rat is a transgenic model developed to express a rhodopsin mutation similar to that found in RP. In this study, the rod's death triggered are organization of the cone mosaic into an orderly array of rings. Four observations were relevant to understand this reorganization. First, rods died in hot spots, which progressively increased as circular waves, leaving rod-less zones behind. Second, rings of cones formed around these zones. Third, remodeled Müller glia processes enveloped cones and filled the center of their rings. Zonula occludens-1 located between the photoreceptor inner segments and the apical processes of Müller cells showed in the rings. Fourth, these rings were formed before the onset of cone cell deaths and were maintained until late stages of RP. From these observations,we hypothesize that cone-Müller-cell interactions mediate and maintain the rings. A test of this hypothesis can be performed by injecting DL-a-aminoadipic acid (AAA), which is known to disrupt Müller cell metabolism. A single intravitreal injection of AAA at P50 disrupted the rings of cones 3 days after the injection. These findings indicate that the rearrangement of cones in rings is modulated by Müller cells in RP. Thus, if the relationship between photoreceptors and Müller glia is better understood, the latter could potentially be manipulated for effective neuroprotection or the restoration of normal cone arrays.

Topics: 2-Aminoadipic Acid; Age Factors; Animals; Animals, Newborn; Cell Communication; Cell Death; Cell Movement; Disease Models, Animal; Excitatory Amino Acid Antagonists; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; In Situ Nick-End Labeling; Intravitreal Injections; Membrane Proteins; Mice; Microscopy, Confocal; Mutation; Neuroglia; Opsins; Phosphoproteins; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Zonula Occludens-1 Protein

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat(-)(/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. Although both M and S cone opsins mistrafficked as reported previously, mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat(-)(/-) cones before the onset of massive ventral/central cone degeneration. As the ventral and central retina express higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65(-)(/-) and Lrat(-)(/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in patients with LCA. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an endoplasmic reticulum stress pathway, as demonstrated in both the Lrat(-)(/-) retina and transfected cells. Replacing rhodopsin with S-opsin in Lrat(-)(/-) rods resulted in mislocalization and aggregation of S-opsin in the inner segment and the synaptic region of rods, ER stress, and dramatically accelerated rod degeneration. Our results demonstrate that cone opsins play a major role in determining the degeneration rate of photoreceptors in LCA.

Topics: Acyltransferases; Animals; Cone Opsins; Endoplasmic Reticulum; Humans; Leber Congenital Amaurosis; Mice; Mice, Knockout; Protein Transport; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Time Factors

To determine the spectrum and frequency of rhodopsin gene (RHO) mutations in Korean patients with retinitis pigmentosa (RP) and to characterize genotype-phenotype correlations in patients with mutations.. The RHO mutations were screened by direct sequencing, and mutation prevalence was measured in patients and controls. The impact of missense mutations to RP was predicted by segregation analysis, peptide sequence alignment, and in silico analysis. The severity of disease in patients with the missense mutations was compared by visual acuity, electroretinography, optical coherence tomography, and kinetic visual field testing.. Five heterozygous mutations were identified in six of 302 probands with RP, including a novel mutation (c.893C>A, p.A298D) and four known mutations (c.50C>T, p.T17M; c.533A>G, p.Y178C; c.888G>T, p.K296N; and c.1040C>T, p.P347L). The allele frequency of missense mutations was measured in 114 ethnically matched controls. p.A298D, newly identified in a sporadic patient, had never been found in controls and was predicted to be pathogenic. Among the patients with the missense mutations, we observed the most severe phenotype in patients with p.P347L, less severe phenotypes in patients with p.Y178C or p.A298D, and a relatively moderate phenotype in a patient with p.T17M.. The results reveal the spectrum of RHO mutations in Korean RP patients and clinical features that vary according to mutations. Our findings will be useful for understanding these genetic spectra and the genotype-phenotype correlations and will therefore help with predicting disease prognosis and facilitating the development of gene therapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Asian People; Case-Control Studies; Child; Electroretinography; Female; Gene Frequency; Genetic Association Studies; Genetic Heterogeneity; Genetic Testing; Genotype; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation, Missense; Pedigree; Phenotype; Republic of Korea; Retinitis Pigmentosa; Rhodopsin; Sequence Alignment; Sequence Analysis, DNA; Severity of Illness Index; Tomography, Optical Coherence; Visual Acuity

The eukaryotic endoplasmic reticulum operates multiple quality control mechanisms to ensure that only properly folded proteins are exported to their final destinations via the secretory pathway and those that are not are destroyed via the degradation pathway. However, molecular mechanisms underlying such regulated exportation to these distinct routes are unknown. In this article, we report the role of Drosophila arf72A--the fly homologue of the mammalian Arl1 - in the quality checks of proteins and in the autosomal-dominant retinopathy. ARF72A localizes to the Golgi membranes of Drosophila photoreceptor cells, consistent with mammalian Arl1 localization in cell culture systems. A loss of arf72A function changes the membrane character of the endoplasmic reticulum and shifts the membrane balance between the endoplasmic reticulum and the Golgi complex toward the Golgi complex, resulting in over-proliferated Golgi complexes and accelerated protein secretion. Interestingly, our study indicated that more ARF72A localized on the endoplasmic reticulum in the ninaE(D1) photoreceptor cell, a Drosophila model of autosomal-dominant retinitis pigmentosa, compared to that in the wild-type. In addition, arf72A loss was shown to rescue the ninaE(D1)-related membrane accumulation and the rhodopsin maturation defect, and suppress ninaE(D1)-triggered retinal degeneration, indicating that rhodopsin accumulated in the endoplasmic reticulum bypasses the quality checks. While previous studies of ARF small GTPases have focused on their roles in vesicular budding and transport between the specific organelles, our findings establish an additional function of arf72A in the quality check machinery of the endoplasmic reticulum distinguishing the cargoes for secretion from those for degradation.

Topics: ADP-Ribosylation Factors; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum; Feedback, Physiological; Golgi Apparatus; Green Fluorescent Proteins; Membrane Proteins; Photoreceptor Cells, Invertebrate; Protein Folding; Protein Transport; Recombinant Fusion Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases.

Topics: Adult; Amino Acid Sequence; Animals; Chromosome Mapping; Cilia; Exons; Female; Genes, Recessive; Genetic Loci; Homeodomain Proteins; Humans; Male; Mice; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Photoreceptor Cells, Vertebrate; Protein Serine-Threonine Kinases; Regulatory Sequences, Nucleic Acid; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Trans-Activators; Transcription, Genetic; Young Adult

To develop a robust ex vivo model for evaluating cone survival in end-stage retinitis pigmentosa (RP) and apply this to quantify the effects of putative neuroprotective compounds.. Rhodopsin knockout mice were crossed with OPN1-GFP reporter mice so that GFP-positive cones could be identified against the background of a rod-specific degeneration. Retinal explants were harvested from 10-week-old mice and maintained in organotypic culture. Ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), or vascular endothelial growth factor 165b (VEGF(165b)) was administered daily to treatment groups at three doses (200 ng/mL, 100 ng/mL, or 50 ng/mL; n = 5 explants per group). Fluorescence microscopy was performed on days 1, 3, 5, 7, 9, and 12 to document the number of GFP-expressing cones.. Cone survival could be assessed reliably and reproducibly in this model, and cone degeneration was significantly greater in the absence of rods, in keeping with clinical observations of RP. Daily administration of 200 ng/mL CNTF led to significantly increased cone survival compared with sham-treated controls. The effect was dose dependent; 100 ng/mL CNTF reduced cone loss but to a lesser extent, and 200 ng/mL GDNF showed significant protection against cone loss at later time points (day 9-12) but was much less effective than CNTF at all doses. VEGF(165b) showed no neuroprotective effect in this model at any dose.. This model allows precise quantification of the neuroprotective effects of various compounds on cone survival and may therefore provide a robust method of screening neuroprotective compounds before application in vivo.

Topics: Animals; Cell Survival; Ciliary Neurotrophic Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Glial Cell Line-Derived Neurotrophic Factor; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Nerve Growth Factors; Neuroprotective Agents; Organ Culture Techniques; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vascular Endothelial Growth Factor A

The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H(2)O and D(2)O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.

Topics: Animals; Cattle; Evolution, Molecular; Eye Diseases; HEK293 Cells; Humans; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Theoretical; Mutation; Retinitis Pigmentosa; Rhodopsin; Schiff Bases; Temperature

Two different mutations at Gly-90 in the second transmembrane helix of the photoreceptor protein rhodopsin have been proposed to lead to different phenotypes. G90D has been classically associated with congenital night blindness, whereas the newly reported G90V substitution was linked to a retinitis pigmentosa phenotype. Here, we used Val/Asp replacements of the native Gly at position 90 to unravel the structure/function divergences caused by these mutations and the potential molecular mechanisms of inherited retinal disease. The G90V and G90D mutants have a similar conformation around the Schiff base linkage region in the dark state and same regeneration kinetics with 11-cis-retinal, but G90V has dramatically reduced thermal stability when compared with the G90D mutant rhodopsin. The G90V mutant also shows, like G90D, an altered photobleaching pattern and capacity to activate Gt in the opsin state. Furthermore, the regeneration of the G90V mutant with 9-cis-retinal was improved, achieving the same A(280)/A(500) as wild type isorhodopsin. Hydroxylamine resistance was also recovered, indicating a compact structure around the Schiff base linkage, and the thermal stability was substantially improved when compared with the 11-cis-regenerated mutant. These results support the role of thermal instability and/or abnormal photoproduct formation in eliciting a retinitis pigmentosa phenotype. The improved stability and more compact structure of the G90V mutant when it was regenerated with 9-cis-retinal brings about the possibility that this isomer or other modified retinoid analogues might be used in potential treatment strategies for mutants showing the same structural features.

Topics: Amino Acid Substitution; Animals; Cattle; Cell Line, Tumor; COS Cells; Diterpenes; Eye Diseases, Hereditary; Genetic Diseases, X-Linked; Humans; Mutation, Missense; Myopia; Night Blindness; Protein Stability; Protein Structure, Tertiary; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

To engineer a knockin mouse model that can be used to monitor the effects of treatments on degradation and mislocalization of proline-to-histidine change at codon 23 (P23H) rhodopsin, a common cause of autosomal dominant retinitis pigmentosa (ADRP). The goal was to introduce a gene that expressed rhodopsin at low levels to avoid rapid retinal degeneration, and with a readily visible tag to make it easy to distinguish from wild type rhodopsin.. One copy of the endogenous mouse rhodopsin gene was replaced with a mutant human rhodopsin gene that encodes P23H-rhodopsin fused to enhanced green fluorescent protein (GFP) at its C terminus. The gene includes a LoxP site in the sequence corresponding to the 5'-untranslated region, which greatly reduces translation efficiency. Characterized are the resulting heterozygous and homozygous P23H-hRho-GFP mouse lines for mRNA and protein expression, P23H-rhodopsin localization in rod cells, effects on visual function, and retinal degeneration.. The retinas of heterozygous P23H-hRho-GFP mice are morphologically and functionally very similar to those of wild type mice, and they display little cell death over time. P23H-hRho-GFP mice transcribe the knockin gene as efficiently as the endogenous mouse allele, but they contain much less of the protein product than do knockin mice expressing nonmutant hRho-GFP, indicating that substantial degradation of P23H-rRho-GFP occurs in mouse rod cells. The remaining P23H-hRho-GFP mislocalizes to the inner segment and outer nuclear layer, with only approximately 20% in rod outer segments.. P23H-hRho-GFP mice provide a valuable tool for evaluating the efficacy of potential therapies for ADRP that influence the levels or localization of P23H-rhodopsin.

Topics: Animals; Blotting, Northern; Codon; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Gene Knock-In Techniques; Genotyping Techniques; Green Fluorescent Proteins; Histidine; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Mutagenesis, Site-Directed; Mutation; Proline; Recombinant Fusion Proteins; Retinal Photoreceptor Cell Inner Segment; Retinal Photoreceptor Cell Outer Segment; Retinitis Pigmentosa; Rhodopsin

Rho GTPases such as RAS-related C3 botulinum substrate 1 (RAC1) and cell division cycle 42 homolog (S. cerevisiae; CDC42) have been linked to cellular processes including movement, development, and apoptosis. Recently, RAC1 has been shown to be a pro-apoptotic factor in the retina during light-induced photoreceptor degeneration. Here, we analyzed the role of CDC42 in the degenerating retina.. Photoreceptor degeneration was studied in a mouse model for autosomal dominant retinitis pigmentosa (VPP) with or without a rod-specific knockdown of Cdc42, as well as in wild-type and Cdc42 knockdown mice after light exposure. Gene and protein expression were analyzed by real-time PCR, western blotting, and immunofluorescence. Retinal morphology and function were assessed by light microscopy and electroretinography, respectively.. CDC42 accumulated in the perinuclear region of terminal deoxynucleotidyl transferase dUTP nick end labeling-negative photoreceptors during retinal degeneration induced by excessive light exposure and in the rd1, rd10, and VPP mouse models of retinitis pigmentosa. The knockdown of Cdc42 did not affect retinal morphology or function in the adult mice and did not influence photoreceptor apoptosis or molecular signaling during induced and inherited retinal degeneration.. Retinal degeneration induces the accumulation of CDC42 in the perinuclear region of photoreceptors. In contrast to RAC1, however, lack of CDC42 does not affect the progression of degeneration. CDC42 is also dispensable for normal morphology and function of adult rod photoreceptor cells. RECEIVED: May 25, 2011 ACCEPTED: November 10, 2011.

Topics: Animals; Apoptosis; Blotting, Western; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique; Gene Expression; Gene Knockdown Techniques; GTPase-Activating Proteins; In Situ Nick-End Labeling; Light; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The Retinitis pigmentosa (RP)-causing mutant of rhodopsin, P23H rhodopsin, is folding-defective and unable to traffic beyond the endoplasmic reticulum (ER). This ER retention, and in some cases aggregation, are proposed to result in ER-stress and eventually cell death. The endogenous rhodopsin ligand 11-cis-retinal and its isomer 9-cis-retinal have been shown to act as pharmacological chaperones, promoting proper folding and trafficking of the P23H rhodopsin. In spite of this promising effect, the development of retinals and related polyenealdehydes as pharmacological agents has been hampered by their undesirable properties, which include chemical instability, photolability, and potential retinoidal actions. Here, we report the design and synthesis of a class of more stable nonpolyene-type rhodopsin ligands, structurally distinct from, and with lower toxicity than, retinals. A structure-activity relationship study was conducted using cell-surface expression assay to quantify folding/trafficking efficiency of P23H rhodopsin.

Topics: Benzaldehydes; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Humans; Ligands; Molecular Structure; Mutation; Protein Folding; Retinitis Pigmentosa; Rhodopsin; Stereoisomerism; Structure-Activity Relationship

Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.

Topics: Amino Acid Substitution; Animals; Base Sequence; Cattle; Cell Line; DNA Primers; Genetic Variation; Humans; Hydroxylamine; In Vitro Techniques; Mutant Proteins; Mutation; Opsins; Photobleaching; Protein Folding; Protein Stability; Recombinant Proteins; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

Purpose. To evaluate the phenotypic effects of two novel frameshift mutations in the RP1 gene in a Chinese pedigree of autosomal recessive retinitis pigmentosa (ARRP). Methods. Family members of a proband with ARRP were screened for RP1, RHO, NR2E3, and NRL mutations by direct sequencing. Detected RP1 mutations were genotyped in 225 control subjects. Since one family member with the RP1 deletion mutation in exon 2 was found to have age-related macular degeneration (AMD) but not RP, exons 2 and 3 of RP1 were screened in 120 patients with exudative AMD. Major AMD-associated SNPs in the HTRA1 and CFH genes were also investigated. Results. Two novel frameshift mutations in RP1, c.5_6delGT and c.4941_4942insT, were identified in the pedigree. They were absent in 225 control subjects. Family members who were compound heterozygous for the nonsense mutations had early-onset and severe RP, whereas those with only one mutation did not have RP. No mutations in RHO, NR2E3, and NRL were identified in the pedigree. Subject I:2 with AMD carried both at-risk genotypes at HTRA1 rs11200638 and CFH rs800292. No mutation in RP1 exons 2 and 3 was identified in 120 AMD patients. Conclusions. This report is the first to associate ARRP with compound heterozygous nonsense mutations in RP1. Identification of the nonsense-mediated mRNA decay (NMD)-sensitive mutation c.5_6delGT provided further genetic evidence that haploinsufficiency of RP1 is not responsible for RP. The authors propose four classes of truncation mutations in the RP1 gene with different effects on the etiology of RP.

Topics: Adolescent; Aged; Aged, 80 and over; Basic-Leucine Zipper Transcription Factors; Child; Eye Proteins; Female; Frameshift Mutation; Genes, Recessive; Genotype; Heterozygote; Humans; Macular Degeneration; Male; Microtubule-Associated Proteins; Middle Aged; Orphan Nuclear Receptors; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Young Adult

Bone spicule pigments (BSP) are a hallmark of retinitis pigmentosa (RP). In this study, we examined the process of BSP formation in the rhodopsin knockout (rho (-/-)) mouse, a murine model for human RP.. In rho (-/-) mice from 2 to 16 months of age, representing the range from early to late stages of degeneration, retinal sections and whole mounts were examined morphologically by light and electron microscopy. The results were compared to scanning laser ophthalmoscopy of BSP degeneration in human RP.. After the loss of all photoreceptor cells in rho-/- mice, the outer retina successively degenerated, leading to approximation and finally a direct contact of inner retinal vessels and the retinal pigment epithelium (RPE). We could show that it was the event of proximity of retinal vessel and RPE that triggered migration of RPE cells along the contacting vessels towards the inner retina. Ultrastructurally, these mislocalized RPE cells partially sealed the vessels by tight junction linkage and deposited extracellular matrix perivascularly. Also, the vascular endothelium developed fenestrations similar to the RPE-choroid interface. In whole mounts, the pigmented cell clusters outlining retinal capillaries correlated well with BSPs in human RP. The structure of the inner retina remained well preserved, even in late stages.. The Rho (-/-) mouse is the first animal model that depicts all major pathological changes, even in the late stages of RP. Using the rho (-/-) mouse model we were able to analyze the complete dynamic process of BSP formation. Therefore we conclude that: (1) In rho (-/-) retinas, BSPs only form in areas devoid of photoreceptors; (2) Direct contact between inner retinal vessels and RPE appears to be a major trigger for migration of RPE cells; (3) The distribution of the RPE cells in BSPs reflects the vascular network at the time of formation. The similarity of the disease process between mouse and human and the possibility to study all consecutive steps of the course of the disease makes the rho (-/-) mouse valuable for further insights in the dynamics of BSP formation in human RP.

Topics: Animals; Cell Movement; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Scanning; Nerve Tissue Proteins; Photoreceptor Cells, Vertebrate; Platelet Endothelial Cell Adhesion Molecule-1; Retinal Pigment Epithelium; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence

Dominant mutations in the visual pigment Rhodopsin (Rh) cause retinitis pigmentosa (RP) characterized by progressive blindness and retinal degeneration. The most common Rh mutation, Rh(P23H) forms aggregates in the endoplasmic reticulum (ER) and impairs the proteasome; however, the mechanisms linking Rh aggregate formation to proteasome dysfunction and photoreceptor cell loss remain unclear. Using mammalian cell cultures, we provide the first evidence that misfolded Rh(P23H) is a substrate of the ERAD effector VCP, an ATP-dependent chaperone that extracts misfolded proteins from the ER and escorts them for proteasomal degradation. VCP co-localizes with misfolded Rh(P23H) in retinal cells and requires functional N-terminal and D1 ATPase domains to form a complex with Rh(P23H) aggregates. Furthermore, VCP uses its D2 ATPase activity to promote Rh(P23H) aggregate retrotranslocation and proteasomal delivery. Our results raise the possibility that modulation of VCP and ERAD activity might have potential therapeutic significance for RP.

Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Cells, Cultured; Endoplasmic Reticulum; Humans; Mice; Molecular Chaperones; Proteasome Endopeptidase Complex; Protein Folding; Recombinant Fusion Proteins; Retina; Retinitis Pigmentosa; Rhodopsin; Valosin Containing Protein

PURPOSE. To identify the prevalence of rhodopsin (RHO) mutations in French patients with autosomal dominant rod-cone dystrophies (adRPs). Methods. Detailed phenotypic characterization was performed, including precise family history, best corrected visual acuity with the ETDRS chart, slit lamp examination, kinetic and static perimetry, full-field and multifocal electroretinography (ERG), fundus autofluorescence imaging (FAF), and optical coherence tomography (OCT). For genetic diagnosis, genomic DNA of 79 families was isolated by standard. The coding exons and flanking intronic regions of RHO were PCR amplified, purified, and sequenced in the index patient. RESULTS. Of this French adRP sample, 16.5% carried an RHO mutation. Three different families showed a novel mutation (p. Leu88Pro, p.Met207Lys and p.Gln344Pro), while ten unrelated families showed recurrent, previously published mutations (p.Asn15Ser, p.Leu131Pro, p.Arg135Trp, p.Ser334GlyfsX21 and p.Pro347Leu). All mutations co-segregated with the phenotype within a family, and the novel mutations were not identified in control samples. CONCLUSIONS. This study revealed that the prevalence of RHO mutations in French adRP patients is in accordance with that in other studies from Europe. Most of the changes identified herein reflect recurrent mutations, within which p.Pro347Leu substitution is the most prevalent. Nevertheless, almost one fourth of the changes are novel, indicating that, although RHO is the first gene implicated and probably the most studied gene in RP, it is still important performing mutation analysis in RHO to detect novel changes. The detailed phenotype-genotype analyses in all available family members deliver the basis for therapeutic approaches in those families.

Topics: Adolescent; Adult; Child; DNA Mutational Analysis; Electroretinography; Female; Fluorescein Angiography; France; Genes, Dominant; Genotype; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Prevalence; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; White People; Young Adult

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

Topics: Amino Acid Substitution; Animals; Apoptosis; Base Sequence; Dependovirus; Disease Models, Animal; Electroretinography; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; HeLa Cells; Humans; Mice; Models, Biological; Multiprotein Complexes; Mutation, Missense; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Recombinant Proteins; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Stress, Physiological; Transcription Factor CHOP; Transfection; Unfolded Protein Response; Vision, Ocular

We have recently developed transgenic X. laevis models of retinitis pigmentosa based on the rhodopsin P23H mutation in the context of rhodopsin cDNAs derived from several different species. The mutant rhodopsin in these animals is expressed at low levels, with levels of export from the endoplasmic reticulum to the outer segment that depend on the cDNA context. Retinal degeneration in these models demonstrates varying degrees of light dependence, with the highest light dependence coinciding with the highest ER export efficiency. Rescue of light dependent retinal degeneration by dark rearing is in turn dependent on the capacity of the mutant rhodopsin to bind chromophore. Our results indicate that rhodopsin chromophore can act in vivo as a pharmacological chaperone for P23H rhodopsin, and that light-dependent retinal degeneration caused by P23H rhodopsin is due to reduced chromophore binding.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum; Light; Models, Biological; Mutant Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis

Disease-causing missense mutations in membrane proteins, such as rhodopsin mutations associated with the autosomal dominant form of retinitis pigmentosa (ADRP), are often linked to defects in folding and/or trafficking. The mechanical unfolding of wild-type rhodopsin was compared with that of 20 selected ADRP-linked mutants more or less defective in folding and retinal binding. Rhodopsin fold is characterized by networks of amino acids in the retinal and G-protein binding sites likely to play a role in the stability and function of the protein. The distribution of highly connected nodes in the network reflects the existence of a diffuse intramolecular communication inside and between the 2 poles of the helix bundle, which makes pathogenic mutations share similar phenotypes irrespective of topological and physicochemical differences between them. Because of this communication, the ADRP-linked rhodopsin mutations share a more or less marked ability to impair selected hubs in the protein structure network. The extent of this structural effect relates to the severity of the biochemical defect caused by mutation. The investigative strategy employed in this study is likely to apply to all structurally known membrane proteins particularly susceptible to misassembly-causing mutations.

Topics: Amino Acid Sequence; Animals; Cattle; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Protein Folding; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin

Autosomal dominant retinitis pigmentosa (adRP) is a common form of RP worldwide. Although rhodopsin (RHO) is the most frequently reported adRP gene in many populations, it has not been detected in patients from the Bai nationality, one of the minority ethnic groups of southwest China. In this study, we used linkage analysis and mutation screening to identify the RHO gene in a Chinese Bai family with adRP. We found that in all affected members of the Bai family, the maximum two-point logarithm of odds score obtained was 3.61 and 4.52 at a recombination fraction (θ) of zero, with markers D3S3606 and D3S1292, respectively. Haplotype analysis showed cosegregation at the 1-c region harboring the RHO gene between the two markers with the disease. Direct sequencing of RHO revealed a c.1040C>T (p.Pro347leu) mutation in exon 5, which was supported by the reaction of the restriction enzyme. Two nonpathogenic single-nucleotide polymorphisms, rs7984 and rs2269736, were found in exon 1. To the best of our knowledge, this is the first genetic analysis of a Chinese Bai family with adRP, and a known missense RHO mutation (p.Pro347leu) is responsible for it.

Topics: China; DNA Mutational Analysis; Family Health; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Haplotypes; Humans; Male; Mutation; Odds Ratio; Polymorphism, Single Nucleotide; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.

Topics: Cell Line; Cell Membrane; Humans; Immunohistochemistry; Models, Biological; Mutation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport; Retinitis Pigmentosa; Rhodopsin; Static Electricity

To report a new genetic variant in the rhodopsin gene (RHO) associated with an unusual autosomal dominant retinal phenotype.. Detailed phenotypic characterization was performed on affected family members spanning 4 generations, including family history, best-corrected visual acuity, fundus examination, kinetic and static perimetry, full-field and multifocal electroretinography, fundus autofluorescence, and optical coherence tomography. For genetic testing, coding exons and flanking intronic regions of RHO were amplified with the use of polymerase chain reaction, purified, and sequenced. Cosegregation and control analysis were performed by direct sequencing of exon 3. Subsequent in silico analysis of the mutational consequence on protein function was undertaken.. The onset of symptoms appeared in the fourth decade of life in this family, with moderate night blindness and asymmetrical visual loss. Affected members showed patchy areas of chorioretinal atrophy with decreased electroretinographic response amplitudes for both scotopic and photopic responses but no implicit time shift, consistent with restricted disease. A novel mutation in exon 3 of RHO was identified and represents a c.620T>A transition leading to a p.Met207Lys substitution. It cosegregated with this phenotype and was not identified in a control population.. We report the phenotype-genotype correlation of an unusual autosomal dominant, late-onset restricted chorioretinal degeneration cosegregating with a novel RHO mutation, p.Met207Lys. A p.Met207Arg substitution has previously been reported to cause a distinct, generalized early-onset rod-cone dystrophy. Clinical Relevance These data outline the phenotypic variability associated with RHO mutations. Depending on the localization and the amino acid substitution, patients may show congenital stationary night blindness, rod-cone dystrophy, sector retinitis pigmentosa, or localized chorioretinal atrophy.

Topics: Adult; DNA Mutational Analysis; Electroretinography; Exons; Female; Fluorescein Angiography; Genes, Dominant; Genotype; Humans; Male; Middle Aged; Mutation, Missense; Pedigree; Phenotype; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; Visual Fields; Young Adult

Retinitis pigmentosa (RP) is the most prevalent cause of registered visual handicap among working aged populations of developed countries. Up to 40% of autosomal dominant cases of disease are caused by mutations within the rhodopsin, RDS-peripherin and inosine 5'-monophosphate dehydrogenase type 1 (IMPDH1) genes, at least 30 mutations within which give rise to proteins that cause disease pathology by misfolding and aggregation. Given the genetic complexity of this disease, therapies that simultaneously target multiple mutations are of substantial logistic and economic significance. We show here, in a murine model of autosomal dominant RP (RP10) involving expression of an Arg224Pro mutation within the IMPDH1 gene, that treatment with the low-molecular-weight drug, 17-allylamino-17-demethoxygeldanamycin (17-AAG), an ansamycin antibiotic that binds to heat shock protein Hsp90, activating a heat shock response in mammalian cells, protects photoreceptors against degeneration induced by aggregating mutant IMPDH1 protein, systemic delivery of this low-molecular-weight drug to the retina being facilitated by RNA interference-mediated modulation of the inner-blood retina barrier. 17-AAG has an orphan drug status and is in current clinical use for the treatment of non-ocular diseases. These data show that a single low-molecular-weight drug has the potential to suppress a wide range of mutant proteins causing RP.

Topics: Animals; Benzoquinones; Drug Delivery Systems; Drug Evaluation, Preclinical; Genes, Dominant; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; IMP Dehydrogenase; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; RNA Interference

Mutations in the rhodopsin gene (RHO) are suggested to be the most common cause of autosomal dominant retinitis pigmentosa (RP). However, the exact spectrum and frequency of RHO mutations in different forms of RP has not been well defined, especially in Chinese populations. In this study, direct cycle sequencing was used to analyze all five coding exons and adjacent intronic regions of RHO in 248 Chinese probands with different forms of non X-linked RP. Eight heterozygous nucleotide changes were detected, including two novel mutations (c.628G>T, p.Val210Phe; c.945C>G, p.Asn315Lys) and six known mutations (c.527C>T, p.Ser176Phe; c.568G>T, p.Asp190Tyr; c.768_770delCAT, p.Ile256del; c.1040C>T, p.Pro347Leu; c.310G>A, p.Val104Ile; c.895G>T, p.Ala299Ser), in 14 probands (nine isolated cases, three from autosomal dominant families, and three from autosomal recessive families). Of the eight mutations, seven were missense changes and one was a small deletion. Six may be pathogenic mutations, and two others (c.310G>A, c.895G>T) may not be causative on their own. The p.Ala299Ser change was present in six of the 248 probands with RP but only in one of 384 normal controls, while the p.Val104Ile was present in two probands but none of the 384 controls. Our results demonstrate an overview of the spectrum and frequency of RP RHO mutations in a Chinese population and emphasize that RHO mutations in isolated RP are not uncommon.

Topics: Adult; Asian People; Child; Child, Preschool; China; Female; Gene Frequency; Humans; Male; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin; Young Adult

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (Rh(P23H)). Unlike the wild-type Rh, mutant Rh(P23H) exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded Rh(P23H) and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that Rh(P23H) is a substrate of the ER-associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1(P37H) (the equivalent of mammalian Rh(P23H)) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1(P37H) toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1(P37H) and further activated the Ire1/Xbp1 ER stress pathway in the Rh1(P37H) retina. Despite this, Rh1(P37H) flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1(P37H) retina. Pharmacological treatment of Rh1(P37H) flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.

Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Disease Models, Animal; Down-Regulation; Drosophila; Drosophila Proteins; Endoplasmic Reticulum; Female; Gene Silencing; Humans; Male; Mutation, Missense; Protein Folding; Retina; Retinitis Pigmentosa; Rhodopsin; Valosin Containing Protein

Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy. Here we report the crystal structures of both wild-type human CRALBP and of its mutant R234W as binary complexes complemented with the endogenous ligand 11-cis-retinal, at 3.0 and 1.7 A resolution, respectively. Our structural model of wild-type CRALBP locates R234 to a positively charged cleft at a distance of 15 A from the hydrophobic core sequestering 11-cis-retinal. The R234W structural model reveals burial of W234 and loss of dianion-binding interactions within the cleft with physiological implications for membrane docking. The burial of W234 is accompanied by a cascade of side-chain flips that effect the intrusion of the side-chain of I238 into the ligand-binding cavity. As consequence of the intrusion, R234W displays 5-fold increased resistance to light-induced photoisomerization relative to wild-type CRALBP, indicating tighter binding to 11-cis-retinal. Overall, our results reveal an unanticipated domino-like structural transition causing Bothnia-type retinal dystrophy by the impaired release of 11-cis-retinal from R234W.

Topics: Amino Acid Substitution; Binding Sites; Carrier Proteins; Crystallography, X-Ray; Humans; Isomerism; Ligands; Light; Mutant Proteins; Mutation, Missense; Protein Structure, Secondary; Retinal Diseases; Retinitis Pigmentosa; Retinoids; Time Factors

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor-specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones.

Topics: Amino Acid Substitution; Animal Nutritional Physiological Phenomena; Animals; Autophagy; Carrier Proteins; Cell Death; Cell Survival; Cyclic Nucleotide Phosphodiesterases, Type 6; Insulin; Mice; Mice, Inbred C57BL; Mice, Knockout; Microarray Analysis; Nutrition Disorders; Phosphotransferases (Alcohol Group Acceptor); Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Time Factors; TOR Serine-Threonine Kinases; Transgenes

To follow the status of cones over the life of the P23H-3 transgenic rat, while the rod population is depleted.. P23H-3 heterozygous and Sprague-Dawley (SD) control rats were raised in dim, cyclic light from postnatal day (P)10 to P540. Retinas were examined for cone density, cone outer segment (OS) length, cone axon and soma morphology, and the amplitude of rod and cone components of the electroretinogram (ERG) were determined.. In the P23H-3 retina, cone density followed a developmental pattern, increasing from P10 until P20, declining during early adult life (to P150), then steadying at levels found in the SD retina until P540. Cone OSs elongated to P30 and then slowly shortened during late adulthood; at P350 and P540, cone OSs were significantly shorter than in the background SD strain. Cone axons shortened slowly throughout adult life as the outer nuclear layer thinned. The rod a-wave declined steadily in the P23H-3 retina from P10, falling below amplitudes seen in the SD strain from early life. By contrast, the cone b-wave maintained amplitude at SD levels, until P380.. Despite the ongoing loss of rod function and numbers, cone numbers in the P23H-3 retina were maintained at levels found in the SD rat to the oldest age examined, and cone function and OS morphology were maintained for approximately 1 year, indicating a long period of cone independence. The long period of cone survival creates an opportunity to induce self-repair, if the stress causing their dysfunction can be reduced.

Topics: Animals; Axons; Cell Count; Cell Survival; Cellular Senescence; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Male; Mutation; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Cone Photoreceptor Cells; Retinal Photoreceptor Cell Inner Segment; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. This is particularly true in ocular diseases such as retinal angiomatous proliferation (RAP) and macular telangiectasia (MacTel), in which, despite the absence of large-scale leakage or hemorrhage, abnormal neovascularization (NV) is associated with local neuronal dysfunction. We describe here a retinal phenotype in mice with dysfunctional receptors for VLDL (Vldlr-/- mice) that closely resembles human retinal diseases in which abnormal intra- and subretinal NV is associated with photoreceptor cell death. Such cell death was evidenced by decreased cone and, to a lesser extent, rod opsin expression and abnormal electroretinograms. Cell death in the region of intraretinal vascular abnormalities was associated with an increased presence of markers associated with oxidative stress. Oral antioxidant supplementation protected against photoreceptor degeneration and preserved retinal function, despite the continued presence of abnormal intra- and subretinal vessels. What we believe to be novel, Müller cell-based, virally mediated delivery of neurotrophic compounds specifically to sites of NV was also neuroprotective. These observations demonstrate that neuronal loss secondary to NV can be prevented by the use of simple antioxidant dietary measures or cell-based delivery of neurotrophic factors, even when the underlying vascular phenotype is not altered.

Topics: Angiogenesis Inhibitors; Animals; Antioxidants; Aptamers, Nucleotide; Disease Models, Animal; Electroretinography; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Growth Factors; Opsins; Oxidative Stress; Receptors, LDL; Retina; Retinal Cone Photoreceptor Cells; Retinal Neovascularization; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vascular Endothelial Growth Factor A

Retinitis pigmentosa (RP), a neurodegenerative disorder, can arise from single point mutations in rhodopsin, leading to a cascade of protein instability, misfolding, aggregation, rod cell death, retinal degeneration, and ultimately blindness. Divalent cations, such as zinc and copper, have allosteric effects on misfolded aggregates of comparable neurodegenerative disorders including Alzheimer disease, prion diseases, and ALS. We report that two structurally conserved low-affinity zinc coordination motifs, located among a cluster of RP mutations in the intradiscal loop region, mediate dose-dependent rhodopsin destabilization. Disruption of native interactions involving histidines 100 and 195, through site-directed mutagenesis or exogenous zinc coordination, results in significant loss of receptor stability. Furthermore, chelation with EDTA stabilizes the structure of both wild-type rhodopsin and the most prevalent rhodopsin RP mutation, P(23)H. These interactions suggest that homeostatic regulation of trace metal concentrations in the rod outer segment of the retina may be important both physiologically and for an important cluster of RP mutations. Furthermore, with a growing awareness of allosteric zinc binding domains on a diverse range of GPCRs, such principles may apply to many other receptors and their associated diseases.

Topics: Amino Acid Motifs; Amino Acid Sequence; Chelating Agents; Conserved Sequence; Crystallography, X-Ray; Histidine; Humans; Hydrogen Bonding; Molecular Sequence Data; Mutant Proteins; Mutation; Opsins; Protein Stability; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, Protein; Temperature; Time Factors; Zinc

Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa. To investigate the role of Tulp1 in the photoreceptor synapse, the authors examined the presynaptic and postsynaptic architecture and retinal function in tulp1(-/-) mice. The authors used immunohistochemistry to examine tulp1(-/-) mice before retinal degeneration and made comparisons with wild-type (wt) littermates and retinal degeneration 10 (rd10) mice, another model of photoreceptor degeneration that has a comparable rate of degeneration. Retinal function was characterized with the use of electroretinography.. In wt mice, Tulp1 is localized to the photoreceptor synapse. In the tulp1(-/-) synapse, the spatial relationship between the ribbon-associated proteins Bassoon and Piccolo are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted. Comparable abnormalities are not seen in rd10 mice. The leading edge of the a-wave had normal kinetics in tulp1(-/-) mice but reduced gain in rd10 mice. The b-wave intensity-response functions of tulp1(-/-) mice are shifted to higher intensities than in wt mice, but those of rd10 mice are not.. Photoreceptor synapses and bipolar cell dendrites in tulp1(-/-) mice display abnormal structure and function. A malformation of the photoreceptor synaptic ribbon is likely the cause of the dystrophy in bipolar cell dendrites. The association of early-onset, severe photoreceptor degeneration preceded by synaptic abnormalities appears to represent a phenotype not previously described. Not only is Tulp1 critical for photoreceptor function and survival, it is essential for the proper development of the photoreceptor synapse.

Topics: Alcohol Oxidoreductases; Animals; Animals, Newborn; Co-Repressor Proteins; Cytoskeletal Proteins; Dendrites; DNA-Binding Proteins; Electroretinography; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Genotype; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neuropeptides; Phosphoproteins; Photoreceptor Cells, Vertebrate; Presynaptic Terminals; Protein Kinase C-alpha; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; Synaptic Vesicles

Great variation exists in the age of onset of symptoms and the severity of disease at a given age in patients with retinitis pigmentosa (RP). The final pathway for this disease may involve apoptotic photoreceptor cell death. Telomere length is associated with biologic aging, senescence, and apoptosis. We evaluated whether the length of telomeres in leukocytes correlated with the severity of RP in patients with the Pro23His rhodopsin mutation who have shown marked heterogeneity in disease severity.. We evaluated 122 patients with the Pro23His rhodopsin mutation. The patients' retinal function was stratified according to their 30-Hz cone electroretinogram (ERG). The length of telomeres in leukocytes was measured by the quantitative real time polymerase chain reaction (qRT-PCR) method in the 15 patients with the highest age-adjusted 30-Hz ERG amplitudes and in the 15 patients with the lowest amplitudes.. Mean leukocyte telomere length was similar in the 15 patients with the highest cone ERG amplitudes (median: 0.40 units; interquartile range 0.36-0.56) and the 15 patients with the lowest cone amplitudes (median: 0.41 units; inter quartile range 0.34 -0.64; p=0.95).. We found no evidence for an association between telomere length and the severity of RP as monitored by the cone ERG in patients with the Pro23His rhodopsin mutation.

Topics: Adult; Apoptosis; Electroretinography; Female; Genes, Dominant; Humans; Male; Middle Aged; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Telomere

To screen mutations in the PRPF31, RHO, and PRPH2 genes in Chinese patients with retinitis pigmentosa (RP).. Patients with RP were recruited from Retina Hong Kong. All the exons of the PRPF31, RHO, and PRPH2 genes were amplified and screened for mutations using single-stranded conformation polymorphism analysis followed by DNA sequencing. Frequencies of sequence changes were determined in patients and controls.. In 76 patients from 54 families, 3 pathogenic mutations and 32 nonpathogenic sequence changes were identified. One family with autosomal dominant RP was found to harbor a novel truncating PRPF31 mutation (p.Phe262SerfsX59) and a known missense RHO mutation (p.Pro347Leu), and 1 affected woman was heterozygous for both mutations. One simplex RP case was caused by a novel truncating PRPH2 mutation (p.Ala78LeufsX99). Thirteen of the 32 nonpathogenic sequence changes were novel and were found in low frequencies in patients with RP and controls.. Mutations in PRPF31, RHO, and PRPH2 were found in low frequencies (1 of 9 autosomal dominant RP families) in Chinese patients, and the PRPF31 and PRPH2 truncating mutations were novel.. A search for a common cause for RP in Chinese patients is needed. The co-occurrence of 2 different gene mutations may modify the phenotype severity.

Topics: Adolescent; Adult; Age of Onset; Asian People; Child; Child, Preschool; DNA Mutational Analysis; Eye Proteins; Female; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Pedigree; Peripherins; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Visual Acuity; Young Adult

The cAMP response element binding protein 1 (CREB1) and activating transcription factor 1 (ATF1) are closely related members of the bZIP superfamily of transcription factors. Both are activated in response to a wide array of stimuli, including cellular stress. This study was conducted to assess the CREB1/ATF1 pathway in photoreceptor disease and protection.. The expression levels of p-CREB1, CREB1, and ATF1 were examined by immunoblot and immunohistochemistry in normal canine retina and retinas of several canine models of retinal degeneration (rcd1, rcd2, erd, prcd, XLPRA1, XLPRA2, T4R RHO). Humans retinas affected with age-related macular degeneration (AMD) were also examined. p-CREB1/ATF1 immunolabeling was assessed in normal and rcd1 dogs treated with ciliary neurotrophic factor (CNTF), to examine the effect of a neuroprotective stimulus on activation of CREB1/ATF1.. Native CREB1 and ATF1 as well as phosphorylated CREB1/ATF1 was examined in normal canine retina by immunoblot. The p-CREB1 antibody identified phosphorylated CREB1 and ATF1 and labeled the inner retina only in normal dogs. In degenerate canine and human retinas, strong immunolabeling appeared in rod and cone photoreceptors, indicating increased expression of native CREB1 and ATF1, as well as increased phosphorylation of these proteins. Retinal protection by CNTF in rcd1 dogs was accompanied by a significant increase in the number of p-CREB1/ATF1-labeled photoreceptor nuclei.. Positive association of CREB1/ATF1 phosphorylation with photoreceptor protection suggests that it may contribute to an innate protective response. These data identify a signaling mechanism in rods and cones of potential importance for therapies of RP and AMD.

Topics: Activating Transcription Factor 1; Aged; Aged, 80 and over; Animals; Arrestin; Cell Count; Ciliary Neurotrophic Factor; Cyclic AMP Response Element-Binding Protein; Dog Diseases; Dogs; Female; Genotype; Humans; Immunoblotting; Immunoenzyme Techniques; Macular Degeneration; Male; Phosphorylation; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. The RP1 gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1. Rp1L1-/- mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than in Rp1-/- mice. In single rods of Rp1L1-/-, photosensitivity is reduced, similar to that of Rp1-/-. While individual heterozygotes are normal, double heterozygotes of Rp1 and Rp1L1 exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or double Rp1 and Rp1L1 mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations in RP1L1 could underlie retinopathy or modify RP1 disease expression in humans.

Topics: Animals; Axoneme; Doublecortin Protein; Electroretinography; Eye Proteins; Genotype; Kinetics; Mice; Mice, Knockout; Microtubule-Associated Proteins; Photic Stimulation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Signal Transduction; Vision, Ocular

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.

Topics: Animals; Blotting, Western; Calnexin; Cell Line; Endoplasmic Reticulum; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Mutant Proteins; Mutation; Protein Binding; Protein Folding; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

Autosomal dominant (AD) inheritance accounts for 15-20% of retinitis pigmentosa (RP) familial cases. The characterization of AD RP-related mutations remains essential because it provides both accurate diagnosis and clinically important prognostic information. Rhodopsin (RHO) and peripherin/RDS are the two most common mutated genes in AD RP in several series. However, the genetic characterization of patients from distinct ethnic groups will help to define the relative contribution of particular AD RP-related genes. In the present study, a search for causal mutations in RHO and peripherin/RDS in a group of 28 Mexican RP probands with AD inheritance was performed.. Methods included complete ophthalmologic examination as well as fluorangiographic and electroretinographic assessment. Molecular analysis included Polymerase (PCR) amplification and direct nucleotide sequencing of the coding exons of RHO and peripherin/RDS in DNA from affected subjects. Mutation-carrying exons were analyzed in a total of 29 first-degree relatives from some of these families.. Five RHO mutations, including two novel ones and three previously reported, were demonstrated in this RP sample. Novel mutations were c.365A>G in exon 2 (Glu122Gly), and c.233A> in exon 1 (Asn78Ile). The other three RHO mutations were Phe45Leu, Arg135Trp, and Ser186Trp. No peripherin/RDS gene mutations were demonstrated in the remaining 23 probands.. Our study adds to the mutational spectrum of adRP by identifying two novel RHO mutations. RHO mutations were responsible of 17% of AD RP Mexican cases, a figure slightly lower to that found in other ethnic groups. Peripherin/RDS mutations are apparently an uncommon cause of AD RP in this population.

Topics: Adolescent; Adult; Aged; Base Sequence; Fundus Oculi; Genes, Dominant; Genetic Testing; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mexico; Mutation; Nerve Tissue Proteins; Peripherins; Retinitis Pigmentosa; Rhodopsin

To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families.. A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene.. In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families.. In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry.

Topics: Alleles; Amino Acid Sequence; Amino Acid Substitution; Asian People; Base Sequence; Chi-Square Distribution; Family; Female; Fundus Oculi; Genes, Recessive; Haplotypes; Homozygote; Humans; Male; Microsatellite Repeats; Molecular Sequence Data; Mutation; Pakistan; Pedigree; Polymorphism, Single Nucleotide; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis

To screen for possible disease-causing mutations in rhodopsin (RHO), pre-mRNA processing factor 31 (PRPF31), retinitis pigmentosa 1 (RP1), and inosine monophosphate dehydrogenase 1 (IMPDH1) genes in Indian patients with isolated and autosomal dominant forms of retinitis pigmentosa (adRP). Information on such data is not available in India and hence this study was undertaken.. Blood samples were obtained from 48 isolated and 53 adRP patients, who were recruited for the study. Each patient underwent a detailed clinical examination. Genomic DNA was extracted from the blood samples and screened for mutations in four genes using an ABI3100 Avant genetic analyzer. Reverse transcriptase polymerase chain reaction was performed to amplify the mutated (IVS6+1G/A) mRNA of PRPF31 in a two-generation adRP family.. Of the 101 probands analyzed, three harbored possible disease-causing mutations. Pathogenic changes were observed in RHO and PRPF31. A RHO mutation, p.Gly106Arg, was found in an isolated RP patient with sectoral RP. Two novel, heterozygous mutations were identified in PRPF31: p.Lys120GlufsX122 in an isolated RP patient and a splice site mutation, IVS6+1G/A in an adRP patient. However, no disease-causing changes were observed in RP1 and IMPDH1.. We screened RHO, PRPF31, RP1, and IMPDH1 and identified causative mutations in 4% of isolated and 2% of adRP patients from India. To the best of our knowledge, this is the first report to identify frequencies of mutations in isolated and adRP patients in India.

Topics: Adolescent; Adult; Aged; Base Sequence; Child; Child, Preschool; DNA Mutational Analysis; Eye Proteins; Female; Fundus Oculi; Humans; IMP Dehydrogenase; India; Introns; Male; Microtubule-Associated Proteins; Middle Aged; Molecular Sequence Data; Pedigree; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

To estimate the mean rates of ocular function loss in patients with autosomal recessive retinitis pigmentosa due to USH2A mutations.. In 125 patients with USH2A mutations, longitudinal regression was used to estimate mean rates of change in Snellen visual acuity, Goldmann visual field area (V4e white test light), and 30-Hz (cone) full-field electroretinogram amplitude. These rates were compared with those of previously studied cohorts with dominant retinitis pigmentosa due to RHO mutations and with X-linked retinitis pigmentosa due to RPGR mutations. Rates of change in patients with the Cys759Phe mutation, the USH2A mutation associated with nonsyndromic disease, were compared with rates of change in patients with the Glu767fs mutation, the most common USH2A mutation associated with Usher syndrome type II (i.e., retinitis pigmentosa and hearing loss).. Mean annual exponential rates of decline for the USH2A patients were 2.6% for visual acuity, 7.0% for visual field area, and 13.2% for electroretinogram amplitude. The rate of acuity loss fell between the corresponding rates for the RHO and RPGR patients, whereas the rates for field and ERG amplitude loss were faster than those for the RHO and RPGR patients. No significant differences were found for patients with the Cys759Phe mutation versus patients with the Glu767fs mutation.. On average, USH2A patients lose visual acuity faster than RHO patients and slower than RPGR patients. USH2A patients lose visual field and cone electroretinogram amplitude faster than patients with RHO or RPGR mutations. Patients with a nonsyndromic USH2A mutation have the same retinal disease course as patients with syndromic USH2A disease.

Topics: Adolescent; Adult; Alleles; Blindness; Child; Disease Progression; Electroretinography; Extracellular Matrix Proteins; Eye Proteins; Follow-Up Studies; Genes, Recessive; Genotype; Humans; Middle Aged; Mutation; Retinitis Pigmentosa; Rhodopsin; Tomography, Optical Coherence; Visual Acuity; Visual Fields; Young Adult

We studied the potential of systemically administered aminoglycosides as a therapy for retinal degeneration resulting from premature termination codon (PTC) mutations. Aminoglycosides were systemically delivered to two rodent models of retinal degeneration: a transgenic rat model of dominant disease caused by a PTC in rhodopsin (S334ter); and a mouse model of recessive disease (rd12) caused by a PTC in the retinoid isomerase Rpe65. Initial luciferase reporter assays were undertaken to measure the efficiency of gentamicin-induced read-through in vitro. These experiments indicated that gentamicin treatment induced on average a 5.3% extra read-through of the S334ter PTC in vitro, but did not affect the rd12 PTC. Beginning at postnatal day 5, animals received daily subcutaneous injections of gentamicin or geneticin at a range of doses. The effect of the treatment on retinal degeneration was examined by histopathology and electroretinography (ERG). Systemic treatment with aminoglycoside significantly increased the number of surviving photoreceptors in the S334ter rat model over several weeks of treatment, but was not effective in slowing the retinal degeneration in the rd12 mouse model. Similarly, ERG recordings indicated better preservation of retinal function in the treated S334ter rats, but no difference was observed in the rd12 mice. Daily subcutaneous injection of 12.5mug/g gentamicin was the only regimen that inhibited retinal degeneration without apparent adverse systemic side effects. Reduced effectiveness beyond postnatal day 50 correlated with reduced ocular penetration of drug as seen in gentamicin-Texas red (GTTR) conjugation experiments. We conclude that, in the rat model, an approximately 5% reduction of abnormal truncated protein is sufficient to enhance photoreceptor survival. Such a change in truncated protein is consistent with beneficial effects seen when aminoglycosides has been used in other, non-ocular animal models. In the rd12 mouse, lack of efficacy was seen despite this particular PTC being theoretically more sensitive to aminoglycoside modification. We conclude that aminoglycoside read-through of PTCs in vitro and in vivo cannot be predicted just from genomic context. Because there is considerable genetic heterogeneity amongst retinal degenerations, pharmacologic therapies that are not gene-specific have significant appeal. Our findings suggest that if adverse issues such as systemic toxicity and limited ocular penetration can be ove

Topics: Aminoglycosides; Animals; Carrier Proteins; cis-trans-Isomerases; Codon, Nonsense; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Electroretinography; Eye Proteins; Fluorescent Dyes; Gentamicins; Mice; Mice, Mutant Strains; Rats; Rats, Transgenic; Retina; Retinitis Pigmentosa; Rhodopsin; Xanthenes

The visual (retinoid) cycle is a fundamental metabolic process in vertebrate retina responsible for production of 11-cis-retinal, the chromophore of rhodopsin and cone pigments. 11-cis-Retinal is bound to opsins, forming visual pigments, and when the resulting visual chromophore 11-cis-retinylidene is photoisomerized to all-trans-retinylidene, all-trans-retinal is released from these receptors. Toxic byproducts of the visual cycle formed from all-trans-retinal often are associated with lipofuscin deposits in the retinal pigmented epithelium (RPE), but it is not clear whether aberrant reactions of the visual cycle participate in RPE atrophy, leading to a rapid onset of retinopathy. Here we report that mice lacking both the ATP-binding cassette transporter 4 (Abca4) and enzyme retinol dehydrogenase 8 (Rdh8), proteins critical for all-trans-retinal clearance from photoreceptors, developed severe RPE/photoreceptor dystrophy at an early age. This phenotype includes lipofuscin, drusen, and basal laminar deposits, Bruch's membrane thickening, and choroidal neovascularization. Importantly, the severity of visual dysfunction and retinopathy was exacerbated by light but attenuated by treatment with retinylamine, a visual cycle inhibitor that slows the flow of all-trans-retinal through the visual cycle. These findings provide direct evidence that aberrant production of toxic condensation byproducts of the visual cycle in mice can lead to rapid, progressive retinal degeneration.

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Bruch Membrane; Lipofuscin; Mice; Mice, Knockout; Pigment Epithelium of Eye; Retinal Cone Photoreceptor Cells; Retinal Drusen; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Perturbed microRNA (miR) expression is a feature of, and may play a fundamental role in, certain disease states such as different forms of cancer. Retinitis pigmentosa (RP) a group of inherited retinal degenerations is characterised by a progressive loss of photoreceptor cells and consequent visual handicap. We have previously reported an altered pan-retinal expression of miR-96, -183, -1 and -133 in a P347S-Rhodopsin transgenic mouse model of RP. As many different mutations in Rhodopsin and other genes such as RDS/Peripherin can lead to RP, it was of interest to explore whether the characterized retinal miR expression signature was observed in three other mouse models of RP linked to rhodopsin and rds/peripherin. Therefore, pan-retinal expression of miR-96, -182, -183, -1, -133 and -142 was analysed using quantitative real-time RT-PCR. A common signature of altered miR expression was found; expression of miR-96, -182 and -183 decreased by 14.1-53.2%, while expression of miR-1, -133 and -142 was up-regulated by 186.1-538.5%. Significantly, the detected pan-retinal miR signature was mirrored by similar miR expression profiles in FACS-isolated rod photoreceptors from these mice. In an attempt to understand the function of these miRs, corresponding target genes were predicted using computational means. Many 'enriched' targets (with binding sites for at least two of the above miRs) were found to be regulatory molecules and members of intracellular signalling circuits. However, further studies are required to highlight which of the large number of in silico predicted targets are actually controlled by these miRs.

Topics: Animals; Disease Models, Animal; Gene Expression; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Knockout; MicroRNAs; Nerve Tissue Proteins; Peripherins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin

To phenotype a family with RHO (Asp190Asn or D190N) dominantly inherited retinitis pigmentosa (RP) and to describe an approach to surveying affected families.. Four patients from a family with a history of autosomal dominant RP had complete clinical examinations and underwent full-field electroretinography (ERG), fundus autofluorescence (AF) imaging, and genetic testing. One patient had microperimetry (MP) mapping.. The patients' ages ranged from 6 years to 47 years. The proband, the father, had fundoscopic findings typical of RP. A small hyperfluorescent ring centered at the fovea was apparent on AF. MP showed preservation of central 7 degrees of visual field within this ring. The three children were all asymptomatic with visual acuity of 20/15 in each eye. One child had mild retinal pigment epithelium migration on fundoscopy; the other two children had normal fundoscopic examinations. Two children showed increased parafoveal AF. In the two affected children, average ERG b-wave implicit times were delayed in scotopic conditions, and maximal ERG tracings had abnormal waveforms. Genetic analysis confirmed that two of three asymptomatic children carried the D190N allele.. Patients with RHO (D190N) autosomal dominant retinitis pigmentosa (adRP) can show classic signs of RP on fundus examination and may be able to maintain good central visual acuity into adulthood. By combining clinical examination with AF imaging and electrophysiology, it is possible to offer presymptomatic clinical evaluation to families with this RP.

Topics: Adolescent; Adult; Asparagine; Aspartic Acid; Child; Electroretinography; Fluorescence; Fundus Oculi; Genes, Dominant; Genotype; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Retinal Pigment Epithelium; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields; Young Adult

Mutational heterogeneity in genes causative of dominantly inherited disorders represents a significant barrier for development of therapies directed towards correction of the primary genetic defect. To circumvent the mutational heterogeneity present in rhodopsin- (RHO-) linked autosomal dominant Retinitis Pigmentosa (adRP), a strategy involving suppression and replacement of RHO has been adopted. RNA interference- (RNAi-) mediated suppression of RHO has been explored as has the generation of an RNAi-resistant replacement gene using the degeneracy of the genetic code. Additionally, the functional equivalence of codon-modified replacement genes has been demonstrated in a transgenic animal (RHO-M). Suppression and replacement, while exemplified by adRP, may also be relevant to many other dominantly inherited diseases with the hallmark of mutational heterogeneity.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Electroretinography; Gene Expression; Genetic Therapy; Mice; Mice, Transgenic; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; RNA, Small Interfering

Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing.. mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors. The mfERG response contributions from different retinal components of in the Tg eyes were estimated and compared with control data from eyes of age-matched wild-type (WT) pigs.. There was a prominent difference in the estimates of the inner retinal response and ON-bipolar cell pathway contribution between the Tg and WT mfERG responses. In particular, the early components of the inner retinal contribution were obviously altered in the Tg mfERG. The inner retinal components at approximately 24 and 40 ms appeared to be inverted. Differences in the estimates of OFF-bipolar cell pathway contributions were minimal. There was no change in cone cell responses in the Tg mfERG.. In Tg retinas, ectopic synapses formed between cones and rod bipolar cells probably altered signal processing of the ON-bipolar cell pathway. In response to the altered visual signal input from the outer retina, signal processing in inner retinal neurons was also modified.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Computers, Handheld; Disease Models, Animal; Electroretinography; Mutation; N-Methylaspartate; Photoreceptor Cells, Vertebrate; Pipecolic Acids; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses; Synaptic Transmission; Tetrodotoxin; Vision, Ocular

Rhodopsin transgenes carrying mutations that cause autosomal dominant retinitis pigmentosa in humans have been used to study rod photoreceptor degeneration in various model organisms including Xenopus laevis. To date, the only transgenes shown to cause rod photoreceptor degeneration in Xenopus laevis have been either mammalian rhodopsins or chimeric versions of rhodopsin based mainly on Xenopus laevis rhodopsin sequences but with a mammalian C-terminus. Since the C-terminal sequence of rhodopsin is highly conserved in mammals and divergent in Xenopus laevis, and mammalian and epitope-tagged rhodopsins may have unexpected properties as transgenes, we decided to test whether a Xenopus laevis rhodopsin transgene carrying only the P23H mutation could also cause rod photoreceptor degeneration. Xenopus laevis tadpoles expressing these transgenes indeed had shortened outer segments and, in severely affected animals, the loss of rod photoreceptors but not the loss of cone photoreceptors. RT-PCR analyses showed that less than 10% of mutant transgenic rhodopsin relative to wild-type endogenous rhodopsin mRNA was sufficient to produce severe rod photoreceptor degeneration. As observed in other animal models as well as humans carrying this particular rhodopsin mutation, the rod photoreceptor degeneration was most severe in the ventral retina and was modified by light. Thus, the rod photoreceptor degeneration produced in Xenopus laevis by the P23H mutation in an otherwise untagged Xenopus laevis rhodopsin is generally similar to that seen with mammalian rhodopsins and epitope-tagged versions of Xenopus laevis rhodopsin, though some differences remain to be explained.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Gene Expression; Genotype; Humans; In Situ Hybridization; Light; Molecular Sequence Data; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Sequence Alignment; Species Specificity; Transgenes; Xenopus laevis

To determine the underlying retinal micropathology in subclasses of autosomal dominant retinitis pigmentosa (ADRP) caused by rhodopsin (RHO) mutations.. Patients with RHO-ADRP (n = 17, ages 6-73 years), representing class A (R135W and P347L) and class B (P23H, T58R, and G106R) functional phenotypes, were studied with optical coherence tomography (OCT), and colocalized visual thresholds were determined by dark- and light-adapted chromatic perimetry. Autofluorescence imaging was performed with near-infrared light. Retinal histology in hT17M-rhodopsin mice was compared with the human results.. Class A patients had only cone-mediated vision. The outer nuclear layer (ONL) thinned with eccentricity and was not detectable within 3 to 4 mm of the fovea. Scotomatous extracentral retina showed loss of ONL, thickening of the inner retina, and demelanization of RPE. Class B patients had superior-inferior asymmetry in function and structure. The superior retina could have normal rod and cone vision, normal lamination (including ONL) and autofluorescence of the RPE melanin; laminopathy was found in the scotomas. With Fourier-domain-OCT, there was apparent inner nuclear layer (INL) thickening in regions with ONL thinning. Retinal regions without ONL had a thick hyporeflective layer that was continuous with the INL from neighboring regions with normal lamination. Transgenic mice had many of the laminar abnormalities found in patients.. Retinal laminar abnormalities were present in both classes of RHO-ADRP and were related to the severity of colocalized vision loss. The results in human class B and the transgenic mice support the following disease sequence: ONL diminution with INL thickening; amalgamation of residual ONL with the thickened INL; and progressive retinal remodeling with eventual thinning.

Topics: Adolescent; Adult; Aged; Animals; Child; Child, Preschool; Dark Adaptation; Electroretinography; Female; Fluorescence; Genes, Dominant; Humans; Male; Mice; Mice, Transgenic; Middle Aged; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Tomography, Optical Coherence; Vision Disorders; Visual Field Tests; Visual Fields

Two types of mutations may lead to deficient pre-mRNA splicing: cis-acting mutations that inactivate a constitutive or alternative splice site within the pre-mRNA, and trans-acting mutations that affect the function of a basal factor of the splicing machinery. Autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in at least 12 genes, with mutations in rhodopsin being the most prevalent. Two cis-acting mutations, g.3811A>G and g.5167G>T at the splice site in the rhodopsin gene (RHO; GenBank U49742.1) are linked to adRP in a Spanish population; while a cis-acting mutation, g.4335G>T, has been linked to recessive RP (arRP). Transcriptional expression analysis showed that the cis-acting splicing mutations linked to adRP promoted alternative splice sites, while the arRP linked mutation results in exclusion of exon 4. Trans-acting splicing mutations associated with adRP have also been found, and mutations in the pre-mRNA splicing factors PRPF3, PRPF8, PRPF31, and RP9 are associated with adRP in several populations. This report describes a new mutation in PRPF3 in a Spanish adRP family. We also investigated the transcriptional patterns in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells from patients carrying a mutation in PRPF8. Despite the role of PRPF8 in the minor U12 splicing processes, microarray analysis revealed that mutations in PRPF8 not only did not result in significant differences in splicing efficiency of rhodopsin, but no apparent changes in expression of U12-type intron genes and splicing processes was observed. Microarray analysis revealed a panel of differentially expressed genes mapped to the RP loci, and future work will determine their role in RP.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cell Line; DNA Mutational Analysis; Female; Genes, Dominant; Humans; Male; Molecular Sequence Data; Mutation; Nuclear Proteins; Pedigree; Retinitis Pigmentosa; Rhodopsin; Ribonucleoprotein, U4-U6 Small Nuclear; RNA Splicing; RNA-Binding Proteins; Sequence Alignment; Spain

A standard operating microscope was modified with a bandpass infrared filter in the light path and infrared image intensifiers for each of the 2 eyepieces. We evaluated this system for subretinal injections in normal control dogs and those with a mutation in the rhodopsin gene. Rhodopsin-mutant dogs are a model for human autosomal dominant retinitis pigmentosa, and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light but not the infrared light. The modified operating microscope provided an excellent view of the ocular fundus under infrared illumination and allowed us to perform subretinal injections in the retinas of the rhodopsin-mutant dogs without any subsequent light-induced retinal degeneration.

Topics: Animals; Dog Diseases; Dogs; Light; Microscopy; Mutation; Ophthalmologic Surgical Procedures; Radiation Injuries; Retina; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse.. For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections.. Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected.. Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Topics: Adenoviridae; Animals; Bone Marrow Cells; Cell Survival; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Gene Silencing; Genetic Vectors; Green Fluorescent Proteins; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Transfection

The purpose of this study was to explore neuroretinal transplantation in a large animal model of severe retinitis pigmentosa and to establish graft development, long-term survival, graft-host integration, and effects on the host retina.. Rhodopsin transgenic pigs, aged 6 months, received in one eye a fetal full-thickness neuroretinal sheet in the subretinal space by means of vitrectomy and retinotomy. Six months postoperatively, eyes were studied in the light microscope and with immunohistochemical markers. Full-field electroretinography (ERG) was performed at 4 and 6 months.. Laminated grafts with well-organized photoreceptors, rod bipolar cells, and Müller cells were found in five of six eyes. Neuronal connections between graft and host retina were not seen. In the five eyes containing a graft, the number of surviving rods in the host retina was significantly higher compared with unoperated eyes. The ERG did not reveal any significant difference in b-wave amplitude between operated and control eyes, but the cone-derived response in operated eyes increased significantly from 4 to 6 months while the rod response in control eyes decreased significantly.. Fetal full-thickness neuroretina can be transplanted safely to an eye with severe retinal degeneration. In their major part, the transplants develop a normal laminated morphology and survive for at least 6 months. Graft and host retinal neurons do not form connections. Retinal function in the host is reduced initially by the surgical trauma, but the presence of a well-laminated graft counteracts this effect and rescues rods from degeneration.

Topics: Animals; Animals, Genetically Modified; Biomarkers; Dark Adaptation; Disease Models, Animal; Electroretinography; Female; Fetal Tissue Transplantation; Fluorescent Antibody Technique, Indirect; Follow-Up Studies; Glial Fibrillary Acidic Protein; Graft Survival; Neuroglia; Pregnancy; Protein Kinase C; Retina; Retinal Bipolar Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Transducin; Transplantation, Homologous; Vitrectomy

To develop an allele independent ribozyme for the treatment of autosomal dominant retinitis pigmentosa (ADRP) associated with mutations in the rhodopsin (RHO) gene, a ribozyme targeting dog, mouse, human but not rat rhodopsin (RHO) mRNA was designed and tested in vitro. Activity of this ribozyme was tested in tissue culture by co-transfection of HEK 293 cells with plasmids expressing opsin mRNA and ribozyme, followed by quantitative RT-PCR to evaluate the level of RHO mRNA. For experiments in vivo, Rz525 driven by the mouse opsin proximal promoter was inserted in plasmids with AAV 2 terminal repeats (TR) and packaged in AAV serotype 5 capsids. AAV-Rz525 was injected subretinally into the right eyes of P23H rat pups. Left eyes were injected with virus expressing GFP from the identical promoter. Animals were analyzed at 4, 8 and 12 weeks post-injection by full field scotopic electroretinography (ERG). After 12 weeks, animals were sacrificed and retinas were dissected, fixed and sectioned. Rz525 had high catalytic activity in vitro and led to a 50% reduction of RHO mRNA in cells. AAV-Rz525 injection into P23H transgenic rats led to significant preservation (about 50%) of scotopic ERG a- and b-wave amplitudes. Histological analysis showed an increased number of ONL nuclei in the central and superior retina of treated eyes relative to control eyes. RT-PCR analysis revealed 46% reduction of transgenic (mouse) RHO mRNA in right eyes relative to left eyes and no change in rat RHO mRNA. AAV5 delivery of Rz525 resulted in a partial rescue of the light response and structural preservation of photoreceptors in transgenic rats. This ribozyme may be a useful component of an RNA replacement gene therapy for ADRP.

Topics: Alleles; Animals; Animals, Genetically Modified; Cells, Cultured; Dependovirus; Electroretinography; Genetic Therapy; Mice; Photic Stimulation; Rats; Retina; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic; RNA, Messenger; Rod Opsins; Species Specificity; Transfection

To measure the rates of visual acuity, visual field, and ERG loss in patients with X-linked retinitis pigmentosa due to RPGR mutations and to determine whether these rates differ from those of patients with dominant retinitis pigmentosa due to RHO mutations.. Snellen visual acuities, Goldmann visual field areas (V4e white test light), and 30 Hz (cone) full-field ERG amplitudes were recorded for an average of 9.8 years in 113 patients with RPGR mutations. After censoring data to eliminate ceiling and floor effects, we used longitudinal regression to estimate mean rates of change and to compare these rates with those of a previously studied cohort of 134 patients with dominant retinitis pigmentosa due to RHO mutations, who were followed for an average of 8.9 years. Survival analysis was used to compare the age distribution of legal blindness in these two groups. To explain group differences in visual acuity, optical coherence tomograms were recorded in some patients to visualize central retinal structure.. Mean annual exponential rates of decline for the patients with RPGR mutations were 4.0% for visual acuity, 4.7% for visual field area, and 7.1% for ERG amplitude. Each of these rates was significantly different from zero (P < 0.001). The rates of visual acuity and visual field loss were significantly faster than the corresponding rates in the RHO patients (1.6%, P < 0.001 and 2.9%, P = 0.002, respectively), whereas the rate of ERG amplitude loss was comparable to that in the RHO patients (7.7%, P = 0.39). The median age of legal blindness was 32 years younger in the RPGR patients than in the RHO patients, due primarily to loss of visual acuity rather than to loss of visual field. Loss of acuity in RPGR patients appeared to be associated with foveal thinning.. Patients with X-linked retinitis pigmentosa due to RPGR mutations lose visual acuity and visual field more rapidly than do patients with dominant retinitis pigmentosa due to RHO mutations.

Topics: Adolescent; Adult; Child; Child, Preschool; Electroretinography; Eye Proteins; Follow-Up Studies; Genetic Diseases, X-Linked; Guanine Nucleotide Exchange Factors; Humans; Male; Middle Aged; Retina; Retinitis Pigmentosa; Rhodopsin; Survival Analysis; Tomography, Optical Coherence; Vision Disorders; Visual Acuity; Visual Fields

To characterize molecular and cellular changes induced by sustained expression of ciliary neurotrophic factor (CNTF) in the rds mutant mouse retina.. Recombinant adeno-associated virus (rAAV) expressing CNTF was injected subretinally, for transduction of peripherin/rds(+/)(-) transgenic mice that carry the P216L mutation found in human retinitis pigmentosa. Characterization of retinal neurons and glia was performed by immunocytochemistry with cell-type-specific markers. Activation of signaling molecules was examined by Western blot and immunostaining. Alterations of gene transcription profiles were studied by microarray analyses.. CNTF viral transduction maintained rhodopsin expression in surviving rod photoreceptors, but greatly reduced both S- and M-opsin normally expressed in cones. In addition, CNTF treatment resulted in increased numbers and dispersion of Müller glia and Chx10-positive bipolar cells within the inner nuclear layer. Persistent CNTF signaling also caused enhanced phosphorylation of STAT1, STAT3, and p42/44 ERK, as well as their levels of expression. Moreover, altered transcription profiles were detected for a large number of genes. Among these, Crx and Nrl involved in photoreceptor differentiation and several genes involved in phototransduction were suppressed.. Despite the rescue from cell death, continuous exposure to CNTF changed photoreceptor cell profiles, especially resulting in the loss of cone immunoreactivity. In addition, the Müller glia and bipolar cells became disorganized, and the number of cells expressing Müller and bipolar cell markers increased. Constitutive CNTF production resulted in sustained activation of cytokine signal transduction and altered the expression of a large number of genes. Therefore, stringent regulation of CNTF may be necessary for its therapeutic application in preventing retinal degeneration.

Topics: Animals; Blotting, Western; Ciliary Neurotrophic Factor; Dependovirus; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Genetic Vectors; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroglia; Oligonucleotide Array Sequence Analysis; Phosphorylation; Photoreceptor Cells, Vertebrate; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; STAT1 Transcription Factor; STAT3 Transcription Factor; Transfection

The transcription factor neural retina leucine zipper (NRL) is required for rod photoreceptor differentiation during mammalian retinal development. NRL interacts with CRX, NR2E3, and other transcription factors and synergistically regulates the activity of photoreceptor-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. Here we report functional analyses of 17 amino acid variations and/or mutations of NRL. We show that 13 of these lead to changes in NRL phosphorylation. Six mutations at residues p.S50 (c.148T>A, c.148T>C, and c.149C>T) and p.P51 (c.151C>A, c.151C>T, and c.152C>T), identified in patients with autosomal dominant retinitis pigmentosa, result in a major NRL isoform that exhibits reduced phosphorylation but enhanced activation of the rhodopsin promoter. The truncated NRL mutant proteins-p.L75fs (c.224_225insC) and p.L160fs (c.459_477dup)-do not localize to the nucleus because of the absence of bZIP domain. The p.L160P (c.479T>C), p.L160fs, and p.R218fs (c.654delC) mutant proteins do not bind to the NRL-response element, as revealed by electrophoretic mobility shift assays. These three and p.S225N (c.674G>A) mutant show reduced transcriptional activity and may contribute to recessive disease. The p.P67S (c.199C>T) and p.L235F (c.703C>T) variations in NRL do not appear to directly cause retinitis pigmentosa, while p.E63K (c.187G>A), p.A76V (c.227C>T), p.G122E (c.365G>A), and p.H125Q (c.375C>G) are of uncertain significance. Our results support the notion that gain-of-function mutations in the NRL gene cause autosomal dominant retinitis pigmentosa while loss-of-function NRL mutations lead to autosomal recessive retinitis pigmentosa. We propose that differential phosphorylation of NRL fine-tunes its transcriptional regulatory activity, leading to a more precise control of gene expression.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Basic-Leucine Zipper Transcription Factors; Cell Line; Chlorocebus aethiops; COS Cells; DNA; DNA-Binding Proteins; Eye Proteins; Gene Expression Regulation; Genes, Dominant; Genes, Recessive; Humans; Molecular Sequence Data; Mutation; Phosphorylation; Promoter Regions, Genetic; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Amino Acid; Transcriptional Activation; Transfection

Many genes from retinoid metabolism cause retinitis pigmentosa. Peropsin, an opsin-like protein with unknown function, is specifically expressed in apical retinal pigment epithelium microvilli. Since rhodopsin and RGR, another opsin-like protein, cause retinitis pigmentosa, we used D-HPLC to screen for the peropsin gene RRH in 331 patients (288 with retinitis pigmentosa and 82 with other retinal dystrophies). We found 13 nonpathogenic variants only, among which a c.730_731delATinsG that truncates the last two transmembrane-spanning fragments and the Lys284 required for retinol binding, but does not segregate with the disease phenotype. We conclude that RRH is not a frequent gene in retinitis pigmentosa.

Topics: Adult; Base Sequence; Case-Control Studies; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Polymorphism, Single Nucleotide; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Nucleic Acid

To examine the clinical picture and molecular genetics of 12 Norwegian families with autosomal dominant retinitis pigmentosa (adRP) in order to achieve a genotype-phenotype correlation.. In addition to a clinical ophthalmological examination, fundus photography, dark adaptometry and electroretinography were performed. Four genes were analysed: rhodopsin (RHO); retinitis pigmentosa 1 (RP1); retinal degeneration slow/peripherin (RDS/peripherin), and inosine monophosphate dehydrogenase 1 (IMPDH1). Seven of the families had been examined about 20 years previously. A total of 63 patients or first-degree relatives (aged 18-79 years) were examined.. Mutations were found only in the RHO gene. Seven families were given a diagnosis of classical RP. Two of them had novel mutation 1003delG, and one family had the mutation V345M. Four families had pericentral retinal dystrophy (PRD), two families with the mutation A164V and one with novel mutation I179F. One family was given a diagnosis of central and pericentral retinal dystrophy (CPRD), a special type of cone/rod dystrophy, and no mutation was found.. Six of 12 families had an RHO mutation. The mutation V345M and the novel mutation 1003delG both caused classical RP, the former indicating the most unfavourable prognosis. Two of the families with PRD had the A164V mutation with a favourable prognosis, whereas the novel mutation I179F caused PRD with extremely variable expressivity.

Topics: Adolescent; Adult; Aged; Dark Adaptation; DNA Mutational Analysis; Electroretinography; Eye Proteins; Female; Follow-Up Studies; Frameshift Mutation; Genes, Dominant; Humans; IMP Dehydrogenase; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Microtubule-Associated Proteins; Middle Aged; Mutation, Missense; Nerve Tissue Proteins; Norway; Pedigree; Peripherins; Phenotype; Photography; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Retinitis pigmentosa (RP) is a heterogeneous group of diseases in which one of a wide variety of mutations selectively causes rod photoreceptor cell death. After rods die, cone photoreceptors gradually die resulting in blindness. Antioxidants reduce cone cell death in rd1/rd1 mice indicating that cones die from oxidative damage in that model of rapidly progressive RP. In this study, we sought to determine if this observation could be generalized to models of other types of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and Q344ter mice, a model of rapidly progressive dominant RP. Compared to appropriate vehicle-treated controls, rd10/rd10 and Q344ter mice treated between P18 and P35 with a mixture of antioxidants previously found to be effective in rd1/rd1 mice showed significantly greater cone survival. Antioxidant-treated rd10/rd10 mice showed preservation of cone function as shown by a significant increase in photopic ERG b-wave amplitudes, and surprisingly showed temporary preservation of scotopic a-wave amplitudes, prolonged rod survival, and slowed depletion of rhodopsin mRNA. These data suggest that oxidative damage contributes to cone cell death regardless of the disease causing mutation that leads to the demise of rods, and that in more slowly progressive rod degenerations, oxidative damage may also contribute to rod cell death. Protection from oxidative damage may be a broadly applicable treatment strategy in RP.

Topics: alpha-Tocopherol; Animals; Antioxidants; Ascorbic Acid; Cell Death; Codon, Nonsense; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Drug Administration Schedule; Electroretinography; Exons; Heterozygote; Homozygote; Injections, Intraperitoneal; Kinetics; Mice; Mice, Mutant Strains; Mutation, Missense; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Thioctic Acid

Mutational heterogeneity represents a significant barrier to development of therapies for many dominantly inherited diseases. For example, >100 mutations in the rhodopsin gene (RHO) have been identified in patients with retinitis pigmentosa (RP). The development of therapies for dominant disorders that correct the primary genetic lesion and overcome mutational heterogeneity is challenging. Hence, therapeutics comprising two elements--gene suppression in conjunction with gene replacement--have been investigated. Suppression is targeted to a site independent of the mutation; therefore, both mutant and wild-type alleles are suppressed. In parallel with suppression, a codon-modified replacement gene refractory to suppression is provided. Both in vitro and in vivo validation of suppression and replacement for RHO-linked RP has been undertaken in the current study. RNA interference (RNAi) has been used to achieve ~90% in vivo suppression of RHO in photoreceptors, with use of adeno-associated virus (AAV) for delivery. Demonstration that codon-modifed RHO genes express functional wild-type protein has been explored transgenically, together with in vivo expression of AAV-delivered RHO-replacement genes in the presence of targeting RNAi molecules. Observation of potential therapeutic benefit from AAV-delivered suppression and replacement therapies has been obtained in Pro23His mice. Results provide the first in vivo indication that suppression and replacement can provide a therapeutic solution for dominantly inherited disorders such as RHO-linked RP and can be employed to circumvent mutational heterogeneity.

Topics: Adenoviridae; Animals; Base Sequence; Genetic Therapy; HeLa Cells; Humans; Mice; Mice, Transgenic; Molecular Sequence Data; Retina; Retinitis Pigmentosa; Rhodopsin; RNA Interference; RNA, Small Interfering; Suppression, Genetic

To investigate macular photoreceptor structure in patients with inherited retinal degeneration using high-resolution images and to correlate the findings with clinical phenotypes and genetic mutations.. Adaptive optics scanning laser ophthalmoscopy (AOSLO) images of photoreceptors were obtained in 16 eyes: five with retinitis pigmentosa (RP), three with cone-rod dystrophy (CRD), and eight without retinal disease. A quadratic model was used to illustrate cone spacing as a function of retinal eccentricity. Cone spacing at 1 degrees eccentricity was compared with standard measures of central visual function, including best-corrected visual acuity (BCVA), foveal threshold, and multifocal electroretinogram (mfERG) amplitude and timing. Intervisit variations were studied in one patient with RP and one patient with CRD. Screening of candidate disease genes identified mutations in two patients, one with RP (a rhodopsin mutation) and the other with CRD (a novel RPGR-ORF15 mutation).. Cone spacing values were significantly different from normal for patients with RP (P = 0.01) and CRD (P < 0.0001) and demonstrated a statistically significant correlation with foveal threshold (P = 0.0003), BCVA (P = 0.01), and mfERG amplitude (P = 0.008). Although many RP patients showed normal cone spacing within 1 degrees of fixation, cones could not be unambiguously identified in several retinal regions. Cone spacing increased in all CRD patients, even those with early disease. Little variation was observed in cone spacing measured during two sessions fewer than 8 days apart.. AOSLO images can be used to study macular cones with high resolution in patients with retinal degeneration. The authors present the first report of cone structure in vivo in patients with mutations in rhodopsin and RPGR-ORF15 and show that macular cones display distinct characteristics, depending on the underlying disease. AOSLO imaging, therefore, can provide new insight into possible mechanisms of cone vision loss in patients with retinal degeneration.

Topics: Adult; Diagnostic Imaging; Electroretinography; Eye Proteins; Female; Humans; Macular Degeneration; Male; Microtubule-Associated Proteins; Middle Aged; Mutation; Ophthalmoscopy; Optics and Photonics; Reproducibility of Results; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

To identify nucleotide sequence variations in the rhodopsin (RHO) gene of Japanese patients with retinitis pigmentosa (RP) in order to search for mutations or haplotypes responsible for RP.. The entire region of RHO locus including a promoter region and introns was sequenced using blood-derived genomic DNA samples donated by 68 patients with RP and 68 control subjects.. We found 39 single nucleotide substitutions including 17 rare substitutions of less than 1% in frequency, one insertion/deletion polymorphism, and one CA-repeat polymorphism in a 7.8 kbp region spanning the promoter, five exons, and four introns of the RHO gene locus. There were no affected subjects with amino acid substitutions in RHO, and there was 1 control subject with a novel substitution (Ala42Thr) who had no symptoms of RP. Fine analysis of single nucleotide polymorphism (SNPs) revealed eight haplotype structures of the Japanese RHO locus. There was no significant difference between RP patients and controls in terms of haplotype frequency.. No mutation causing an amino acid substitution of RHO was observed in 68 Japanese patients with RP, but 1 control subject did have a novel amino acid substitution. The Japanese RHO locus is comprised of eight major haplotypes. The RP-associated haplotype was not identified. The haplotype-tagging SNPs identified in this study will be useful as markers for the linkage-based screening of RP patients.

Topics: Asian People; Chromosome Mapping; DNA Mutational Analysis; DNA Transposable Elements; Exons; Female; Gene Deletion; Gene Frequency; Genetic Variation; Haplotypes; Humans; Introns; Linkage Disequilibrium; Male; Phylogeny; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Retinitis Pigmentosa; Rhodopsin

Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations.. Metabolic labeling with 33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis.. Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX-mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells.. Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by disease-associated NRL mutations. As inhibition of MAPK signaling pathways decreases NRL-mediated transactivation of rhodopsin promoter, we propose that phosphorylation changes associated with NRL mutations perturb gene expression in rods, leading to photoreceptor degeneration in retinopathies.

Topics: Animals; Animals, Newborn; Chlorocebus aethiops; COS Cells; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Leucine Zippers; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Promoter Regions, Genetic; Retina; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Transcriptional Activation; Transfection

To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic approximately 27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

Topics: Animals; Animals, Genetically Modified; Cattle; Cell Line, Transformed; Darkness; Disease Models, Animal; Gene Expression Regulation; Histidine; Humans; Mice; Microscopy, Electron, Scanning; Mutation; Peptide Fragments; Proline; Retinal Rod Photoreceptor Cells; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Transfection; Xenopus laevis

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.

Topics: Activating Transcription Factor 6; Animals; Animals, Genetically Modified; Apoptosis; Cell Line; Cell Survival; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum; Endoribonucleases; Humans; Kinetics; Membrane Proteins; Mice; Mutation; Protein Folding; Protein Serine-Threonine Kinases; Proteins; Rats; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Retinitis pigmentosa is a very heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasian population. To investigate the frequency and pattern of RHO point mutations in Chinese patients with ADRP, we have screened the five coding exons and splice sites of the RHO gene in 50 unrelated probands from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified each in one family, thus the frequency of RHO mutations among ADRP families in this study is less than 14% (2/50=4%, 95% confidence interval: 1-14%), lower than that in Europe and North America, which may reflect an ethnic difference between Chinese and Caucasian populations. Loss of all phosphorylation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del). CSGE was found to be a sensitive, simple and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Topics: Base Sequence; China; DNA Mutational Analysis; DNA Primers; Genes, Dominant; Humans; Mutation, Missense; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

Mutations in the C terminus of rhodopsin disrupt a rod outer segment localization signal, causing rhodopsin mislocalization and aggressive forms of retinitis pigmentosa (RP). Studies of cultured photoreceptors suggest that activated mislocalized rhodopsin can cause cell death via inappropriate G-protein-coupled signaling. To determine whether this pathway occurs in vivo, we developed a transgenic Xenopus laevis model of RP based on the class I rhodopsin mutation Q344Ter (Q350Ter in X. laevis). We used a second mutation, K296R, to block the ability of rhodopsin to bind chromophore and activate transducin. We compared the effects of expression of both mutants on X. laevis retinas alone and in combination. K296R did not significantly alter the cellular distribution of rhodopsin and did not induce retinal degeneration. Q350Ter caused rhodopsin mislocalization and induced an RP-like degeneration, including loss of rods and development of sprouts or neurites in some remaining rods, but did not affect the distribution of endogenous rhodopsin. The double mutant K296R/Q350Ter caused a similar degeneration and neurite outgrowth. In addition, we found no protective effects of dark rearing in these animals. Our results demonstrate that the degenerative effects of mislocalized rhodopsin are not mediated by the activated form of rhodopsin and therefore do not proceed via conventional G-protein-coupled signaling.

Topics: Animals; Animals, Genetically Modified; Chlorocebus aethiops; COS Cells; Female; Male; Neurites; Point Mutation; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Xenopus laevis; Xenopus Proteins

Identification of a novel rhodopsin mutation in a family with retinitis pigmentosa and comparison of the clinical phenotype to a known mutation at the same amino acid position.. Screening for mutations in rhodopsin was performed in 78 patients with retinitis pigmentosa. All exons and flanking intronic regions were amplified by PCR, sequenced, and compared to the reference sequence derived from the National Center for Biotechnology Information (NCBI, Bethesda, MD) database. Patients were characterized clinically according to the results of best corrected visual acuity testing (BCVA), slit lamp examination (SLE), funduscopy, Goldmann perimetry (GP), dark adaptometry (DA), and electroretinography (ERG). Structural analyses of the rhodopsin protein were performed with the Swiss-Pdb Viewer program available on-line (http://www.expasy.org.spdvbv/ provided in the public domain by Swiss Institute of Bioinformatics, Geneva, Switzerland).. A novel rhodopsin mutation (Gly90Val) was identified in a Swiss family of three generations. The pedigree indicated autosomal dominant inheritance. No additional mutation was found in this family in other autosomal dominant genes. The BCVA of affected family members ranged from 20/25 to 20/20. Fundus examination showed fine pigment mottling in patients of the third generation and well-defined bone spicules in patients of the second generation. GP showed concentric constriction. DA demonstrated monophasic cone adaptation only. ERG revealed severely reduced rod and cone signals. The clinical picture is compatible with retinitis pigmentosa. A previously reported amino acid substitution at the same position in rhodopsin leads to a phenotype resembling night blindness in mutation carriers, whereas patients reported in the current study showed the classic retinitis pigmentosa phenotype. The effect of different amino acid substitutions on the three-dimensional structure of rhodopsin was analyzed by homology modeling. Distinct distortions of position 90 (shifts in amino acids 112 and 113) and additional hydrogen bonds were found.. Different amino acid substitutions at position 90 of rhodopsin can lead to night blindness or retinitis pigmentosa. The data suggest that the property of the substituted amino acid distinguishes between the phenotypes.

Topics: Adult; Amino Acid Substitution; DNA Mutational Analysis; Electroretinography; Female; Genes, Dominant; Humans; Male; Middle Aged; Mutation, Missense; Night Blindness; Pedigree; Phenotype; Polymerase Chain Reaction; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Field Tests; Visual Fields

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited diseases that are characterised by primary degeneration of rod photoreceptors and secondary degeneration of cone photoreceptors in the retina. Additional pathological changes include vascular changes and invasion of the inner retina by retinal pigment epithelial (RPE) cells. RP represents a major cause of progressive retinal disease worldwide. Using a mouse model of autosomal dominant Retinitis pigmentosa (adRP) with retinopathy induced by targeted disruption of the rhodopsin gene Rho(-/-), we have analysed the levels of expression of a range of tight and adherens junction associated proteins, in order to further elucidate the pathogenic mechanisms occurring at an early stage of this condition. Using western blot analysis and indirect immunostaining of retinal cryosections from 6-week-old mice from a C-129 background we have determined changes, if any, in the levels of expression and localisation of a series of tight and adherens junction associated proteins, including Zonula Occludens-1 (ZO-1), occludin, N-Cadherin, p120-Catenin, alpha-Catenin, gamma-Catenin, beta-Catenin, and E-Cadherin. We have found an up-regulation of the tight junction and adherens junction associated protein Zonula Occludens-1 (ZO-1) in the neural retina of 6-week-old Rho(-/-) knockout mice compared with 6-week-old Wild-Type (WT) mice. Following immunohistochemistry, however, it appears, that ZO-1, beta-Catenin and p120-Catenin expression at the Outer Limiting Membrane (OLM) of the Rho(-/-) retina is compromised, in part, compared to WT animals of the same age. We hypothesise that these retinal changes following photoreceptor cell death may contribute to the pathogenesis of adRP. Our findings of changes in the levels of expression of ZO-1 and associated adherens junction proteins beta-Catenin and p120-Catenin at the OLM in 6-week-old Rho(-/-) mice provide evidence for tight junction and adherens junction associated protein modifications in an animal model of autosomal dominant RP (adRP).

Topics: Adherens Junctions; alpha Catenin; Animals; beta Catenin; Biomarkers; Blotting, Western; Cadherins; gamma Catenin; Gene Expression Regulation; Genes, Dominant; Immunohistochemistry; Intercellular Junctions; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Confocal; Models, Animal; Occludin; Phosphoproteins; Protein Processing, Post-Translational; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Zonula Occludens-1 Protein

Retinitis pigmentosa (RP) describes a group of inherited disorders characterised by progressive retinal dysfunction, cell loss and atrophy of retinal tissue. RP demonstrates considerable clinical and genetic heterogeneity, with wide variations in disease severity, progression, and gene involvement. We studied a large family with RP to determine the pattern of inheritance and identify the disease-causing mutation, and then to describe the phenotypic presentation of this family.. Ophthalmic examination was performed on 46 family members to identify affected individuals and to characterise the disease phenotype. Family pedigree was obtained. Some family members also had fundus photographs, fluorescein angiography, and/or optical coherence tomography (OCT) analysis performed. Genetic linkage was performed using short tandem repeat (STR) polymorphic markers encompassing the known loci for autosomal dominant RP. Finally, DNA sequencing was performed to identify the mutation present in this family.. Clinical features included nyctalopia, constriction of visual fields and eventual loss of central vision. Sequence analysis revealed a G-to-T nucleotide change in the Rhodopsin gene, predicting a Gly-51-Val substitution.. This large multi-generation family demonstrates the phenotypic variability of a previously identified autosomal dominant mutation of the Rhodopsin gene.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Female; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

To investigate the pathogenic mechanisms that underlie retinal degeneration induced by the rhodopsin mutation P23H in a Xenopus laevis model of RP.. Transgenic X. laevis were generated that expressed the rhodopsin mutants rhoP23H and rhoP23H/K29R (a variant incapable of transducin activation). Using quantitative dot blot assay, transgenic rhodopsin levels and the extent of retinal degeneration were determined. The contribution of rhodopsin signal transduction to cell death was assessed by comparison of rhoP23H and rhoP23H/K296R effects and by dark rearing of rhoP23H tadpoles. Intracellular localization and the oligomeric state of rhoP23H were determined by confocal immunofluorescence microscopy and Western blot analysis.. RhoP23H induced retinal degeneration in a dose-dependent manner whereas expression of a control rhodopsin did not, indicating that rod photoreceptor death was specific to the P23H mutation and was not caused by the overexpression of rhodopsin. Neither abolishment of rhoP23H photosensitivity and ability to activate transducin nor dark rearing rescued rod viability. RhoP23H was localized primarily to the endoplasmic reticulum (ER) of inner segments. Western blot analysis of transgenic retinas showed that rhoP23H was prone to form dimers and higher molecular weight oligomers. However, aggresomes were not observed in rhoP23H transgenic retinal sections, despite their being reported in cultured cells expressing rhoP23H.. These results support a role for rhoP23H misfolding and inner segment accumulation in rod death, possibly by ER overload or other cellular stress pathways rather than by altered rhodopsin signal transduction or aggresome formation.

Topics: Animals; Animals, Genetically Modified; Blotting, Western; Cell Death; Cell Line; Dark Adaptation; Disease Models, Animal; Dose-Response Relationship, Drug; Fluorescent Antibody Technique, Indirect; Kidney; Microscopy, Confocal; Mutation; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transducin; Transfection; Transgenes; Xenopus laevis

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca2+/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca2+/recoverin than wild-type rhodopsin. This suggests an involvement of Ca2+/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca2+/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.

Topics: Animals; Calcium; Cattle; Chlorocebus aethiops; COS Cells; Dose-Response Relationship, Drug; G-Protein-Coupled Receptor Kinase 1; Genes, Dominant; Mutation; Phosphorylation; Recoverin; Retinitis Pigmentosa; Rhodopsin; Transducin

We compare the known retinitis pigmentosa (RP) mutations in rhodopsin with mutational data obtained for the complement factor 5a receptor (C5aR), a member of the rhodopsin-like family of G protein-coupled receptors (GPCRs). We have performed genetic analyses that define residues that are required for C5aR folding and function. The cognate residues in rhodopsin are not preferentially mutated in RP, suggesting that the predominant molecular defect in RP involves more than simple misfolding or inactivation. Energy calculations are performed to elucidate the structural effects of the RP mutations. Many of these mutations specifically disrupt the environment of the retinal prosthetic group of rhodopsin, and these do not correspond to essential residues in C5aR. This may be because a retinal group is present in rhodopsin but not in C5aR. Another subset of RP mutations is more generally important for receptor structure, and these mutations correlate with essential residues of C5aR.

Topics: Amino Acid Sequence; Animals; Cattle; Humans; Models, Molecular; Molecular Sequence Data; Night Blindness; Protein Structure, Quaternary; Receptor, Anaphylatoxin C5a; Retinitis Pigmentosa; Rhodopsin; Sequence Alignment; Structure-Activity Relationship; Vision, Ocular

Similar retinitis pigmentosa (RP) phenotypes can result from mutations affecting different rhodopsin regions, and distinct amino acid substitutions can cause different RP severity and progression rates. Specifically, both the R135L and R135W mutations (cytoplasmic end of H3) result in diffuse, severe disease (class A), but R135W causes more severe and more rapidly progressive RP than R135L. The P180A and G188R mutations (second intradiscal loop) exhibit a mild phenotype with regional variability (class B1) and diffuse disease of moderate severity (class B2), respectively. Computational and in vitro studies of these mutants provide molecular insights into this phenotypic variability.

Topics: Adolescent; Adult; Age Factors; Amino Acid Substitution; Child; Child, Preschool; Computational Biology; Disease Progression; DNA Mutational Analysis; Electroretinography; Female; Humans; Male; Mutation; Pedigree; Peptide Fragments; Phenotype; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Vision, Ocular

Over 100 rhodopsin mutation alleles have been associated with autosomal dominant retinitis pigmentosa (ADRP). These mutations appear to cause photoreceptor cell death through diverse molecular mechanisms. We show that K296E, a rhodopsin mutation associated with ADRP, forms a stable complex with arrestin that is toxic to mouse rod photoreceptors. This cell death pathway appears to be conserved from flies to mammals. A genetics approach to eliminate arrestin unmasked the constitutive activity of K296E and caused photoreceptor cell death through a transducin-dependent mechanism that is similar to light damage. Expressing K296E in the arrestin/transducin double knock-out background prevented transducin signaling and led to substantially improved retinal morphology but did not fully prevent cell death caused by K296E. The adverse effect of K296E in the arrestin/transducin knock-out background can be mimicked by constant exposure to low light. Furthermore, we found that arrestin binding causes K296E to mislocalize to the wrong cellular compartment. Accumulation of stable rhodopsin/arrestin complex in the inner segment may be an important mechanism for triggering the cell death pathway in the mammalian photoreceptor cell.

Topics: Animals; Arrestin; Cell Compartmentation; Cell Death; Chromosome Disorders; Disease Models, Animal; Female; Genes, Dominant; Humans; Light; Macromolecular Substances; Male; Mice; Mice, Knockout; Mice, Transgenic; Mutation; Protein Binding; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transducin

Diabetic patients who also have retinitis pigmentosa (RP) appear to have fewer and less severe retinal microvascular lesions. Diabetic retinopathy may be linked to increased inner retinal hypoxia, with the possibility that this is exacerbated by oxygen usage during the dark-adaptation response. Therefore, patients with RP with depleted rod photoreceptors may encounter proportionately less retinal hypoxia, and, when diabetes is also present, there may be fewer retinopathic lesions. This hypothesis was tested in rhodopsin knockout mice (Rho-/-) as an RP model in which the diabetic milieu is superimposed. The study was designed to investigate whether degeneration of the outer retina has any impact on hypoxia, to examine diabetes-related retinal gene expression responses, and to assess lesions of diabetic retinopathy.. Streptozotocin-induced diabetes was created in male C57Bl6 (wild-type; WT) and Rho-/- mice, and hyperglycemia was maintained for 5 months. The extent of diabetes was confirmed by measurement of glycated hemoglobin (%GHb) and accumulation of advanced glycation end products (AGEs). Retinal hypoxia was assessed using the bioreductive drug pimonidazole. The retinal microvasculature was studied in retinal flatmounts stained by the ADPase reaction, and the outer retina was evaluated histologically in paraffin-embedded sections. Retinal gene expression of VEGF-A, TNF-alpha, and mRNAs encoding basement membrane component proteins were quantified by real-time RT-PCR.. The percentage GHb increased significantly in the presence of diabetes (P < 0.001) and was not different between WT or Rho-/- mice. Hypoxia increased in the retina of WT diabetic animals when compared with controls (P < 0.001) but this diabetes-induced change was absent in Rho-/- mice. Retinal gene expression of VEGF-A was significantly increased in WT mice with diabetes (P < 0.05), but was unchanged in Rho-/- mice. TNF-alpha gene expression significantly increased (4.9-fold) in WT mice with diabetes (P < 0.05) and also increased appreciably in Rho-/- mice but to a reduced extent (1.5 fold; P < 0.05). The outer nuclear layer in nondiabetic Rho-/- mice was reduced to a single layer after 6 months, but when diabetes was superimposed on this model, there was less degeneration of photoreceptors (P < 0.05). Vascular density was attenuated in diabetic WT mice compared with the nondiabetic control (P < 0.001); however, this diabetes-related disease was not observed in Rho-/- mice.. Loss of the outer retina reduces the severity of diabetic retinopathy in a murine model. Oxygen usage by the photoreceptors during dark adaptation may contribute to retinal hypoxia and exacerbate the progression of diabetic retinopathy.

Topics: Animals; Animals, Genetically Modified; Apyrase; Basement Membrane; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Glycated Hemoglobin; Glycation End Products, Advanced; Histocytochemistry; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Vessels; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

To investigate the peropsin gene (RRH), encoding a retinal pigment epithelium homolog of the rod-expressed opsin (rhodopsin), for the presence of pathogenic mutations causing retinitis pigmentosa (RP) or other retinal degenerations.. All seven exons composing the RRH open reading frame and the immediate intron sequences were analyzed by direct nucleotide sequencing of 613 patients with forms of retinal degeneration.. One patient with retinitis punctata albescens was a heterozygote with the missense change Cys98Tyr (TGT>TAT, c.293G>A). This change affects the homologous residue that is the target of the rhodopsin mutation Cys110Tyr, a reported cause of dominant RP. Unfortunately, none of the patient's relatives were available for a segregation analysis to determine if this change is unambiguously associated with disease. No definite pathogenic mutation was found in any of the other 612 patients who were evaluated.. The Cys98Tyr is a possible cause of retinitis punctata albescens, although this conclusion is tentative because the change was found in only one patient. Our results indicate that the peropsin gene is not a common cause of RP or some related retinal degenerations, at least in the set of patients we analyzed.

Topics: Cysteine; DNA Mutational Analysis; Heterozygote; Humans; Mutation, Missense; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tyrosine

Autosomal dominant retinitis pigmentosa (ADRP) has been linked to mutations in the gene encoding rhodopsin. Most RP-linked rhodopsin mutants are unable to fold correctly in the endoplasmic reticulum, are degraded by the ubiquitin proteasome system, and are highly prone to forming detergent-insoluble high molecular weight aggregates. Here we have reported that coexpression of folding-deficient, but not folding-proficient, ADRP-linked rhodopsin mutants impairs delivery of the wild-type protein to the plasma membrane. Fluorescence resonance energy transfer and co-precipitation studies revealed that mutant and wild-type rhodopsins form a high molecular weight, detergent-insoluble complex in which the two proteins are in close (<70 A) proximity. Co-expression of ARDP-linked rhodopsin folding-deficient mutants resulted in enhanced proteasome-mediated degradation and steady-state ubiquitination of the wild-type protein. These data suggested a dominant negative effect on conformational maturation that may underlie the dominant inheritance of ARDP.

Topics: Cell Line; Genes, Dominant; Glycosylation; Humans; Mutation; Proteasome Endopeptidase Complex; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Retinitis Pigmentosa; Rhodopsin; Ubiquitin

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cells, Cultured; Eye Proteins; Gene Expression Regulation; Humans; Mice; Mutation; Neurons; Retina; Retinitis Pigmentosa; Rhodopsin; RNA Splicing

Retinitis pigmentosa (RP) is a group of hereditary retinal diseases in which photoreceptor cells degenerate. It is both clinically and genetically heterogenous. Using a two-stage approach by combining linkage analysis with mutation detection, we have rapidly identified the gene locus and the mutation site of a Chinese Singaporean family with autosomal dominant RP.. Three Chinese Singaporean families were tested. One family showed autosomal dominant inheritance pattern, while the other two could be recessive or sporadic. Twelve di-nucleotide markers tightly linked to 6 genes known to be responsible for either autosomal dominant or recessive RP were selected for linkage analysis. Cosegregation of marker and disease inheritance pattern permits identification of the target candidate gene. RFLP (restriction fragment length polymorphism) markers were added to confirm the linkage result prior to the detailed mutation detection study.. With this two-stage strategy, the autosomal dominant RP family showed the rhodopsin locus segregating concordantly with the disease. Mutation screening later identified a nonsense mutation 5261C>T in the last exon of rhodopsin gene. It predicted a Q344X changes at the C-terminus of the gene product, truncating it by 5 amino acids.. This systematic approach facilitates molecular diagnosis of a genetically heterogenous disease like RP. This is the first report of an RP mutation in Singapore. This 5261C>T mutation has been reported in the Caucasian, but not the Chinese population. The relatively milder phenotype in this family showed similarity to the reported US family, indicating the correlation of mutation site to severity of disease regardless of ethnicity.

Topics: Asian People; DNA; Family; Female; Genes, Dominant; Genes, Recessive; Genetic Linkage; Haplotypes; Heterozygote; Humans; Locus Control Region; Male; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Retinitis Pigmentosa; Rhodopsin; Singapore; Tandem Repeat Sequences

Genetic and environmental factors modify the severity of human neurodegenerations. Retinal degenerations caused by rhodopsin gene mutations show severity differences within and between families and even within regions of the same eye. Environmental light is thought to contribute to this variation. In the naturally occurring dog model of the human disorder, we found that modest light levels, as used in routine clinical practice, dramatically accelerated the neurodegeneration. Dynamics of acute retinal injury (consisting of abnormal intraretinal light scattering) were visualized in vivo in real time with high-resolution optical imaging. Long term consequences included fast or slow retinal degeneration or repair of injury depending on the dose of light exposure. These experiments provide a platform to study mechanisms of neuronal injury, repair, compensation, and degeneration. The data also argue for a gene-specific clinical trial of light reduction in human rhodopsin disease.

Topics: Animals; Base Sequence; Disease Models, Animal; DNA; Dogs; Humans; Light; Mutation; Nerve Regeneration; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Transcription Factor AP-1

To detect mutation in the rhodopsin gene (RHO) in a Chinese family with autosomal dominant retinitis pigmentosa (ADRP).. A total of 25 family members from a Chinese family were investigated. All the subjects were examined clinically by direct funduscopy, perimetry and vision test. Evaluation of the proband included electroretinography (ERG). Genomic DNA was extracted using standard method. The complete coding regions of RHO were amplified by polymerase chain reaction (PCR) and the PCR products were subjected to automatic DNA sequencing.. 512 C>T (P171L), a recurrent missense mutation was detected in the proband. All 12 affected subjects in the family were heterozygous for the mutation. The affected individuals had night blindness at the age of 5-6 years. They had relatively severe impairment of visual acuity and suffered a gradual loss of peripheral visual field at the age of 20-30 years. And they went blind at the age of 40-50 years. Rod and cone ERG were not detectable in the proband.. A recurrent missense mutation, 512C>T (P171L), was detected in a Chinese family with ADRP.

Topics: Adolescent; Adult; Base Sequence; China; DNA Mutational Analysis; Family Health; Female; Humans; Male; Middle Aged; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing.. We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families.. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls.. The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Topics: Asian People; Base Sequence; DNA Mutational Analysis; DNA, Antisense; Electrophoresis, Polyacrylamide Gel; Exons; Female; Genotype; Humans; Middle Aged; Molecular Sequence Data; Mutation, Missense; Phenotype; Retinitis Pigmentosa; Rhodopsin

More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases. We describe use of RNA interference (RNAi) and specifically short hairpin RNAs (shRNAs) expressed from DNA templates to silence both normal and mutant (P23H) human rhodopsin alleles by 94.34+/-2.17 and 94.9+/-1.9%, respectively, in human embryonic retinoblasts. Degeneracy of the genetic code was used to engineer a codon-exchanged mRNA (cmRNA) that demonstrated complete resistance to silencing by the shRNA. Simulation of autosomal dominant retinitis pigmentosa in cell culture through triple transfection of DNAs expressing a cmRNA, a P23H mRNA and an shRNA revealed shRNA-mediated silencing, specifically of P23H rhodopsin by 90.64+/-5.19% and no loss of rhodopsin translation from the cmRNA in those cells. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin. Our results have implications for the treatment of a very large variety of retinal degenerations in a mutation-independent manner.

Topics: Base Sequence; Cells, Cultured; Cloning, Molecular; Codon; Gene Expression; Genetic Therapy; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Retina; Retinitis Pigmentosa; Rhodopsin; RNA Interference; RNA, Small Interfering; Transfection; Transgenes

The intragenic heterogeneity encountered in many dominant disease-causing genes represents a significant challenge with respect to development of economically viable therapeutics. For example, 25% of autosomal dominant retinitis pigmentosa is caused by over 100 different mutations within the gene encoding rhodopsin, each of which could require a unique gene therapy. We describe here an RNA interference (RNAi)-based mutation-independent approach, targeting as an example murine rhodopsin. Native transcripts are suppressed by a single RNAi molecular species, whereas transcripts from replacement genes engineered at degenerate third-codon wobble positions are resistant to suppression. We demonstrate suppression of murine rhodopsin transcript by up to 90% with full concomitant expression of replacement transcript and establish the validity of this approach in cell culture, retinal explants, and mouse liver in vivo.

Topics: Animals; Cell Separation; Cells, Cultured; Chlorocebus aethiops; COS Cells; DNA, Complementary; Dose-Response Relationship, Drug; Electroporation; Flow Cytometry; Gene Silencing; Genes, Dominant; Genetic Therapy; Liver; Mice; Models, Genetic; Mutation; Pressure; Retina; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA; RNA Interference; RNA, Messenger; RNA, Small Interfering; Time Factors; Transfection

Retinitis pigmentosa is the most common form of retinal degeneration and is heterogeneous both clinically and genetically. The autosomal dominant forms (ADRP) can be caused by mutations in 12 different genes. This report describes the first simultaneous mutation analysis of all the known ADRP genes in the same population, represented by 43 Italian families. This analysis allowed the identification of causative mutations in 12 of the families (28% of the total). Seven different mutations were identified, two of which are novel (458delC and 6901C-->T (P2301S), in the CRX and PRPF8 genes, respectively). Several novel polymorphisms leading to amino acid changes in the FSCN2, NRL, IMPDH1, and RP1 genes were also identified. Analysis of gene prevalences indicates that the relative involvement of the RHO and the RDS genes in the pathogenesis of ADRP is less in Italy than in US and UK populations. As causative mutations were not found in over 70% of the families analysed, this study suggests the presence of further novel genes or sequence elements involved in the pathogenesis of ADRP.

Topics: Adolescent; Adult; Age of Onset; Basic-Leucine Zipper Transcription Factors; Carrier Proteins; Child; Child, Preschool; DNA Mutational Analysis; DNA-Binding Proteins; Eye Proteins; Family; Gene Frequency; Genes, Dominant; Homeodomain Proteins; Humans; Italy; Microtubule-Associated Proteins; Middle Aged; Mutation; Prevalence; Retinitis Pigmentosa; Rhodopsin; RNA-Binding Proteins; Trans-Activators

rhodopsin mutations result in autosomal dominant retinitis pigmentosa (ADRP), the most frequent being Proline-23 substitution by histidine (RhoP23H). Although cellular and rodent animal models have been developed, the pathogenic mechanisms leading to RhoP23H-induced cell death are still poorly understood. For this, we have used a Drosophila model by introducing a mutation in the fly rhodopsin-1 gene (Rh1P37H) that corresponds to human RhoP23H. Rh1P37H transgenic flies show dominant photoreceptor degeneration that mimics age-, light-dependent and progressive ADRP. Moreover, we clarify the pathogenic mechanism of Rh1P37H mutation that acts as an antimorph. First, we show the dual-localization of mutant Rhodopsin since most of Rh1P37H accumulates in endoplasmic reticulum. Second, expression of mutant, mislocalized, Rhodopsin leads to cytotoxicity, via the activation of two stress-specific mitogen-activated protein kinases (MAPKs), p38 and JNK, which are known to control stress-induced apoptosis. In Rh1P37H flies, visual loss and degeneration are indeed accompanied by apoptotic features and prevented by expression of p35 apoptosis inhibitor. Finally, we show for the first time that properly localized, mutant, Rhodopsin is active. Thus, the development of a fly model that faithfully reproduces the human disease sheds light onto the molecular defects causing ADRP thereby making it possible to devise potential therapeutic approaches.

Topics: Amino Acid Substitution; Animals; Apoptosis; Base Sequence; Cell Death; Diptera; Disease Models, Animal; Humans; Molecular Sequence Data; Mutation, Missense; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

To develop a hammerhead ribozymes (Rz) that might be exploited in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rhodopsin (RHO).. A ribozyme (Rz397) was designed to hybridize with an accessible region in rhodopsin mRNA. It was tested in vitro to determine the kinetics of cleavage of a target oligonucleotide. Following transfection of cultured cells, reduction of rhodopsin mRNA in response to Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) was inserted in an adeno-associated virus (AAV2) vector and packaged in AAV2 capsids. The virus was injected subretinally in the eyes of C57BL/6J (RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice at postnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-field electroretinography (ERG). The reduction of opsin protein was measured by western blot analysis and visualized by immunocytochemistry. Reduction of rhodopsin mRNA was assessed using in situ hybridization. Morphometric microscopy of fluorescent antibody-antigen complexes and autoradiography of retinas were used to quantify levels of rhodopsin protein and mRNA, respectively.. Transient co-transfection of HEK 293 cells with a wild-type rhodopsin cDNA and Rz397 resulted in an approximately 60% reduction of RHO mRNA one day after transfection. RHO+/- -mice injected with AAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injected eyes relative to saline injected contralateral eyes. However, injection of RHO+/- -animals at one month and of RHO+/+-animals at either age had no impact on ERG. Nevertheless, we detected an 80% reduction of opsin protein in ribozyme-injected eyes of hemizygous mice (by western blot) and a 50% reduction in opsin content in RHO+/+ mice (by morphometry). These reductions were confirmed by in situ hybridization.. AAV2-Rz397 led to significant (greater than or equal to 50%) reduction of rhodopsin mRNA and protein in mice. It affected ERG amplitudes only when injected in hemizygous RHO knockout pups. This RNA inhibitor may prove useful in treating animal models of ADRP as part of an RNA replacement approach.

Topics: Animals; Blotting, Western; Cells, Cultured; Dependovirus; Electroretinography; Genetic Therapy; Genetic Vectors; Immunohistochemistry; In Situ Hybridization; Kidney; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Catalytic; RNA, Messenger; Rod Opsins; Transfection

To identify novel or rare rhodopsin gene mutations in patients with autosomal dominant retinitis pigmentosa and description of their clinical phenotype.. The complete rhodopsin gene was screened for mutations by DNA sequencing in index patients. Mutation specific assays were used for segregation analysis and screening for controls. Eight patients from five families and their relatives were diagnosed with autosomal dominant retinitis pigmentosa (adRP) by means of clinical evaluation.. Mutation screening identified five different rhodopsin mutations including three novel mutations: Ser176Phe, Arg314fs16, and Val20Gly and two missense mutations, Pro215Leu and Thr289Pro, that were only reported once in a mutation report. Electrophysiological and psychophysical testings provide evidence of an impaired rod system with additionally affected cone system in subjects from each genotype group. Visual function tended to be less affected in subjects with the Arg314fs16 and Val20Gly mutations than in the Ser176Phe phenotype. In contrast, Pro215Leu and Thr289Pro mutations caused a remarkably severe phenotype.. The ophthalmic findings support a correlation between disease expression and structural alteration: (1) extracellular/intradiscal Val20Gly and cytoplasmic Arg314fs16 mutation-mild adRP phenotype; (2) Ser176Phe mutation-"mostly type 1" disease; (3) predicted alteration of transmembrane domains TM V and TM VII induced by Pro215Leu and Thr289Pro-severe phenotype. However, variation of phenotype expression in identical genotypes may still be a typical feature of RHO mutations.

Topics: Adolescent; Adult; Age of Onset; Amino Acid Sequence; Child; Child, Preschool; DNA Mutational Analysis; Female; Genes, Dominant; Genotype; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Mutation, Missense; Pedigree; Phenotype; Polymorphism, Restriction Fragment Length; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields

To identify the mutation patterns of RHO and RP1 genes in the Chinese patients with retinitis pigmentosa (RP) and to explore their potential interactions in the pathogenesis of RP.. Sequence alterations in the entire coding region and splice sites of RHO and RP1 gene were screened in 151 RP affected probands and 150 unrelated controls who were all Hong Kong Chinese. Additional 46 relatives of 12 RP probands carrying possible mutations in RHO or RP1 were recruited for segregation analysis. Univariate analysis, multivariate analysis and genotype-pedigree disequilibrium test were used to examine the associations of polymorphisms in these two genes with RP.. Two mutations in the RHO gene, 5211delC and P347L, were identified each in one proband from the 151 probands, accounting for 1.3% of the RP patients. Two mutations in the RP1 gene, R677X and D984G, were identified each in one proband from the 151 probands, also accounting for 1.3% of the RP patients. In univariate analysis, non-coding sequence variants in the RHO gene, -26G > A, was found to increase the risk of RP, while R872H in the RP1 gene was likely to be a protective factor for RP. Multivariable logistic regression analysis and haplotype analysis confirmed these associations.. The prevalences of RHO and RP1 mutations among the RP patients in Chinese population are both less than reported in other populations. Besides the disease-causing mutations, non-coding sequence alterations may also be a modifier for RP. The potential interactions between RHO and RP1 suggest a digenic etiology for RP.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Asian People; Child; Eye Proteins; Female; Hong Kong; Humans; Male; Microtubule-Associated Proteins; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.

Topics: Humans; Mutation; Protein Conformation; Retinitis Pigmentosa; Retinoids; Rhodopsin; Rod Opsins; Temperature

The purpose of this study was to determine the QTL that influence acute, light-induced retinal degeneration differences between the BALB/cByJ and 129S1/SvImJ mouse strains. Five- to 6-week-old F(2) progeny of an intercross between the two strains were exposed to 15,000 LUX of white light for 1 h after their pupils were dilated, placed in the dark for 16 h, and kept for 10-12 days in dim cyclic light before retinal rhodopsin was measured spectrophotometrically. This was used as the quantitative trait for retinal degeneration. Neither gender nor pigmentation had a significant influence on the amount of rhodopsin after light exposure in the F(2) progeny. For genetic study, DNAs of the 27-36 F(2) progeny with the highest and 27-36 F(2) with the lowest levels of rhodopsin after light exposure were genotyped with 71 dinucleotide repeat markers spanning the genome. Any marker with a 95% probability of being associated with phenotype was tested in all 289 F(2) progeny. Data were analyzed with Map Manager QTX. Significant QTL were found on mouse Chrs 1 and 4, and suggestive QTL on Chrs 6 and 2. The four QTL together equal an estimated 78% of the total genetic effect, and each of the QTL represents a gene with BALB/c susceptible alleles. The Chr 6 QTL is in the same region as a highly significant age-related retinal degeneration QTL found previously. Identification of these QTL is a first step toward identifying the modifier genes/alleles they represent, and identification of the modifiers may provide important information for human retinal diseases that are accelerated by light exposure.

Topics: Alleles; Animals; Carrier Proteins; Chromosome Mapping; cis-trans-Isomerases; Crosses, Genetic; Dinucleotide Repeats; DNA; Eye Proteins; Female; Leucine; Light; Male; Mice; Mice, Inbred BALB C; Proteins; Quantitative Trait Loci; Retina; Retinitis Pigmentosa; Rhodopsin

To evaluate the retina in autopsy eyes from patients over age 60 with autosomal dominant retinitis pigmentosa and a mutation in the RP13 gene (designated as PRPC8, Arg2310Gly), rhodopsin Pro23His, rhodopsin Cys110Arg, or rhodopsin Glu181Lys.. Histologic study of the retina.. All eyes were prepared for electron microscopy within 12 hours after death.. All eyes showed loss of rod photoreceptors. Remaining cones showed perinuclear membranous swirls, inclusion bodies in the inner segments, and shortened or absent outer segments despite causation by various gene defects.. The comparable histologic findings in these four cases suggest a final common pathway leading to photoreceptor cell death in these dominant forms of retinitis pigmentosa.

Topics: Aged; Aged, 80 and over; Carrier Proteins; Genes, Dominant; Humans; Middle Aged; Mutation, Missense; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; RNA-Binding Proteins

Mutations in rhodopsin, the visual pigment found in rod cells, account for a large fraction of genetic changes underlying the human retinal diseases, Retinitis Pigmentosa (RP). The availability of rhodopsin sequences from a large number of vertebrates has allowed us to investigate factors important in the development of RP by contrasting interspecific differences (long-term evolutionary patterns) with RP disease mutation data. We find that disease mutations in rhodopsin are overabundant in highly conserved sites and that amino acid positions with any potential of variability among vertebrates are likely to harbour disease mutations less frequently. At any amino acid position in rhodopsin, the set of disease-associated amino acids does not show any commonality with the set of amino acids present among species. The disease mutations are biochemically four times more radical than the interspecific (neutral) variation. This pattern is also observed when disease mutations are categorized based on clinical classifications that reflect biochemical, physiological and psychophysical traits such as protein folding, cone electroretinogram (ERG) amplitude, pattern of visual field loss, and equivalent field diameter. We also found that for artificial mutations (those not observed in nature interspecifically), there was a positive relationship between the biochemical distance and the magnitude of blue shift in the absorption spectrum maximum. We introduce the concept of the expected chemical severity based on the normal human codon at a position. Results reveal that the analysis of disease mutations in the context of the original codon is very important for the practical application of evolutionary principles when comparing original and disease amino acid mutations. We conclude that the analysis of rhodopsin data clearly demonstrates the usefulness of molecular evolutionary analyses for understanding patterns of clinical as well as artificial mutations and underscores the biomedical insights that can be gained by using simple measures of biochemical difference in the context of evolutionary divergence.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Codon; Conserved Sequence; Evolution, Molecular; Gene Frequency; Genetic Variation; Genotype; Humans; Molecular Sequence Data; Mutation; Phenotype; Phylogeny; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Species Specificity

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.

Topics: Amino Acid Substitution; Arrestin; Cell Line; Cells, Cultured; Endocytosis; Humans; Kidney; Mutation, Missense; Phosphorylation; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin; Transfection

To correlate the functional outcomes with histologic findings following transplantation of fetal retinal sheets in rd mice, and to investigate the mechanisms of visual function restoration.. Twenty-one postnatal day 31-38 rd/rd (C3H/HeJ) mice were transplanted in one eye with retinal sheets (1.0 x 0.4 mm) obtained from embryonic day (E) 17 enhanced-green-fluorescent protein (eGFP) mice. Five mice underwent sham surgery without insertion of tissue. Four to five weeks after transplantation, visual responses to a light flash were recorded across the superior colliculus (SC) in seven eyes of seven transplanted mice that had clear corneas and lenses, and in all five sham surgery mice. Following the SC recording, the eyes were enucleated and processed for immunohistochemistry and examined using confocal microscopy.. In three out of the seven eyes (43%), positive responses were recorded in the SC in an area topographically corresponding to the placement of the transplant in the host retina. No responses were recorded in the untreated eyes of 5-week-old and 9-week-old rd/rd mice, and in the 9-week-old sham surgery mice. In contrast, visual responses were recorded over the entire SC in normal eyes. The response onset latencies of the 3 transplanted mice with responses were similar to those of normal control mice. The organization of the graft did not appear to correlate as expected with the electrophysiology results, as eyes with well-organized, laminated grafts showed no response whereas the three light-responsive eyes had rosetted or disorganized grafts. All three light-responsive eyes demonstrated much higher levels of recoverin immunoreactivity in the host retina overlying the graft compared with untreated age-matched rd/rd mice.. Restoration of the SC visual response does not appear to depend on a well-organized transplant in the rd mouse. Increased recoverin-staining in the host retina in light-responsive animals suggested that host cone rescue was the likely mechanism of vision restoration in this transplant model.

Topics: Animals; Calcium-Binding Proteins; Coloring Agents; Evoked Potentials, Visual; Eye Proteins; Fetal Tissue Transplantation; Immunohistochemistry; Lipoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Photic Stimulation; Photoreceptor Cells; Recoverin; Retina; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Visual Perception

Changes induced by mutations in rhodopsin that are associated with the degenerative visual disease retinitis pigmentosa result in an altered pattern of light absorption according to quantum mechanical simulations and reference experimental works. Eleven single-point mutations associated with retinitis pigmentosa at and in the proximity to the retinal binding pocket of rhodopsin have been modeled in silico and their spectra calculated with the NDOL (Neglect of Differential Overlap accounting L azimuthal quantum number) a priori method. The altered pattern of absorption found would lead to cumulative consequences in energy dissipation with aging. Different energy balances in the case of mutants at the very molecular level, compared to native nonmutated rhodopsin, can cause permanent cellular stress and would play a role in the progression of the retine degenerative process. It could explain the worsening of the pathological condition mostly in adults and suggests the probable beneficial effects of using quenching drugs and protection devices against excess of light in the early stages of life for avoiding or reducing potential damage.

Topics: Absorption; Amino Acid Sequence; Computational Biology; Humans; Isomerism; Light; Models, Structural; Molecular Sequence Data; Point Mutation; Predictive Value of Tests; Retina; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Spectrum Analysis; Ultraviolet Rays

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

Topics: Alleles; Amino Acid Sequence; Animals; Chromatography, Liquid; Cytoplasm; Detergents; Disease Models, Animal; Dogs; Electrophoresis, Polyacrylamide Gel; Glycosylation; Immunoblotting; Immunohistochemistry; Ligands; Light; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Mutation; Peptides; Protein Structure, Tertiary; Proteins; Receptors, G-Protein-Coupled; Retina; Retinitis Pigmentosa; Retinoids; rho GTP-Binding Proteins; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Time Factors; Ultraviolet Rays

Bardet-Biedl syndrome (BBS) is a heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, hypogenitalism, and an increased incidence of diabetes and hypertension. No information is available regarding the specific function of BBS2. We show that mice lacking Bbs2 gene expression have major components of the human phenotype, including obesity and retinopathy. In addition, these mice have phenotypes associated with cilia dysfunction, including retinopathy, renal cysts, male infertility, and a deficit in olfaction. With the exception of male infertility, these phenotypes are not caused by a complete absence of cilia. We demonstrate that BBS2 retinopathy involves normal retina development followed by apoptotic death of photoreceptors, the primary ciliated cells of the retina. Photoreceptor cell death is preceded by mislocalization of rhodopsin, indicating a defect in transport. We also demonstrate that Bbs2(-/-) mice and a second BBS mouse model, Bbs4(-/-), have a defect in social function. The evaluation of Bbs2(-/-) mice indicates additional phenotypes that should be evaluated in human patients, including deficits in social interaction and infertility.

Topics: Animals; Apoptosis; Bardet-Biedl Syndrome; Cilia; Disease Models, Animal; Gene Targeting; Humans; Kidney Diseases, Cystic; Male; Mice; Mice, Knockout; Obesity; Phenotype; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin; Sensation Disorders; Social Dominance; Spermatogenesis

To describe the clinical characteristics and disease course of a large family with retinitis pigmentosa (RP) from an Arg135Leu change in rhodopsin.. 29 patients in this family were evaluated. Goldmann visual fields were performed on 14 affected individuals, Ganzfeld electroretinography (ERG) on eight individuals (11-56 years), and blood samples collected on 10 individuals (11-58 years). Patient visual field data were compared with previously reported patients with different rhodopsin mutations using linear regression.. An Arg135Leu mutation was identified in rhodopsin. Distinct stages of clinical evolution were identified for this family ranging from normal, white dots, classic bone spicules and, finally, ending with extensive retinal pigment epithelium (RPE) atrophy. 9/16 patients over the age of 20 years also demonstrated marked macular atrophy. All patients who underwent full field ERG testing demonstrated non-recordable ERGs. The overall regression model comparing solid angles of visual fields from patients with rhodopsin mutations (Pro23His, Pro347Ala, Arg135Leu) shows significant effects for age (p = 0.0005), mutation (p = 0.0014), and interaction between age and mutation (p = 0.018) with an R(2) of 0.407.. An Arg135Leu change in rhodopsin results in a severe form of RP that evolves through various fundus appearances that include white dots early in life and classic appearing RP later. This transmembrane change in rhodopsin proves to be more severe than in a family with an intradiscal change and a family with a cytoplasmic change.

Topics: Adolescent; Adult; Arginine; Child; Electroretinography; Family Health; Female; Fluorescein Angiography; Genotype; Humans; Leucine; Male; Middle Aged; Mutation; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Field Tests; Visual Fields

The aim of that study was to identify the mutations in rhodopsin and peripherin genes in Polish families with autosomal dominant form of retinitis pigmentosa and determine the population polymorphism in both genes in adRP families.. We performed ERG, visual acuity, Goldman visual fields, intraocular pressure measurements and fundoscopy in all the patients included in the study. On the basis of disease history, the families pedigree was made and the mode of inheritance was analyzed. The molecular analysis of DNA for each family with adRP was conducted. Genomic DNA was obtained from leucocytes by phenol-chloroform procedure according to Maniatis protocol. DNA was amplified by the PCR reaction in a volume of 50 microl containing 100 ng/microl of genomic DNA, water, Cetus buffer pH 8.4 (1 n Tris, 1 n MgCl, 1 n KCl, 2% gelatin), 0.25 microM of each primer, 200 microM of each of dATP, dTTP, dCTP, and dGTP and 2.5 U Taq polymerase (Promega). For amplification of rhodopsin gene 30 cycles of PCR were carried out. Each cycle consisting of denaturation at 95 degrees C for 5 min, annealing: at 58 degrees C (exon 1), 63 degrees C (exon 2 and 3), 68 degrees C (exon 4) and 2 min extension at 72 degrees C min. For amplification of peripherin gene 30 cycles of PCR were carried out with annealing at 60 degrees C. The entire PCR product was in electrophoresis on 8% PAA. The PCR-RFLP PCR-HD PCR-SSCP and analysis of polymorphism (CA)n dinucleotide repetition was performed.. Molecular study demonstrated, that mutations in rhodopsin gene were cause of retinitis pigmentosa in case of two families. In any study families mutations in peripherin gene were not identified. Two kinds of bases polymorphism were identified: restriction fragments length polymorphism (RFLP) in rhodopsin gene in exon 1 and 3 and single strand conformation polymorphism (SSCP) in exon 1 and 3 in rhodopsin gene and in exon 3 in peripherin gene. The confirmed mutations in rhodopsin gene, cosegregation with adRP, whereas two kinds of population polymorphism did not correlate with clinical symptoms. Natural polymorphism appeared to be a frequent feature in rhodopsin gene while a less frequent feature in peripherin gene.. Genetic investigations in patients with adRP allow to confirm the diagnosis and evaluate the prognosis. The mutation in rhodopsin gene should be confirmed in directly sequencing reaction in next study.

Topics: DNA Mutational Analysis; DNA Primers; Exons; Eye Proteins; Haplotypes; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Microtubule-Associated Proteins; Nerve Tissue Proteins; Pedigree; Peripherins; Point Mutation; Poland; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

To determine relative susceptibility to, and regional variation of, light-induced retinal damage in two rhodopsin-mutant rat models of retinitis pigmentosa, using slow- and fast-degenerating lines.. Transgenic S334ter (lines 4 and 9) and P23H (lines 2 and 3) rats were reared in dim cyclic light or darkness and then exposed to intense green light for 1 to 8 hours. Sections along the vertical meridian were collected for retinal morphology and photoreceptor morphometry 2 weeks later. Unexposed transgenic and normal Sprague-Dawley rats served as the control. Mean outer segment lengths and outer nuclear layer thicknesses were analyzed as a function of position along the vertical meridian and as averages across that vector.. Rapidly degenerating S334ter-4 retinas, reared in dim cyclic light, exhibited no light-induced damage, whereas retinas in the other sublines sustained damage within a sensitive region in the superior hemisphere. Light-induced damage always involved loss of outer segment membrane and photoreceptors. In some cases, the retinal pigment epithelium and inner nuclear layer were also affected. Potentiation of light-induced damage by dark-rearing was increased by at least a factor of three, and in some sublines the sensitive region was enlarged to include the entire vertical meridian.. A complex pattern of light-induced damage outcomes was identified in S334ter (sublines 4 and 9) and P23H (sublines 2 and 3) rats. The relative susceptibilities of each subline to damage by light were different, even within the same transgene, but consistent factors included a sensitive region in the superior hemisphere and potentiation by dark-rearing.

Topics: Animals; Animals, Genetically Modified; Light; Pigment Epithelium of Eye; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Ganglion Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

Two previous studies have shown that N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of neuronal nitric oxide synthase, protects retinas of albino rats and mice from damaging levels of light. The aims of the present study were two-fold: (1) to confirm the protective effect of L-NAME on wild type albino rats and (2) to determine if L-NAME protects the retinas of transgenic rats with P23H and S334ter rhodopsin mutations. In the first study, albino rats born and raised in 5-10 lux cyclic light were injected intraperitoneally with either L-NAME or its inactive isomer D-NAME 30 min before being placed in bright light (2700 lux) for 24hr. Electroretinograms (ERGs) were recorded before light treatment and 2 days after cessation of exposure, and eyes were enucleated for morphologic evaluation. L-NAME, but not D-NAME provided structural protection of photoreceptor cells from light damage. The functional rescue was not statistically significant between the drug treated groups. In the second study, albino WT, P23H transgenic, and S334ter transgenic rats were born and raised in 400 lux cyclic light. Three week old animals received daily intraperitoneal injections of L-NAME or D-NAME for 4 weeks, and the same drugs were added to their drinking water. At 7 weeks of age, the ERG sensitivity curves and the outer nuclear layer thickness of both transgenic groups were significantly reduced compared to WT controls. However, administration of L-NAME did not protect against retinal degeneration caused by the rhodopsin mutation in either strain of transgenic (P23H and S334ter) rats. Thus, although photoreceptor cell death in light damage and inherited retinal degenerations share a common apoptotic mechanism, there must be significant 'up-stream' differences that allow selective neuroprotection by L-NAME.

Topics: Albinism, Ocular; Animals; Animals, Genetically Modified; Electroretinography; Enzyme Inhibitors; Female; Light; Male; Mutation; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retinitis Pigmentosa; Rhodopsin

Point mutations found in rhodopsin associated with the retinal degenerative disease retinitis pigmentosa have been expressed in mammalian COS-1 cells, purified, and characterised. The mutations characterised-most of them for the first time-have been Met44Thr, Gly114Asp, Arg135Leu, Val137Met, and Pro171Leu in the transmembrane domain; Leu328Pro and Ala346Pro in the C-terminal tail of the cytoplasmic domain; and Gly106Trp in the intradiscal domain. Several of these mutations cause misfolding which results in impaired 11-cis-retinal binding. Two of them, Met44Thr and Val137Met, show spectral and structural features similar to those of wild type rhodopsin (Type I mutants) but significantly increased transducin initial activation rates. We propose that, in the case of these mutants, abnormal functioning resulting in faster activation kinetics could also play a role in retinitis pigmentosa by altering the stoichiometric balance of the different proteins involved in the phototransduction biochemical reactions.

Topics: Amino Acid Sequence; Animals; Blotting, Western; COS Cells; Cytoplasm; Enzyme Activation; Humans; Hydroxylamines; Molecular Sequence Data; Mutation; Point Mutation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Spectrophotometry; Transducin; Ultraviolet Rays

To determine the causative mutation in a 5 generation pedigree with autosomal dominant retinitis pigmentosa (ADRP).. Genomic DNA from four patients and 4 normal persons in the same pedigree suffering ADRP were extracted, and subsequently eight exons of three ADRP candidate genes were screened for mutations by a combined polymerase chain reaction-single strand conformation polymorphism and DNA sequencing techniques.. A new point mutation in rhodopsin gene at codon 52 of exon 1 (TTC to TAC) that resulted in a substitution of Tyr to Phe was detected in the four affected family members, but not in the four control individuals from the same pedigree.. A causative mutation of rhodopsin gene was identified in a large Chinese pedigree with ADRP. The present study confirmed the molecular genetic heterogeneity of ADRP.

Topics: Adult; Base Sequence; DNA; DNA Mutational Analysis; Family Health; Female; Genes, Dominant; Genetic Predisposition to Disease; Genetic Testing; Humans; Male; Middle Aged; Mutation, Missense; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

This publication focuses on the inhibition of gene expression by triplex-forming oligonucleotides, which is a promising approach to the treatment of dominant genetic diseases. The most common cause for autosomal dominant retinitis pigmentosa is a mutation in the rhodopsin gene.. Two psoralen-linked triplex-forming oligonucleotides were used to inhibit the expression from a plasmid carrying the rhodopsin and green fluorescent protein fusion gene. Following in vitro triplex formation, UVA irradiation was used to activate the psoralen moiety to form covalent photoadducts between the three strands. The samples where then transfected into human fibrosarcoma cells and analysed for green fluorescence.. Photoadducted triple helix formation resulted in reduction of gene expression by as much as 90% and this effect persisted for at least 72 hours. However, expression levels from a cotransfected control plasmid were unaffected. Mutations at one of the triplex binding sites within the rhodopsin gene also abolished the effect of the corresponding triplex forming oligonucleotide, without diminishing the inhibition by the other oligo. Northern blots indicated that photoadducted triplex formation blocked the progression of the RNA polymerase, resulting in truncated transcripts.. The authors conclude, that psoralen linked triplex forming oligonucleotides are efficient and specific tools for blocking gene expression from the human rhodopsin gene.

Topics: DNA Adducts; Fibrosarcoma; Ficusin; Gene Expression Regulation; Genes, Dominant; Genetic Therapy; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mutation; Oligonucleotides; Photosensitizing Agents; Plasmids; Retinitis Pigmentosa; Rhodopsin; Time Factors; Transcription, Genetic; Transfection; Ultraviolet Rays

L125R, a severe retinitis pigmentosa rhodopsin missense mutation, results in rhodopsin protein misfolding, retinal degeneration, and ultimately blindness. The initiating structural events leading to this protein misfolding are unknown. Through the use of compensatory mutations, in conjunction with crystal structure-based molecular analysis, we established that the larger and positively charged Arg replacing Leu125 sterically hinders both the adjacent Trp126 and a critical interhelical interaction between transmembrane III (TM III) and transmembrane V (TM V; Glu122 and His211 salt bridge). Further, analysis of another retinitis pigmentosa mutation, A164V (TM IV), indicates that the larger Val interferes with residues Leu119 and Ile123 on TM III, leading to the disruption of the same critical Glu122-His211 salt bridge (TM III-TM V interaction). Combined, these localized defects in interhelical interactions cause structural changes that interfere with the ability of opsin to bind 11-cis-retinal. These distortions ultimately lead to the formation of an abnormal disulfide bond, severe protein instability, aggregation, and endoplasmic reticulum retention. In the absence of a crystal or NMR structure of each retinitis pigmentosa mutation, compensatory mutagenesis and crystal structure-based analysis are powerful tools in determining the localized molecular disturbances. A detailed understanding of the initiating local perturbations created by missense mutations such as these, not only identifies critical factors required for correct folding and stability, but additionally opens avenues for rational drug design, mimicking the compensatory mutations and stabilizing the protein.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Membrane; COS Cells; Disulfides; Electrophoresis, Polyacrylamide Gel; Humans; Leucine; Microscopy, Fluorescence; Models, Molecular; Molecular Sequence Data; Mutation; Mutation, Missense; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Software; Spectrophotometry; Temperature; Time Factors; Transfection; Tryptophan; Ultraviolet Rays

To study the relationship between the rhodopsin gene on chromosome 3 and autosomal dominant retinitis pigmentosa (ADRP) in a Chinese kindred.. Sixteen normal persons and 18 RP patients in a ADRP family were recruited. Genome scan method based on fluorescence labeled (using 3 different labels: 6-FAM, HEX, and NED) microsatellite markers with multiplex PCR system was used to identify loci influencing susceptibility to ADRP. Fourteen microsatellites (D3S1297, D3S1263, D3S1266, D3S1289, D3S1300, D3S3681, D3S1271, D3S1292, D3S1569, D3S1279, D3S1614, D3S1262, D3S1580 and D3S1311) on chromosome 3 were used as genetic markers. Linkage analysis (using Genescan3.0, GeneScan Analysis 2.1, Genotyper 2.1 and Designer sofe system) was performed using these markers.. The LOD value was

Topics: Asian People; Chromosomes, Human, Pair 3; Humans; Microsatellite Repeats; Pedigree; Retinitis Pigmentosa; Rhodopsin

To examine the difference in expression of retinitis pigmentosa from mutations at codon 23 and codon 347 or rhodopsin; to report a novel mutation in rhodopsin.. Goldmann perimetry (solid angle of I4e isopter) and electroretinographic amplitudes (square root transform of a response ratio) were analyzed for 24 patients with mutations at codon 347 (15 with Pro347Ala, 2 with Pro347Gln, 6 with Pro347Leu, and 1 with a novel Pro347Cys change) and 41 patients with mutations at codon 23 (6 with Pro23Ala; 35 with Pro23His).. When all patients with mutations at codons 347 and 23 were compared, loss of visual fields was significantly worse in patients with codon 347 changes (P =.0003). Only rod responses of the electroretinograms were significantly different between the two groups (P =.048). Specific comparison of Pro347Ala with Pro23Ala using regression analysis demonstrated significant differences in severity between codon 23 and codon 347 patients for b-wave amplitudes of rod (P =.0069), cone (P =.039) and maximum combined response (P =.049). The solid angle of the I4e isopter was also significantly different (P =.025) between the groups after controlling for age. Modeling age by group for Pro347Ala comparison produced an R(2) of.44.. We reconfirmed that rhodopsin-related retinitis pigmentosa from mutations involving codon 347 produces a more severe phenotype than that involving codon 23. Accurate modeling of disease was shown to be possible by incorporating the effects of a patient's age and specific genotype. Therefore, both of these variables must be considered in prognostic counseling and subject recruitment for future therapeutic trials.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Codon; DNA Mutational Analysis; Electroretinography; Humans; Middle Aged; Mutation; Phenotype; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Vision Disorders; Visual Field Tests; Visual Fields

To study rhodopsin (Rho) phosphorylation and dephosphorylation in Royal College of Surgeons (RCS) rat retina, specific antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser, were prepared by immunization of authentic phosphorylated peptides in rabbit. Enzyme-linked immunosorbent assay identified that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immunofluorescence labeling, both antibodies recognized photoreceptor outer segments in light-adapted retinas from Sprague-Dawley (SD), Brown-Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward phosphorylated 338Ser and 334Ser sites were diminished within several hours (0.2-2 h) in SD and BN rat retinas. However, those toward phosphorylated 338Ser and 334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly affected than the phosphorylation reaction. Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat.

Topics: Adaptation, Physiological; Animals; Antibodies; Darkness; Kinetics; Phosphorylation; Phosphoserine; Rats; Rats, Inbred BN; Rats, Mutant Strains; Rats, Sprague-Dawley; Retinitis Pigmentosa; Rhodopsin

Having identified a disease-associated rhodopsin mutation in a patient with retinitis pigmentosa (RP), the issue is to address the question of whether to offer genetic testing to at-risk family members. Two members of a South African (SA) family, one of whom suffers from RP, as well as 54 unrelated SA RP patients from the same population group were investigated using single-stranded conformational polymorphism analysis followed by DNA sequencing. A rare homozygous mutation at the intron-exon boundary of exon 4 in the rhodopsin gene was identified in the proband. One of his siblings was found to be heterozygous for the same mutation. The mutation was not detected in the 54 unrelated SA RP patients examined, 11 of whom were sporadic cases. A low incidence of RP amongst heterozygous carriers of this mutation has been reported; however, in the past it has been unclear whether the mutation has an effect in single copy or dual copy. To the best of our knowledge, this is the first time that this mutation has been reported as homozygous in an affected individual, thereby resolving the issue and confirming that it is a recessive disease-associated mutation. This is also the first autosomal recessive RP disease-causing rhodopsin mutation that has been identified in Southern Africa. Analysis of the extended pedigree indicated obligate heterozygous carriers of the mutation, without obvious signs of visual impairment in early adulthood. The extent to which potential heterozygous carriers should be pursued and clinically examined is discussed and the question is addressed as to whether to inform the family of these molecular findings.

Topics: Aged; DNA; Electroretinography; Exons; Female; Genes, Recessive; Genetic Carrier Screening; Heterozygote; Humans; Introns; Male; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; RNA Splicing

Topics: Carrier Proteins; Chromosome Mapping; Chromosomes, Human, Pair 17; Cohort Studies; Genes, Dominant; Genes, Recessive; Genetic Linkage; Genetic Testing; Humans; Molecular Biology; Mutation; Prevalence; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; RNA-Binding Proteins; South Africa

To detect rhodopsin (RHO) mutation in Chinese families with autosomal dominant retinitis pigmentosa (ADRP) and study on the association of RHO gene mutations with clinical phenotype.. Twenty-seven members from 13 Chinese families with ADRP and 30 normal subjects were recruited. The complete coding regions of the rhodopsin gene were amplified with polymerase chain reaction (PCR) and then DNA single-strand conformation polymorphism (SSCP) technique was used to screen RHO gene mutations. When a variant band was observed after the SSCP electrophoresis, the variant band was analyzed by sequencing PCR-amplified DNA. All subjects were examined clinically by slit-lamp, direct funduscopy, Goldmann kinetic perimetry, Humphrey threshold perimetry and electroretinogram.. Nine affected subjects and 2 boys (11 and 9 years old respectively) in one pedigree among 13 families were found to have three DNA single strand bands by SSCP analysis. Results of assaying sequence showed the 11 members were heterozygous for rhodopsin E341ter mutation. The codon 341 is changed from GAG to TAG, resulting in a stop codon mutation. Thirty normal controls and unaffected subjects in this family were the wild type of RHO gene. Affected individuals reported night blindness in the second decade, showed optic atrophy, vessel attenuation and a few bone spicule-like pigments in the peripheral retina. The impairment of visual acuity was relatively severe, loss of peripheral visual field was greatly considerable after 30 years of age, rod and cone ERG were not detectable in the second decade, and only slight cone response was left.. The natural history of RP in this family begins with a loss of rod function, progresses to involve the cone system, and leads eventually to a severe loss of visual function. A novel rhodopsin gene mutation E341ter is responsible for a Chinese family with ADRP.

Topics: Adult; Asian People; Codon, Terminator; DNA, Single-Stranded; Female; Genetic Predisposition to Disease; Heterozygote; Humans; Male; Middle Aged; Night Blindness; Optic Atrophy; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

To compare histopathologic findings in an autopsy eye of an 87-year-old woman with retinitis pigmentosa and the rhodopsin mutation Pro23His with findings in an autopsy eye of a 77-year-old female relative (first cousin) with retinitis pigmentosa and the same mutation.. Histopathologic study.. One eye from each patient was prepared for light and electron microscopy within 5 hours after death. Photoreceptor nuclear counts were performed.. Photoreceptor degeneration and intraretinal bone spicule pigmentation were evident in both cases. The younger patient had more extensive photoreceptor loss and more intraretinal pigmentation than her older relative.. A marked variation in the extent of retinal degeneration can be seen in two relatives with retinitis pigmentosa and rhodopsin, Pro23His. This study supports the idea that factors other than the primary gene defect are responsible for the severity of this condition.

Topics: Aged; Aged, 80 and over; Female; Genes, Dominant; Humans; Photoreceptor Cells, Vertebrate; Point Mutation; Retinitis Pigmentosa; Rhodopsin

To measure the rate of progression of retinal degeneration in patients with retinitis pigmentosa due to dominant rhodopsin mutations and to determine whether the rate of progression correlates with the location of the altered amino acid in the rhodopsin molecule.. Change in ocular function was observed for an average of 8.7 years in 140 patients. After censoring data to eliminate "ceiling" and "floor" effects, longitudinal rates of change were compared, after weighting by follow-up time and number of visits, with rates inferred from cross-sectional analyses of the data from baseline visits. Mean rates of change were compared among groups of patients with mutations affecting the globule, plug, or C-terminal region of the protein after adjusting for age, gender, and baseline function.. Mean annual exponential rates of decline were 1.8% for visual acuity, 2.6% for visual field area, and 8.7% for ERG amplitude. The rates of visual acuity and ERG amplitude decline were significantly faster, and the rate of visual field area decline was significantly slower, than those inferred from baseline visits. Rates of acuity loss did not vary significantly with the region affected by the mutation. In contrast, the mean annual rate of field loss in the C terminus group (7.4%) was significantly faster than that in the globule (1.7%) or plug (1.1%) group. The mean annual rate of ERG decline was also significantly faster in the C terminus group (13.5%) than in the globule (8.5%) or plug (3.7%) groups and significantly faster in the globule group than in the plug group.. Rates of decline in visual function for groups of patients with rhodopsin mutations cannot be accurately inferred from cross-sectional analyses of baseline visits. Average rates of decline of visual field area and ERG amplitude are fastest in patients with mutations affecting the C-terminal region.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Child; Disease Progression; Electroretinography; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields

Genetic characterization of a series of patients with autosomal dominant retinitis pigmentosa (ADRP).. All patients underwent complete ophthalmological examination including computerized perimetry, electroretinography and occasionally fluorescein angiography. Blood samples were drawn for genetic analysis of candidate genes namely rhodopsin (RHO), peripherin-RDS, ROM-1, CRX, RP1 and NRL.. 148 ADRP index cases were examined at our hospital from June 1991 to September 2001. Genetic analysis detected the following mutations: 29 different families (19.5%) carried a RHO mutation among which the Pro-347-Leu was the most frequent one, five different RP-1 mutations (3.3%), 2 RDS mutations and one NRL mutation, which is the second reported in the world literature.. RHO followed by RP1 are the most frequent ADRP-causing genes in our series as in other published ones, and RDS causes mainly macular dystrophies. Molecular characterization was possible in 37 families (25%) which is of great interest for visual prognosis and genetic counselling.

Topics: Amino Acid Substitution; Basic-Leucine Zipper Transcription Factors; DNA Mutational Analysis; DNA-Binding Proteins; Eye Proteins; Genes, Dominant; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Proteins; Microtubule-Associated Proteins; Mutation; Mutation, Missense; Nerve Tissue Proteins; Peripherins; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Tetraspanins; Trans-Activators

To determine whether dietary-induced alterations in the long-chain polyunsaturated fatty acid content of retinal rod outer segments (ROS) of P23H rats, a transgenic model of retinitis pigmentosa (RP), prolongs photoreceptor cell life.. Heterozygous P23H and normal Sprague-Dawley rats were fed a standard house diet or a diet deficient in 18:3n-3. Diet-deficient rats were given supplements of either linseed oil (high in 18:3n-3) or fish oil (high in 20:5n-3). ROS fatty acid profiles and serum fatty acids were determined by gas chromatography. Serum cholesterol was evaluated by HPLC. Retinal damage was assessed by measuring whole-retina rhodopsin and DNA content before and after exposure to high-intensity light.. The retinas of 60 day old, cyclic-light-reared, P23H transgenic rats contained 50% of the rhodopsin and 75% of the DNA content found in control Sprague-Dawley rats. Eight hours of intense light had little effect on the rhodopsin or DNA content in the Sprague-Dawley rats, but resulted in rhodopsin and DNA losses of nearly 70%, compared to controls, in P23H animals fed either a standard or an 18:3n-3-deficient diet. Supplementation with linseed oil resulted in small, statistically insignificant, increases in the rhodopsin and DNA losses, which occurred after exposure to intense light, in P23H transgenics. In unexposed animals, supplementation with linseed oil or fish oil had no effect on either rhodopsin or DNA levels in P23H rats or in Sprague-Dawley controls. On standard diet, the ROS 22:6n-3 (DHA) content in P23H rats was lower than that of control animals. DHA decreased in both groups when an 18:3-deficient diet was fed. The reduction was greater in controls than in P23H transgenics, but a concomitant increase in 22:5n-6 was nearly the same in both groups. Supplementation of the 18:3-deficient diet with linseed oil or fish oil in P23H rats resulted in a ROS fatty acid profile comparable to that of Sprague-Dawley rats raised on a standard diet. Serum DHA and 22:5n-6 levels were low in both groups. No significant differences in serum cholesterol were observed as a function of genotype or diet.. Heterozygous P23H rats are capable of forming ROS DHA from dietary fatty acid precursors found in linseed oil (18:3n-3) or fish oil (20:5n-3). Under all dietary conditions, P23H transgenics are highly susceptible to retinal damage from exposure to intense light. Although levels of DHA in the ROS of P23H rats could be altered by dietary manipulation, only small changes in photoreceptor cell survival, as measured by whole-retina rhodopsin and DNA content, were observed. The lower-than-normal levels of ROS DHA may reflect an adaptive, possibly protective, mechanism in the P23H transgenic rat model of RP.

Topics: Animals; Animals, Genetically Modified; Cholesterol; Chromatography, Gas; Chromatography, High Pressure Liquid; Dietary Fats, Unsaturated; DNA; Fatty Acids; Fatty Acids, Unsaturated; Light; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

A large number of mutations in rhodopsin are associated with autosomal dominant retinitis pigmentosa (ADRP). We analyzed the biochemical phenotypes of the ADRP-associated cysteine mutants C167R, C222R, and C264del. C222R behaved as wild type in every aspect testable and is classified as a class I mutant. C167R produced intact protein but did not regenerate with 11-cis retinal and was not transported to the plasma membrane. We confirm its classification as a class IIa mutant. C264del represents a novel phenotype, which we propose to call class III. It produced a truncated protein of 27kDa that failed to regenerate with 11-cis retinal and was not targeted to the plasma membrane.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cell Line; Cloning, Molecular; Cysteine; Darkness; Gene Expression; Humans; Light; Molecular Sequence Data; Phenotype; Protein Conformation; Protein Folding; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry; Spodoptera; Transfection

To reexamine a large Albertan family previously reported with a progressive cone dystrophy with variable phenotype and to map the disorder using molecular genetic techniques.. Observational case series.. Twenty-nine subjects (10 affected) from four generations of a large kindred were clinically examined. Twenty-three of these individuals, as well as two unaffected spouses, were included in the molecular genetic study. Subject ages ranged from 17 to 91 years of age.. Disease status and associated ocular abnormalities were assessed primarily by measurement of visual acuity, color vision, fundus photography, and both full-field and multifocal electroretinography (ERG and mfERG). Linkage of the disorder to the rhodopsin gene was studied using microsatellites. A mutational screen of the CRX gene was performed to identify coding sequence changes.. Visual acuity and color discrimination were reduced in clinically affected individuals; full-field flash ERG was used to measure function of both cones and rods. mfERG and fundus photography allowed documentation of the observed macular changes.. We noted a variable, adult-onset macular dystrophy, progressing in some cases to a retinitis pigmentosa-like phenotype. Both photopic and scotopic full-field ERG amplitudes were reduced by approximately 50%, demonstrating involvement of both photoreceptor systems. A reduced b-wave amplitude with a relatively preserved a-wave was observed at both cone and rod levels. Macular involvement was confirmed by mfERG. The rhodopsin locus was excluded by haplotype analysis. A novel frameshift mutation was detected in exon III of the CRX retinal homeobox gene. ERG and molecular genetic findings were consistent with the reclassification of this disease as an autosomal dominant cone-rod dystrophy (CRD) CONCLUSIONS: We report a novel CRX mutation causing autosomal dominant CRD. Observed ERG changes suggest that this mutation primarily impairs inner retinal function. Because retinal expression of CRX is limited to photoreceptors, this dysfunction may be the result of faulty photoreceptor communication with second-order retinal neurons. We propose misexpression of gated cation channels caused by altered CRX activity as one putative mechanism by which a sole photoreceptor defect may selectively impair neurotransmission without disrupting the upstream events of phototransduction.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Chromosome Mapping; Color Perception; Electroretinography; Female; Frameshift Mutation; Fundus Oculi; Genes, Dominant; Genetic Linkage; Haplotypes; Homeodomain Proteins; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Phenotype; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Trans-Activators; Visual Acuity

To test the frequency and pattern of rhodopsin (RHO) mutations in Chinese retinitis pigmentosa (RP) patients and to evaluate their effects in the pathogenesis of RP.. Genomic DNA was extracted from peripheral blood samples of 100 Hong Kong Chinese RP patients. Sequence variants of the entire coding exons of the RHO gene were tested using PCR, conformation sensitive gel electrophoresis and DNA sequencing.. Totally six nucleotide changes were identified, among which three were silent mutations, two missense mutations and one deletion mutation. P347L was found in one RP proband and her three children who also had RP. P327(1 bp del) was novel and detected in a late-onset RP patient of 53 years. Her 26-year-old daughter, also carrying the identified mutation, had no RP phenotypes except for the mottled retinal pigment epithelium (RPE) revealed by fundal examination. Neither of the two mutations was detected in normal controls.. Two patients had disease-causing mutations in the RHO gene, thus RHO mutations cause about 2.0% (95% confidence interval: 0.2%-7.0%) of all RP among Chinese in Hong Kong. A highly conserved C-terminal sequence QVS(A)PA was altered due to P347L and thereby resulting in an aberrant subcellular localization of rhodopsin. Loss of all six phosphorylatable residues at the C-terminus and the highly conserved C-terminal sequence QVS(A)PA may occur because of P327(1 bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying P327(1 bp del) would be of greatest value.

Topics: Adolescent; Adult; Aged; Child; China; DNA; DNA Mutational Analysis; Female; Gene Frequency; Genetic Testing; Humans; Male; Middle Aged; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

To report the localization of a gene for an autosomal dominant retinitis pigmentosa (ADRP) family.. We studied a large ADRP family, collected 3 - 5 ml of venous blood samples from some family members, and genomic DNA was extracted from the blood. Then two-point linkage analysis between the known markers and the disease locus was performed.. Linkage analysis showed the maximum LOD score reaches 2.732852 at marker D3S1292 (at recombination fraction theta = 0.1).. Since D3S1292 was localized at 3q21, we roughly localized the disease gene on 3q21. In the mean time, the rhodopsin gene had been reported as linked to 3q21 adjacent to D3S1292, thus we think the disease gene may be the rhodopsin gene.

Topics: Chromosome Mapping; Chromosomes, Human, Pair 3; DNA; Family Health; Female; Genes, Dominant; Genetic Linkage; Genetic Predisposition to Disease; Humans; Lod Score; Male; Microsatellite Repeats; Pedigree; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Ciliary Neurotrophic Factor; Disease Progression; Fibroblast Growth Factors; Genetic Vectors; Humans; Mutation; Photoreceptor Cells; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; X Chromosome

Previously we have shown that a subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Here we demonstrate that both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease.

Topics: Animals; Arrestin; Drosophila melanogaster; Endocytosis; Immunohistochemistry; Microscopy, Confocal; Models, Animal; Phosphatidylinositol Diacylglycerol-Lyase; Photoreceptor Cells, Invertebrate; Retinitis Pigmentosa; Rhodopsin; Type C Phospholipases

Comparative analysis of the transcriptional profiles of approximately 6000 genes in the retinas of wild-type mice with those carrying a targeted disruption of the rhodopsin gene was undertaken by microarray analysis. This revealed a series of transcripts, of which some were derived from genes known to map at retinopathy loci, levels of which were reduced or elevated in the retinas of Rho(-/-) mice lacking functional photoreceptors. The human homologue of one of these genes, encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), maps to the region of 7q to which an adRP gene (RP10) had previously been localized. Mutational screening of DNA from the Spanish adRP family, originally used to localize the RP10 gene, revealed an Arg224Pro substitution co-segregating with the disease phenotype. The amino acid at position 224 of the IMPDH1 protein is conserved among species and the substitution is not present in healthy, unrelated individuals of European origin. These data provide strong evidence that mutations within the IMPDH1 gene cause adRP, and validate approaches to mutation detection involving comparative analysis of global transcription profiles in normal and degenerating retinal tissues. Other genes showing significant alterations in expression include some with anti-apoptotic functions and many encoding components of the extracellular matrix or cytoskeleton, a possible reflection of a response by Muller cells to preserve the remaining outer nuclear layer of the retina. We suggest that those genes identified are prime candidates for etiological involvement in degenerative retinal disease.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Animals; Arginine; Chromosomes, Human, Pair 7; Conserved Sequence; Gene Expression Profiling; Genes, Dominant; Humans; IMP Dehydrogenase; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Oligonucleotide Array Sequence Analysis; Phenotype; Retina; Retinitis Pigmentosa; Rhodopsin; Transcription, Genetic

To search for mutations in the neural retina leucine zipper (NRL) gene in patients with dominant retinitis pigmentosa and to compare the severity of disease in these patients with that observed previously in patients with dominant rhodopsin mutations.. Single-strand conformation analysis was used to survey 189 unrelated patients for mutations. The available relatives of index patients with mutations were also evaluated. In our clinical examination of patients, we measured visual acuity, final dark-adaptation threshold equivalent visual field diameter, and electroretinogram amplitudes among other parameters of visual function. We compared the clinical findings with those obtained earlier from similar evaluations of a group of 39 patients with the dominant rhodopsin mutation Pro23His and a group of 25 patients with the dominant rhodopsin mutation Pro347Leu.. We identified 3 novel missense mutations in a total of 4 unrelated patients with dominant retinitis pigmentosa: Ser50Pro, Ser50Leu (2 patients), and Pro51Thr. Each mutation cosegregated with dominant retinitis pigmentosa. None of these mutations were found among 91 unrelated control individuals. The visual acuities among the 4 index patients and 3 relatives with NRL mutations who were clinically evaluated ranged from 20/20 (in a 9-year-old patient) to 20/200 (in a 73-year-old patient). All patients had bone-spicule pigment deposits in their fundi. Average rod-plus-cone and cone-isolated electroretinogram amplitudes were both decreased by 99% or more compared with normal amplitudes. The dark-adaptation thresholds, equivalent visual field diameters, and electroretinogram amplitudes (all corrected for age and refractive error) indicated that the disease caused by the NRL mutations was more severe than that caused by the dominant rhodopsin mutation Pro23His and was similar in severity to that produced by the rhodopsin mutation Pro347Leu.. The 3 novel NRL mutations we discovered bring the total number of reported mutations in this gene to 6. Five of the 6 mutations affect residues 50 or 51, suggesting that these residues are important in a structural or functional domain of the encoded protein.. Rod and cone function is affected to a similar degree in patients with these mutations. The disease caused by NRL mutations found in this study appears to be more severe than that caused by the rhodopsin mutation Pro23His and is similar in severity to that caused by the rhodopsin mutation Pro347Leu, even after correcting for age.

Topics: Adult; Aged; Basic-Leucine Zipper Transcription Factors; Child; Dark Adaptation; DNA Mutational Analysis; DNA Primers; DNA-Binding Proteins; Electroretinography; Eye Proteins; Female; Humans; Leucine Zippers; Male; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin (RHO) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the RHO gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with RHO mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in RHO mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The RHO mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies.

Topics: Animals; Cell Membrane; DNA Mutational Analysis; Dogs; Electroretinography; Genes, Dominant; Genetic Linkage; Humans; Immunohistochemistry; Molecular Sequence Data; Mutation; Phenotype; Photoreceptor Cells; Point Mutation; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Time Factors; Tomography; X Chromosome

Mutations of the rhodopsin gene are responsible for autosomal dominant or recessive retinitis pigmentosa (RP). The present study reports the first prenatal diagnosis performed on chorionic villi biopsy of a pregnant woman affected by a severe form of autosomal dominant transmitted RP, due to the Arg135Trp substitution.. The rhodopsin gene was analysed by automated direct sequencing and, for the first time, by fluorescence-assisted mismatch analysis (FAMA). The latter is an inexpensive, rapid and particularly sensitive method, based on the chemical cleavage of the mismatch in heteroduplex DNA molecules marked with strand-specific fluorophores.. FAMA is a feasible procedure for prenatal molecular diagnosis of rhodopsin mutations. The redundancy of signals obtained by FAMA and its sensitivity make it suitable for identifying exactly the position of the mutation and the nucleotide substitution.. An association is proposed between FAMA and automated direct sequencing procedures, in order to achieve optimal results in terms of reliability for prenatal diagnosis of rhodopsin mutations.

Topics: Adult; Base Pair Mismatch; Chorionic Villi Sampling; DNA; DNA Mutational Analysis; Female; Heteroduplex Analysis; Humans; Mutation; Polymerase Chain Reaction; Pregnancy; Retinitis Pigmentosa; Rhodopsin

Because of a previous report suggesting that D-cis-diltiazem slows retinal degeneration in rd mice, this study was undertaken to examine the effect of D-cis-diltiazem on photoreceptor structure and function in this line of mice.. Mice were randomly assigned to daily intraperitoneal injections of D-cis-diltiazem or saline between postnatal days 9 and 24. On postnatal day 26 or 27, retinal function was assessed by recording dark-adapted bright-flash ERGs in all animals. Retinal morphology was examined in fixed sections and in immunolabeled frozen sections. Examiners were masked to the treatment group assignment.. On postnatal days 26 and 27, diltiazem- and saline-treated mice had only one row of remaining photoreceptor cells throughout most of the central retina. Cone cells in the periphery had remnants of inner segments. Total cell counts and separate counts of rod and cone photoreceptor cells by immunostaining were similar in the diltiazem- versus saline-treated mice. Both groups of mice had, on average, comparable subnormal ERG amplitudes.. D-cis-Diltiazem had no detectable effect on preservation of photoreceptor structure and function in rd mice.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium Channel Blockers; Cell Count; Cyclic Nucleotide Phosphodiesterases, Type 6; Dark Adaptation; Diltiazem; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Injections, Intraperitoneal; Mice; Mice, Inbred C3H; Photic Stimulation; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Rhodopsin

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.

Topics: Animals; Cell Membrane; COS Cells; Cysteine Endopeptidases; Diterpenes; Eukaryotic Cells; Glycosylation; Golgi Apparatus; Inclusion Bodies; Microscopy, Electron; Molecular Chaperones; Multienzyme Complexes; Mutation; Organelles; Proteasome Endopeptidase Complex; Protein Folding; Protein Transport; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Tunicamycin; Ubiquitin

The inherited retinal degenerations are typified by retinitis pigmentosa (RP), a heterogeneous group of inherited disorders that causes the destruction of photoreceptor cells, the retinal pigmented epithelium, and choroid. This group of blinding conditions affects over 1.5 million persons worldwide. Approximately 30-40% of human autosomal dominant (AD) RP is caused by dominantly inherited missense mutations in the rhodopsin gene. Here we show that P23H, the most frequent RP mutation in American patients, renders rhodopsin extremely prone to form high molecular weight oligomeric species in the cytoplasm of transfected cells. Aggregated P23H accumulates in aggresomes, which are pericentriolar inclusion bodies that require an intact microtubule cytoskeleton to form. Using fluorescence resonance energy transfer (FRET), we observe that P23H aggregates in the cytoplasm even at extremely low expression levels. Our data show that the P23H mutation destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. P23H is stabilized by proteasome inhibitors and by co-expression of a dominant negative form of ubiquitin. We show that expression of P23H, but not wild-type rhodopsin, results in a generalized impairment of the ubiquitin proteasome system, suggesting a mechanism for photoreceptor degeneration that links RP to a broad class of neurodegenerative diseases.

Topics: Cysteine Endopeptidases; Humans; Multienzyme Complexes; Mutation; Proteasome Endopeptidase Complex; Protein Folding; Retinitis Pigmentosa; Rhodopsin; Ubiquitin

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas-Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease-causing mutation in approximately one-third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone-rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone-rod homeobox gene, CRX. Based on this large survey, the prevalence of disease-causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation-specific studies.

Topics: Amino Acid Substitution; Animals; Arginine; Cysteine; Genetic Variation; Glutamine; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Leucine; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Optic Atrophies, Hereditary; Peripherins; Prevalence; Proline; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Trans-Activators; Tyrosine

To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina.. Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age.. Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities.. From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.

Topics: Animals; Animals, Newborn; Electroretinography; Evaluation Studies as Topic; Fundus Oculi; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Photic Stimulation; Photography; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Time Factors

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsin(-/-)) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration.

Topics: Animals; Animals, Newborn; Biological Assay; Blotting, Western; Cell Line; Cell Survival; Cells, Cultured; Chick Embryo; Ciliary Neurotrophic Factor; Dependovirus; Enzyme-Linked Immunosorbent Assay; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Humans; Immunohistochemistry; Luminescent Proteins; Mice; Mice, Knockout; Microscopy, Fluorescence; Mutagenesis, Insertional; Neurons; Open Reading Frames; Photoreceptor Cells, Vertebrate; Retina; Retinitis Pigmentosa; Rhodopsin; Time Factors; Transduction, Genetic

The goal of these experiments was to evaluate the potential of the fibroblast growth factor family members FGF-5 and FGF-18 to rescue photoreceptors from cell death in retinal degenerative disease. Two strains of transgenic rats, expressing either a P23H or an S334ter rhodopsin mutation, were used as model systems. The neurotrophic growth factors were delivered by subretinal injection of adeno-associated virus vectors, driving expression of the genes with a constitutive CMV promoter. Morphological and functional analyses were performed to determine whether FGF-5 or FGF-18 overexpression could ameliorate cell death in the retina. Immunocytochemistry was used to determine the cellular sites of expression of the factors and to test for up-regulation of FGF receptors due to injection. Significant rescue from photoreceptor cell death was found after injections of vectors expressing either FGF-5 or FGF-18 in the animal models. Increased survival of photoreceptors did not produce a significant increase in electroretinographic responses, however, reflecting either trauma due to the surgery or a suppression of signaling due to expression of proteins. Three weeks after injections, both growth factors were localized to the inner and outer segments of photoreceptors, and the receptors FGFR1 and FGFR2 were also found to be up-regulated in these regions. No visible pathological changes were seen in the FGF-5- or FGF-18-treated eyes. These results indicate that the delivery of either FGF-5 or FGF-18 with adeno-associated virus protects photoreceptors from apoptosis in transgenic rat models of retinitis pigmentosa and that the rescue is probably mediated by conventional receptor tyrosine kinase pathways in photoreceptors.

Topics: Animals; Animals, Genetically Modified; Blotting, Western; Cell Death; Cell Line; Cell Survival; Cytomegalovirus; Dependovirus; Disease Models, Animal; Electroretinography; Fibroblast Growth Factor 5; Fibroblast Growth Factors; Genetic Vectors; Humans; Immunohistochemistry; Models, Genetic; Plasmids; Point Mutation; Promoter Regions, Genetic; Rats; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Transfection; Up-Regulation

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110-Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Topics: Amino Acid Sequence; Animals; COS Cells; Cysteine; Disulfides; Endopeptidase K; Humans; Lysine; Maleimides; Models, Molecular; Molecular Sequence Data; Mutation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship

To determine the pattern of rhodopsin mutations in Chinese retinitis pigmentosa (RP) patients.. The rhodopsin gene was examined in 101 RP patients and 190 controls from Hong Kong.. Three coding changes were identified: Pro347Leu, Ala299Ser, and 5211delC. Each protein sequence alteration was found in one patient. Ala299Ser also existed in two controls.. The C-terminal nonsense mutation may cause mis-sorting of rhodopsin protein. The finding of controls with Ala299Ser suggests this is only the third missense alteration reported that does not cause RP. The expected frequency of rhodopsin mutations in RP is <7% (2/101=2.0%, 95% confidence interval: 0.2%-7.0%).

Topics: Adult; Amino Acid Sequence; Base Sequence; Codon, Nonsense; Female; Humans; Middle Aged; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin

To survey patients with dominant retinitis pigmentosa (RP) for mutations in the RP1 gene to determine the spectrum of dominant mutations in this gene, to estimate the proportion of dominant RP caused by this gene, and to determine whether the clinical features of patients with RP1 mutations differ from features of those with rhodopsin mutations.. A set of 241 patients who did not have mutations in the rhodopsin gene (based on previous work) formed the basis for the study. Of these patients, 117 had also been previously evaluated and were found not to carry mutations in the RDS gene. The single-strand conformation polymorphism (SSCP) method was used to search for sequence variants, which were then directly sequenced. The relatives of selected patients were recruited for segregation analyses. Clinical evaluations of patients included a measurement of Snellen visual acuity, final dark adaptation thresholds, visual fields, and ERGs. Clinical data were compared with those obtained earlier from a study of 128 patients with dominant rhodopsin mutations.. Of the 241 patients, all were screened for the most common RP1 mutation (Arg677Ter), and 10 patients were found to have this mutation. In addition, an evaluation of a subset of 189 patients in whom the entire coding sequence was evaluated revealed the following mutations: Gln679Ter (1 case), Gly723Ter (2 cases), Glu729(1-bp del) (1 case), Leu762(5-bp del) (2 cases), and Asn763(4-bp del) (1 case). All of these mutations cosegregated with RP in the families of the index patients. Nine missense mutations that were each found in six or fewer patients were encountered. The segregation of eight of these was evaluated in the respective patients' families, and only one segregated with dominant RP. This cosegregating missense change was in cis with the nonsense mutation Gln679Ter. Although patients with RP1 mutations had, on average, slightly better visual acuity than patients with rhodopsin mutations, there was no statistically significant difference in final dark-adaptation thresholds, visual field diameters, or cone electroretinogram (ERG) amplitudes. Comparably aged patients with RP1 mutations had visual function that varied by approximately two orders of magnitude, based on visual fields and ERG amplitudes.. Dominant RP1 alleles typically have premature nonsense codons occurring in the last exon of the gene and would be expected to encode mutant proteins that are only approximately one third the size of the wild-type protein, suggesting that a dominant negative effect rather than haploinsufficiency is the mechanism leading to RP caused by RP1 mutations. On average, patients with RP1 mutations have slightly better visual acuity than patients with dominant rhodopsin mutations; otherwise, they have similarly severe disease. The wide range in severity among patients with RP1 mutations indicates that other genetic or environmental factors modulate the effect of the primary mutation.

Topics: Adolescent; Adult; Aged; Dark Adaptation; Electroretinography; Eye Proteins; Female; Frameshift Mutation; Genes, Dominant; Humans; Male; Microtubule-Associated Proteins; Middle Aged; Mutation, Missense; Pedigree; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Visual Acuity; Visual Fields

To identify mutations in the rhodopsin (RHO) gene in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) and to measure the prevalence rate of RHO mutations in Chinese ADRP cases.. Thirteen Chinese families with ADRP were clinically characterized. The complete coding region and intron splice sites of RHO were analyzed for mutations with single-strand conformation polymorphism (SSCP) analysis and direct genomic sequencing.. One of the 13 Chinese families with ADRP was found to have a new, previously unidentified RHO mutation, a change from GAG to TAG at codon 341. The mutation (E341X) results in an in-frame stop codon, leading to the truncation of the rhodopsin protein. Mutation E341X was not detected in 100 normal control individuals. Patients carrying mutation E341X reported night blindness and showed optic atrophy, vessel attenuation, and a few bone spicule-like pigments in peripheral retina at the age of 23-25 years. At the age of 30 years, visual acuity was severely impaired, peripheral visual field was greatly constricted, rod and cone ERG was not detectable, and only a slight left cone response remained.. We have identified a novel rhodopsin mutation (E341X) in a Chinese family with ADRP. The location and character of the mutation expand the spectrum of RHO mutations causing RP. Identification of a RHO mutation in one of the 13 ADRP families studied suggests that only 7.7% of the ADRP cases in a Chinese population were caused by RHO mutations, a ratio significantly lower than that from North America or Europe.

Topics: Adult; Amino Acid Sequence; Base Sequence; China; Codon, Terminator; DNA Primers; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; X Chromosome

Physiological consequences of early stages of photoreceptor degeneration were examined in heterozygous P23H rhodopsin transgenic (Tg) and in aging normal Sprague-Dawley rats. Rod photoreceptor and rod bipolar (RB) cell function were estimated with maximum value and sensitivity parameters of P3 and P2 components of the electroretinogram. In both Tg and aging normal rats, the age-related rate of decline of P3 amplitude was steeper than that of the P2 amplitude. Tg rats showed greater than normal sensitivity of the rods. A new model of distal RB pathway connectivity suggested photoreceptor loss could not be the sole cause of physiological abnormalities; there was an additional increase of post-receptoral sensitivity. We propose that changes at rod-RB synapses compensate for the partial loss of rod photoreceptors in senescence and in early stages of retinal degeneration.

Topics: Aging; Animals; Animals, Genetically Modified; Confidence Intervals; Disease Models, Animal; Electroretinography; Linear Models; Rats; Rats, Sprague-Dawley; Regression Analysis; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Visual Pathways

More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P --> S/A/R/Q/L/T) were absent while the codon 345 mutation V --> M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.

Topics: Apoptosis; Codon; DNA Mutational Analysis; Female; Humans; India; Male; Mutation, Missense; Pedigree; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

To evaluate changes in the rods and amacrine cells and horizontal cells in human retinas with retinitis pigmentosa.. Seven retinas from patient donors with retinitis pigmentosa and 14 age- and postmortem-matched normal human retinas were processed for immunocytochemistry and confocal microscopy. The following cell-specific antibodies were used: anti-rhodopsin (rods), anti-gamma-aminobutyric acid (amacrine cells), anticalbindin (cones and horizontal cells), anti-glial fibrillary acidic protein (astrocytes and reactive Müller cells), and anti-synaptophysin and anti-SV2 (synaptic vesicles).. In retinal regions with significant photoreceptor loss, the rods, gamma-aminobutyric acid-positive amacrine cells, and calbindin-positive horizontal cells had undergone neurite sprouting. The rod, amacrine and horizontal cell neurites were associated with the surfaces of glial fibrillary acidic protein-immunoreactive Müller cells. Most rod neurites that projected into the inner retina contacted the somata of gamma-aminobutyric acid-positive amacrine cells.. Rods, amacrine and horizontal cells undergo neurite sprouting in human retinas with retinitis pigmentosa. These changes in the retinal neurons may contribute to the electroretinographic abnormalities and progressive decline in vision noted by patients with retinitis pigmentosa. These alterations may also complicate strategies for treatment of retinitis pigmentosa.

Topics: Adult; Aged; Aged, 80 and over; Calbindins; Female; Fluorescent Antibody Technique, Indirect; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Humans; Male; Microscopy, Confocal; Middle Aged; Neurites; Neurons, Afferent; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; S100 Calcium Binding Protein G; Synaptic Vesicles; Synaptophysin

To determine the extent to which rhodopsin mis-sorting and constitutive activation of the phototransduction cascade contribute to retinal degeneration in a transgenic rat model of retinitis pigmentosa.. Retinas from transgenic rats expressing truncated rhodopsin (Ser334ter) were examined by light and electron microscopic immunocytochemistry at several time points. Retinal degeneration in transgenic rats raised in darkness was evaluated by quantification of outer nuclear layer thickness and by electroretinography.. Mutant rhodopsin was found at inappropriately high levels in the plasma membrane and cytoplasm of Ser334ter rat photoreceptors. When the cell death rate was high this mis-sorting was severe, but mis-sorting attenuated greatly at later stages of degeneration, as the cell death rate decreased. The distributions of two other outer segment proteins (the cGMP-gated channel and peripherin) were examined and found to be sorted normally within the photoreceptors of these rats. Raising Ser334ter transgenic rats in darkness resulted in minimal rescue from retinal degeneration.. Because dark rearing Ser334ter rats results in little rescue, it is concluded that constitutive activation of the phototransduction cascade does not contribute significantly to photoreceptor cell death in this rat model. The nature of the rhodopsin sorting defect and the correlation between the severity of mis-sorting and rate of cell death indicate that truncated rhodopsin may cause apoptosis by interfering with normal cellular machinery in the post-Golgi transport pathway or in the plasma membrane.

Topics: Animals; Animals, Genetically Modified; Apoptosis; Biological Transport; Blotting, Western; Disease Models, Animal; Electroretinography; Fluorescent Antibody Technique, Indirect; Point Mutation; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

Ectopic expression of Bcl-2 in photoreceptors of mice with retinal degenerative disease slows progression of the disease. BAG-1 has previously been shown to augment the inhibitory effect of Bcl-2 on programmed cell death in cultured cell systems. This study was designed to determine whether the coexpression of BAG-1 and Bcl-2 in the photoreceptors of mice with an autosomal dominant form of retinitis pigmentosa (RP) would enhance the protective effect provided by Bcl-2 alone.. An expression vector using the 5' regulatory region of the murine opsin gene was used to target the expression of BAG-1 specifically to photoreceptor cells of mice. The BAG-1 transgenic mice were crossed to Bcl-2 transgenics to obtain animals that coexpress the two transgenes in photoreceptor cells. BAG-1/Bcl-2 animals were then crossed to an RP mouse model (a transgenic line overexpressing the S334ter rhodopsin mutant) to assess the effect of coexpression of BAG-1 and Bcl-2 on retinal degeneration. Morphologic analysis was performed on retinas isolated at various times after birth to monitor disease progression.. High levels of BAG-1 expression resulted in retinal degeneration that was not prevented by Bcl-2 expression. However, coexpression of appropriate levels of BAG-1 and Bcl-2 was found to have a profound inhibitory effect on retinal degeneration caused by overexpression of a mutant rhodopsin transgene. Whereas expression of Bcl-2 alone was previously found to delay degeneration of the retina from 2 weeks to approximately 4 weeks of age, coexpression of BAG-1 and Bcl-2 inhibited photoreceptor cell death for as long as 7 to 9 weeks.. The synergistic effect against photoreceptor cell death produced by the coexpression of Bcl-2 and BAG-1 indicates that these proteins can function in concert to prevent cell death. At the correct dosage, coexpression of Bcl-2 and BAG-1 may serve as a potential means to treat retinal degenerative diseases.

Topics: Animals; Apoptosis; Blotting, Western; Carrier Proteins; Cell Survival; DNA-Binding Proteins; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; Male; Mice; Mice, Mutant Strains; Mice, Transgenic; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-bcl-2; Retinitis Pigmentosa; Rhodopsin; Transcription Factors

Topics: Amino Acid Substitution; Genes, Dominant; Humans; Membrane Proteins; Mutation, Missense; Perilipin-2; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin; Spain

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Topics: Animals; Cell Line; Cricetinae; Disease Models, Animal; Genetic Therapy; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred CBA; Mice, Transgenic; Nerve Tissue Proteins; Peripherins; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.

Topics: Actins; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Biological Transport; Cattle; Cilia; Dyneins; Female; Humans; Immunoblotting; Immunohistochemistry; Mice; Mice, Inbred C57BL; Microscopy, Immunoelectron; Middle Aged; Molecular Sequence Data; Myosin VIIa; Myosins; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Rod Opsins

Autosomal dominant retinitis pigmentosa (adRP) may be caused by point mutations in the rhodopsin gene in up to 20% of Spanish families. Most of the rhodopsin mutations causing adRP have been reported in the heterozygous state. We describe a patient with adRP who is homozygous for a missense mutation at codon 188 in the second intradiscal domain of rhodopsin. All her sons are heterozygous for the mutation and show an RP phenotype suggesting complete penetrance for this mutation. The homozygous carrier of the mutation Gly-188-Arg in the rhodopsin gene showed a later subjective onset of symptoms than the heterozygotes, suggesting that the photoreceptor degeneration induced by the mutation is not dramatically influenced by mutant allele dosage.

Topics: Adult; Child, Preschool; Consanguinity; Disease Progression; DNA Mutational Analysis; Electrooculography; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Fluorescein Angiography; Heterozygote; Homozygote; Humans; Male; Middle Aged; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Topics: Amino Acid Substitution; Codon; DNA Mutational Analysis; Genes, Dominant; Humans; Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; South Africa

To design, generate, and compare in vitro a range of hammerhead ribozymes targeting retinal transcripts implicated in autosomal dominant retinitis pigmentosa (adRP) and thereby identify ribozymes that may be valuable as therapeutic agents for adRP. To address mutational heterogeneity in rhodopsin and peripherin-linked adRP using mutation-independent ribozyme-based therapeutic approaches.. Ribozyme and cDNAs constructs were cloned into pcDNA3 and expressed in vitro from the T7 promoter. Cleavage reactions were separated on polyacrylamide gels, visualized by autoradiography, and quantified using an instant imager. Ribozymes targeting rhodopsin and peripherin transcripts in a mutation-independent manner (Rz9, Rz10, and Rz40) and a multimeric ribozyme (RzMM) targeting rhodopsin transcripts were evaluated for in vitro activity. Parameters such as V:(max), K:(m), k(2) and k(-1) were established for each ribozyme.. Four ribozymes targeting retinal transcripts were evaluated. Mutation-independent ribozymes targeting degenerate sites or untranslated regions in retinal transcripts resulted in cleavage products of predicted size, whereas transcripts from modified replacement genes remained intact. Detailed kinetic evaluation of ribozymes revealed substantial differences in cleavage rates between ribozymes.. Mutation-independent hammerhead ribozymes targeting rhodopsin and peripherin have been screened in vitro, and a number of extremely efficient ribozymes identified subsequent to detailed kinetic analyses, suggesting that these ribozymes may provide mutation-independent methods of treating adRP. These are the first ribozymes reported that potentially will provide benefit for inherited retinopathies.

Topics: Autoradiography; Base Sequence; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Gene Expression; Gene Expression Profiling; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Peripherins; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic

To identify mutations in the rhodopsin gene in North American patients with autosomal dominant retinitis pigmentosa (ADRP) and to measure the proportion of cases with rhodopsin mutations.. Single-strand conformation polymorphism (SSCP) analysis and direct genomic sequencing were used to evaluate the coding region and intron splice sites of the rhodopsin gene for mutations in 91 unrelated patients.. Nineteen patients heterozygously carried a missense change in the rhodopsin gene (six with Pro23His, two with Pro347Leu, and one each with Thr17Met, Phe45Leu, Gly51Arg, Gly89Asp, Gly114Val, Arg135Trp, Pro171Leu, Gln184Pro, Phe220Leu, Ser297Arg, and Pro347Thr). All these missense changes were previously reported as causes for ADRP except for Gly114Val, Gln184Pro, and Phe220Leu, which were evaluated further by examining the relatives of index patients. The Gly114Val and Gln184Pro alleles cosegregated with ADRP as expected if they were pathogenic. Phe220Leu did not, indicating that it is not a cause of ADRP.. Summation of the results of cases in this study with those of 272 unrelated cases of ADRP previously evaluated by our group shows that 90 of 363 (25%) of cases were caused by rhodopsin mutations.

Topics: DNA Mutational Analysis; Female; Glutamine; Glycine; Humans; Male; Mutation, Missense; Pedigree; Polymorphism, Single-Stranded Conformational; Proline; Retinitis Pigmentosa; Rhodopsin; Valine

To correlate retinal functional changes with structural changes in P23H rhodopsin transgenic rats as a model of autosomal dominant retinitis pigmentosa.. P23H heterozygote (lines 1 and 3) and Sprague-Dawley control rats were studied at 4 to 29 weeks by retinal histology, electroretinogram (ERG), and a-wave transduction modeling.. Both line 1 (faster degeneration) and line 3 (slower degeneration) showed progressive rod outer segment (ROS) shortening and outer nuclear layer (ONL) cell loss with age. ERG b-wave maximum amplitude (Vb(max)) decreased with age, but b-wave threshold remained constant within each line despite progressive ONL thinning and ROS shortening. The only exception was in line 1 at 29 weeks, which showed a slight threshold change relative to earlier ages. Va(max) and a-wave threshold changed more rapidly and were more sensitive than the b-wave in reflecting histologic degeneration. Va(max) was linearly proportional to the product of (ROS x ONL) across a two log unit range of data combined from both lines. The photopic b-wave was normal for both lines until the ONL thinned beyond 50%. Phototransduction sensitivity was normal for both lines, and dark-adaptation recovery after bleaching rhodopsin was normal.. The P23H transgenic rat has a slow rod degeneration with initially normal cone function, consistent with clinical findings of P23H patients. However, the normal bleach recovery and the normal phototransduction sensitivity in this rat model are different from human P23H disease. a-Wave measures were more sensitive than the b-wave for tracking changes. b-Wave threshold was inexplicably poor for tracking degeneration. Although line 1 degenerated faster than line 3, the functional-structural correlates were the same. The tight linear relationship between saturated a-wave amplitude and the product of (ROS x ONL) indicates that the density of cGMP-gated channels per unit ROS plasma membrane area remains constant over a wide range of degenerations.

Topics: Animals; Animals, Genetically Modified; Dark Adaptation; Electroretinography; Female; Male; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retina; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

A combined total of approximately 100 mutations have been encountered within the rhodopsin gene in retinitis pigmentosa (RP) and congenital night blindness. Mice carrying a targeted disruption of the rhodopsin gene phenotypically mimic RP, losing their photoreceptors over a period of 3 months and having no recordable rod electroretinogram. These animals will serve as a model for both recessive and dominant disease (in the latter case, the presence of normal and mutant human rod opsin transgenes on the murine Rho(-/-)background). Precise knowledge of apoptotic photoreceptor cell death, together with factors which may influence apoptosis will be required for optimum utility of Rho(-/-)mice as a model for therapeutic genetic intervention. A peak phase of apoptosis of the photoreceptors of Rho(-/-)mice was shown to occur at 24 days post-birth. The extent of apoptosis appeared to be similar, irrespective of whether or not the rod opsin knockout was present on a c-fos(+/+)or c-fos(-/-)genetic background, the latter known to favor survival of photoreceptors following exposure of mouse retinas to excessive light. These data clearly support the existence in animals of distinct apoptotic pathways in light-induced, as opposed to mutation-induced apoptosis, and together with similar observations recently reported in studies of the naturally occurring rd mouse, may assist in focusing future research on precisely defining the distinct molecular pathways giving rise to such dichotomy.

Topics: Animals; Apoptosis; Disease Models, Animal; Genes, fos; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Knockout; Photoreceptor Cells, Vertebrate; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

To report the clinical characteristics of a family with autosomal dominant retinitis pigmentosa caused by a proline-to-alanine mutation at codon 23 (Pro23Ala) of the rhodopsin gene and to compare this phenotype with that associated with the more common proline-to-histidine mutation at codon 23 (Pro23His).. We examined 6 patients within a single pedigree. The electroretinograms (ERGs) of 35 patients with known Pro23His mutations and of 22 healthy individuals were reviewed. Scotopic dim flash-response amplitudes, maximum combined-response amplitudes, and photopic-response amplitudes from the ERGs of these patients were plotted against age. The ERG indices of 5 individuals in the Pro23Ala family were compared with those of the patients with Pro23His mutations and of healthy individuals. Multiple linear regression was performed to evaluate the effect of age and mutation type on amplitudes. Mutation detection was performed using single-strand conformation polymorphism analysis, followed by automated DNA sequencing.. Patients with the Pro23Ala mutation have a clinical phenotype characterized by onset of symptoms in the second to fourth decades of life, loss of superior visual field with relatively well-preserved inferior fields, and mild nyctalopia. Comparison with patients with the Pro23His mutation demonstrates statistically significant differences (P<.001) in responses to dim flash, maximum combined, and photopic responses between patients with these mutations after controlling for the effects of age. Patients with Pro23Ala mutations were less affected by ERG criteria than patients with Pro23His mutations. Patients with Pro23Ala mutations also differed significantly from healthy patients in all ERG indices examined (P<.001), after controlling for age.. We describe a rare mutation in codon 23 of rhodopsin causing autosomal dominant retinitis pigmentosa. The retinal dystrophy associated with the Pro23Ala mutation is characteristically mild in presentation and course, with greater preservation of ERG amplitudes than the more prevalent Pro23His mutation. Arch Ophthalmol. 2000;118:1269-1276

Topics: Adolescent; Adult; Aged; Child; Codon; DNA Mutational Analysis; Electroretinography; Female; Humans; Male; Middle Aged; Pedigree; Phenotype; Photic Stimulation; Point Mutation; Polymorphism, Single-Stranded Conformational; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Field Tests; Visual Fields

The aim of this work was to identify NRL mutations in a panel of 200 autosomal dominant retinitis pigmentosa (adRP) families. All samples were subjected to heteroduplex analysis of the three exons of the NRL gene, and HphI restriction digest analysis of exon 2 (to identify the S50T mutation). Families found to have the S50T mutation, and six additional larger pedigrees (which had previously been excluded from the other nine adRP loci) underwent linkage analysis using polymorphic markers located in the region of 14q11. HphI restriction analysis followed by direct sequencing of the amplified NRL exon 2 product demonstrated the presence of the NRL S50T sequence change in three adRP families. Comparison of marker haplotypes in affected individuals from these families with those of affected members of the original 14q11 linked family revealed a common disease haplotype for markers within the adRP locus. Recombination events observed in these families define an adRP critical interval of 14.9 cM between D13S72 and D14S1041. Linkage analysis enabled all six of the larger adRP pedigrees to be excluded from the 14q11 locus. The NRL S50T mutation represents another example of a 'founder effect' in a dominantly inherited retinal dystrophy. Identification of such 'founder effects' may greatly simplify diagnostic genetic screening and lead to better prognostic counselling. The exclusion of several adRP families from all ten adRP loci indicates that at least one further adRP locus remains to be found.

Topics: Basic-Leucine Zipper Transcription Factors; Chromosome Mapping; DNA Mutational Analysis; DNA Primers; DNA-Binding Proteins; Eye Proteins; Female; Founder Effect; Genes, Dominant; Haplotypes; Heteroduplex Analysis; Humans; Male; Microsatellite Repeats; Mutation; Pedigree; Polymerase Chain Reaction; Restriction Mapping; Retinitis Pigmentosa; Rhodopsin

To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin-EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin-GFP expression by 70-80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen-TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin-EGFP. We conclude that directing DNA damage with psoralen-TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene.

Topics: Base Sequence; Binding Sites; Cross-Linking Reagents; DNA; DNA Damage; Down-Regulation; Ficusin; Flow Cytometry; Fluorescence; Genes, Reporter; Genetic Therapy; Humans; Mutation; Oligodeoxyribonucleotides; Photosensitizing Agents; Plasmids; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger; Substrate Specificity; Thermodynamics; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

To examine the effect of rhodopsin mutations on cone photoreceptors in human retinas with retinitis pigmentosa (RP).. Four RP retinas with rhodopsin mutations and four normal retinas were examined by immunofluorescence with a battery of cell-specific antibodies against cone and rod cytoplasmic and outer segment membrane proteins. Areas of the retinas were studied that showed maximal preservation of photoreceptor structure.. All four RP retinas showed loss of rods, ranging from mild (T-17-M), to more severe (P-23-H), to advanced degeneration (Q-64-ter and G-106-R). The majority of cones in the T-17-M and P-23-H retinas were cytologically normal but showed loss of immunoreactivity for the cytoplasmic proteins 7G6, calbindin, and X-arrestin. The cone outer segments (OS) remained positive for cone opsins and peripherin-2 (rds/peripherin). All remaining cones in the Q-64-ter and G-106-R retinas were degenerate, with short to absent OS, but had strong reactivity for these cytoplasmic and OS membrane markers. Cones in the maculas of the RP retinas were degenerate, with short to absent OS, but retained strong labeling for the cytoplasmic and OS proteins.. Even before cones show cytologic changes in response to rod cell degeneration, they lose immunoreactivity for certain cytoplasmic proteins. These cones later show shortening and loss of OS, although their OS membrane proteins remain well labeled. Cones may down regulate expression of both cytoplasmic and outer segment membrane proteins in response to mutant rod cell dysfunction and/or cell death in human RP retinas. Such cytologic and immunocytochemical changes in the cones may presage death of these critical cells in the later stages of RP.

Topics: Aged; Aged, 80 and over; Biomarkers; Eye Proteins; Female; Fluorescent Antibody Technique, Indirect; Humans; Male; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Point Mutation; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP), a type of retinal degeneration involving first rod and then slow cone photoreceptor degeneration, can be caused by any of a number of mutations in different genes. In the cases of mutations affecting rod-specific genes such as rhodopsin, it is unclear how the mutations may cause degeneration of cones. We have used the porcine retina, which is rod-dominated and has an abundance of cones, to study the mutation-induced changes in both rod and cone photoreceptors. Like patients with the same mutation, rhodopsin P347L transgenic swine manifest rod-cone degeneration. In addition, the rod bipolar cells fail to form synaptic connections with rods; instead, they form ectopic synapses with cones. The mechanisms that prevent the formation of the rod-rod bipolar cell synaptic connection are not known. We used specific antibodies and immunocytochemistry to show that the synaptic protein, PSD-95, is present in both normal and transgenic porcine retinas. During neonatal development, however, PSD-95 is lost from rod terminals in the transgenic swine. This loss is virtually complete (90%) by postnatal day 5, at a time when greater than 80% of rod cell bodies still remain. Furthermore, the remaining rods retain their outer segments and their gross morphology appears relatively normal. In contrast, PSD-95 expression continues in cone terminals, even in 10-month-old transgenic swine, where the rods have all disappeared and the cones show signs of severe degeneration. These results suggest that loss of PSD-95 may not be a general consequence of the deteriorating cell. Rather, the very early and selective loss of PSD-95 from the rod terminals may be causally related to the absence of rod-rod bipolar cell synapses in the rhodopsin P347L transgenic retina.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Antibodies; Disease Models, Animal; Nerve Tissue Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses

To compare histologic findings in an autopsy eye of an 84-year-old man with advanced retinitis pigmentosa and rhodopsin, Glu181Lys, with two cases of autosomal dominant retinitis pigmentosa (one with rhodopsin, Pro23His, and one with rhodopsin, Cys110Arg) and with a normal control, all of comparable age.. All eyes were prepared for light and electron microscopy within 6 hours after death.. Extensive photoreceptor degeneration was revealed in the eyes with retinitis pigmentosa. Some macular cones showed membranous swirls only in the eye with rhodopsin, Glu181Lys.. The retinal degeneration caused by rhodopsin, Glu181Lys, can feature membranous swirls in the inner segments of cones in the macula. These swirls have not been reported in other cases of dominant retinitis pigmentosa studied so far, and their pathogenesis remains to be defined.

Topics: Adult; Aged; Aged, 80 and over; Autopsy; DNA Mutational Analysis; Family; Female; Humans; Male; Pedigree; Photoreceptor Cells, Vertebrate; Point Mutation; Retinitis Pigmentosa; Rhodopsin

A Spanish family affected with autosomal dominant retinitis pigmentosa (ADRP) with a diffuse phenotype showed a mutation in the rhodopsin gene. The mutation was the transition T-->C in codon 186, which has been reported once before in an American patient (Dryja et al., Proc Natl Acad Sci USA 1991;88:9370-9374). This change replaces a serine by a proline in the second intradiscal loop of the protein, generating a molecule that is probably folding- and transport-defective.

Topics: Adult; DNA Mutational Analysis; Electroretinography; Female; Fundus Oculi; Genes, Dominant; Genotype; Humans; Male; Middle Aged; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Proline; Retinitis Pigmentosa; Rhodopsin; Serine; Spain; Visual Fields

Mutations in the rhodopsin cause of retinitis pigmentosa autosomal dominant (ADRP). We report a large family affected with ADRP. Analysis by denaturant gradient gel electrophoresis and direct DNA sequence detected an heterozygous G to T transversion in the exon 3 of the rhodopsin gene. This mutation damages a restriction site for Taq I enzyme and produces the change Asp-190-Tyr in rhodopsin. All carriers of the mutation show a regional RP phenotype. This mutation is responsible for the disease in this family.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child, Preschool; Chromosome Aberrations; Chromosome Disorders; Cytogenetics; Female; Gene Expression; Humans; Male; Middle Aged; Pedigree; Point Mutation; Retinitis Pigmentosa; Rhodopsin; X Chromosome

Several families have been described in which a variety of retinal dystrophies were apparently caused by a mutation in the peripherin/RDS gene. We present clinical and genetic findings in a new family affected with a retinal dystrophy with features of retinosis pigmentosa, pattern dystrophy and fundus flavimaculatus in which a mutation in the peripherin/RDS gene has been ruled out.. A screening in the rhodopsin, peripherin/RDS and ROM1 genes was done in the affected members of the family by PCR amplification and SSCP (single strand conformation polymorphism) analysis.. No mutation was found in any of the family members.. Mutations in other genes may be involved in retinal dystrophies.

Topics: Adult; Aged; Female; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Middle Aged; Mutation; Nerve Tissue Proteins; Nuclear Family; Pedigree; Peripherins; Pigment Epithelium of Eye; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is the most prevalent inherited degeneration in the retina. The clinical manifestations are variable in terms of severity, age of onset and progression. The clinical variation is paralleled by genetic heterogeneity (more than 20 different loci have been described to date). The aim of this work was to identify mutations in rhodopsin gene and to determine the frequencies of the different genetic forms of RP in the Galician population.. 47 previously diagnosed RP patients and their relatives were studied. Genetic forms of RP were identified by recording full family history and clinical examination. DNA samples from patients with RP and control individuals were screened for point mutations in the rhodopsin gene by using PCR SSCPs (Single Strand Conformation Polymorphisms) and direct sequencing in 36 unrelated nonsyndromic RP patients.. We report the frequency distribution of the different genetic RP forms. In the SSCPs analysis of rhodopsin gene we found different mobility shifts: one variant in the 5'-untranslated region of the gen and one variant in the third intron. Direct sequencing revealed an A269-->G and an C3982-->T transitions, respectively. Additionally, we observed a single base change in codon 160 (C-->A) of this gene.. Polymorphisms are commom findings in the exon 1 and 3 of rhodopsin gene. They are neutral variations and do not represent a change in the protein. No significant differences in the frecuencies of A269-->G and C3982-->T polymorphisms among the three groups of RP patients (ADRP, ARRP, Esporadic RP) and normal individuals were found. There was no significant deviation from Hardy-Weinberg's equilibrium in each genotype in any group.

Topics: Adolescent; Adult; Aged; Child; Female; Humans; Male; Middle Aged; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Spain

The elegant architecture of photoreceptor cells in the retina is dependent on organization of the actin cytoskeleton during eye development. But what drives this organization? In an equally elegant Perspective, Colley explains new findings in fruit flies (Chang and Ready) that point to the photopigment rhodopsin and its signaling molecule the Rho GTPase Drac1 as the orchestrators of actin organization and the consequent assembly of the sensory membrane in the photoreceptor cell.

Topics: Actin Cytoskeleton; Amino Acid Motifs; Animals; Drosophila; Drosophila Proteins; Enzyme Activation; Humans; Models, Biological; Morphogenesis; Photoreceptor Cells, Invertebrate; rac GTP-Binding Proteins; Retina; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Topics: Adult; Codon; Female; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

To investigate the genotype-phenotype correlation in Chinese patients with retinitis pigmentosa caused by rhodopsin gene mutation.. On the basis of the onset of symptoms, degree of morphological changes and progression of visual disability in three (3/83) patients with identified mutations, the correlation of the phenotype with the corresponding mutations was assessed.. There was a certain degree of allele-specificity. Severe form of retinitis pigmentosa was found in patients with mutation in the cytoplasmic domain and mild form of retinitis pigmentosa in patients with mutation in the intradiscal domain.. Although there is a certain relation between the mutant rhodopsin and ocular manifestation, we need to accumulate more materials before relating a rhodopsin mutation to a specific phenotype.

Topics: Adult; Alleles; Asian People; Female; Genes, Dominant; Genotype; Humans; Male; Pedigree; Phenotype; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110-C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cattle; Cysteine; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Spectrophotometry

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene - Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene - Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.

Topics: Exons; Genes, Dominant; Heteroduplex Analysis; Humans; Intermediate Filament Proteins; Lithuania; Membrane Glycoproteins; Mutation; Nerve Tissue Proteins; Peripherins; Phenotype; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA

We performed histopathologic and immunofluorescence studies of autopsy eyes from a 73-year-old woman with autosomal dominant retinitis pigmentosa from a family with reduced penetrance. Light microscopic examination showed extensive photoreceptor loss in most regions. In the temporal midperiphery of the retina, there were patches of remaining photoreceptors, some arranged in rosettes. Electron microscopic examination showed that these rosettes were composed mostly of rods, with a few cone-like inner segments. The malformed photoreceptor elements in the rosette lumens stained positively with anti-rhodopsin, but not with anti-red- and green-cone opsin or anti-blue-cone opsin. To our knowledge, this is the first report of photoreceptor rosettes containing rod photoreceptors in a case of retinitis pigmentosa. Future studies of additional patients will be needed to determine if the rod-abundant rosettes seen in our patient are a characteristic finding of autosomal dominant retinitis pigmentosa with reduced penetrance.

Topics: Aged; Aged, 80 and over; Electroretinography; Female; Fluorescent Antibody Technique, Indirect; Humans; Pedigree; Photoreceptor Cells, Vertebrate; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Mutations in the rhodopsin gene are reported to be responsible for approximately 25% of all cases of autosomal dominant Retinitis pigmentosa (adRP). Affected individuals from a large family with an unusually severe form of adRP were screened for mutations in the rhodopsin gene. Direct sequencing of exon 5 revealed a TAA to GAA transversion at nucleotide 5276/codon 349, which was confirmed by Dde1 restriction digest analysis. This change would replace the normal termination codon with a glutamic acid residue (Ter-349-Glu, or X349E). The next predicted termination codon (TAA) lies 153bp downstream at nucleotides 5429 to 5431. Termination of transcription at this point would add an additional 51 amino-acid residues to the carboxy terminus of the rhodopsin molecule. This mutation is unique in producing a mutant rhodopsin in which all of the normal 348 amino-acid residues remain intact. It produces one of the most severe adRP phenotypes ever observed in a family with a rhodopsin mutation. In view of this the Ter-349-Glu mutation is worthy of further investigation to determine how the presence of this particular mutant opsin leads to rod photoreceptor degeneration.

Topics: Amino Acid Substitution; Codon, Terminator; DNA Mutational Analysis; Genes, Dominant; Glutamic Acid; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Animals; Basic-Leucine Zipper Transcription Factors; Cell Line; DNA-Binding Proteins; Eye Proteins; Female; Gene Expression Regulation; Genes, Dominant; Genetic Linkage; Genetic Markers; Heteroduplex Analysis; Humans; Leucine Zippers; Male; Mutation; Pedigree; Promoter Regions, Genetic; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin; Transcription Factors; Transfection

Mice carrying a targeted disruption of the rhodopsin gene develop a severe degenerative retinopathy, failing to elaborate rod photoreceptor outer segments (ROS), having no recordable rod electroretinogram (ERG) and losing all of their rod cells over a period of approximately 12 weeks. Murine and human rhodopsins differ in their amino acid sequences. Whether, or to what extent, such variability might influence the ability of human rhodopsin to serve as an adequate structural and functional substitute for the endogenous protein in mouse rod cells bears direct relevance to exploiting the full utility of Rho-/-animals as a model of degenerative retinal disease in man. We crossed Rho-/-mice with mice expressing a wild-type human rhodopsin transgene at levels approximating to those of the endogenous protein. Immunohistological examination of retinal selections from such animals demonstrated ROS of normal number and length and temporal expression of rhodopsin similar to that observed in wild-type animals; that is, immunoreactivity to an anti-rhodopsin antibody became clearly evident by day 3 post-partum. Whereas Rho-/-mice never display a rod ERG response, and even lose cone responses by 12 weeks of age, rescued mice showed 75% normal maximum amplitudes and had ERG b-wave thresholds (based on a 50 microV criterion) within 0.1 log unit of normal wild-type at 20 weeks, and cone amplitudes remained normal at this age. These data demonstrate very substantial structural and functional rescue of the rod photoreceptors of Rho-/-mice and long-term preservation by the human rhodopsin transgene.

Topics: Animals; Genetic Therapy; Humans; Mice; Mice, Transgenic; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Transgenes

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell-autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild-type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild-type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival-promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival-promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival-promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Brain-Derived Neurotrophic Factor; Cell Survival; Cells, Cultured; Ciliary Neurotrophic Factor; Culture Media, Conditioned; Female; Fibroblast Growth Factor 2; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutagenesis; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Tretinoin

Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.

Topics: Amino Acid Substitution; Animals; Animals, Genetically Modified; Dark Adaptation; Electroretinography; Gene Expression Regulation, Developmental; Humans; Membrane Potentials; Neural Pathways; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses

To assess visual acuity recovery times and cone photopigment regeneration kinetics after a bleach in the fovea of patients with dominant retinitis pigmentosa due to rhodopsin mutations.. The authors measured acuity recovery times by computerized photostress testing in 13 patients with dominant retinitis pigmentosa and one of eight rhodopsin mutations. The authors also measured their time constants of cone photopigment regeneration with a video imaging fundus reflectometer to determine whether acuity recovery time depended on pigment regeneration kinetics. These values were compared with those of normal subjects, by the Mann-Whitney U test. The relationship between acuity recovery time and the time constant of cone photopigment regeneration among the patients was quantified by the Spearman rank correlation.. The visual acuity recovery times, which averaged 22.0 seconds for the patients with retinitis pigmentosa and 11.2 seconds for the normal subjects, were significantly slower for the patient group (P < 0.001). The time constants of cone pigment regeneration, which averaged 172 seconds for the patients with retinitis pigmentosa and 118 seconds for the normal subjects, also were significantly slower for the patient group (P = 0.043). The authors also found a significant, positive correlation between the visual acuity recovery time and the time constant of pigment regeneration for the patients with retinitis pigmentosa (r = 0.65, P = 0.017).. A slowing of foveal visual acuity recovery and cone pigment regeneration, which are related to each other, can occur in patients with retinitis pigmentosa, due to a rod-specific gene defect.

Topics: Adult; Female; Humans; Light; Male; Middle Aged; Point Mutation; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins; Visual Acuity

Hereditary retinal degenerations may be subdivided into those affecting predominantly the central (macular) or peripheral regions of the retina. Retinitis pigmentosa (RP) affects the photoreceptors; death of the rod cells is followed by a progressive loss of cone cells, resulting in relatively early loss of peripheral vision and progressive constriction of the visual fields. A mutation in the gene encoding the photoreceptor protein, rhodopsin, was the first molecular defect identified as a potential cause of inherited retinal degeneration (RD). In the study reported here, simple tests for rhodopsin involvement in 194 southern African patients with a history of retinal degeneration, including 14 black African patients, were performed. Two RP patients were identified with disease-causing mutations in the rhodopsin gene: one from a black African family in which a codon 347 mutation resulted in a Pro-Leu substitution, and one in a family of Caucasian origin where a codon 58 alteration resulted in a Thr-Arg substitution. This is the first report of a disease-causing rhodopsin mutation in an indigenous black African family with retinitis pigmentosa.

Topics: Black People; Codon; Humans; Mutation; Photoreceptor Cells; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; South Africa

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, were shown to be the most common cause of autosomal retinitis pigmentosa (RP). In order to determine the prevalence of rhodopsin alterations in southern French populations, we examined 52 unrelated patients/families with autosomal dominant RP (adRP=29), RP simplex (6), or unclassified RP (17).. The full coding and flanking sequences of the rhodopsin (RHO) gene were scanned using an improved DGGE (denaturing gradient gel electrophoresis) assay, followed by sequencing of abnormal fragments.. This study revealed three RHO mutations in patients with adRP (G106R, R135W, and c.998999ins4) and a number of frequent or rare polymorphisms. No disease-causing sequence variation was found in simplex and unclassified RP pedigrees. Mutation c.998999ins4 has not been previously reported, and appears as the first duplication identified so far in the RHO gene. This frameshift mutation, which is associated with a severe RP, alters the carboxy terminus and predicts a 353-amino acid mutant rhodopsin instead of 348.. Our study demonstrates that rhodopsin mutations are responsible for only 10.3% of adRP in French populations living in the Mediterranean area in contrast to the 25-35% reported in other populations.

Topics: Adult; Amino Acid Sequence; Base Sequence; DNA Mutational Analysis; Female; France; Gene Duplication; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

Topics: Aged; Humans; Male; Mutation, Missense; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

To disclose the mutation of rhodopsin and peripherin/RDS genes among Chinese patients with retinitis pigmentosa as there was no identified mutation through sequencing reported in Chinese.. Genomic DNA was prepared from the peripheral lymphocytes. Gene fragments of the rhodopsin and peripherin/RDS genes were amplified by the polymerase chain reaction. The PCR products were analyzed by Heteroduplex-SSCP technique. PCR samples with aberrant migrational bands were identified through direct sequencing or cloning sequencing.. Three different mutations in the rhodopsin gene were found in 3 of the 83 patients with retinitis pigmentosa(Va1104Phe, Lys311Glu, Pro347Leu). Two of the three mutations have not been reported before. One of the two (heterozygous, Va1104Phe) was found in an isolated patient and the other (homozygous, Lys311Glu) in a family with autosomal recessive retinitis pigmentosa. Mutation of peripherin/RDS gene was not found in the 83 patients.. Mutation in the rhodopsin gene is the common cause in Chinese patients with retinitis pigmentosa, either autosomal dominant, recessive or sporadic.

Topics: Base Sequence; Genes, Dominant; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Pedigree; Peripherins; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA

Topics: Amino Acid Substitution; Deoxyribonucleases, Type II Site-Specific; DNA; DNA Mutational Analysis; Family Health; Genes, Dominant; Humans; Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; DNA; DNA Mutational Analysis; DNA, Complementary; Exons; Humans; Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; RNA, Messenger

Autosomal dominant retinitis pigmentosa (ADRP) is caused by mutations in two known genes, rhodopsin and peripherin/Rds, and seven loci identified only by linkage analysis. Rhodopsin and peripherin/Rds have been estimated to account for 20-31% and less than 5% of ADRP, respectively. No estimate of frequency has previously been possible for the remaining loci, since these can only be implicated when families are large enough for linkage analysis. We have carried out such analyses on 20 unrelated pedigrees with 11 or more meioses. Frequency estimates based on such a small sample provide only broad approximations, while the above estimations are based on mutation detection in much larger clinic based patient series. However, when markers are informative, linkage analysis cannot fail to detect disease causation at a locus, whereas mutation detection techniques might miss some mutations. Also diagnosing dominant RP from a family history taken in a genetic clinic may not be reliable. It is therefore interesting that 10 (50%) of the families tested have rhodopsin-RP, suggesting that, in large clearly dominant RP pedigrees, rhodopsin may account for a higher proportion of disease than had previously been suspected. Four (20%) map to chromosome 19q, implying that this is the second most common ADRP locus. One maps to chromosome 7p, one to 17p, and one to 17q, while none maps to 1cen, peripherin/Rds, 8q, or 7q. Three give exclusion of all of these loci, showing that while the majority of dominant RP maps to the known loci, a small proportion derives from loci yet to be identified.

Topics: Chromosome Mapping; Female; Gene Frequency; Genes, Dominant; Genetic Heterogeneity; Genetic Linkage; Humans; Male; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

To determine the phenotype of a Japanese family in which retinitis pigmentosa cosegregates with a rhodopsin gene mutation, i.e. an asparagine-to-serine change at codon 15 (Asn-15-Ser), 5 affected and 5 unaffected members of one pedigree underwent several ophthalmic examinations as well as Ganzfeld electroretinography (ERG) and multifocal ERG. Genomic DNA samples were analyzed by PCR amplification, sequencing and restriction enzyme digestion. A codon 15 rhodopsin gene mutation (Asn-15-Ser) was found in all affected members. The region of pigmentary degeneration was localized in the lower hemiretina, and visual field defects corresponded to the retinal pigmentary changes. Scotopic ERG amplitudes, rather than photopic ERG amplitudes, were reduced. Multifocal ERG revealed a low magnitude of response density, even for the upper hemiretina, which showed no bony corpuscle pigmentation. Visual function in sectorial retinitis pigmentosa associated with rhodopsin gene codon 15 mutation is on the basis of the rod-cone dystrophy, regardless of differences in phenotypic expression.

Topics: Adult; Aged; Codon; Dark Adaptation; DNA; DNA Primers; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Humans; Male; Middle Aged; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

To further characterize the retinas of Pro3471Leu rhodopsin transgenic pigs, a model for human retinitis pigmentosa.. Retinas from normal and transgenic pigs, newborn to 20 months old, were processed for light and electron microscopic immunocytochemical examination.. At birth, rod numbers were normal in the transgenic retinas, but their outer segments were short and disorganized and their inner segments contained stacks of rhodopsin-positive membranes. The newborn rod synapses lacked synaptic vesicles and ribbons and had numerous rhodopsin-positive, filopodia-like processes that extended past the cone synapses into the outer plexiform layer. Rod cell death was apparent by 2 weeks and was pronounced in the mid periphery and central regions by 6 weeks. Far peripheral rods were initially better preserved, but by 9 months virtually all rods had degenerated. Cones degenerated more slowly than rods, but by 4 weeks the cone synapses were shrunken and some mid peripheral cones had lost their immunoreactivity for phosphodiesterase-gamma, arrestin, and recoverin. From 9 months to 20 months, the cone outer segments shortened progressively, and more cones lost immunoreactivity for these proteins.. The rhodopsin transgenic pig retina shares many cytologic features with human retinas with retinitis pigmentosa and provides an opportunity to examine the earliest stages in photoreceptor degeneration, about which little is known in humans. The finding of abnormal rhodopsin localization in newborn rods is consistent with misrouting of mutant rhodopsin as an early process leading to rod cell death. Novel changes in the photoreceptor synapses may correlate with early electrophysiological abnormalities in these retinas.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Cell Count; Cell Death; Disease Models, Animal; Eye Proteins; Fluorescent Antibody Technique, Indirect; Neuroglia; Photoreceptor Cells; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Swine

Retinitis pigmentosa (RP) is a degenerative disorder affecting the outer segment of the retina and leading to night blindness and progressive visual field loss. The rhodopsin gene encodes a photolabile pigment located in the rod outer segments constituting around 80-90% of its protein content and is the initiation point for the visual cascade upon absorption of a single photon. Seventy-five unrelated, isolated RP families in the Basque Country, with at least one affected member, were diagnosed at our hospital after ophthalmic examination and electroretinogram analysis. The patients received genetic counselling according to their individual case based on their clinical diagnosis. The modes of inheritance found from pedigree studies were the following: 20% (15/75) were classified as autosomal dominant retinitis pigmentosa (ADRP), 17.33% (13/75) were autosomal recessive (ARRP), 2.66% (2/75) were unclassified (NC), and 60% (45/75) were sporadic cases (SCRP). From these families, 75 unrelated and affected index cases together with 22 affected relatives and 42 unaffected relatives were screened for mutations in the rhodopsin gene by GC clamped denaturing gradient gel electrophoresis. Our results showed that five ADRP, three ARRP, 15 SCRP, and one NC families had alterations in this gene. Only three of these alterations, that is 4% (3/75) (95% CL 0-8), appeared to be responsible for the disease. This represents a lower percentage than the 10% previously reported.

Topics: Electrophoresis, Polyacrylamide Gel; Female; Humans; Male; Mutation; Pedigree; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin; Spain

We present a Spanish family affected with autosomal dominant pigmentary retinosis in which we have identified the mutation responsible for the disease (Pro347Leu) within the rhodopsin (RHO) gene. Complete ophthalmological and electrophysiological studies were performed in 14 members of this family. The molecular study, performed by SSCP analysis of the 5 exon and the promotor region of the rhodopsin gene, direct sequentiation and restriction analysis with the enzyme Mspl, showed a C-->T change in the second base of 347 codon of RHO gene. This mutation predicts a change of proline by leucine at this position. Every patient with the mutation showed a phenotype of diffuse, early onset and severe pigmentary retinosis with a little intrafamiliar variation. The Pro347Leu mutation, that has been very frequently described among all the populations, has been identified as a cause of RP in an Spanish family.

Topics: Adolescent; Adult; Child; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Spain

A couple requested preimplantation genetic analysis for a dominant form of retinitis pigmentosum caused by a C-->A transversion in the rhodopsin gene. Since this point mutation does not alter a restriction endonuclease site we designed two separate analytical systems, one involving site-specific mutagenesis and the other involving allele-dependent length polymorphism. After establishing the accuracy and robustness of these assay systems we utilized both systems simultaneously in a heminested polymerase chain reaction (PCR) system. This allowed accurate preimplantation diagnosis to be performed. One embryo was transferred but a pregnancy did not occur.

Topics: Cells, Cultured; Female; Genes, Dominant; Humans; Point Mutation; Polymerase Chain Reaction; Preimplantation Diagnosis; Retinitis Pigmentosa; Rhodopsin

Phenotypic, genetic and molecular characterization of 69 index patients with retinitis pigmentosa (RP) and various inherited retinal diseases.. patients went through complete ocular examination and blood samples were drawn for mutational screening of three candidate genes: rhodopsin (RHO), peripherin/RDS, and ROM-1.. the most frequent type of RP among our population was the autosomal dominant (43.6%). Three RHO mutations were found among the RP patients. A RDS mutation was detected in three unrelated families segregating dominant macular dystrophy.. 18% of the autosomal dominant RP patients presented a RHO mutation; RDS R172W mutation was present in 25% of the dominant macular dystrophies.

Topics: Adult; Aged; Aged, 80 and over; DNA Mutational Analysis; Eye Proteins; Female; Fluorescein Angiography; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Membrane Proteins; Middle Aged; Nerve Tissue Proteins; Pedigree; Peripherins; Phenotype; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Tetraspanins

We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd

Topics: Binding Sites; DNA; Gene Targeting; Humans; Nucleic Acid Conformation; Oligonucleotides; Plasmids; Retinitis Pigmentosa; Rhodopsin; Sensitivity and Specificity

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Topics: Animals; Animals, Genetically Modified; Dependovirus; Disease Models, Animal; Genes, Dominant; Genetic Therapy; Histidine; Photoreceptor Cells; Point Mutation; Proline; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA, Catalytic; Rod Opsins

A family with 1 case of retinitis pigmentosa (III-1) and 2 cases of Oguchi's disease (III-2, 3) was examined in terms of electrophysiology as well as molecular biology. The proband (III-3), a 42-year-old female, and 2 older brothers (III-1, 2, aged 52 and 45 years) and 2 unaffected members in the same family participated in this study. Corrected visual acuities of the individuals with Oguchi's disease (III-2, 3) were 1.2. On funduscopy, blood vessels stood out in relief against a metallic-appearing background and a Mizuo-Nakamura phenomenon was evident. Full-field electroretinograms (ERGs) recorded from the proband were indicative of rod dystrophy, but results of other electrophysiological examinations (multifocal ERG, pattern ERG and visual-evoked cortical potential recordings) were within normal limits. Patient III-1 had corrected visual acuities of RE 20 cm/m.m. and LE 30 cm/n.d., severe chorioretinal atrophy in both fundi, and full-field ERG revealed rod-cone dystrophy. Mutation of the arrestin gene (1147de1A) was detected in all 3 patients. Visual function in each patient coincides with that of retinitis pigmentosa or Oguchi's disease, respectively.

Topics: Adult; Arrestin; Base Sequence; Electroretinography; Evoked Potentials, Visual; Eye Diseases, Hereditary; Female; Fundus Oculi; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

A therapeutic effect of vitamin A supplementation on the course of photoreceptor degeneration, previously reported for patients with retinitis pigmentosa, was tested in two transgenic mouse models of this disease, each carrying a dominant rhodopsin mutation. The threonine-17 --> methionine (T17M) mutation is a class II rhodopsin mutation, characterized by a thermal instability/folding defect and minimal regeneration with the chromophore. The proline-347 --> serine (P347S) mutation belongs to class I, comprised of a smaller number of mutations that exhibit no recognized biochemical abnormality in vitro. In the present study, each of the two mouse models was fed a diet containing 2.5 mg of vitamin A palmitate (control) or 102.5 mg of vitamin A palmitate (high vitamin A) per kilogram of diet. Dark-adapted, full-field electroretinograms showed that the high vitamin A diet significantly reduced the rate of decline of a-wave and b-wave amplitudes in the T17M mice but had no significant effect on the decline of electroretinogram amplitude in the P347S mice. Correspondingly, histologic evaluation revealed that the treatment was associated with significantly longer photoreceptor inner and outer segments and a thicker outer nuclear layer in the T17M mice but had no effect on photoreceptor morphology in the P347S mice. In a separate series of experiments, the instability defect of the T17M mutant opsin expressed in vitro was partially alleviated by inclusion of 11-cis-retinal in the culture media. These results show that vitamin A supplementation slows the rate of photoreceptor degeneration caused by a class II rhodopsin mutation. Vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of the chromophore.

Topics: Animals; Cells, Cultured; Diterpenes; Electroretinography; Gene Expression; Humans; Liver; Mice; Mice, Transgenic; Microscopy, Electron; Photoreceptor Cells; Point Mutation; Retina; Retinaldehyde; Retinitis Pigmentosa; Retinyl Esters; Rhodopsin; Transfection; Vitamin A

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11-cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Disease Models, Animal; Genes, Dominant; Mice; Mice, Transgenic; Photochemistry; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry; Vision, Ocular

Topics: Adolescent; Child; Female; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.

Topics: Adenoviridae; Animals; Apoptosis; Ciliary Neurotrophic Factor; Disease Models, Animal; Electroretinography; Gene Transfer Techniques; Homozygote; Lac Operon; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Nerve Growth Factors; Nerve Tissue Proteins; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

The RHO C110Y mutation has been recently reported to cause a phenotypically unspecified form of autosomal dominant retinitis pigmentosa (adRP). The study of a family affected with this mutation allowed us to hereby describe the genotype/phenotype correlation associated with the RHO C110Y mutation.. A six-generation pedigree cosegregating adRP and RHO C110Y in ten accessible individuals was ophthalmologically investigated. All family members affected with RP went through complete eye examination and ERG testing.. The disease first manifested with nyctalopia during adulthood and slowly progressed over the next decades towards tubular visual field defects and relatively preserved central vision. Ophthalmoscopically, the fundus remained almost unaltered until the end of the third decade of life, and then slowly progressed towards typical RP changes with minimal macular involvement by the eighth decade. Color vision remained unaltered. Earliest ERG alteration was limited to the rod system followed by a rod-cone pattern. Scotopic and photopic ERG were recordable until the fourth and sixth decades, respectively.. RHO C110Y-associated adRP is characterized by a late onset and a mild progression compatible with type 2 or regional RP with little intrafamilial phenotypic variability and complete penetrance. Characterization of genotype-phenotype correlations plays a role in the improvement of genetic and prognostic counselling.

Topics: Adult; Aged; Amino Acid Substitution; Disease Progression; Electroretinography; Female; Fluorescein Angiography; Fundus Oculi; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Vision, Ocular

The authors report two new rare DNA sequence variants in the Rhodopsin gene. This gene is involved in the pathogenesis of some retinal hereditary disorders as Retinitis Pigmentosa. These rare variants are G-->A at nucleotide 721 of the non-coding region and C-->T at nucleotide 5200 within codon 323 which does not alter the aminoacid cysteine. Therefore, they are not implicated in the development of the Retinitis Pigmentosa disease.

Topics: Adenine; Cytosine; Guanine; Humans; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Thymine

Topics: Animals; Arrestin; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; Genotype; Humans; Mice; Mice, Knockout; Mice, Transgenic; Models, Molecular; Protein Kinases; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Signal Transduction; Transducin

Retinitis pigmentosa (RP) represents the most common mendelian degenerative retinopathy of man, involving death of rod photoreceptors, cone cell degeneration, retinal vessel attenuation and pigmentary deposits. The patient experiences night blindness, usually followed by progressive loss of visual field. Genetic linkage between an autosomal dominant RP locus and rhodopsin, the photoreactive pigment of the rod cells, led to the identification of mutations within the rhodopsin gene in both dominant and recessive forms of RP. To better understand the functional and structural role of rhodopsin in the normal retina and in the pathogenesis of retinal disease, we generated mice carrying a targeted disruption of the rhodopsin gene. Rho-/- mice do not elaborate rod outer segments, losing their photoreceptors over 3 months. There is no rod ERG response in 8-week-old animals. Rho+/- animals retain the majority of their photoreceptors although the inner and outer segments of these cells display some structural disorganization, the outer segments becoming shorter in older mice. These animals should provide a useful genetic background on which to express other mutant opsin transgenes, as well as a model to assess the therapeutic potential of re-introducing functional rhodopsin genes into degenerating retinal tissues.

Topics: Age Factors; Animals; Electroretinography; Gene Targeting; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Pigment Epithelium of Eye; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment

We here present the clinical phenotype in 6 patients from a family with autosomal dominant retinitis pigmentosa found to carry a point mutation in the rhodopsin gene (arginine-135-tryptophan). The mutation is the second found by mutation screening of DNA from 20 Swedish families with dominant retinitis pigmentosa. With full-field electroretinography we could document a severe form of retinitis pigmentosa in patients belonging to the family, similar to the phenotype associated with the previously reported mutation (arginine-135-leucine). Our results indicate that different point mutations in the same region of the rhodopsin gene, resulting in amino acids with similar properties (both hydrophobic), may cause a similar clinical phenotype. Further, point mutations in this specific region seem to cause an agressive form of retinitis pigmentosa.

Topics: Adolescent; Adult; Arginine; Child; DNA Mutational Analysis; DNA Primers; Electroretinography; Female; Fundus Oculi; Genes, Dominant; Genetic Testing; Humans; Leucine; Male; Middle Aged; Molecular Sequence Data; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Fields

By screening blood samples from patients with autosomal dominant retinitis pigmentosa, we found in one of the families a rhodopsin mutation (Pro-267-Leu), which segregates with the disease in two affected and five unaffected family members. Here, we present the results of the clinical evaluation of the family, including full-field electroretinography from the two affected family members. A 25-year-old family member with the mutation had an almost normal electrophysiological retinal response. The patient's father, who was also heterozygous for the mutation and had mild subjective symptoms of retinitis pigmentosa, demonstrated a substantially preserved retinal function. Our results suggest that the Pro-267-Leu rhodopsin mutation is associated with a very mild phenotype of retinitis pigmentosa. Young patients with the disease may have minimal pathological changes in the electroretinogram and some patients with few symptoms may be affected without acquiring a diagnosis of eye disease.

Topics: Adult; Chromosome Aberrations; Chromosome Disorders; Electroretinography; Exons; Eye Diseases, Hereditary; Family Health; Female; Genes, Dominant; Genetic Testing; Heterozygote; Humans; Male; Middle Aged; Pedigree; Phenotype; Point Mutation; Proline; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Sweden; Visual Acuity

Topics: Animals; Disease Models, Animal; Genetic Engineering; Humans; Mutation; Phenotype; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin; Rodentia; Swine

Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Topics: Animals; Animals, Genetically Modified; Blotting, Southern; Disease Models, Animal; Electroretinography; Embryo Transfer; Gene Expression Regulation; Genetic Engineering; Microscopy, Electron; Molecular Sequence Data; Phenotype; Polymerase Chain Reaction; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Swine; Transgenes

This report describes ocular findings obtained in four patients from three families with autosomal dominant retinitis pigmentosa (adRP) due to missense mutations in the rhodopsin gene. Phenotypes were characterized by standard ophthalmologic examinations, visual fields, electroretinography (ERG), dark adaptation, and two-color dark-adapted threshold perimetry. Two patients aged 38 and 45 years, respectively, from a family with the Cys110Phe mutation showed mild fundus changes without bone spicules as well as small arcuate scotomas in the inferior quadrants of their visual fields but displayed severe functional loss of rods and cones in the ERG. Two-color dark-adapted threshold perimetry revealed a regional type of degeneration. A 48-year-old patient with an Arg135Gly mutation had typical RP with concentrically narrowed visual fields and nondetectable ERG responses. Central visual functions were well preserved for a long time. Two-color dark-adapted threshold perimetry indicated a diffuse type of retinal degeneration. An 18-year-old patient with a Gln344stop mutation has been followed for 13 years. His ERG was clearly reduced at the age of 5 years; since that time, disease progression has been very slow. Currently, there are relatively mild alterations in visual acuity, rod sensitivity, and visual fields. Our findings confirm that there is a large phenotypic variety among patients with adRP and different rhodopsin mutations.

Topics: Adolescent; Adult; Child; Child, Preschool; Dark Adaptation; Disease Progression; Electroretinography; Female; Humans; Male; Middle Aged; Pedigree; Phenotype; Photoreceptor Cells; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests; Visual Fields

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; Chromosome Mapping; Chromosomes, Human, Pair 19; Cloning, Molecular; Genes; Homeodomain Proteins; Humans; Mice; Molecular Sequence Data; Photoreceptor Cells; Pineal Gland; Promoter Regions, Genetic; Retinitis Pigmentosa; Rhodopsin; Trans-Activators; Transcriptional Activation

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

Topics: Animals; Binding Sites; COS Cells; Diterpenes; Genes, Dominant; Humans; In Vitro Techniques; Phosphorylation; Point Mutation; Recombinant Proteins; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin

To screen for mutations in the rhodopsin, peripherin/RDS, and ROM1 genes in a family affected with retinitis punctata albescens. Because clinical heterogeneity was observed in this family, with some members affected with retinitis punctata albescens and one member affected with features typical of retinitis pigmentosa, we analyzed the apolipoprotein E gene to elucidate this unusual intrafamilial heterogeneity.. The coding sequences of these genes were analyzed with a combination of single-strand conformation polymorphism and direct sequence analysis. Haplotypes of the apolipoprotein E gene were analyzed by polymerase chain reaction and enzymatic digestion.. The Arg135Trp mutation in the rhodopsin gene was observed in all affected members of this family, but no mutation was detected in the peripherin/RDS or ROM1 genes. The e4 allele of the apolipoprotein E gene apparently cosegregated with the albescens phenotype in this family.. The albescent phenotype in retinal dystrophy appears to not be caused exclusively by a peripherin/RDS gene mutation, and we suggest that the apolipoprotein E gene may play a role in the albescent phenotype.

Topics: Adolescent; Adult; Amino Acid Sequence; Apolipoproteins E; Arginine; Base Sequence; DNA; Eye Proteins; Female; Fundus Oculi; Humans; Infant; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Membrane Proteins; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Pedigree; Peripherins; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Tetraspanins; Tryptophan

To describe the clinical features of a Japanese family with autosomal dominant retinitis pigmentosa, the locus of which has been mapped on chromosome 19q.. Ophthalmologic testing, including visual acuity, slit-lamp biomicroscopy, and fundus examinations, for all family members examined. Selected members underwent kinetic visual field testing, electroretinography, and fluorescein angiography.. Eleven symptomatic members, two asymptomatic obligate carriers, and nine nonaffected members in four generations of a single family with autosomal dominant retinitis pigmentosa.. Asymptomatic carriers showed mildly affected fundus and fluorescein angiographic images. Visual field testing disclosed restricted central and midperipheral fields. Electroretinograms disclosed reduced amplitudes of rod-isolated responses in both of these family members, indicating functional abnormalities.. Marked variability in expressivity of the retinitis pigmentosa phenotype was found in a family with autosomal dominant retinitis pigmentosa linked to chromosome 19q.

Topics: Aged; Chromosomes, Human, Pair 19; DNA; Electroretinography; Eye Proteins; Female; Fluorescein Angiography; Fundus Oculi; Genetic Linkage; Genetic Variation; Heterozygote; Humans; Intermediate Filament Proteins; Japan; Male; Membrane Glycoproteins; Middle Aged; Nerve Tissue Proteins; Pedigree; Peripherins; Phenotype; Retina; Retinitis Pigmentosa; Rhodopsin; Visual Fields

To correlate retinal histopathology with functional changes caused by the rhodopsin Q64ter mutation.. A 50-year-old female heterozygote was evaluated clinically and with psychophysical and electroretinographic measurements of rod and cone function. The retinas obtained after death were examined microscopically, including immunolabeling with antibodies against the C- and N-termini of rhodopsin.. On clinical examination 4 months before death, patient's acuity was 20/60, and she had midperipheral scotomas with retained function centrally and in the far periphery. The rod electroretinogram (ERG) was undetectable, and the cone ERG was reduced in amplitude with abnormal receptoral and postreceptoral responses. A previous study of the phenotype of mildly affected family members of the donor suggested that the rod outer segments (ROS) were shortened and that only wild-type rhodopsin was functional. The retinas contained only scattered cones in the midperiphery; the maculas and far peripheral regions contained reduced numbers of rods and cones with short to absent outer segments. The ROS appeared to contain wild-type, but not mutant, rhodopsin, and many peripheral rods had sprouted long rhodopsin-positive neurites that projected into the inner retina. Many cone synapses were abnormal, and the axons of some peripheral cones reached the inner plexiform layer.. Microscopic changes in the donor retinas correlated well with the abnormalities in visual function in the patient donor and other family members. Postreceptoral ERG defects may relate to the abnormal photoreceptor processes found in the inner retina.

Topics: Aged; Aged, 80 and over; Electroretinography; Female; Fluorescent Antibody Technique; Glutamine; Humans; Immunohistochemistry; Male; Middle Aged; Pedigree; Photoreceptor Cells; Point Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Scotoma; Visual Acuity; Visual Field Tests; Visual Fields; Visual Perception

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line; Gene Expression; Genes, Dominant; Humans; Molecular Sequence Data; Molecular Structure; Point Mutation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Retinitis Pigmentosa; Rhodopsin

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Topics: Amino Acid Sequence; Animals; Arginine; Binding Sites; Cattle; Cell Line; Conserved Sequence; Gene Expression; Genes, Dominant; Humans; Molecular Sequence Data; Point Mutation; Protein Folding; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Transfection

Mutations in the rhodopsin gene have been sought in a family with autosomal dominant retinitis pigmentosa. Screening for mutations in the rhodopsin gene was carried out by polimerase chain reaction and denaturant gradient gel electrophoresis. Direct DNA sequencing was performed for the characterization of punctual mutations. A base substitution in the exon 2 of the rhodopsin gene was detected. Direct DNA sequencing revealed a CGC to CTG change in codon 135, that substitutes arginine for leucine residue in rhodopsin. The mutation segregates with the disease phenotype in the family. The mutation Arg-135-Leu causes the retinitis pigmentosa phenotype in the family, where the disease is inherited following an autosomal dominant pattern.

Topics: Adult; Arginine; Base Sequence; Child; Codon; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Exons; Female; Genes, Dominant; Humans; Leucine; Male; Molecular Sequence Data; Pedigree; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

A family affected with autosomal dominant retinitis pigmentosa (RP) is presented. Two clinically affected patients (mother and daughter) were heterozygous for the same novel missense mutation (Val137Met) of the rhodopsin gene (RHO). Both heterozygous and homozygous cases were observed among their few symptomatic relatives. Wide clinical variation was exhibited among the individuals with mutations in this family. None of the controls showed this change in RHO, nor has it been previously reported in other RP families. No other RHO mutation was observed. Additional genetic or environmental factors could play a role in modulating the penetrance and clinical expression of this RHO mutation.

Topics: Base Sequence; Cataract; DNA Primers; Exons; Female; Genes, Dominant; Heterozygote; Homozygote; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Phenotype; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis; Visual Acuity

Topics: Female; Genetic Markers; Histidine; Humans; Leucine; Male; Mutation; Pedigree; Point Mutation; Polymorphism, Restriction Fragment Length; Proline; Retinitis Pigmentosa; Rhodopsin

We have previously identified rhodopsin gene mutations in France in autosomal dominant (ADRP) retinitis pigmentosa in France, using a combination of SSCP (single-strand conformation polymorphism) and direct sequence analysis. The aim of this study was to perform a more rapid tool to identify a mutation in a ADRP family, when this mutation has been identified for one affected member of this family.. We looked for a restriction site, created or abolished by a mutation in the rhodopsin gene. We performed in vitro DNA amplification using PCR (polymerase chain reaction), enzymatic digestion, and migration on agarose gel.. Abnormal patterns of migration were observed for affected ADRP members.. This technique is useful and rapid (less than 5 hours) to recognize a previously identified mutation. However, previous precise identification of the mutation in one member of the family is needed.

Topics: DNA Restriction Enzymes; Electrophoresis, Agar Gel; Genes, Dominant; Humans; Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Risk

A study was made of two families with autosomal dominant retinitis pigmentosa (ADRP) from Valencia (Spain). One family (ADRP15) was found to have mutation in codon 114 of the rhodopsin gene that led to a substitution of a glycine for an aspartic acid. The second family (ADRP7) substituted an aspartic acid for valine in codon 173 of the peripherin-RDS gene. Rhodopsin is involved in 25% of ADRP cases and many mutations of this gene have been described as causing different forms of the disease, with variable severity and age at onset. ADRP has been classified as RP with a milder symptom evolution, a typical RP fundus pattern, and macular involvement occurring after the second decade of life. Peripherin-RDS gene mutations lead to RP or other retinopathies. Furthermore, two mutations in codon 172 have been described as causing macular dystrophy. In ADRP7, a mutation in neighboring codon 173 produced RP with an atypical fundus pattern and macular involvement within the first decade of life. These observations confirm the established clinical and genetic heterogeneity involved in this form of RP.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 6; Female; Genes, Dominant; Genetic Linkage; Humans; Male; Middle Aged; Pedigree; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

Topics: Female; Genes, Dominant; Humans; Introns; Male; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; RNA Splicing; Spain

Topics: Base Sequence; Codon; Genes, Dominant; Humans; Molecular Sequence Data; Point Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

Topics: Base Sequence; Codon; Genes, Dominant; Humans; Molecular Sequence Data; Point Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

To report the clinical and functional characteristics of patients affected with autosomal-dominant transmitted retinitis pigmentosa (adRP) from a large Italian pedigree in which a point mutation predicting the Arg-135-Trp change of rhodopsin was identified by polymerase chain reaction-single-strand conformation polymorphism analysis.. Seven patients, ranging in age from 6 to 41 years, underwent a full clinical ophthalmologic evaluation, kinetic visual field testing, and electroretinographic testing.. In agreement with previous reports, this rhodopsin mutation yielded a particularly severe phenotype, both clinically and functionally. The evaluation of patients from this pedigree in the first and second decade of life demonstrated that retinal function is still electroretinographically measurable at least until 18 years of age, although reduced to 2% to 4% of normal. Longitudinal measures showed that the rate of progression of the disease was unusually high, with an average 50% loss per year of electroretinographic amplitude and visual field area with respect to baseline. Later in the course of the disease, macular function is also severely compromised, leaving only residual central vision by the fourth decade of life.. The phenotype associated with mutations in codon 135 of the rhodopsin molecule appears to have an unusually high progression rate and yields an extremely poor prognosis. These distinctive features make the Arg-135-Trp phenotype substantially different from the general RP population, and also from many of the other adRP pedigrees with known rhodopsin mutations reported to date.

Topics: Adolescent; Adult; Arginine; Child; DNA; Electroretinography; Female; Humans; Longitudinal Studies; Male; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retina; Retinitis Pigmentosa; Rhodopsin; Tryptophan; Visual Fields

A large family affected with autosomal dominant retinitis pigmentosa (ADRP) with a sectorial phenotype showed a previously described (G to A) mutation in the rhodopsin gene resulting in the substitution of a glycine residue by an arginine in codon 106 of rhodopsin. This mutation shows some unusual characteristics, such as initial pathology of the inferior retina, superior visual field with normal disc and retinal vessels, and ERG findings that show a modest reduction in both cone and rod amplitudes with normal implicit times. The Gly 106 Arg mutation has been previously reported in American and British patients. Its presence in a Spanish ADRP family confirms that it and its homogeneous associated phenotype are geographically widespread.

Topics: Adolescent; Adult; Aged; Arginine; Child; Child, Preschool; DNA; Electroretinography; Female; Glycine; Humans; Male; Pedigree; Point Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Spain; Visual Fields

Expression of a mouse opsin transgene containing three point mutations (V20G, P23H, and P27L; termed VPP) causes a progressive photoreceptor degeneration that resembles in many important respects that seen in patients with autosomal dominant retinitis pigmentosa caused by a P23H point mutation. We have attempted to determine whether the degree of degeneration induced by expression of the transgene is influenced by albinism, a genetically mediated recessive trait that results in a deficiency in melanin formation in pigmented tissues throughout the body. Litters of albino and pigmented mice (normal as well as transgenic) were reared in either darkness or cyclic light. Retinal structure and function were evaluated by light microscopy, electroretinography (ERG), and retinal densitometry. The data were consistent in demonstrating that at similar ages, the extent of photoreceptor degeneration was greater in transgenic albino animals than in their pigmented counterparts. The albino VPP mice had significantly fewer cell bodies in the outer nuclear layer of the retina, a larger reduction in ERG amplitude, and a lower rhodopsin content in the rod photoreceptors. These structural and functional differences could not be attributed to the greater level of retinal illumination experienced by the albino retina under normal ambient conditions, because they persisted when pigmented and albino mice were reared in darkness from birth. Although the explanation remains unclear, our findings indicate that the rate of photoreceptor degeneration in VPP mice is adversely affected by the existence of the albino phenotype, a factor that may have implications for the counseling of human patients with retinitis pigmentosa and a familial history of other genetic disorders.

Topics: Albinism; Animals; Densitometry; Electroretinography; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Photoreceptor Cells; Reference Values; Retina; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the beta-subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease-causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.

Topics: Calcium-Binding Proteins; Chromosome Mapping; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 6; Eye Proteins; Genes; Genes, Recessive; Guanylate Cyclase-Activating Proteins; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Membrane Proteins; Nerve Tissue Proteins; Peripherins; Phosphoric Diester Hydrolases; Retinitis Pigmentosa; Rhodopsin; Spain; Tetraspanins

A novel rhodopsin missense mutation (M216R) was found in a Danish patient with autosomal dominant retinitis pigmentosa. Clinical examination of the proband disclosed a phenotype of intermediate severity. In view of the predicted amino acid substitution in the 5th transmembrane domain of rhodopsin, the clinical picture of the proband is in keeping with the data from the literature on patients carrying similar mutations.

Topics: Adolescent; Adult; Denmark; DNA; Female; Follow-Up Studies; Genetic Linkage; Humans; Male; Middle Aged; Nucleic Acid Heteroduplexes; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

Transgenic mice expressing the rhodopsin mutant Pro347Ser (Serine 6) display retinal degeneration through apoptosis that is characteristic of the disease retinitis pigmentosa. By 5 weeks after birth, these mice have lost approximately 35% of their photoreceptor cells. Retinas from these mice showed higher levels of cAMP compared to the levels in retinas of normal mice. Our studies provide evidence that elevated cAMP is common to the apoptotic process that occurs in retinitis pigmentosa. In addition, in vitro studies demonstrate no differences in the ability of the mutant and the wild-type rhodopsin to activate transducin, the rod cell G protein, to be phosphorylated by rhodopsin kinase or to bind arrestin. Mutants of rhodopsin, including Pro347Ser, are mistargeted to the rod inner segment, raising the possibility that rhodopsin triggers apoptosis through activation of signaling pathways not normally under its control.

Topics: Animals; Apoptosis; Cyclic AMP; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Mice, Transgenic; Mutation; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Serine; Signal Transduction

Topics: Animals; Cyclic GMP; Humans; Ion Channel Gating; Ion Channels; Mice; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

To determine whether a rhodopsin splice donor site mutation at the 5' end of intron 4 is a cause of autosomal dominant retinitis pigmentosa.. Heterozygous carriers of the same rhodopsin splice site mutation in two pedigrees were identified using single-strand conformation polymorphism analysis. Twelve heterozygous carriers were evaluated by ophthalmoscopy. Goldmann kinetic visual fields, dark adaptation thresholds, and full-field electroretinograms including rod intensity-response functions. Clinical findings from the heterozygous carriers of the splice site mutation were compared with those from heterozygous carriers from a separate family with a known recessive rhodopsin null mutation, Glu249X.. Analysis of DNA from 48 members of two pedigrees revealed 25 heterozygous carriers of the splice site mutation, ranging in age from 14 to 82 years. There were no homozygotes with the rhodopsin splice site mutation. Of the 25 heterozygous carriers, 24 were asymptomatic. Eleven asymptomatic heterozygotes were examined, including four older than 65 years of age. They were found to have normal fundi, full visual fields, and slightly elevated final rod dark adaptation thresholds. Their rod electroretinographic b-wave amplitudes were slightly diminished over the full range of blue light intensities. Rod a-wave implicit times were slightly but significantly prolonged in response to the brightest blue flash of light. These subtle abnormalities in rod function were similar to those found in asymptomatic heterozygous carriers of the recessive Glu249X mutation. Only one of the 25 heterozygous carriers of the splice site mutation had symptoms and signs of retinitis pigmentosa.. Because 96% of these heterozygous carriers do not have retinitis pigmentosa, it is unlikely that this mutation in intron 4 is a dominant allele. The subtle abnormalities of rod function found in asymptomatic carriers are similar to those found in heterozygous carriers of a recessive rhodopsin allele. The one heterozygous carrier with retinitis pigmentosa probably has a second mutation in the rhodopsin gene or has a defect or defects in another gene that causes his disease.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Dark Adaptation; DNA; Electroretinography; Female; Heterozygote; Humans; Incidence; Male; Middle Aged; Pedigree; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA Splicing; Sensory Thresholds; Visual Fields

The leading edge of the rod a-wave in normal human subjects can be fit with a computational model of the activation phase of transduction to provide parameters analogous to those obtained from individual photoreceptors. The authors extend this work to the kinetics of recovery after saturating flashes.. Electroretinograms were recorded from three patients with autosomal dominant retinitis pigmentosa and the pro-23-his rhodopsin mutation, two patients with rod monochromatism, and five normal subjects. Rod-only a-waves were obtained for a series of flashes ranging from 4.4 to 10.1 ln (1.9 to 4.4 log) scot td-sec. One set of parameters describing the activation process was derived from fits to the a-wave model. A double-flash paradigm was used to study inactivation mechanisms. The first flash was achromatic and varied in intensity (I(f)) from 6.1 to 13.9 ln (2.6 to 6.0 log) scot td-sec. The second flash was a short-wavelength probe held constant at 9.3 ln (4.0 log) scot td-sec. Cone components were elicited with a photopically matched long-wavelength stimulus and were computer subtracted. Recovery at each I(f) was followed by measuring the amplitude to the probe flash at various interstimulus intervals (ISI). The critical time (Tc) before the initiation of rod recovery was determined from the function relating relative rod amplitude to ISI.. Recovery from activation was similar in normal subjects and in patients with rod monochromatism. Over a large range of I(f) above rod saturation, Tc increased in proportion to ln I(f). The mean slope of the function relating Tc to I(f) was 2.3 s/ln I(f) when I(f) varied between 11 and 13.9 ln scot td-sec. Patients with retinitis pigmentosa and the pro-23-his rhodopsin mutation had a decrease in the gain of activation. They also had significantly slower than normal recovery after high test flash intensities, such that the slope of the function relating Tc to ln I(f) was 12.1 seconds.. Available data from other species imply that complete, transient activation of transducin (T saturation) occurs within or below the investigated range of flash intensities. Based on the slope of the delay function (delta Tc/ delta ln I(f)) above 11 ln scot td-sec, the authors hypothesize that the lifetime of activated rhodopsin (R) in normal human rods is approximately 2.3 seconds. In patients with the pro-23-his mutation, the gain of the activation mechanism is reduced and the reaction determining the delta Tc/ delta ln I(f) slope is markedly slowed. The activated species that exhibits this prolonged lifetime could be the mutant rhodopsin itself.

Topics: Adolescent; Adult; Dark Adaptation; Electroretinography; Female; Histidine; Humans; Male; Middle Aged; Mutation; Photic Stimulation; Proline; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; Structure-Activity Relationship

Topics: Alanine; Amino Acid Sequence; Base Sequence; DNA; Female; Genes, Dominant; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Proline; Retinitis Pigmentosa; Rhodopsin

To determine whether severity of retinitis pigmentosa caused by dominant rhodopsin mutations depends on the location altered by the mutation.. Data from 128 patients (age range, 7 to 73 years), each with 1 to 27 rhodopsin mutations, were analyzed. To approximate normal distributions, visual acuities were converted to ranks and then to the normal form, kinetic visual fields to a V4e test light were converted to equivalent diameters, and dark-adapted sensitivities to an 11 degrees diameter stimulus and electroretinogram (ERG) amplitudes to full-field 0.5-Hz and 30-Hz flashes were converted to common logarithms. Each of these measures was then regressed on age, refractive error (for the ERG), and domain (intradiscal, transmembrane, or cytoplasmic) or codon number of the opsin molecule altered by the mutation.. All five measures of function varied significantly with the domain (P < or = 0.0007) or codon number (P < 0.0001) altered by a mutation; visual acuity, visual field diameter, dark-adapted sensitivity, and ERG amplitudes were highest for mutations altering the intradiscal domain or low-numbered codons and lowest for mutations altering the cytoplasmic domain or high-numbered codons.. These data indicate that severity of disease correlates with the location of the amino acid residue altered by a rhodopsin mutation in dominant retinitis pigmentosa.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Child; Codon; Dark Adaptation; Electroretinography; Humans; Middle Aged; Molecular Sequence Data; Mutation; Proline; Retina; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship; Visual Acuity; Visual Fields

In animal models for retinitis pigmentosa (RP), rod photoreceptors show abnormal distribution of rhodopsin prior to undergoing cell death. To elucidate the steps in degeneration of human photoreceptors, immunocytochemistry was performed on donor retinas from 15 RP patients and five normal subjects. Rhodopsin immunolabeling in the normal retinas was restricted to the rod outer segments. In the RP retinas, rhodopsin was present in shortened rod outer segments and in the surface membranes of the rod inner segments and somata. In regions of photoreceptor death, the surviving rods had sprouted rhodopsin-positive neurites that were closely associated with gliotic Müller cell processes and extended to the inner limiting membrane. Rods and cones in the RP maculas did not form neurites, but the axons of peripheral cones were abnormally elongated and branched. Double immunofluorescence labeling showed that the rod neurites bypassed the horizontal and rod bipolar cells that are normally postsynaptic to rod axons. To our knowledge, this is the first report of rod neurite sprouting in vivo. We were unable to find neurites on degenerate rods in old rds mice, an animal model for RP. The rod neurites in the human RP retinas resemble the long, branched processes formed by rods cultured on Müller cells or purified N-CAM. Neurite growth by surviving rods in the RP retinas may be a response to neurotrophic factor upregulation, loss of inhibitory factors, or changes in molecules associated with reactive Müller cells. Such changes in the retinal microenvironment may impede functional integration of transplanted photoreceptors. The contributions of the rhodopsin-positive rod neurites and abnormal cone axons to the functional abnormalities observed in RP are unknown.

Topics: Adult; Aged; Aged, 80 and over; Animals; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Mutant Strains; Microscopy, Electron; Middle Aged; Neurites; Reference Values; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Topics: Base Sequence; Gene Frequency; Humans; Japan; Molecular Sequence Data; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

Topics: Alleles; Amino Acid Sequence; Animals; Biological Transport; Cloning, Molecular; Drosophila; Electroretinography; Endoplasmic Reticulum; Ethyl Methanesulfonate; Genes, Dominant; Molecular Sequence Data; Mutagenesis; Photoreceptor Cells; Photoreceptor Cells, Invertebrate; Point Mutation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

To determine the molecular basis of an early and severe form of autosomal dominant retinitis pigmentosa and to characterize the associated phenotype.. Visual function evaluation included electrophysiologic and psychophysical testing. Molecular genetic analysis included determining the DNA sequence of sections of the rhodopsin gene amplified by polymerase chain reaction and screening for changes single-nucleotide by allele-specific oligonucleotide hybridization.. Affected family members are heterozygous for a unique Cys187Tyr rhodopsin mutation which disrupts a highly conserved disulfide bond essential to normal rhodopsin function. The retinitis pigmentosa (RP) phenotype includes early and severe retinal dysfunction. The full-field electroretinogram showed only negligible remaining rod and cone responses by 22 years of age. Visual fields were constricted severely by early middle-age years. Macular dysfunction caused reduced visual acuity in early adult years, and macular atrophy was present in older age. The severity of phenotype generally correlated with age, with the exception of an affected 44-year-old patient who had better visual acuity, fields, electroretinogram, and dark-adapted thresholds than did three younger affected relatives, ranging in age from 22 to 38 years.. An early onset, blinding form of autosomal dominant RP results from a rhodopsin Cys187Tyr mutation that eliminates a residue necessary for the formation of a highly conserved disulfide bond essential to normal rhodopsin function. The fact that one family member is significantly less affected than his younger relatives suggests that genetic or environmental factors can modulate the phenotype.

Topics: Adolescent; Adult; Amino Acid Sequence; Base Sequence; Child; Cysteine; Dark Adaptation; Disulfides; DNA Primers; Electroretinography; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Phenotype; Point Mutation; Retina; Retinitis Pigmentosa; Rhodopsin; Tyrosine; Visual Fields

Two autosomal dominant retinitis pigmentosa families of different origin were screened for rhodopsin mutations using the method of single strand conformation polymorphism and direct sequencing. We found a CGG-CAG substitution in codon 114 of rhodopsin in both families. This change predicted the replacement of a glycine by an aspartic acid and suggested that this change is the cause of the disease in these families.

Topics: Adolescent; Adult; Amino Acid Sequence; Aspartic Acid; Base Sequence; Codon; Female; Genes, Dominant; Glycine; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Point Mutation; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

Abnormalities in lipid metabolism have been reported in numerous patients with retinitis pigmentosa. As an initial step in evaluating these anomalies, two trials of fatty acid intervention were conducted with autosomal dominant retinitis pigmentosa (adRP) patients and controls. The first trial addressed absorption and incorporation of omega 3 long-chain fatty acids from a fish-oil concentrate into red blood cell (RBC) lipids. The utilization of omega 3 long-chain fatty acids by adRP patients was found to be equivalent to that of controls. The second trial addressed the conversion of precursor, eicosapentaenoic acid (EPA, 20:5 omega 3), to end-product, docosahexaenoic acid (DHA, 22:6 omega 3), following oral supplementation of EPA ethyl ester. Although the levels of EPA and the intermediate, docosapentaenoic acid (22:5 omega 3), were both elevated by EPA supplementation in RBCs of adRP patients with rhodopsin gene mutations and controls, DHA production was elevated only in controls. Based on these results, we suggest the presence of a metabolic defect in the final stages of DHA biosynthesis.

Topics: Adult; Docosahexaenoic Acids; Eicosapentaenoic Acid; Electroretinography; Erythrocytes; Fatty Acids, Omega-3; Fatty Acids, Unsaturated; Female; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Retina; Retinitis Pigmentosa; Rhodopsin

To evaluate the consequences of the expression of a mutant mouse opsin gene on rod- and cone-mediated function. Experimental conditions were chosen to provide a basis of comparison to the results reported for patients with autosomal dominant retinitis pigmentosa (ADRP) in whom the proline at position 23 has been replaced by a histidine (P23H).. The mutated gene product resulted in three substitutions in the rhodopsin molecule: P23H, glycine for valine at position 20 (V20G), and leucine for proline at position 27 (P27L). Mice positive for the transgene were differentiated from normal littermates by the polymerase chain reaction. Electroretinograms (ERGs) were obtained from anesthetized mice between 1 and 9 months of age. After photically bleaching approximately 18% of the available rhodopsin, the time course of rod dark adaptation was examined by monitoring rod ERG amplitude recovery. Rhodopsin densitometry was used to determine the relative amounts of rhodopsin in the retinae of normal and transgenic mice.. ERGs obtained from transgenic mice showed a significant reduction in rod-mediated response amplitude at 1 month of age and a relatively slow progressive decrease thereafter. Cone-mediated ERGs, on the other hand, were nearly normal in amplitude for approximately the first 5 months after birth, but at later ages response amplitudes also underwent a progressive decline. In the normal retina, rod ERG amplitudes returned to prebleach levels within 30 minutes, whereas in transgenic mice response amplitudes did not recover within a 2-hour test period. The age-related decline in rod-mediated electroretinal potentials seen in transgenic mice was paralleled by a concomitant fall in rhodopsin density, and the sensitivity losses obtained electroretinographically could be accounted for solely on the basis of reduced quantal absorption.. The pattern of functional changes seen in the transgenic mice are in good agreement with those reported in patients with ADRP with the P23H mutation in the rhodopsin gene. Particularly noteworthy is the fact that the changes in rhodopsin density and visual sensitivity are associated with a progressive shortening of the rod outer segments; the histologic changes induced by the disease process in patients with ADRP have yet to be determined.

Topics: Animals; Dark Adaptation; Densitometry; Disease Models, Animal; Electroretinography; Gene Expression; Mice; Mice, Mutant Strains; Mice, Transgenic; Mutation; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

We identified genetic mutations and characterized their associated phenotypes in patients with retinitis pigmentosa.. Patients with retinitis pigmentosa were prospectively examined and screened for genetic mutations.. A 46-year-old man with retinitis pigmentosa was found to have a heterozygous mutation in the peripherin/RDS gene (arginine-46-stop). He had late onset of symptoms and demarcated peripheral retinal atrophy. All five first-degree relatives including his parents had no detectable mutations or retinitis pigmentosa. Genotypic data were consistent with reported family structure.. This study shows that new dominant mutations are a rare cause of isolated, or simplex, cases of retinitis pigmentosa. Identification of these mutations is helpful for genetic counseling.

Topics: Adult; Amino Acid Sequence; Arginine; Base Sequence; Codon; DNA; Electroretinography; Fundus Oculi; Genes, Dominant; Humans; Intermediate Filament Proteins; Male; Membrane Glycoproteins; Middle Aged; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Neuropeptides; Pedigree; Peripherins; Polymerase Chain Reaction; Prospective Studies; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Heterozygous missense mutation in codon 15 of the rhodopsin gene was detected in a patient with autosomal dominant retinitis pigmentosa (ADRP), where a transition of adenine to guanine at the second nucleotide in codon 15 (AAT-->AGT), corresponding to a substitution of serine residue for asparagine residue (Asn-15-Ser) was detected. None of the remaining unrelated 42 ADRP, 24 autosomal recessive RP (ARRP) and 34 normal individuals had this alteration. Her funduscopic findings were sectorial in type similar to that of the patients with the same mutation found in an Australian pedigree (Sullivan et al., 1993). This study shows phenotypic similarities in patients with the same mutation of a different ancestry.

Topics: Amino Acid Sequence; Base Sequence; Codon; Female; Humans; Japan; Male; Middle Aged; Molecular Sequence Data; Pedigree; Point Mutation; Retinitis Pigmentosa; Rhodopsin

We analyzed 11 sites of the rhodopsin gene using polymerase chain reaction (PCR) amplification and restriction endonucleases in 30 unrelated Japanese patients with autosomal dominant retinitis pigmentosa (ADRP). No point mutation was found in any patient. The frequencies of the single nucleotide (nt) substitution at nt 269, nt 5145 and nt 5321 were examined in three groups, 38 unrelated patients with ADRP, 23 patients with autosomal recessive retinitis pigmentosa (ARRP), and 67 normal controls. There was no significant difference in the frequencies of substitution among these three groups. The frequencies of A269G, G5145A, and C5321A were 52%, 36%, and 5%, respectively. These values were different from those of the American population. The polymorphisms, A269G and G5145A, are useful as DNA makers for linkage analysis.

Topics: Asian People; Base Sequence; DNA; DNA Restriction Enzymes; Gene Frequency; Humans; Japan; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

Recent reports have linked numerous point mutations in the human genome to retinitis pigmentosa (RP), suggesting that in the near future molecular biology based genetic counselling for patients with RP might become a reality. In this paper we assess the viability of a DNA-based screening method for RP. Specifically, we screened rhodopsin and the beta-subunit of phosphodiesterase (B-PDE) genes for the presence of abnormalities in West Australian RP patients.. Blood was collected from 27 patients. Leukocyte DNA was extracted from patients and 50 randomly selected controls. Exons 1-19 and 21 of B-PDE and exons 1, 3 and 5 of rhodopsin were analysed using single-strand conformational polymorphism (SSCP) following DNA amplification. The nature of anomalies detected by SSCP was classified with DNA sequencing.. In RP patient samples, we found anomalous bands in exons 5, 9, 10, 15 and 16 and 17 of B-PDE and in exons 1 and 3 of rhodopsin genes. In B-PDE none of the anomalous bands represented mutations. Some of the anomalous bands in the rhodopsin gene, however, corresponded to silent mutations at nucleotide positions 269 and 3982 in four patients.. In this study we have demonstrated that SSCP in combination with DNA sequencing is a powerful tool to identify new mutations and to provide information for a 'mutational panel' for future screening.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cyclic Nucleotide Phosphodiesterases, Type 6; DNA; DNA Mutational Analysis; DNA Primers; Gene Amplification; Humans; Mutation; Phosphoric Diester Hydrolases; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Sequence Analysis, DNA; Western Australia

Over 45 mutations in the rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa, including a cluster near the extreme carboxy-terminus, a region of the protein for which no function has yet been assigned. To elucidate the biochemical defect(s) in this group of mutants, we have studied a naturally occurring stop codon mutation that removes the last five amino acids of rhodopsin (Q344ter). When produced in transfected tissue culture cells, the mutant protein is indistinguishable from the wild type in light-dependent activation of the photoreceptor G-protein (transducin), and in serving as a light-dependent substrate for rhodopskin kinase. Mice that express a Q344ter transgene in rod photoreceptors show nearly normal light responses as determined by suction electrode recordings of the membrane current from single rod outer segments; the main difference between transgenic and nontransgenic responses is a 15% longer time-to-peak in the response of transgenic rods. In the Q344ter transgenic retina, direct immunofluorescent staining with antibodies specific for either wild-type or Q344ter rhodopsin shows abnormal accumulation of the Q344ter, but not the endogenous rhodopsin, in the plasma membrane of the photoreceptor cell body. These data indicate that rhodopsin's carboxy-terminus is required for efficient transportation to or retention in the outer segment.

Topics: Animals; Base Sequence; Cell Membrane; Codon, Terminator; DNA Mutational Analysis; DNA, Complementary; Fluorescent Antibody Technique; Gene Amplification; Gene Expression; Humans; Mice; Mice, Transgenic; Microscopy, Confocal; Molecular Sequence Data; Point Mutation; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA; Transducin

Retinitis pigmentosa (RP) is the name given to a group of disorders, both clinically and genetically heterogeneous, that primarily affect the photoreceptor function of the eye. Mutations in the genes encoding for rhodopsin, RDS-peripherin, or the beta subunit of the cGMP phosphodiesterase enzyme can be responsible for the phenotype. In this study the rhodopsin gene has been screened for mutations in a panel of RP individuals and five different sequence changes have been detected to date in three dominantly inherited and two unclassified families. One of these, a base substitution in the 3'UTR, has not yet been confirmed as disease specific, while three missense substitutions have previously been reported and are likely to be responsible for the phenotype. The fifth change, a base substitution at the intron 4 acceptor splice site, represents a novel mutation and is assumed to be the causative mutation.

Topics: Base Sequence; Exons; Female; Humans; Introns; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Phenotype; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin; Scotland

A mother and daughter with autosomal dominant retinitis pigmentosa (adRP) were found to carry a cytosine-to-adenine transversion mutation at codon 4 of the rhodopsin gene. This mutation predicts a substitution of lysine for threonine at one of the glycosylation sites in the rhodopsin molecule (Thr4Lys). Both patients presented with a similar phenotype including a tigroid pattern of the posterior pole and a regional predilection for degenerative pigmentary changes in the inferior retina with corresponding visual field defects. The electroretinographic pattern was suggestive of RP of the cone-rod type. This report documents the clinical findings associated with this defined mutation of the rhodopsin gene.

Topics: Adult; Codon; Electroretinography; Female; Fundus Oculi; Humans; Lysine; Middle Aged; Netherlands; Pedigree; Photoreceptor Cells; Point Mutation; Retinitis Pigmentosa; Rhodopsin; Threonine; Vision Tests; Visual Fields

The PCR/restriction endonuclease digestion (RE) assay and PCR/SSCP analysis of the rhodopsin gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ad RP) revealed a G-A substitution of the first nucleotide of codon 181, replacing Glu (GAG) with Lys (AAG), in one family. The proband showed an early onset of symptoms in childhood with a diffuse loss of rod and cone function and a relatively good preservation of cone function, corresponding to the type with relatively rapid progression to blindness (type I category of ad RP).

Topics: Adult; Base Sequence; Codon; DNA Mutational Analysis; Female; Fluorescein Angiography; Fundus Oculi; Glutamic Acid; Humans; Japan; Lysine; Molecular Sequence Data; Photoreceptor Cells; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Fields

The retinas from a 68-year-old man with autosomal dominant retinitis pigmentosa caused by the threonine-17-methionine rhodopsin mutation were studied. Patients with this mutation have a characteristic clinical phenotype that shows intra- and interfamilial consistency. The retinas were examined by light and electron microscopy, including immunocytochemistry with markers for rods, cones, the retinal pigment epithelium, the interphotoreceptor matrix and Müller cells, and the results were correlated with those from visual function tests performed fourteen months before death. Grossly, both retinas had heavy deposits of bone spicule-like pigmentation inferiorly, but the superior retinas showed much less pigmentation. The maculas contained no rods and an incomplete monolayer of cone somata, consistent with the patient's severely reduced central vision. The inferior retinas contained no rods and rare cone somata, correlating with an absolute scotoma in the superior field. The superior retinas had near-normal-appearing rods and cones in the far periphery and a gradient from the midperipheral to central retina of progressively shortened outer segments and loss of photoreceptors, consistent with the patient's reduced rod and cone electroretinograms and retained rod and cone sensitivities in the inferior peripheral field by perimetry. Immunocytochemistry with rod-specific markers was qualitatively normal in the superior mid to far peripheral retina. Electron microscopic immunogold labeling with anti-rhodopsin revealed similar densities of gold particles over rod outer segments of the RP and a normal donor retina. Degenerate photoreceptors in the superior equatorial region of the RP retina had short outer segments, and some rods had surface labeling with anti-rhodopsin of the inner segments, somata and synapses. The results indicate regional retinal differences of disease severity in this genotype and that rods in the superior peripheral retina have normal morphology despite the presence of the mutant allele. To the authors' knowledge, this is the first detailed study of rod photoreceptors in the retina of an RP patient with a rhodopsin mutation.

Topics: Aged; Fluorescent Antibody Technique; Genes, Dominant; Humans; Male; Methionine; Microscopy, Electron; Microscopy, Immunoelectron; Mutation; Photoreceptor Cells; Retina; Retinitis Pigmentosa; Rhodopsin; Threonine; Vision, Ocular

Mutations of the gene encoding rhodopsin are responsible for 30% of the cases autosomal dominant retinitis pigmentosa. Rhodopsin molecules are key G-coupled transmembrane proteins initiating the visual transduction cascade in rods. These cells are specialized retinal cells allowing the detection of low intensity lights. Mutations in the rhodopsin gene lead to the progressive destruction of rods, clinically translated by night blindness, a progressive bilateral loss of the peripheral visual field, and predominant alterations of the rod component of the electroretinograms. Inherited colour vision deficiencies are mainly caused by alterations of the genes encoding coloured opsins. These proteins are G-coupled receptors specialized in visual transduction in the cones. These sensorial cells are localized in the center of the neural retina where they allow the detection of refined details and chromatic lights. Rearrangements of the genes encoding the green and the red color pigments are responsible for daltonism.

Topics: Color Vision Defects; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous retinopathies, some of which have been shown to result from mutations in two different known retinal genes, rhodopsin (3q) and peripherin-rds (6p). Three additional anonymous loci at 7p, 7q and pericentric 8 have been implicated by linkage studies. There are still, however, a few families in which all known loci have been excluded. In this report we present data indicating a location, on the short arm of chromosome 17, for the autosomal dominant RP (ADRP) locus in a large South African (SA) family of British ancestry. Positive two-point lod scores have been obtained for nine markers (D17S938, Z = 5.43; D17S796, Z = 4.82; D17S849, Z = 3.6; D17S786, Z = 3.55; TP53, Z = 3.55; D17S578, Z = 3.29; D17S960, Z = 3.16; D17S926, Z = 1.51; D17S804, Z = 0.47 all at theta = 0.10 except D17S804 and D17S926, theta = 0.20). These data provide definitive evidence for the localization of an ADRP gene on chromosome 17p. The human recoverin gene has been localized to 17p13.1 and was consequently a prime candidate for ADRP in the family studied. However, mutation screening of the three exons of this gene failed to produce any evidence of recoverin being the gene involved in the pathogenesis of ADRP in this SA family.

Topics: Calcium-Binding Proteins; Chromosome Mapping; Chromosomes, Human, Pair 17; DNA; Exons; Eye Proteins; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Hippocalcin; Humans; Intermediate Filament Proteins; Lipoproteins; Lod Score; Male; Membrane Glycoproteins; Nerve Tissue Proteins; Pedigree; Peripherins; Recoverin; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; South Africa

Topics: Base Sequence; Codon; Genes; Genes, Dominant; Humans; Molecular Sequence Data; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Adult; Base Sequence; Codon; Genes, Dominant; Humans; Molecular Sequence Data; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Codon; Genes, Dominant; Genotype; Humans; Point Mutation; Polymorphism, Single-Stranded Conformational; Retinitis Pigmentosa; Rhodopsin

Topics: Genes, Recessive; Humans; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

A lysine to glutamic acid substitution at codon 296 in the rhodopsin gene has been reported in a family with autosomal dominant retinitis pigmentosa. This mutation is of particular functional interest as this lysine molecule is the binding site of 11-cis-retinal. The clinical features of a family with this mutation have not been reported previously. We examined 14 patients with autosomal dominant retinitis pigmentosa and a lysine-296-glutamic acid rhodopsin mutation. Four had detailed psychophysical and electrophysiological testing. Most affected subjects had severe disease with poor night vision from early life, and marked reduction of visual acuity and visual field by their early forties. Psychophysical testing showed no demonstrable rod function and severely reduced cone function in all patients tested.

Topics: Adolescent; Adult; Aged; Binding Sites; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Genes, Dominant; Humans; Lysine; Male; Middle Aged; Mutation; Night Blindness; Pedigree; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Vision Disorders; Visual Fields

To characterize the pedigree, visual function phenotype, and responsible mutation in a large family with autosomal dominant retinitis pigmentosa.. Pedigree data were obtained by personal interviews and corroborated with community records. One hundred twenty-eight members of the family were examined clinically, and a subset of 12 affected subjects was further studied with dark- and light-adapted static perimetry and electroretinography. The coding region of the rhodopsin gene was polymerase chain reaction (PCR) amplified and resolved by denaturing gradient gel electrophoresis. Genomic DNA samples from nine affected and five unaffected family members were analyzed by PCR amplification and restriction enzyme digestion.. A 14-generation pedigree was identified in which retinitis pigmentosa (RP) was inherited in an autosomal dominant fashion. Affected individuals reported early night blindness and showed vessel attenuation and bone spicule-like pigmentary changes. In these individuals, the rod electroretinogram (ERG) was not detectable, and the cone ERG was reduced in amplitude and delayed in timing. With dark-adapted perimetry, rod function could be detected in only one young patient, and it was markedly abnormal. Light-adapted perimetry indicated that cone sensitivity could be relatively well preserved in the central field, but it was diminished in the periphery even in the most mildly affected subjects. A valine345-to-leucine mutation was identified in the rhodopsin gene and shown to cosegregate in the heterozygous condition with the disease.. The natural history of RP in this family begins with a loss of rod function, progresses to involve the cone system, and leads eventually to a severe loss of visual function. The invariance of valine345 in all functional vertebrate visual pigments sequenced to date, and the unusually conservative nature of the valine345-to-leucine mutation suggests that the carboxy terminus of rhodopsin is involved in a highly specific interaction with one or more rod proteins.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Dark Adaptation; DNA Mutational Analysis; Electroretinography; Female; Humans; Leucine; Male; Molecular Sequence Data; Pedigree; Photoreceptor Cells; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Valine; Visual Field Tests

To investigate functional abnormalities in mutations in the peripherin (RDS) gene leading to different clinical types of autosomal dominant retinal disease--macular degeneration and retinitis pigmentosa.. Patients from two families, one with a mutation in codon 167 (Gly167Asp) leading to macular degeneration and another with a mutation in codon 210 (Pro210Ser) leading to retinitis pigmentosa, were studied with clinical examinations and measurements of rod and cone sensitivities and dark adaptation, electroretinography, and rhodopsin levels.. Mildly affected patients had sizable rod and cone electroretinograms, reduced levels of rhodopsin, and minor losses of sensitivity. In both mutations, there were delays of rod and cone dark adaptation after bleaching, and the adaptational abnormalities were observed in peripheral and central retinal locations. Analysis of the kinetics of rod adaptation indicates that the underlying abnormalities are similar in both mutations and that the effects of the mutations are similar to those caused by mild systemic vitamin A deficiency.. Patients with the Gly167Asp and Pro210Ser mutations in the peripherin/RDS gene have widely different clinical phenotypes but show the same abnormality, slowed dark adaptation, of rod and cone photoreceptor function. The similarities of the characteristics of the adaptational abnormalities in the two genotypes suggest that, in addition to the structural roles normally assumed for it, peripherin influences or participates in the function of the visual cycle.

Topics: Adolescent; Adult; Dark Adaptation; Electroretinography; Eye Proteins; Female; Fundus Oculi; Humans; Intermediate Filament Proteins; Macular Degeneration; Male; Membrane Glycoproteins; Middle Aged; Mutation; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal degenerations that can be autosomal dominant (ADRP), autosomal recessive (ARRP), or X-linked. Approximately 30% of ADRP patients show point mutations or small deletions in the rhodopsin gene. However, over 50% of the RP patients are simplex cases (sporadic). Screening for mutations in the rhodopsin gene of 33 patients with simplex RP by denaturing gradient gel electrophoresis (DGGE) was carried out. One patient, with D-type (diffuse) RP and consanguineous parents, showed an altered electrophoretic pattern for the 5' half of exon 1. Direct sequencing revealed a new mutation ATG to ACG in codon 44; this predicts a change of Met-44-Thr in rhodopsin. The position and amino acid substitution suggest that this mutation causes the RP phenotype. Implications for genetic counselling are discussed.

Topics: Adolescent; Aged; DNA Mutational Analysis; Electrophoresis, Agar Gel; Exons; Female; Humans; Male; Methionine; Middle Aged; Pedigree; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Threonine

Topics: Arginine; Genes, Dominant; Humans; Leucine; Lysine; Methionine; Point Mutation; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Base Sequence; Codon; Female; Genes, Dominant; Humans; Male; Pedigree; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Recently, mutations of the active site Lys296 residue in rhodopsin (Lys296-->Glu and Lys296-->Met) have been found as the cause of disease in some patients with autosomal dominant retinitis pigmentosa. In vitro, these mutations result in constitutive activation of the protein. In an effort to develop a potential therapeutic agent for treatment of the disease, we have examined various amine derivatives of 11-cis- and 9-cis-retinal for ability to irreversibly inactivate a related constitutively active mutant, K296G. Three amines were prepared by reductive amination of retinal: 11-cis-retinylpropylamine, 11-cis-retinylamine, and 9-cis-retinylamine. All three compounds inactivated K296G, and the inactivation could not be reversed upon exposure to light. None of the compounds inactivated the wild-type protein. Although the amines were not effective on the naturally occurring retinitis pigmentosa mutants, presumably because of unfavorable steric interactions with the bulky Glu and Met side chains at position 296, the success with K296G makes it highly encouraging that this approach will evolve related compounds that are capable of inactivating the naturally occurring mutants as well.

Topics: Amines; Amino Acid Sequence; Animals; Binding Sites; Cattle; Humans; Kinetics; Lysine; Molecular Structure; Point Mutation; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship; Transducin

Topics: Amino Acid Sequence; Arginine; Base Sequence; DNA; Exons; Female; Genes, Dominant; Histidine; Humans; Male; Molecular Sequence Data; Pedigree; Point Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Spain; Threonine

Topics: Adult; Amino Acid Sequence; Base Sequence; DNA Transposable Elements; Exons; Female; Genes, Dominant; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein.. Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels.. There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes.. There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Topics: Adolescent; Adult; Aged; Child; Codon; Dark Adaptation; Electroretinography; Female; Humans; Male; Middle Aged; Mutation; Phenotype; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; RNA Splicing; Sensory Thresholds; Visual Field Tests; Visual Fields

Autosomal dominant retinitis pigmentosa (ADRP) is a hereditary form of retinitis pigmentosa which accounts for about 15% of all types of the latter disease. Recently, close to 50 mutations, mostly point mutations, have been identified in the rhodopsin gene in ADRP patients. We have introduced these mutations in the synthetic bovine rhodopsin gene and herein report on the expression of the mutant genes in COS-1 cells and studies in vitro of the properties of the expressed opsins. The mutant phenotypes fall into three classes: Class I mutants are expressed in COS-1 cells at wild-type levels, form the normal rhodopsin chromophore with 11-cis-retinal, and are transported to the cell surface. However, on illumination, they activate transducin inefficiently. Class II mutants remain in the endoplasmic reticulum and do not bind 11-cis-retinal to form the chromophore. Class III mutants are expressed at low levels and form rhodopsin chromophore only poorly. They also remain in the endoplasmic reticulum and, as expected, show high mannose glycosylation. Nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently. We conclude that the majority of the ADRP mutants have folding defects.

Topics: Amino Acid Sequence; Animals; Biological Transport; Cattle; Cell Line; Cell Membrane; Endoplasmic Reticulum; Gene Transfer Techniques; Glycosylation; Mannose; Molecular Sequence Data; Mutagenesis; Photochemistry; Point Mutation; Protein Folding; Protein Structure, Secondary; Retinaldehyde; Retinitis Pigmentosa; Rhodopsin; Structure-Activity Relationship

Retinitis pigmentosa (RP) is a group of inherited human diseases in which photoreceptor degeneration leads to visual loss and eventually to blindness. Although mutations in the rhodopsin, peripherin, and cGMP phosphodiesterase genes have been identified in some forms of RP, it remains to be determined whether these mutations lead to photoreceptor cell death through necrotic or apoptotic mechanisms. In this paper, we report a test of the hypothesis that photoreceptor cell death occurs by an apoptotic mechanism in three mouse models of RP: retinal degeneration slow (rds) caused by a peripherin mutation, retinal degeneration (rd) caused by a defect in cGMP phosphodiesterase, and transgenic mice carrying a rhodopsin Q344ter mutation responsible for autosomal dominant RP. Two complementary techniques were used to detect apoptosis-specific internucleosomal DNA fragmentation: agarose gel electrophoresis and in situ labeling of apoptotic cells by terminal dUTP nick end labeling. Both methods showed extensive apoptosis of photoreceptors in all three mouse models of retinal degeneration. We also show that apoptotic death occurs in the retina during normal development, suggesting that different mechanisms can cause photoreceptor death by activating an intrinsic death program in these cells. These findings raise the possibility that retinal degenerations may be slowed by interfering with the apoptotic mechanism itself.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Apoptosis; Disease Models, Animal; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Mutant Strains; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

A 49-year-old Japanese man had autosomal dominant retinitis pigmentosa with a point mutation in codon 17 of the rhodopsin gene, resulting in a threonine-to-methionine change, and retinal neovascularization in both eyes. Pigmentary degeneration mainly in the inferior area of the fundus, and severe loss in the upper portion of the visual field were observed. Moderately preserved rod and cone functions were demonstrated by electroretinograms. These findings differed from those of Japanese and white patients with autosomal dominant retinitis pigmentosa with a codon 347 mutation and were almost the same as those of white patients with the codon 17 mutation. Our study indicates that phenotypic similarities exist among patients with the same mutation, but of different racial backgrounds. The neovascularization in the right eye diminished over a two-year period in conjunction with the progression of retinal degeneration.

Topics: Adaptation, Ocular; Base Sequence; Codon; DNA; Electroretinography; Female; Fluorescein Angiography; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Neovascularization, Pathologic; Oligonucleotide Probes; Pedigree; Phenotype; Polymerase Chain Reaction; Retinal Degeneration; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin

Topics: Humans; Retinitis Pigmentosa; Rhodopsin

Twenty five symptomatic individuals and six asymptomatic obligate gene carriers from four families with autosomal dominant retinitis pigmentosa (adRP) showing apparent incomplete penetrance have been studied. Symptomatic individuals from three families showed early onset of night blindness, non-recordable rod electroretinograms, and marked elevation of both rod and cone thresholds in all subjects tested. In the fourth family, there was more variation in the age of onset of night blindness and some symptomatic individuals showed well preserved rod and cone function in some retinal areas. All asymptomatic individuals tested had evidence of mild abnormalities of rod and cone function, indicating that these families show marked variation in expressivity rather than true non-penetrance of the adRP gene. No mutations of the rhodopsin or RDS genes were found in these families and the precise genetic mutation(s) remain to be identified.

Topics: Adolescent; Adult; Chromosomes, Human, Pair 7; Electrophysiology; Electroretinography; Female; Genetic Linkage; Heterozygote; Humans; Male; Middle Aged; Night Blindness; Pedigree; Photoreceptor Cells; Psychophysics; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Visual Fields

A family is described in which an 8 base pair deletion (nucleotides 5252-5259, codons 341-343) of the rhodopsin gene cosegregates with autosomal dominant retinitis pigmentosa (adRP). The deletion results in a shift in the reading frame, causing a rhodopsin molecule extended by one residue and substantially altered at the carboxyl terminus. Phenotypic expression is relatively mild. In affected members, night blindness did not occur before the age of 16, and late onset of visual field loss was consistently reported. Even older individuals (59 and 76 years) had preserved central islands in the visual field; a younger female patient had normal visual fields until the age of 34. ERG and psychophysical tests showed well preserved cone function at stages of virtually abolished rod function. Phenotypic differences and similarities between this form of adRP and others associated with mutations at the carboxyl terminus of the rhodopsin molecule are discussed. The cause of RP by mutations in this region remains to be clarified.

Topics: Adult; Aged; Amino Acid Sequence; Base Sequence; Carboxylic Acids; Child, Preschool; Electroretinography; Female; Fluorescein Angiography; Frameshift Mutation; Fundus Oculi; Gene Deletion; Humans; Male; Middle Aged; Molecular Sequence Data; Night Blindness; Pedigree; Phenotype; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Vision Disorders; Visual Fields

To ascertain and characterize rhodopsin gene mutations in autosomal dominant retinitis pigmentosa and to correlate these mutations with the clinical phenotypes.. DNA was extracted from leukocytes, and the rhodopsin gene was amplified and analyzed using molecular-biological methods. Clinical and electrophysiological data were collected from patient charts.. We found a disease-causing mutation that was previously undescribed, to our knowledge, for autosomal dominant retinitis pigmentosa within codon 15 of exon 1 of the rhodopsin gene. It was a single base-pair transversion (AAT to AGT) leading to a serine-for-asparagine substitution. This altered a glycosylation site in the intradiscal portion of the rhodopsin molecule. The pedigree examined demonstrated an inferior distribution of retinal pigmentary changes and predominantly superior visual field loss with relative preservation of electroretinographic amplitudes and good vision, which is consistent with sectorial or sectorial-like retinitis pigmentosa.. A codon 15 rhodopsin gene mutation caused retinitis pigmentosa in the pedigree studied. There may be an association between intradiscal rhodopsin gene mutations and sectorial forms of retinitis pigmentosa.

Topics: Adult; Aged; Base Sequence; Chromosome Aberrations; Chromosome Disorders; Chromosomes, Human, Pair 3; Codon; DNA Primers; Electroretinography; Female; Fundus Oculi; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Visual Fields

To determine the phenotypes of two families in which retinitis pigmentosa cosegregates with a rhodopsin (RHO) gene mutation: a leucine-to-arginine change at codon 40 (Leu-40-Arg) in one family, and a 150-base pair insertion that disrupts the RHO 5'-splice junction of exon 5 in another.. Three affected members of each family.. The Leu-40-Arg mutation was associated with the onset of night blindness in the first decade of life. By the fourth decade, severe retinal functional loss was evident on dark-adapted static threshold perimetry, and electroretinographic responses were absent or barely detectable. In contrast, the RHO 150-base pair insertion was associated with the later onset of mild night vision difficulties; in two individuals, mild night vision difficulties were first noticed in the second decade while a third, a 25-year-old woman, was asymptomatic. Dark-adapted static threshold perimetry of this latter individual revealed a "regional" or class 2 pattern of retinal functional loss associated with equal loss of rod and cone electroretinographic responses.. The RHO Leu-40-Arg mutation causes symptomatic retinal dysfunction by the end of the first decade while the insertion disrupting the 5'-splice junction of RHO exon 5 causes later onset "regional" or class 2 retinal dysfunction.

Topics: Adult; Arginine; Dark Adaptation; Electroretinography; Exons; Female; Fundus Oculi; Humans; Leucine; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; RNA Splicing; Visual Acuity; Visual Fields

Over 40 mutations in the rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa. Twenty-one of these mutations have been introduced into a human rhodopsin cDNA by site-directed mutagenesis, and the encoded proteins have been produced by transfection of a human embryonic kidney cell line (293S). Three of the mutant proteins (G51V, V345M, and P347S) resemble the wild type in yield, regenerability with 11-cis-retinal, and accumulation in the plasma membrane (class I). The remaining 18 mutant proteins are produced at lower levels, regenerate variably or not at all with 11-cis-retinal, and accumulate partially or predominantly in the endoplasmic reticulum (class II). Together with an earlier analysis of 13 mutant rhodopsins (Sung, C.-H., Schneider, B., Agarwal, N., Papermaster, D.S., and Nathans, J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8840-8844), these experiments define distinct classes of biochemical defects in human rhodopsin and further show that amino acid substitutions in class II reside within the transmembrane and extracellular domains, whereas class I mutants cluster in the first transmembrane domain and at the extreme carboxyl terminus.

Topics: DNA, Complementary; Genes, Dominant; Humans; Membrane Proteins; Mutation; Peptides; Retinitis Pigmentosa; Rhodopsin

Ten rhodopsin mutations have been found in a screen of 282 subjects with retinitis pigmentosa (RP), 76 subjects with Leber congenital amaurosis, and 3 subjects with congenital stationary night blindness. Eight of these mutations (gly51-to-ala, val104-to-ile, gly106-to-arg, arg135-to-gly, cys140-to-ser, gly188-to-glu, val209-to-met, and his211-to-arg) produce amino acid substitutions, one (gln64-to-ter) introduces a stop codon, and one changes a guanosine in the intron 4 consensus splice donor sequence to thymidine. Cosegregation of RP with gln64-to-ter, gly106-to-arg, arg135-to-gly, cys140-to-ser, gly188-to-glu, his211-to-arg, and the splice site guanosine-to-thymidine indicates that these mutations are likely to cause retinal disease. Val104-to-ile does not cosegregate and is therefore unlikely to be related to retinal disease. The relevance of gly51-to-ala and val209-to-met remains to be determined. The finding of gln64-to-ter in a family with autosomal dominant RP is in contrast to a recent report of a recessive disease phenotype associated with the rhodopsin mutation glu249-to-ter. In the present screen, all of the mutations that cosegregate with retinal disease were found among patients with RP. The mutations described here bring to 35 the total number of amino acid substitutions identified thus far in rhodopsin that are associated with RP. The distribution of the substitutions along the polypeptide chain is significantly nonrandom: 63% of the substitutions involve those 19% of amino acids that are identical among vertebrate visual pigments sequenced to date.

Topics: Alleles; Blindness; DNA Mutational Analysis; Female; Gene Frequency; Humans; Male; Mutation; Night Blindness; Pedigree; Polymerase Chain Reaction; Protein Conformation; Retinitis Pigmentosa; Rhodopsin

Topics: Chromosomes, Human, Pair 3; Genes, Dominant; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Base Sequence; Female; Genes, Dominant; Humans; Lod Score; Male; Molecular Sequence Data; Pedigree; Phenotype; Point Mutation; Retinitis Pigmentosa; Rhodopsin

To evaluate the extent to which rhodopsin mutations are involved in autosomal dominant forms of retinitis pigmentosa (adRP) we collected DNAs from patients with adRP and screened the rhodopsin coding sequence with single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. This screening revealed a thymidine to guanine transversion at nucleotide 431 (nucleotide sequence numbers as per Genebank) in affected members of one family (RFS04). The nucleotide substitution leads to a missense mutation at the 46th amino acid of rhodopsin. The mutation occurs at an amino acid conserved in mammals and changes the hydrophobic nature of the protein at a transmembrane-spanning region. The mutation causes the substitution of a non-polar hydrophobic amino acid, leucine, for the basic amino acid arginine (Leu46Arg). This nucleotide substitution is unique to the family studied and occurs in the affected individuals in the family. Full-field electroretinograms (ERGs) in four affected members of the family showed nondetectable rod responses at an early age, with markedly reduced cone responses, and a faster than average rate of progression of the phenotype as measured by yearly ERGs.

Topics: Adolescent; Adult; Amino Acid Sequence; Base Sequence; Child; Codon; DNA; Electroretinography; Female; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Nucleic Acid Conformation; Pedigree; Point Mutation; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

We have found four mutations in the human gene encoding the beta-subunit of rod cGMP phosphodiesterase (PDE beta) that cosegregate with autosomal recessive retinitis pigmentosa, a degenerative disease of photoreceptors. In one family two affected siblings both carry allelic nonsense mutations at codons 298 and 531. Affected individuals have abnormal rod and cone electroretinograms. PDE beta is the second member of the phototransduction cascade besides rhodopsin that is absent or altered as a cause of retinitis pigmentosa, suggesting that other members of this pathway may be defective in other forms of this disease.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Animals; Base Sequence; Codon; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; DNA Mutational Analysis; DNA, Single-Stranded; Dog Diseases; Dogs; Electroretinography; Eye Proteins; Female; Genes, Recessive; Humans; Male; Mice; Mice, Mutant Strains; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Pedigree; Phosphoric Diester Hydrolases; Photoreceptor Cells; Polymorphism, Genetic; Retinal Degeneration; Retinal Dysplasia; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Eighty-eight patients/families with autosomal dominant retinitis pigmentosa (RP) were screened for rhodopsin mutations. Direct sequencing revealed 13 different mutations in a total of 14 (i.e., 16%) unrelated patients. Five of these mutations (T4K, Q28H, R135G, F220C, and C222R) have not been reported so far. In addition, multipoint linkage analysis was performed on two large families with autosomal dominant RP due to rhodopsin mutations by using five DNA probes from 3q21-q24. No tight linkage was found between the rhodopsin locus (RHO) and D3S47 (theta max = 0.08). By six-point analysis, RHO was localized in the region between D3S21 and D3S47, with a maximum lod score of 13.447 directly at D3S20.

Topics: Australia; Base Sequence; DNA Mutational Analysis; Europe; Genes; Genes, Dominant; Genetic Linkage; Genetic Testing; Humans; Molecular Sequence Data; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Since the initial report of linkage of autosomal dominant retinitis pigmentosa (adRP) to the long arm of chromosome 3, several mutations in the gene encoding rhodopsin, which also maps to 3q, have been reported in adRP pedigrees. However, there has been some discussion as to the possibility of a second adRP locus on 3q. This suggestion has important diagnostic and research implications and must raise doubts about the usefulness of linked markers for reliable diagnosis of RP patients. In order to address this issue we have performed an admixture test (A-test) on 10 D3S47-linked adRP pedigrees and have found a likelihood ratio of heterogeneity versus homogeneity of 4.90. We performed a second A-test, combining the data from all families with known rhodopsin mutations. In this test we obtained a reduced likelihood ratio of heterogeneity versus homogeneity, of 1.0. On the basis of these statistical analyses we have found no significant support for two adRP loci on chromosome 3q. Furthermore, using 40 CEPH families, we have localized the rhodopsin gene to the D3S47-D3S20 interval, with a maximum lod score (Zm) of 20 and have found that the order qter-D3S47-rhodopsin-D3S20-cen is significantly more likely than any other order. In addition, we have mapped (Zm = 30) the microsatellite marker D3S621 relative to other loci in this region of the genome.

Topics: Chromosome Mapping; Chromosomes, Human, Pair 3; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Genetic Variation; Humans; Lod Score; Male; Probability; Retinitis Pigmentosa; Rhodopsin

Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It was hypothesized that amino acid changes at these sites would result in mutant rhodopsins with normal spectral properties but defects in their ability to interact with the rod outer segment G protein, transducin. A set of 12 mutant opsin genes was prepared. Four of the mutants were known to cause ADRP: Thr-58 replaced by Arg, a four-amino acid deletion (Leu-68/Arg-69/Thr-70/Pro-71), Arg-135 replaced by Leu, and Arg-135 replaced by Trp. Eight additional mutants were prepared to provide complementary structure-function information. The four-amino acid deletion mutant failed to bind 11-cis-retinal. However, each of the Thr-58 and Arg-135 mutants bound 11-cis-retinal to form a pigment with a visible absorbance maximum (lambda max) of 500 nm. Upon illumination, each pigment was converted to a metarhodopsin II-like spectral form (lambda max = 380 nm). However, each of these spectrally normal ADRP mutants was defective in activating guanine nucleotide exchange by transducin. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line; Genes, Dominant; Genes, Synthetic; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Protein Structure, Secondary; Retinitis Pigmentosa; Rhodopsin; Rod Cell Outer Segment; Sequence Homology, Amino Acid; Transducin; Transfection

Topics: Aged; Aged, 80 and over; Amino Acid Sequence; Base Sequence; Child, Preschool; Female; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

Autosomal dominant retinitis pigmentosa (adRP) is known to result from mutations in two different retinal genes--rhodopsin and peripherin--while a third locus has been implicated by linkage data. However, families have been reported in which all three known loci have been excluded. We report linkage of adRP in one such family to two microsatellite markers on chromosome 7p. D7S435 has previously been localized to 7p13-15.1; D7S460, previously only localized to chromosome 7, maps to within 2 cM of D7S435 with a lod score of 12.15. Two point linkage analysis between these markers and adRP gave lod scores of 5.65 (theta = 0) and 4.19 (theta = 0.046) for D7S460 and D7S435, respectively. Multipoint analysis gave a maximum lod score of 8.22. These data strongly suggest a new adRP locus on chromosome 7p.

Topics: Base Sequence; Chromosomes, Human, Pair 7; DNA, Satellite; Female; Genes, Dominant; Genetic Markers; Humans; Intermediate Filament Proteins; Lod Score; Male; Membrane Glycoproteins; Molecular Sequence Data; Nerve Tissue Proteins; Pedigree; Peripherins; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin; United Kingdom

Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 68-71, 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.

Topics: Amino Acid Sequence; Base Sequence; Codon; DNA; Exons; Genes, Dominant; Genetic Testing; Humans; Lymphocytes; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Point Mutation; Polymerase Chain Reaction; Protein Conformation; Retinitis Pigmentosa; Rhodopsin; Sequence Deletion

Retinitis Pigmentosa (RP) is a group of inherited retinopathies which affect approximately 1 in 4,000 individuals. The disorder can be classified on the basis of inheritance; dominant, recessive and X-linked forms have been well documented. The existence of genetic heterogeneity within autosomal dominant RP (adRP) had been previously demonstrated. As a result of extensive linkage studies in 2 large Irish families and 1 American pedigree three adRP genes have been mapped. adRP genes have been localised to chromosome 3q close to the rod photoreceptor gene, rhodopsin; to chromosome 6p close to another transmembrane photoreceptor gene, peripherin/RDS and to the pericentric region of chromosome 8, although the causative gene in this region has not yet been identified. Here we report the results of a linkage study in a Spanish family, who exhibit an early-onset form of adRP. The adRP gene segregating in this family has been excluded from the three known adRP loci on chromosomes 3q, 6p and 8 using a series of both intragenic microsatellite markers from the rhodopsin and peripherin/RDS genes and markers flanking the three known loci. These results provide definitive evidence for the existence of a fourth adRP locus, further emphasising the genetic heterogeneity that exists within adRP.

Topics: Chromosome Mapping; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 6; Chromosomes, Human, Pair 8; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Humans; Lod Score; Male; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Topics: Amino Acid Sequence; Animals; Exons; Female; Genes, Dominant; Humans; Lod Score; Male; Models, Molecular; Molecular Sequence Data; Mutation; Pedigree; Protein Conformation; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Sequence Homology, Amino Acid

Topics: Alleles; Animals; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin

Mutations within the rhodopsin gene are known to give rise to autosomal dominant retinitis pigmentosa (RP), a common hereditary form of retinal degeneration. We now describe a patient with autosomal recessive RP who is homozygous for a nonsense mutation at codon 249 within exon 4 of the rhodopsin gene. This null mutation, the first gene defect identified in autosomal recessive retinitis pigmentosa, should result in a functionally inactive rhodopsin protein that is missing the sixth and seventh transmembrane domains including the 11-cis-retinal attachment site. We also found a different null mutation carried heterozygously by an unrelated unaffected individual. Heterozygous carriers of either mutation had normal ophthalmologic examinations but their electroretinograms revealed an abnormality in rod photoreceptor function.

Topics: Adaptation, Ocular; Adult; Amino Acid Sequence; Base Sequence; Consanguinity; Darkness; DNA; DNA Mutational Analysis; Electroretinography; Female; Genes, Recessive; Heterozygote; Homozygote; Humans; Male; Molecular Sequence Data; Pedigree; Photoreceptor Cells; Point Mutation; Retinitis Pigmentosa; Rhodopsin

The mutations of codon 17, 23, 58, and 347 of rhodopsin gene were investigated in 24 unrelated Japanese families including 33 patients with autosomal dominant retinitis pigmentosa (ADRP). A patient with codon 17 mutation (Thr-17-Met, ACG-->ATG) and a family including 4 patients with codon 347 mutation (Pro-347-Leu, CCG-->CTG) were detected among them. Their clinical findings were extremely different between the two mutations. The former showed type 2 and the latter showed type 1 ADRP. No mutation of codon 23 and 58 was detected in any families so far analyzed in the present study. Clinical findings associated with the mutation in codon 17 and 347 of the rhodopsin gene show an existence of allelic heterogeneity.

Topics: Adolescent; Adult; Base Sequence; Codon; Deoxyribonucleotides; DNA Mutational Analysis; Female; Genes, Dominant; Humans; Japan; Male; Molecular Sequence Data; Pedigree; Point Mutation; Retinitis Pigmentosa; Rhodopsin

Topics: Female; Genes, Dominant; Humans; Male; Pedigree; Retinitis Pigmentosa; Rhodopsin; Scotland

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.

Topics: Animals; Codon; Disease Models, Animal; Electroretinography; Fundus Oculi; Humans; Mice; Mice, Transgenic; Microscopy, Electron; Mutation; Ophthalmoscopy; Photoreceptor Cells; Proline; Restriction Mapping; Retina; Retinal Vessels; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Topics: Humans; Mutation; Retinitis Pigmentosa; Rhodopsin

Using single-strand conformation polymorphism electrophoresis, heteroduplex analysis, and direct sequencing, we have searched for possible disease-causing mutations in the adRP family in which we originally found tight linkage of the disease to 6p. We have now identified a single base change in exon 2, which results in the replacement of a serine residue at codon 212 for a glycine residue. The mutation cosegregates with the disease with a lod score of 12.1 at theta = 0.0.

Topics: Amino Acid Sequence; Base Sequence; Chromosomes, Human, Pair 6; DNA, Single-Stranded; Genes, Dominant; Genetic Linkage; Humans; Intermediate Filament Proteins; Membrane Glycoproteins; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Pedigree; Peripherins; Retinitis Pigmentosa; Rhodopsin

Recent evidence suggesting the involvement of mutant rhodopsin proteins in the pathogenesis of autosomal recessive retinitis pigmentosa has prompted us to investigate whether this form of the disease shows non-allelic genetic heterogeneity, as has previously been shown to be the case in autosomal dominant retinitis pigmentosa. The availability of a unique inbred Dutch pedigree has enabled us to address this question. We have used an intragenic polymorphism to exclude the possibility that a mutation in the rhodopsin gene is responsible for the disease in this patient population. These data provide evidence for the involvement of at least two loci in autosomal recessively inherited retinitis pigmentosa.

Topics: Base Sequence; Consanguinity; DNA, Single-Stranded; Female; Genes, Recessive; Humans; Intermediate Filament Proteins; Lod Score; Male; Membrane Glycoproteins; Molecular Sequence Data; Nerve Tissue Proteins; Pedigree; Peripherins; Retinitis Pigmentosa; Rhodopsin

Two members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-thymine mutation in the second nucleotide of codon 267 in the rhodopsin gene that resulted in a proline-to-leucine change. Two members of another family with autosomal dominant retinitis pigmentosa showed a guanine-to-thymine mutation in the first nucleotide of codon 190 in the rhodopsin gene that resulted in an aspartate-to-tyrosine change. Three members from a third family with autosomal dominant retinitis pigmentosa were also found to have a mutation in codon 190; however, this guanine-to-adenine mutation in the first nucleotide of codon 190 resulted in an aspartate-to-asparagine change. The relatively less severe functional retinal impairment in our patients with a transmembrane codon 267 rhodopsin gene mutation is generally comparable with that observed in a previously described codon 58 transmembrane mutation. The two families with different intradiscal codon 190 mutations showed a considerable difference in severity of their disease.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Codon; Dark Adaptation; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Photic Stimulation; Retinitis Pigmentosa; Rhodopsin; Visual Acuity

There is considerable variety among the clinical features of autosomal dominant retinitis pigmentosa (ADRP). This is probably at least in part due to genetic heterogeneity. Recently, various mutations of the rhodopsin gene have been detected in some ADRP families. We report on six patients from two families with ADRP who were investigated by means of psychophysical and electrophysiological methods. All displayed the same rhodopsin gene mutation at codon 347, which exchanges the amino acid proline for leucine (pro-347-leu). The patients had early-onset night blindness and impaired side vision as of the end of their second life decade. They produced monophasic dark-adaptation curves, showing a lack of rod function and elevated cone thresholds. Dark-adapted two-color threshold perimetry using 500- and 650-nm stimuli revealed a diffuse loss of rod function and centrally preserved cone function. The electroretinogram was nonrecordable at the age of about 30 years. A certain variability of visual function loss was noted among patients in the overall severe course of the disease, but the clinical findings of this genotype corresponded to type 1 ADRP of Massof and Finkelstein in all cases.

Topics: Adult; Aged; Dark Adaptation; DNA Mutational Analysis; Electroretinography; Female; Fundus Oculi; Humans; Leucine; Male; Middle Aged; Mutation; Pedigree; Photoreceptor Cells; Proline; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Visual Fields

This study documents the ophthalmological findings in a six-generation. Swedish family with autosomal dominant retinitis pigmentosa with a previously unknown rhodopsin, exon 2, mutation, Arg-135-Leu (CGG to CTG). Six affected patients from the family were available for analysis and were all found to be heterozygous for the mutation, whereas eight clinically normal family members and 29 unrelated normal individuals did not have it. The disease appears to be of a type with comparatively rapid progression to blindness.

Topics: Adult; Aged; Arginine; Base Sequence; DNA; DNA Probes; Electrophoresis, Agar Gel; Electroretinography; Female; Fundus Oculi; Humans; Leucine; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

By screening patients with autosomal dominant retinitis pigmentosa for mutations in the rhodopsin gene, two deletions (8 bp and 1 bp) have been identified in exon 5; these deletions cause a shift in the reading frame. The predicted proteins should be radically altered with translation continuing past the normal stop signal and resulting in a rhodopsin molecule that is, respectively, 1 and 10 amino acids longer. The clinical phenotype of the patients is described and is compared with that associated with other mutations in the same region of the gene.

Topics: Adult; Aged; Amino Acid Sequence; Base Sequence; Child, Preschool; DNA; Exons; Female; Gene Deletion; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Open Reading Frames; Pedigree; Retinitis Pigmentosa; Rhodopsin

Autosomal dominant retinitis pigmentosa (adRP) has shown linkage to the chromosome 3q marker C17 (D3S47) in two large adRP pedigrees known as TCDM1 and adRP3. On the basis of this evidence the rhodopsin gene, which also maps to 3q, was screened for mutations which segregated with the disease in adRP patients, and several have now been identified. However, we report that, as yet, no rhodopsin mutation has been found in the families first linked to C17. Since no highly informative marker system is available in the rhodopsin gene, it has not been possible to measure the genetic distance between rhodopsin and D3S47 accurately. We now present a linkage analysis between D3S47 and the rhodopsin locus (RHO) in five proven rhodopsin-retinitis pigmentosa (rhodopsin-RP) families, using the causative mutations as highly informative polymorphic markers. The distance, between RHO and D3S47, obtained by this analysis is theta = .12, with a lod score of 4.5. This contrast with peak lod scores between D3S47 and adRP of 6.1 at theta = .05 and 16.5 at theta = 0 in families adRP3 and TCDM1, respectively. These data would be consistent with the hypothesis that TCDM1 and ADRP3 represent a second adRP locus on chromosome 3q, closer to D3S47 than is the rhodopsin locus. This result shows that care must be taken when interpreting adRP exclusion data generated with probe C17 and that it is probably not a suitable marker for predictive genetic testing in all chromosome 3q-linked adRP families.

Topics: Chromosome Mapping; Chromosomes, Human, Pair 3; DNA Probes; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Humans; Lod Score; Male; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Genetic; Recombination, Genetic; Retinitis Pigmentosa; Rhodopsin

Topics: Base Sequence; Chromosome Deletion; Codon; Family; Genes, Dominant; Germany; Humans; Molecular Sequence Data; Recurrence; Retinitis Pigmentosa; Rhodopsin

We studied rod and cone function in 13 patients from four families with autosomal dominant retinitis pigmentosa and the proline-23-histidine rhodopsin mutation. In patients with early stages of this disease, rod sensitivity was mildly abnormal throughout the retina and cone sensitivity was normal. In more severely affected patients, sensitivity loss varied with retinal region, some regions showing mild rod loss only and other regions having pronounced rod and cone dysfunction. Rhodopsin levels were decreased below normal by amounts that indicated the rod sensitivity loss was determined by the reduced ability to absorb light. The most characteristic abnormality of this genotype was a slowed rod branch of dark adaptation, which was present regardless of the extent or severity of disease. The time required for recovery of rod sensitivity was more than twice the normal time. These findings with dark-adapted perimetry, fundus reflectometry, and dark adaptometry showed intrafamilial and interfamilial consistency.

Topics: Adolescent; Adult; Aged; Codon; Dark Adaptation; Electroretinography; Female; Histidine; Humans; Male; Middle Aged; Mutation; Pedigree; Photoreceptor Cells; Proline; Retinitis Pigmentosa; Rhodopsin; Visual Fields

The author analyzed codon 347 of the rhodopsin gene using PCR (polymerase chain reaction) amplification and restriction enzymes in 19 unrelated Japanese families including 28 patients with autosomal dominant retinitis pigmentosa (ADRP). An allele of codon 347 mutation was found in a family (father and daughter). Sequence analysis shows that the mutation is from CCG to CTG. This mutation appears to be the cause of one form of ADRP, since it was also found in Japanese cases of ADRP which have a different racial background from families reported by Dryja et al.

Topics: Codon; DNA Mutational Analysis; Genes, Dominant; Humans; Japan; Mutation; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Three members of one family and one person from another family were found to have a guanine-to-adenine transition mutation in the first nucleotide of codon 106 in the rhodopsin gene that results in a glycine-to-arginine change. All affected members presented with a similar phenotype that included a regional predilection for pigmentary changes to occur in the inferior retina as well as visual field impairment predominantly in the superior hemisphere. The distribution of pigmentary changes, pattern of visual field loss, and substantial remaining electroretinographic amplitudes with normal implicit times were consistent with a form of "sector" retinitis pigmentosa. We documented the association of a distinct phenotype of autosomal dominant retinitis pigmentosa with a better visual prognosis and a specific rhodopsin gene mutation.

Topics: Adult; Amino Acid Sequence; Codon; DNA; Fundus Oculi; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Visual Fields

In a family with autosomal dominant retinitis pigmentosa, documented over six generations, a previously undescribed point mutation in the rhodopsin gene could be identified. The mutation found in the six affected members examined but in none of the controls, including healthy members of the family, was a point mutation in codon 347 predicting a substitution of the amino acid arginine for proline, designated Pro-347-Arg. Six affected members from two generations were examined clinically and with ganzfeld rod and cone electroretinography. The cone and, more dramatically, the rod electroretinograms were reduced to residual b-wave amplitudes or were non-detectable as early as ages 18 to 22 years. The Pro-347-Arg mutation resulted in a subjectively and clinically homogeneous phenotype: early onset of night blindness before age 11, relatively preserved usable visual fields until about age 30, blindness at ages 40 to 60, and change from an initial apparently sine pigmento to a hyperpigmented and atrophic fundus picture between 30 and 50 years of age.

Topics: Adolescent; Adult; Arginine; Electroretinography; Humans; Male; Middle Aged; Mutagenesis, Site-Directed; Pedigree; Photoreceptor Cells; Proline; Retinitis Pigmentosa; Rhodopsin

Four members in a Japanese family had autosomal dominant retinitis pigmentosa caused by a single point mutation in codon 347 of the rhodopsin gene. The youngest, an 11-year-old girl, had an abnormal electroretinographic response, although her fundus appeared normal. The other affected family members noticed night blindness in the second decade. Their fundi showed diffuse pigmentation with concentric visual field loss, and there was no recordable electroretinographic response. Cataract developed in the fourth decade in the older patients. Good visual acuity was retained however, even in the fifth decade, after cataract extraction. These clinical features were similar to those of American patients (European family origin) with the same mutation of the rhodopsin gene reported previously.

Topics: Adult; Aged; Cataract; Child; Codon; DNA; Electroretinography; Female; Fundus Oculi; Humans; Japan; Male; Mutagenesis, Site-Directed; Night Blindness; Pedigree; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Six members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-thymine transition mutation in the second nucleotide of codon 17 in the rhodopsin gene that resulted in a threonine to methionine change. Three members from another family with autosomal dominant retinitis pigmentosa showed a guanine-to-adenine transition mutation in the first nucleotide of codon 182 in the rhodopsin gene that resulted in a glycine to serine change. Each of these two mutations presented with a similar phenotype because both showed a regional predilection for pigmentary changes to occur in the inferior part of the retina as well as field impairment predominantly in the superior hemisphere. Electroretinographic amplitudes were more substantial than usually encountered in other forms of retinitis pigmentosa, a finding consistent with the better visual prognosis in patients with either of these two mutations. This article documents the association of two similar phenotypes of autosomal dominant retinitis pigmentosa with specific gene defects at a molecular level.

Topics: Adolescent; Adult; Aged; Amino Acids; Codon; Dark Adaptation; DNA; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Fundus Oculi; Genes, Dominant; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields

Point mutations within the rhodopsin gene have been found recently in some patients with autosomal dominant retinitis pigmentosa (ADRP). Currently, four types of point mutations at codons 23, 58 and 347 have been identified. The purposes of this study were to establish simple methods for screening patients with retinitis pigmentosa (RP) to detect these point mutations, and to apply these methods to determine if these mutations are found in Japanese patients with RP. Utilizing the polymerase chain reaction (PCR), a one-step method was developed to detect point mutations at codon 23. This method was then applied to screen genomic DNAs from 30 patients with various types of RP, including ADRP, autosomal recessive RP, simplex RP, Leber's congenital amaurosis or Usher's syndrome. Subsequently, point mutations at codons 58 and 347 were detected by restriction enzyme digestion (Dde I or Msp I) of exons 1 and 5 amplified by PCR. To date, no mutations have been found in codons 23 and 58 in Japanese patients. By using the allele-specific PCR, however, two patients from one pedigree of ADRP were confirmed to have a C-to-T transition at the second nucleotide of codon 347, which results in the substitution of leucine for proline. Our findings indicated the availability of this simple method for detecting these point mutations.

Topics: Alleles; Antisense Elements (Genetics); Base Sequence; Codon; DNA; DNA Restriction Enzymes; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Topics: Base Sequence; Humans; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin

Detection of small alterations or abnormalities in genomic DNA (eg, point mutations or small deletions) has become increasingly important in the diagnosis of genetic disease and polymorphism. When a mutation or polymorphism creates a new restriction endonuclease site, it can easily be identified by polymerase chain reaction (PCR) amplification of the DNA region of interest, followed by digestion with the restriction endonuclease. However, useful restriction sites are the exception, and a variety of specialised techniques have been developed to identify subtle DNA abnormalities. We have shown that where a DNA mutation does not create a useful novel restriction site, such a site can be introduced by PCR and specially chosen primers. The approach is simple and inexpensive and should be broadly applicable in the diagnosis of genetic polymorphism and mutation. The technique is illustrated here by the three base-pair deletion responsible for most cases of cystic fibrosis and by detection of the point mutation in the rhodopsin gene that has been associated with some cases of autosomal dominant retinitis pigmentosa.

Topics: Alleles; Base Sequence; Blood Proteins; Calgranulin A; Chromosome Deletion; Cystic Fibrosis; DNA, Single-Stranded; Evaluation Studies as Topic; Humans; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping; Retinitis Pigmentosa; Rhodopsin

Topics: Calcium-Binding Proteins; Eye Proteins; Hippocalcin; Humans; Lipoproteins; Nerve Tissue Proteins; Radiation; Recoverin; Retinitis Pigmentosa; Rhodopsin; Sodium Channels

Topics: Codon; Humans; Mutation; Retinitis Pigmentosa; Rhodopsin; United Kingdom

There is considerable clinical variability in autosomal dominant retinitis pigmentosa (ADRP). The underlying biochemical defect had remained unknown until recently, so that it was not possible to determine the primary cause(s) of this phenotypic diversity. Recently, different point mutations and base pair deletions have been identified in the rhodopsin gene in a proportion of patients with ADRP, providing convincing evidence for allelic genetic heterogeneity in this disease. We screened a total of 65 patients with ADRP in Germany, Austria, and Switzerland for the presence of the point mutations described recently at codons 58 and 347 in patients in the USA. Our results show that the frequency of point mutations at codon 347 in the patients studied here is about 3%, a figure similar to that found in the USA. The frequency of the mutation at codon 58 seems to be generally low. The identification of patients with point mutations in the rhodopsin gene offers the possibility, for the first time, of studying the correlation between genotype and disease phenotype.

Topics: Chromosome Aberrations; Chromosome Disorders; Codon; Genes, Dominant; Genetic Carrier Screening; Genetic Markers; Humans; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Several mutations in the rhodopsin gene in patients affected by autosomal dominant retinitis pigmentosa (ADRP) have recently been described. We report four new rhodopsin mutations in ADRP families, initially identified as hetero-duplexed PCR fragments on hydrolink gels. One is an in-frame 12-bp deletion of codons 68 to 71. The other three are point mutations involving codons 190, 211, and 296. Each alters the amino acid encoded. The codon 190 mutation has been detected in 2 from a panel of 34 ADRP families, while the remaining mutations were seen in single families. This suggests that, consistent with a dominant condition, no single mutation will account for a large fraction of ADRP cases. The base substitution in codon 296 alters the lysine residue that functions as the attachment site for 11-cis-retinal, mutating it to glutamic acid. This mutation occurs in a family with an unusually severe phenotype, resulting in early onset of disease and cataracts in the third or fourth decade of life. This result demonstrates a correlation between the location of the mutation and the severity of phenotype in rhodopsin RP.

Topics: Amino Acid Sequence; Base Sequence; Deoxyribonucleotides; Female; Genes, Dominant; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Retina; Retinitis Pigmentosa; Rhodopsin

Topics: Humans; Molecular Biology; Retinitis Pigmentosa; Rhodopsin

The authors report a family in which a Thr58Arg rhodopsin mutation co-segregates with the disease phenotype of autosomal dominant retinitis pigmentosa (RP) in 16 family members. DNA sequence determination confirms the presence of the same mutation reported previously for one family apparently unrelated to the pedigree now reported. Features of RP in this family included a later onset of symptoms, with night blindness first noticed between ages 12 to 24 years. Although symptoms worsened with age, no complete blindness was observed even with advanced age. Results of psychophysical and electrophysiologic testing showed that a 19-year-old affected woman and her 65-year-old affected uncle had relatively similar extent of visual dysfunction, and that the vision of both was better than 2 of their relatives aged 37 and 53 years. This study presents a range of phenotypic similarities and differences observed between individuals whose RP appears to be caused by the same mutation.

Topics: Adult; Aged; Arginine; Base Sequence; Dark Adaptation; DNA; Electroretinography; Female; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Threonine; Visual Fields

Mutations in the rhodopsin gene are associated with as many as one quarter of all cases of autosomal dominant retinitis pigmentosa (RP). A number of different rhodopsin mutations have been reported but only the proline to histidine mutation in codon 23 (Pro-23-His) has been well characterized clinically. One recent report described a "sectoral" distribution of the retinal degeneration associated with this mutation, while another reported only that pigment was present in all four quadrants in 13 of 17 patients. This asymmetric distribution of pigmentation and visual field loss may prove to be an important clinical sign of a type of RP with a relatively good visual prognosis. The authors present a family with Pro-23-His rhodopsin-associated RP in which all six affected individuals had a regional distribution of the retinal degeneration in which the inferior hemisphere of the retina was most severely affected.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Base Sequence; Codon; DNA; Female; Histidine; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Proline; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Topics: Codon; Female; Genes, Dominant; Humans; Ireland; Male; Mutation; Pedigree; Retinitis Pigmentosa; Rhodopsin

Rhodopsin gene mutations appear to cause some forms of autosomal dominant retinitis pigmentosa. In the family described, the mutation called rhodopsin, Val345Met segregated perfectly with the disease. All affected individuals had abnormal ERGs; the two oldest members of this family had more loss of function than the two youngest members. Some intra-familial variability existed as an older member showed larger visual fields and ERG amplitudes than a younger member. This mutation was not seen in 106 control subjects nor in any other patients yet described with other rhodopsin gene mutations. Patients so far studied with rhodopsin, Val345Met, have smaller 0.5-Hz full-field ERG amplitudes, on average, than those with Pro23His or Thr58Arg and larger ERG amplitudes than those with Pro347Leu or Pro347Ser. These forms of retinitis pigmentosa can now be detected through analysis of leukocyte DNA.

Topics: Adult; Amino Acid Sequence; Child; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Humans; Male; Methionine; Middle Aged; Molecular Sequence Data; Mutagenesis, Site-Directed; Pedigree; Retinitis Pigmentosa; Rhodopsin; Valine; Visual Fields

We searched for point mutations in every exon of the rhodopsin gene in 150 patients from separate families with autosomal dominant retinitis pigmentosa. Including the 4 mutations we reported previously, we found a total of 17 different mutations that correlate with the disease. Each of these mutations is a single-base substitution corresponding to a single amino acid substitution. Based on current models for the structure of rhodopsin, 3 of the 17 mutant amino acids are normally located on the cytoplasmic side of the protein, 6 in transmembrane domains, and 8 on the intradiscal side. Forty-three of the 150 patients (29%) carry 1 of these mutations, and no patient has more than 1 mutation. In every family with a mutation so far analyzed, the mutation cosegregates with the disease. We found one instance of a mutation in an affected patient that was absent in both unaffected parents (i.e., a new germ-line mutation), indicating that some "isolate" cases of retinitis pigmentosa carry a mutation of the rhodopsin gene.

Topics: Amino Acid Sequence; Base Sequence; Cell Membrane; Codon; Exons; Eye Proteins; Female; Genes, Dominant; Humans; Male; Models, Structural; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Pedigree; Protein Conformation; Restriction Mapping; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients' DNA revealed a previously undescribed CCG----CGG transversion in codon 347 predicting a Pro----Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene.

Topics: Base Sequence; DNA Probes; Humans; Molecular Sequence Data; Mutation; Oligonucleotides; Pedigree; Phenotype; Polymerase Chain Reaction; Restriction Mapping; Retinitis Pigmentosa; Rhodopsin

DNA samples from 161 unrelated patients with autosomal dominant retinitis pigmentosa were screened for point mutations in the rhodopsin gene by using the polymerase chain reaction and denaturing gradient gel electrophoresis. Thirty-nine patients were found to carry 1 of 13 different point mutations at 12 amino acid positions. The presence or absence of the mutations correlated with the presence or absence of retinitis pigmentosa in 174 out of 179 individuals tested in 17 families. The mutations were absent from 118 control subjects with normal vision.

Topics: Base Sequence; DNA; Female; Genes; Genes, Dominant; Humans; Male; Molecular Sequence Data; Mutation; Night Blindness; Nucleic Acid Hybridization; Oligonucleotide Probes; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Fields

We studied rod and cone function in 20 patients from six families with autosomal dominant retinitis pigmentosa, who represented five different point mutations in the gene encoding rhodopsin. In a family with a stop codon mutation at the carboxyl end of the molecule (glutamine-344), young members with the mutation were asymptomatic and clinically unaffected but showed about 1 log unit of rod sensitivity loss across the visual field and decreased rhodopsin levels; at this stage, cone function was essentially normal. In three families with mutations at the border of a transmembrane segment (arginine-135-leucine and arginine-135-tryptophan), there was neither detectable rod function nor measurable rhodopsin; cone function was variably impaired. Two families carrying different mutations (threonine-17-methionine and threonine-58-arginine) had altitudinal visual field defects with less impaired rod and cone function in the inferior than in the superior field. Rod adaptation was abnormal in both families, but the time course of adaptation differed between patients with the two mutations. Differences in the pattern of retinal dysfunction were therefore discernible in patients with different rhodopsin mutations.

Topics: Adolescent; Adult; Aged; Dark Adaptation; Electrophysiology; Female; Humans; Male; Middle Aged; Mutation; Pedigree; Phenotype; Photoreceptor Cells; Protein Conformation; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests; Visual Fields

Retinitis pigmentosa (RP) is a group of disorders characterized by progressive degeneration of the outer retina, resulting in night blindness, visual field loss, an abnormal electroretinogram, and characteristic retinal pigmentary changes. An important step in the understanding of RP has been the recognition that some cases of autosomal dominant RP (ADRP) are caused by mutations in the rhodopsin gene. Multiple different point mutations within the coding sequence of the rhodopsin gene have been associated with ADRP. We have developed a GC-clamped denaturing-gradient-gel electrophoresis (DGGE) assay for the coding region of the rhodopsin gene and have used this assay to screen ADRP patients for mutations. The assay consists of amplifying with PCR the five exons of the rhodopsin gene and then analyzing each PCR product by DGGE. We have used this assay to detect three previously unreported rhodopsin base substitutions associated with ADRP. The use of this assay to identify ADRP patients who have various rhodopsin mutations has allowed us to begin studies seeking to correlate molecular genotype with clinical phenotype. Furthermore, GC-clamped DGGE has allowed us to identify families with ADRP not caused by a rhodopsin mutation. Such families will be important in the search for other genes involved in ADRP.

Topics: Amino Acid Sequence; Base Sequence; Electrophoresis, Polyacrylamide Gel; Exons; Female; Genetic Testing; Humans; Male; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Pedigree; Polymerase Chain Reaction; Polymorphism, Genetic; Retinitis Pigmentosa; Rhodopsin

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.

Topics: Blotting, Western; Cell Compartmentation; Cells, Cultured; Cloning, Molecular; DNA Mutational Analysis; Genes, Dominant; Glycosylation; Humans; Microscopy, Electron; Molecular Structure; Protein Processing, Post-Translational; Retinitis Pigmentosa; Rhodopsin; Spectrum Analysis

Eight members of a family with autosomal dominant retinitis pigmentosa were found to have a cytosine-to-guanine (C-to-G) transversion mutation in the second nucleotide of codon 58 of the rhodopsin gene, causing a substitution of the amino acid arginine for threonine. Five of these individuals were examined clinically. There was a distinct phenotypic expression of the gene defect within this family that included a regional predilection for pigmentary changes in the inferior and inferonasal parts of the retina, as well as field impairment predominantly in the superior hemisphere. Characteristic electroretinographic recordings and psychophysical threshold profiles also helped to identify this phenotype that, on a relative basis, causes less severe photoreceptor cell functional impairment than often occurs in other subtypes of retinitis pigmentosa. This report documents the association of a clinically recognizable phenotype in a family with autosomal dominant retinitis pigmentosa and a specific gene defect at the molecular level.

Topics: Adult; Arginine; Base Sequence; Chromosome Aberrations; Chromosome Disorders; Codon; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Electroretinography; Female; Fundus Oculi; Gene Expression; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutagenesis; Pedigree; Phenotype; Retinitis Pigmentosa; Rhodopsin; Threonine; Visual Fields

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.

Topics: Amino Acid Sequence; Base Sequence; Chromosome Deletion; Chromosomes, Human, Pair 3; DNA Probes; Electrophoresis, Polyacrylamide Gel; Female; Genes, Dominant; Genetic Linkage; Humans; Male; Molecular Sequence Data; Nucleic Acid Hybridization; Pedigree; Recombination, Genetic; Retinitis Pigmentosa; Rhodopsin

A cytosine-to-adenine transversion in codon 23 of rhodopsin, the rod visual pigment gene, was reported recently by Dryja et al in 17 of 148 unrelated patients with autosomal dominant retinitis pigmentosa, but the clinical findings associated with this deletion have not been reported in detail. In screening our patients with autosomal dominant retinitis pigmentosa for the codon 23 transversion, we found positive results in four affected individuals from two families with sectoral retinitis pigmentosa, while 12 patients with sectoral retinitis pigmentosa from different families had negative results, suggesting that other gene sites or locations may give this same phenotypic change. From our patients' history of light exposure and the location of degeneration in the retina, we hypothesize that light phototoxicity may be playing an expressive role in this point mutation of the rhodopsin gene. This is the first report in which a type of retinitis pigmentosa has been associated with a specific molecular gene defect, although the actual pathophysiologic mechanism currently is unknown.

Topics: Adult; Base Sequence; Codon; DNA; Electroretinography; Female; Fluorescein Angiography; Fundus Oculi; Genes, Dominant; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Ocular findings are presented from 17 unrelated patients with a form of autosomal dominant retinitis pigmentosa and the same cytosine-to-adenine transversion in codon 23 of the rhodopsin gene corresponding to a substitution of histidine for proline in the 23rd amino acid of rhodopsin (designated rhodopsin, Pro-23-His). On average, these patients (mean age, 37 years) had significantly better visual acuity and larger electroretinographic amplitudes than 131 unrelated patients (mean age, 32 years) with autosomal dominant retinitis pigmentosa without this mutation. However, these 17 patients from separate families, as well as 12 relatives with the mutation from four of these families, showed interfamilial and intrafamilial variability with respect to severity of their ocular disease, suggesting that some factor(s) other than this gene defect itself is involved in the expression of their condition. This form of retinitis pigmentosa can now be detected by testing leukocyte DNA from peripheral blood. Some mechanisms by which this mutation in the rhodopsin gene could lead to rod photoreceptor cell death are suggested.

Topics: Adolescent; Adult; Base Sequence; Codon; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Histidine; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Pedigree; Polymerase Chain Reaction; Proline; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Visual Fields

A linkage analysis has been performed on three Australian families segregating for autosomal dominant retinitis pigmentosa (ADRP). No evidence of linkage has been found in any of the pedigrees studied between the locus D3S47 and the gene for ADRP. The D3S47 locus was found to show very close linkage with the ADRP gene in a large Irish pedigree. Our study together with a similar report on a British family indicates that there is genetic heterogeneity in this disease.

Topics: Adolescent; Adult; Australia; Child; Child, Preschool; Female; Genes, Dominant; Genetic Linkage; Genetic Markers; Genotype; Humans; Male; Pedigree; Recombination, Genetic; Retinitis Pigmentosa; Rhodopsin

We studied the ocular findings in eight unrelated patients with a form of autosomal dominant retinitis pigmentosa and the same cytosine-to-thymine transition in the second nucleotide of codon 347 of the rhodopsin gene. This mutation, detected in leukocyte DNA, corresponds to a substitution of leucine for proline in amino acid 347 of the rhodopsin protein, and, therefore, we designated this form of retinitis pigmentosa as rhodopsin, proline-347-leucine. On average, these patients had significantly smaller visual field areas and smaller electroretinogram amplitudes than 140 unrelated patients of comparable age with dominant retinitis pigmentosa without this mutation. The findings in eight relatives with this mutation from three of these families are presented to provide examples of the variability that exists in the clinical severity of this disease.

Topics: Adolescent; Adult; Amino Acid Sequence; Chromosome Aberrations; Chromosome Disorders; Dark Adaptation; Electroretinography; Female; Fundus Oculi; Humans; Leucine; Male; Middle Aged; Molecular Sequence Data; Mutation; Night Blindness; Pedigree; Proline; Retinitis Pigmentosa; Rhodopsin; Visual Fields

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Southern; Chromosomes, Human, Pair 3; Data Interpretation, Statistical; DNA; Female; Gene Amplification; Genes, Dominant; Genetic Linkage; Genetic Markers; Humans; Male; Molecular Sequence Data; Pedigree; Polymerase Chain Reaction; Retinitis Pigmentosa; Rhodopsin

Topics: Blindness; Eye Proteins; Humans; Mutation; Photoreceptor Cells; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

The gene for autosomal dominant retinitis pigmentosa in a large pedigree of Irish origin has recently been found to be linked to an anonymous polymorphic sequence, D3S47 (C17), from the long arm of chromosome 3. As the gene coding for rhodopsin is also assigned to the long arm of chromosome 3 and is expressed in rod photoreceptors that are affected early in this blinding disease, we searched for a mutation of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. We found a C----A transversion in codon 23 (corresponding to a proline----histidine substitution) in 17 of 148 unrelated patients and not in any of 102 unaffected individuals. This result, coupled with the fact that the proline normally present at position 23 is highly conserved among the opsins and related G-protein receptors, indicates that this mutation could be the cause of one form of autosomal dominant retinitis pigmentosa.

Topics: Adult; Amino Acid Sequence; Base Sequence; Chromosomes, Human, Pair 3; Codon; Electroretinography; Eye Proteins; Humans; Molecular Sequence Data; Mutation; Pedigree; Photoreceptor Cells; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod Opsins

Night blindness is an early symptom of retinitis pigmentosa. The rod photoreceptors are responsible for night vision and use rhodopsin as the photosensitive pigment.. We found three mutations in the human rhodopsin gene; each occurred exclusively in the affected members of some families with autosomal dominant retinitis pigmentosa. Two mutations were C-to-T transitions involving separate nucleotides of codon 347; the third was a C-to-G transversion in codon 58. Each mutation corresponded to a change in one amino acid residue in the rhodopsin molecule. None of these mutations were found in 106 unrelated normal subjects who served as controls. When the incidence of these three mutations was added to that of a previously reported mutation involving codon 23, 27 of 150 unrelated patients with autosomal dominant retinitis pigmentosa (18 percent) were found to carry one of these four defects in the rhodopsin gene. All 27 patients had abnormal rod function on monitoring of their electroretinograms. It appears that patients with the mutation involving codon 23 probably descend from a single ancestor.. In some patients with autosomal dominant retinitis pigmentosa, the disease is caused by one of a variety of mutations of the rhodopsin gene.

Topics: Base Sequence; Codon; Electroretinography; Genes, Dominant; Humans; Molecular Sequence Data; Mutation; Retinitis Pigmentosa; Rhodopsin

In exon 1 at codon 23 of the rhodopsin gene, a mutation resulting in a proline-to-histidine substitution has previously been observed in approximately 12% of American autosomal dominant retinitis pigmentosa (ADRP) patients. The region around the site of this mutation in the rhodopsin gene has been amplified and analyzed in affected individuals from 91 European ADRP pedigrees. The codon 23 mutation has been found to be absent in all cases, including a large Irish pedigree in which the disease gene has previously been shown to be closely linked to the rhodopsin locus. This indicates the presence of either allelic or nonallelic heterogeneity in ADRP.

Topics: Amino Acid Sequence; Base Sequence; Codon; Europe; Exons; Female; Genes, Dominant; Histidine; Humans; Male; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Proline; Retinitis Pigmentosa; Rhodopsin

Topics: Base Sequence; Histidine; Humans; Molecular Conformation; Molecular Sequence Data; Photoreceptor Cells; Proline; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin

Recently Dryja and his co-workers observed a mutation in the 23d codon of the rhodopsin gene in a proportion of autosomal dominant retinitis pigmentosa (ADRP) patients. Linkage analysis with a rhodopsin-linked probe C17 (D3S47) was carried out in two large British ADRP families, one with diffuse-type (D-type) RP and the other with regional-type (R-type) RP. Significantly positive lod scores (lod score maximum [Zmax] = +5.58 at recombination fraction [theta] = .0) were obtained between C17 and our D-type ADRP family showing complete penetrance. Sequence and oligonucleotide analysis has, however, shown that no point mutation at the 23d codon exists in affected individuals in our complete-penetrance pedigree, indicating that another rhodopsin mutation is probably responsible for ADRP in this family. Significantly negative lod scores (Z less than -2 at theta = .045) were, however, obtained between C17 and our R-type family which showed incomplete penetrance. Previous results presented by this laboratory also showed no linkage between C17 and another large British R-type ADRP family with incomplete penetrance. This confirms genetic heterogeneity. Some types of ADRP are being caused by different mutations in the rhodopsin locus (3q21-24) or another tightly linked gene in this region, while other types of ADRP are the result of mutations elsewhere in the genome.

Topics: Base Sequence; Codon; Female; Genes, Dominant; Genetic Linkage; Genetic Variation; Humans; Lod Score; Male; Molecular Sequence Data; Mutation; Pedigree; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin

New methods using computer based measurements and image analysis techniques can improve and expand our ability to investigate non-invasively the function of the retina in patients. These can provide insight into the underlying mechanism of an abnormality and further our understanding of disease processes.

Topics: Dark Adaptation; Humans; Image Processing, Computer-Assisted; Photochemistry; Photoreceptor Cells; Psychophysics; Retina; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Visual Field Tests; Visual Perception

We used reflection retinal densitometric, psychophysical, and electroretinographic techniques to study the scotopic retinal function of a mother and her three daughters who had clinical evidence of a sectoral type of retinitis pigmentosa. Retinal regions with, and those without, ophthalmoscopic signs of degeneration were investigated. During dark adaptation, the time courses of rhodopsin regeneration and recovery of scotopic sensitivity were similar to normal as was the relation of rhodopsin to scotopic threshold. In dark-adapted eyes, threshold increases were not proportional to rhodopsin loss. The results of psychophysical tests of background adaptation and temporal summation, and analysis of the relation of electroretinographic a- to b-wave amplitudes, led to the conclusion that abnormalities of photoreceptor cell function central to the rhodopsin-bearing outer segments accounted for the increased thresholds.

Topics: Adult; Dark Adaptation; Densitometry; Electroretinography; Female; Humans; Ophthalmoscopy; Photoreceptor Cells; Regeneration; Retina; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Visual Field Tests

Visual thresholds and rhodopsin levels were determined in nine subjects with autosomal dominant retinitis pigmentosa. The subjects fell into two groups, corresponding to two subtypes of the disease revealed by two-color, dark-adapted static perimetry. In the first of these subtypes, rod-mediated function was variably reduced and was accompanied by a corresponding reduction in cone function in the same retinal region. Dark-adapted threshold elevations varied in a way consistent with decreased quantal absorption by the rods as a result of reduced rhodopsin levels. In the second subtype, rod function was greatly reduced or absent throughout the retina, while cone function was much less severely affected. Although the levels of rhodopsin were only about half of normal, they were much too great to account for the visual threshold elevations on the basis of decreased probabilities of absorption by the visual pigment. Rhodopsin regeneration appeared to follow normal kinetics in patients from both groups. The results indicate that the examples of the two psychophysical subtypes of AD RP investigated here have very different disease manifestations.

Topics: Adult; Dark Adaptation; Electroretinography; Female; Genes, Dominant; Humans; Male; Middle Aged; Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Vision Disorders; Visual Field Tests

An imaging fundus reflectometer for in vivo mapping of rhodopsin levels is described. The instrument is based on a high-sensitivity television camera attached to a Zeiss fundus camera, which enables areas of retina of angular subtense 25 degrees to be examined at a resolution of about 1 degree. Digital processing techniques are used to average the video signal spatially and temporally and to analyse the spatial information. Measurements with an artificial eye indicate that performance is comparable to that of photomultiplier-based systems. Rhodopsin levels and regeneration data for a normal human subject are presented; these are consistent with published values. The map of visual pigment levels derived from these normal data is contrasted with that for a subject with a patchy retinal dysfunction (autosomal dominant retinitis pigmentosa).

Topics: Adult; Dark Adaptation; Fundus Oculi; Humans; Male; Methods; Ophthalmology; Retina; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Spectrophotometry; Television

A broad-field imaging fundus reflectometer was used to determine the levels of visual pigment and their relationship to rod-mediated sensitivity in 3 patients with autosomal dominantly inherited retinitis pigmentosa. In each case the loss of sensitivity could be accounted for wholly by the decreased probability of light absorption by the rod photoreceptors resulting from the decreased levels of rhodopsin they contained. In contrast, in a subject whose night blindness was due to vitamin A deficiency, the large sensitivity loss was accompanied by a relatively small reduction in the rhodopsin level.

Topics: Female; Fundus Oculi; Humans; Ophthalmoscopes; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Vision, Ocular; Vitamin A Deficiency

Night blindness is a frequent concomitant of retinal disorders, many of which are of genetic origin. Through the use of quantitative noninvasive test procedures it has been possible to study patients with these hereditary conditions and to show that the visual abnormalities often result from defects in the functional properties of the rod photoreceptors. More important, the uniqueness of the functional disturbance in the various types of night-blinding disorders suggests that each involves a specific aspect of the rod's internal machinery, i.e., the molecular processes devoted to transduction, intercellular communication, and the renewal of cellular components. Knowledge gained from the study of these clinical entities and from the investigation of experimental animals regarding the cellular events involved in these vital processes have enabled us to formulate tentative hypothesis as to the molecular bases of the hereditary defects.

Topics: Genetic Diseases, Inborn; Horseradish Peroxidase; Humans; Neuromuscular Junction; Neurotransmitter Agents; Night Blindness; Photoreceptor Cells; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Synapses; Synaptic Transmission

Rhodopsin density and absolute threshold were determined in 11 patients with retinitis pigmentosa (RP) and nine normal subjects. In eight patients with familial histories suggesting recessive inheritance, the retinal areas studied showed marked decrease in sensitivity which was related to rhodopsin loss, probably via a log-linear relationship. The other three patients showed absolute thresholds that were linearly related to rhodopsin density, suggesting that sensitivity loss was determined by the decrease in quantal absorption resulting from rhodopsin loss. The results indicate that RP patients can be classified into two categories according to the effects of rhodopsin loss on rod sensitivity. These categories may correspond to different genetic groups. Furthermore, this differentiation may, in fact, reflect different underlying disease mechanisms.

Topics: Adolescent; Adult; Densitometry; Female; Genes, Recessive; Humans; Male; Retina; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Sensory Thresholds; Visual Perception

The immune status of patients suffering from different types of retinitis pigmentosa has been investigated. The lymphocytes of these patients could be stimulated by incubation with human soluble retinal antigens as well as with bovine rod outer segments. The results suggest the involvement of the cellular immune system in retinitis pigmentosa. The leukocyte migration inhibition test also pointed in that way, especially if bovine rhodopsin was used as the antigen. The complement fixation test suggested the presence of a nonspecific weak antibody activity in the blood of retinitis pigmentosa patients as well as of controls. This activity seemed predominantly to be directed to the insoluble fraction of human retinas. On the basis of the findings we conclude that patients suffering from retinitis pigmentosa may become sensitized to retinal antigens, especially to those localized in the rod outer segments. This sensitization concerns the cell-mediated immune system and seems not to be correlated with a special type of the disease.

Topics: Adolescent; Adult; Antibodies; Antigens; Female; Humans; Immunity, Cellular; Immunologic Techniques; Leukocytes; Lymphocytes; Male; Retina; Retinitis Pigmentosa; Rhodopsin

Rhodopsin kinetics and visual threshold were determined in three subjects with a dominant form of retinitis pigmentosa. The retinal areas studied showed varying loss of sensitivity, which correlated well with the reduction in the measured density of rhodopsin in the test region. Rhodopsin photosensitivity was normal, and there was no evidence that either rhodopsin or the cone pigments regenerated more rapidly than normal. The findings in these cases of retinitis pigmentosa, when compared with the threshold changes induced by vitamin A deficiency or photic bleaching, suggest that the disease produces an imbalance between disc removal and new disc formation, which results in a progressive shortening of the photoreceptor outer segments and, eventually, in their complete disappearance.

Topics: Adult; Female; Humans; Light; Male; Middle Aged; Ophthalmology; Retina; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Visual Acuity; Vitamin A Deficiency

Topics: Animals; Darkness; Disease Models, Animal; Fatty Acids; Haplorhini; Hyperbaric Oxygenation; Iodoacetates; Light; Macaca mulatta; Phospholipids; Photoreceptor Cells; Rabbits; Rats; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Temperature

Of 107 consecutive patients with genetically-determined retinitis pigmentosa, 23 were provisionally diagnosed as having inherited the disease in an X-linked fashion. 42 affected males and 61 females were examined, and from the data obtained the following conclusions were drawn: (1) X-linked retinitis pigmentosa exists and is distinct from choroideremia. (2) In contrast to the results of previous surveys, X-linked retinitis pigmentosa is a common form of this disease and over 20 per cent. of retinitis pigmentosa is probably transmitted in an X-linked manner. (3) (a) In contradistinction to the findings of previous investigators, most if not all adult heterozygous females have detectable degenerative changes in the ocular fundus. (b) The ocular changes in heterozygous females are most easily detected by fundus examination, visual field testing, dark adaptation measurements, and estimation of retinal rhodopsin concentration. The single most frequent abnormality is peripheral retinal pigment epithelial atrophy, which is found in all adult heterozygous females. (c) The pattern of retinal dysfunction in heterozygous females, and in particular preservation of the ocular electrical responses, suggests that the disease in women is qualitatively different from that in men and in other genetic forms of retinitis pigmentosa. There is some evidience that the disease in heterozygous women is patchy. (d) Degeneration in heterozygous females is usually symmetrical, but great variation was found in the severity of degeneration amongst heterozygotes of similar ages. No non-genetic influences were found to account for this. No evidence came to light by which the importance of X-chromosome inactivation could be assessed in determining the phenotype of heterozygous women. (4) No evidience is available to determine the number of X-linked genes transmitting the disease.

Topics: Adult; Child; Dark Adaptation; Electrooculography; Female; Fluorescein Angiography; Genetic Linkage; Genetic Variation; Heterozygote; Humans; Male; Pedigree; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Sex Chromosomes; Sex Factors; Visual Fields