calpain and Muscular-Dystrophies

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Eukaryota; Humans; Mice; Muscular Dystrophies; Plants; Protein Structure, Tertiary; Stomach Ulcer

The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.

Topics: Animals; Calcium; Calpain; Humans; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies

Muscular dystrophy (MD), a group of inherited disorders characterized by progressive skeletal muscle wasting and weakness, can be classified into several groups according to Mendelian inheritance patterns and clinical features. Many genes related to MD have been identified and cloned by genetic linkage analysis and positional cloning strategy. Our understanding of the molecular mechanisms giving rise to muscular dystrophy have made a progress by the functional analysis of proteins encoded by candidate genes for MD. This article reviews genes and their functional mechanisms in the pathogenesis of muscular dystrophy.

Topics: Calpain; Dystrophin; Humans; Lamin Type A; Muscle Proteins; Muscular Dystrophies; Myostatin; Transforming Growth Factor beta; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Mutations in the calpain 3 (CAPN3) gene are responsible for limb-girdle muscular dystrophy (LGMD) type 2A. We report five causal mutations: 550delA, DeltaFWSAL, R541W, Y357X and R49H found on 45/50 of alleles studied in 25 unrelated families from Croatia. The 550delA mutation was present on 76% of CAPN3 chromosomes that led us to screen general population for this mutation; 532 random blood samples from three different regions were analyzed using allele-specific PCR. Four healthy 550delA heterozygous were found suggesting a frequency of 1 in 133. All four carriers detected originated from an island and mountain region close to the Adriatic Sea. These findings combined with haplotype analysis confirm that our general population is rather "closed" with a probable founder effect in some parts of the country. In addition, the high frequency of 550delA mutation found in some neighboring European countries together with the easy detection of the 550delA mutation should streamline genetic analysis, especially bearing in mind the geographic and ethnic origin of the patients. Our results, combined with published haplotype studies suggest that 550delA originated in the Eastern Mediterranean from which it has probably spread widely across Europe. Extending this study to other areas would help to address this epidemiological question. Our data are relevant to accurate genetic counseling and patient testing since we lack sensitive and specific biopsy screening methods for detecting patients with calpainopathy. Thus, detection of patients relies on the direct detection of gene mutation and our findings may be helpful in establishing diagnostic screening strategy.

Topics: Alleles; Calpain; Croatia; Female; Gene Deletion; Gene Frequency; Haplotypes; Heterozygote; Homozygote; Humans; Isoenzymes; Male; Muscle Proteins; Muscular Dystrophies; Mutation; Pedigree; Polymerase Chain Reaction; Prevalence

Calpains are calcium-modulated proteases which respond to Ca2+ signals by removing limited portions of protein substrates, thereby irreversibly modifying their function(s). Members of this protease family are present in a variety of organisms ranging from mammals to plants; some of them are ubiquitously expressed, while others are tissue specific. Although calpains are apparently involved in a multitude of physiological and pathological events, their functions are still poorly understood. In two cases, however, the alteration of a member of the calpain family has been clearly identified as being responsible for a human disease: the loss of function of calpain 3 causes limb girdle muscular dystrophy type 2A, and mutations in the gene coding for calpain 10 have been shown to correlate with non-insulin-dependent diabetes.

Topics: Calpain; Diabetes Mellitus, Type 2; Humans; Isoenzymes; Muscle Proteins; Muscular Dystrophies; Mutation

Calpain, a Ca(2+)-requiring cytoplasmic cysteine protease, plays indispensable roles in various cellular functions such as signal transduction, cell growth and differentiation, apoptosis, necrosis, and so on. Although most of the detailed physiological functions of calpains have not yet been elucidated, the importance of calpain is obvious from the increasing numbers of papers describing relationships between human disease states (such as Alzheimer's disease, cataract, and muscular dystrophies) and malfunction of calpain. One of the recent remarkable topics of calpain is that a single nucleotide polymorphism of CAPN10, the gene for calpain 10, is related to type 2 diabetes. However, physiological functions of calpain 10 and its relation to diabetes are still unclear. Among 14 human calpain genes, mutations in CAPN3, the gene for p94/calpain 3a and Lp82/calpain 3b, are the only example that genetically connects the calpain gene and human disease, in this case, limb-girdle muscular dystrophy type 2A (LGMD2A). p94 has unique characteristics such as apparent Ca(2+)-independent activation and very rapid autolytic activity, which are dependent on p94-specific regions, NS, IS1, and IS2. Based on the 3D structures of micro - and m-calpain, molecular functions of p94 in relation to LGMD2A are discussed, with the hope of providing us with some clues to understand calpain functions and its relationships to human diseases.

Topics: Animals; Calpain; Humans; Isoenzymes; Muscular Dystrophies; Protein Conformation

Muscular dystrophy includes many genetically distinct disorders. The list of causative genes for muscular dystrophy has been expanding rapidly, including those for congenital muscular dystrophies.. We review the newly identified causative genes and suggested molecular mechanisms, focusing on glycosylation abnormality of alpha-dystroglycan, collagen VI deficiency, four allelic diseases of caveolin-3 gene, and titin gene mutations.. Several possible mechanisms causing muscular dystrophy were discussed. Defects in extracellular molecules have more significant effects resulting mainly in congenital muscular dystrophy, while intracellular molecular defects show milder effect on the phenotype. These hypotheses may provide a new paradigm in understanding the pathomechanism of muscular dystrophies.

Topics: Alleles; Animals; Calpain; Caveolin 3; Caveolins; Collagen Type VI; Connectin; Cytoskeletal Proteins; Dystroglycans; Dystrophin; Glycosylation; Humans; Isoenzymes; Membrane Glycoproteins; Muscle Proteins; Muscular Dystrophies; Mutation; Protein Kinases

In our laboratory, limb-girdle muscular dystrophy (LGMD) accounted for 20% of all patients with muscular dystrophy. To determine the incidence of various forms of LGMD phenotypes, we looked for mutations in the calpain 3 gene and, for deficiencies in dysferlin and sarcoglycan by immunohistochemical studies with specific antibodies on muscle biopsies from patients with probable autosomal recessive inheritance (LGMD2), which were mostly sporadic cases of LGMD. Fourteen of 276 (5%) patients examined had sarcoglycan complex deficiency (sarcoglycanopathy) and 21 of 80 (26%) had mutations in the calpain 3 gene. Although we have not performed gene analysis in all patients, 10 of 64 (15%) patients examined had no apparent immunoreactivity against the dysferlin antibody. Thus, approximately 46% of LGMD2 patients had the above 3 distinct disorders, but in 54% the causative defects remain unknown.

Topics: Calpain; Cytoskeletal Proteins; Dysferlin; Humans; Isoenzymes; Membrane Proteins; Muscle Proteins; Muscular Dystrophies; Mutation

Limb girdle muscular dystrophies (LGMDs) are a group of clinically heterogeneous genetic diseases characterized by progressive weakness and atrophy of scapular and pelvic muscles, with either a dominant or recessive autosomic mode of inheritance. The first symptoms of the disorder appear during the first 20 years of life and progresses gradually, and a walking disability develops 10-20 years later. The gene responsible for LGMD2A has been identified and encodes calpain 3, a protease expressed mainly in skeletal muscle. Apoptotic myonuclei were recently detected in muscular biopsy specimens of LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of inhibitory protein kappaBalpha (IkappaBalpha) and nuclear factor kappaB (NF-kappaB), resulting in sarcoplasmic sequestration of NF-kappaB. Calpain 3 dependent IkappaBalpha degradation was reconstituted in vitro, supporting a possible in vivo sequence of events leading from calpain 3 deficiency to IkappaBkappa accumulation, prevention of nuclear translocation of NF-kappaB, and ultimately apoptosis. Therefore calpain 3, present in healthy muscle as sarcoplasmic and nuclear forms, may control IkappaBalpha turnover and indirectly regulate NF-kappaB dependent expression of survival genes. Recent data reported from a new model of LGMD2A in mice and from other muscular disorders strengthen understanding of the molecular links between calpain 3 and the Ikappaalpha/NF-kappaB pathway. Finally, in light of the lack of apoptosis observed in inflammatory myopathies, a unifying model for the control of cell survival in muscle is proposed and discussed

Topics: Animals; Apoptosis; Calpain; Cell Survival; DNA-Binding Proteins; Humans; I-kappa B Proteins; Isoenzymes; Models, Biological; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myositis; NF-kappa B; NF-KappaB Inhibitor alpha

The number of mammalian calpain protease family members has grown to 14 on last count. Overactivation of calpain 1 and calpain 2 (and their small subunit) has long been tied to acute neurological disorders (e.g. stroke and traumatic brain injury) and recently to Alzheimer's disease. Loss-of-function mutations of the calpain 3 gene have now been identified as the cause of limb-girdle muscular dystrophy 2A. Calpain 10 was recently identified as a susceptibility gene for type 2 diabetes, whereas calpain 9 appears to be a gastric cancer suppressor. This review describes our current understanding of the calpain family members and their mechanistic linkages to the aforementioned diseases as well as other emerging pathological conditions.

Topics: Alzheimer Disease; Animals; Calpain; Cataract; Diabetes Mellitus, Type 2; Disease; EF Hand Motifs; Humans; Multigene Family; Muscular Dystrophies; Nervous System Diseases; Stomach Neoplasms

Topics: Calpain; Diagnosis, Differential; Genes, Recessive; Humans; Isoenzymes; Muscle Proteins; Muscular Dystrophies; Mutation; Prognosis

Limb-girdle muscular dystrophy is a class of disorders encompassing many forms of this disease. Variation exists between the inheritance patterns, genes responsible, course of disease and symptoms, with the cohesive factor among these disorders being the predominance of proximal muscle weakness. Here we review each form of limb-girdle muscular dystrophy with attention to molecular genetics, clinical features, inheritance, and diagnostic issues pertaining to each primary genetic cause.

Topics: Calpain; Cytoskeletal Proteins; Humans; Membrane Glycoproteins; Muscular Dystrophies; Sarcoglycans

Topics: Amino Acid Sequence; Animals; Calpain; Humans; Molecular Sequence Data; Muscle, Skeletal; Muscular Dystrophies; Mutation; Organ Specificity; Structure-Activity Relationship

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.

Topics: Animals; Binding Sites; Calcium; Calpain; Disease Models, Animal; Dystrophin; Humans; Muscular Dystrophies

The skeletal muscle-specific calpain homologue, p94 (also called calpain 3), is essential for normal muscle function. A mutation of the p94 gene causes limb-girdle muscular dystrophy type 2A (LGMD2A), which is one type of autosomal recessive inherited disease characterized by progressive muscular degeneration. In myofibrils, p94 specifically binds to connectin/titin, and the activity of p94 is probably suppressed by this binding. Thus, we postulate that a signal transduction pathway exists, involving p94 and connectin/titin to modulate functions of skeletal muscle, and LGMD2A occurs when this signalling pathway is not properly regulated by p94. LGMD2A mutants of p94 also reveal significant information on the factors that relate structure to function in this molecule.

Topics: Animals; Calpain; Connectin; Humans; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myofibrils; Protein Kinases

Muscular dystrophy is a group of genetically determined muscular disorders marked by progressive wasting and weakness of the skeletal muscle, but which often affect cardiac and smooth muscles or other tissues. The patterns of inheritance are either dominant or recessive although the gene may be defective because of a new mutation. Growing evidence revealed the marked heterogeneity of the muscle disorders, and considerable numbers of Japanese scientists and physicians have contributed to the research progress in muscular dystrophy. Among these the discovery of an increased serum creatine kinase activity in muscular dystrophy opened the way for the most reliable laboratory test for muscular dystrophy in 1959, and subsequently accelerated progress in a broad range of research areas in medicine. Progress in modern genetics and molecular pathology provided another breakthrough in muscular dystrophy research and, in 1987, dystrophin was identified, a deficiency of which causes DMD. The present review article highlights contributions of Japanese scientists to muscular dystrophy research.

Topics: Calpain; Cell Membrane; Creatine Kinase; Dystrophin; Humans; Japan; Muscle, Skeletal; Muscular Dystrophies

Topics: Animals; Apoptosis; Calpain; Humans; Muscle, Skeletal; Muscular Dystrophies; Organ Specificity; Protein Conformation; Signal Transduction; Structure-Activity Relationship

The clinical heterogeneity which has long been recognized in the limb-girdle muscular dystrophies (LGMD) has been shown to relate to the involvement of a large number of different genes. At least eight forms of autosomal recessive LGMD and three forms of autosomal dominant disease are now recognized and can be defined by the primary gene or protein involved, or by a genetic localization. These advances have combined the approaches of positional cloning and candidate gene analysis to great effect, with the pivotal role of the dystrophin-associated complex confirmed through the involvement of at least four dystrophin-associated proteins in different subtypes of autosomal recessive LGMD (the sarcoglycanopathies). Two novel mechanisms may have to be postulated to explain the involvement of the calpain 3 and dysferlin genes in other forms of LGMD. Using the diagnostic tools which have become available as a result of this increased understanding, the clinical features of the various subtypes are also becoming clearer, with useful diagnostic and prognostic information at last available to the practising clinician.

Topics: Calpain; Chromosome Mapping; Cytoskeletal Proteins; Dysferlin; Dystroglycans; Genes, Recessive; Humans; Isoenzymes; Membrane Glycoproteins; Membrane Proteins; Muscle Proteins; Muscular Dystrophies; Peptide Fragments; Sarcoglycans

In the field of muscular dystrophy, advances in understanding the molecular basis of the various disorders in this group have been rapidly translated into readily applicable diagnostic tests, allowing the provision of more accurate prognostic and genetic counselling. The limb-girdle muscular dystrophies (LGMD) have recently undergone a major reclassification according to their genetic basis. Currently 13 different types can be recognized. Amongst this group, increasing diversity of the mechanisms involved in producing a muscular dystrophy phenotype is emerging. Recent insights into the involvement of the dystrophin glycoprotein complex in muscular dystrophy suggests that its members may play distinct or even multiple roles in the maintenance of muscle fibre integrity. In other forms of LGMD, proteins have been implicated which may be important in intracellular signalling, vesicle trafficking or the control of transcription. As these various mechanisms are more fully elucidated, further insights will be gained into the pathophysiology of muscular dystrophy. At a practical level, despite the marked heterogeneity of this group real progress can at last be made in determining a precise diagnosis.

Topics: Calpain; Caveolin 3; Caveolins; Dysferlin; Genes, Dominant; Genes, Recessive; Genetic Variation; Humans; Membrane Proteins; Muscle Proteins; Muscular Dystrophies; Mutation; Phenotype

p94, a muscle-specific member of the calpain family, also called calpain3 (CAPN3), has been identified as the gene product responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). To elucidate the molecular mechanism of LGMD2A, the effects of missense point mutations found in LGMD2A on the unique properties of p94 were studied. All of the mutants examined to date lose their proteolytic activity against fodrin, a cytoskeletal protein, strongly suggesting that of the specific properties of p94, the loss of protease activity is the prime cause of LGMD2A. Studies of LGMD2A and p94 suggest a novel molecular mechanism for muscular dystrophy, showing that a combined pathologic and biochemical approach is effective.

Topics: Animals; Calpain; Carrier Proteins; COS Cells; Humans; Hydrolysis; Microfilament Proteins; Muscle Proteins; Muscular Dystrophies; Mutation, Missense

Topics: Alzheimer Disease; Amino Acid Sequence; Animals; Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Cataract; Cell Cycle; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Long-Term Potentiation; Muscular Dystrophies; Parkinson Disease; Substrate Specificity

To review the up-dated classification of limb girdle muscular dystrophies (LGMDs) in relation to the defective protein and the genetic abnormality. To explain how these proteins are related to dystrophin and to the proteins of the extracellular matrix. To show that an accurate diagnosis is necessary and that it can be adequately made in neuromuscular pathology laboratories.. We present a study of the different types of LGMDs, dystrophinopathies and congenital muscular dystrophy. We emphasize the recent events which concluded in the identification of these disorders, the genetic alteration, the defective proteins and, briefly, the clinical features.. The recent identification of numerous skeletal muscle proteins and of the codifying genes made possible a new classification of a large group of muscular dystrophies. The possibility to study these proteins on the muscle biopsy with immunohistochemistry and Western blot techniques indicates the need of an accurate diagnosis in specialized neuromuscular laboratories. Since there is a great number of genes discovered and of mutations within the same gene, and the clinical picture of different diseases can be similar, a previous study of the protein is advisable as a guide for a further genetic study.

Topics: Calpain; Child, Preschool; Chromosome Mapping; Chromosomes, Human; Cytoskeletal Proteins; Dystroglycans; Dystrophin; Female; Humans; Infant; Infant, Newborn; Laminin; Macromolecular Substances; Male; Membrane Glycoproteins; Muscle Proteins; Muscular Dystrophies; Sarcoglycans

Topics: Animals; Calcium; Calpain; Cataract; Central Nervous System Diseases; Humans; Muscular Dystrophies; Myocardial Ischemia; Proteins

Topics: Calpain; Dystrophin; Glycoproteins; Humans; Laminin; Muscular Dystrophies; Myotonin-Protein Kinase; Protein Kinases; Protein Serine-Threonine Kinases

Topics: Calpain; Chromosome Mapping; Chromosomes, Human; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 4; Chromosomes, Human, Pair 5; Cytoskeletal Proteins; Diagnosis, Differential; Dystroglycans; Dystrophin; Genes, Dominant; Genes, Recessive; Humans; Membrane Glycoproteins; Muscular Dystrophies; Sarcoglycans

Topics: Animals; Calcium; Calpain; Chaperonins; Connectin; Humans; Isoenzymes; Membrane Proteins; Models, Structural; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Muscular Dystrophy, Animal; Protein Conformation; Protein Kinases; Recombinant Proteins

Both, recessive (LGMD R1) and dominant (LGMD D4) inheritance occur in calpain 3-related muscular dystrophy. We report a family with calpain-related muscular dystrophy caused by two known variants in the calpain 3 gene (CAPN3, NM_000070.3; (I) c.700G>A, p.Gly234Arg and (II) c.1746-20C>G, p.?). Three family members are compound heterozygous and exhibit a relatively homogeneous phenotype characterized by progressive proximal weakness starting in the third to fourth decade of life in the shoulder girdle and spreading to the legs. Two family members affected only by the p.Gly234Arg heterozygous missense variants show a different phenotype characterized by severe exertional myalgia without overt pareses. We conclude that in our family, the missense variant causes a severe myalgic phenotype without pareses that is aggravated by the second intronic variant and put these findings in the context of previous studies of the same variants.

Topics: Calpain; Humans; Muscle Proteins; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Mutation; Paresis; Phenotype

Aberrant expression of dystrophin, utrophin, dysferlin, or calpain-3 was originally identified in muscular dystrophies (MDs). Increasing evidence now indicates that these proteins might act as tumor suppressors in myogenic and non-myogenic cancers. As DNA damage and somatic aneuploidy, hallmarks of cancer, are early pathological signs in MDs, we hypothesized that a common pathway might involve the centrosome. Here, we show that dystrophin, utrophin, dysferlin, and calpain-3 are functional constituents of the centrosome. In myoblasts, lack of any of these proteins caused excess centrosomes, centrosome misorientation, nuclear abnormalities, and impaired microtubule nucleation. In dystrophin double-mutants, these defects were significantly aggravated. Moreover, we demonstrate that also in non-myogenic cells, all four MD-related proteins localize to the centrosome, including the muscle-specific full-length dystrophin isoform. Therefore, MD-related proteins might share a convergent function at the centrosome in addition to their diverse, well-established muscle-specific functions. Thus, our findings support the notion that cancer-like centrosome-related defects underlie MDs and establish a novel concept linking MDs to cancer.

Topics: Calpain; Centrosome; Dysferlin; Dystrophin; Humans; Membrane Proteins; Muscular Dystrophies; Neoplasms; Utrophin

A Dutch cohort of 105 carefully selected limb girdle muscular dystrophy (LGMD) patients from 68 families has been subject to genetic testing over the last 20 years. After subsequent targeted gene analysis around two thirds (45/68) of the families had received a genetic diagnosis in 2013.. To describe the results of further genetic testing in the remaining undiagnosed limb girdle muscular dystrophy families in this cohort.. In the families of the cohort for whom no genetic diagnosis was established (n = 23) further testing using Sanger sequencing, next generation sequencing with gene panel analysis or whole-exome sequencing was performed. In one case DNA analysis for facioscapulohumeral dystrophy type 1 was carried out.. In eight families no additional genetic tests could be performed. In 12 of the remaining 15 families in which additional testing could be performed a genetic diagnosis was established: two LGMDR1 calpain3-related families with CAPN3 mutations, one LGMDR2 dysferlin-related family with DYSF mutations, three sarcoglycanopathy families (LGMDR3-5 α-, β- and γ-sarcoglycan-related) with SGCA/SGCB/SGCG mutations, one LGMDR8 TRIM 32-related family with TRIM32 mutations, two LGMDR19 GMPPB-related families with GMPPB mutations, one family with MICU1-related myopathy, one family with FLNC-related myopathy and one family with facioscapulohumeral dystrophy type 1. At this moment a genetic diagnosis has been made in 57 of the 60 families of which DNA was available (95%).. A genetic diagnosis is obtained in 95% of the families of the original Dutch LGMD cohort of which DNA was available.

Topics: Adolescent; Adult; Calcium-Binding Proteins; Calpain; Cation Transport Proteins; Child; Dysferlin; Exome Sequencing; Female; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Mitochondrial Membrane Transport Proteins; Muscle Proteins; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Mutation; Netherlands; Phenotype; Sarcoglycanopathies; Sequence Analysis, DNA; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Young Adult

Presented here are five members of a family that was ascertained from an isolated, consanguineous, indigenous Amerindian community in Colombia that was affected with calpain 3-related, limb-girdle muscular dystrophy type R1. These patients are homozygous for a unique and novel deletion of four bases (TGCC) in exon 3 of the calpain 3 gene (

Topics: Adolescent; Adult; Calpain; Child; Exons; Family; Female; Homozygote; Humans; Male; Muscle Proteins; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Mutation; Pedigree; Phenotype; Sequence Deletion

Dysferlin is a large transmembrane protein that plays a key role in cell membrane repair and underlies a recessive form of inherited muscular dystrophy. Dysferlinopathy is characterized by absence or marked reduction of dysferlin protein with 43% of reported pathogenic variants being missense variants that span the length of the dysferlin protein. The unique structure of dysferlin, with seven tandem C2 domains separated by linkers, suggests dysferlin may dynamically associate with phospholipid membranes in response to Ca

Topics: Amino Acid Substitution; Biopsy; C2 Domains; Calpain; Cells, Cultured; Dysferlin; HEK293 Cells; Humans; Molecular Weight; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies; Mutation, Missense; Peptide Fragments; Proteasome Endopeptidase Complex; Protein Conformation; Protein Folding; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; Proteolysis; Recombinant Fusion Proteins; Recombinant Proteins

Myopathies encompass a wide variety of acquired and hereditary disorders. The pathomechanisms include structural and functional changes affecting, e.g., myofiber metabolism and contractile properties. In this study, we observed increased passive tension (PT) of skinned myofibers from patients with myofibrillar myopathy (MFM) caused by FLNC mutations (MFM-filaminopathy) and limb-girdle muscular dystrophy type-2A due to CAPN3 mutations (LGMD2A), compared to healthy control myofibers. Because the giant protein titin determines myofiber PT, we measured its molecular size and the titin-to-myosin ratio, but found no differences between myopathies and controls. All-titin phosphorylation and site-specific phosphorylation in the PEVK region were reduced in myopathy, which would be predicted to lower PT. Electron microscopy revealed extensive ultrastructural changes in myofibers of various hereditary myopathies and also suggested massive binding of proteins to the sarcomeric I-band region, presumably heat shock proteins (HSPs), which can translocate to elastic titin under stress conditions. Correlative immunofluorescence and immunoelectron microscopy showed that two small HSPs (HSP27 and αB-crystallin) and the ATP-dependent chaperone HSP90 translocated to the titin springs in myopathy. The small HSPs, but not HSP90, were upregulated in myopathic versus control muscles. The titin-binding pattern of chaperones was regularly observed in Duchenne muscular dystrophy (DMD), LGMD2A, MFM-filaminopathy, MFM-myotilinopathy, titinopathy, and inclusion body myopathy due to mutations in valosin-containing protein, but not in acquired sporadic inclusion body myositis. The three HSPs also associated with elastic titin in mouse models of DMD and MFM-filaminopathy. Mechanical measurements on skinned human myofibers incubated with exogenous small HSPs suggested that the elevated PT seen in myopathy is caused, in part, by chaperone-binding to the titin springs. Whereas this interaction may be protective in that it prevents sarcomeric protein aggregation, it also has detrimental effects on sarcomere function. Thus, we identified a novel pathological phenomenon common to many hereditary muscle disorders, which involves sarcomeric alterations.

Topics: alpha-Crystallin B Chain; Animals; Calpain; Connectin; Disease Models, Animal; Filamins; Fluorescent Antibody Technique; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Mice, Inbred C57BL; Mice, Inbred mdx; Microscopy, Immunoelectron; Molecular Chaperones; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Myofibrils; Myosins; Phosphorylation; Protein Kinases; Sarcomeres

The muscular dystrophies (MDs) result from perturbations in the myofibers. These alterations are induced in part by mechanical stress due to membrane cell fragility, disturbances in mechanotransduction pathways, muscle cell physiology, and metabolism.. We analyzed 290 biopsies of patients with a clinical diagnosis of muscular dystrophy. Using immunofluorescence staining, we searched for primary and secondary deficiencies of 12 different proteins, including membrane, costamere, cytoskeletal, and nuclear proteins. In addition, we analyzed calpain-3 by immunoblot.. We identified 212 patients with varying degrees of protein deficiencies, including dystrophin, sarcoglycans, dysferlin, caveolin-3, calpain-3, emerin, and merosin. Moreover, 78 biopsies showed normal expression of all investigated muscle proteins. The frequency rates of protein deficiencies were as follows: 52.36% dystrophinopathies; 18.40% dysferlinopathies; 14.15% sarcoglycanopathies; 11.32% calpainopathies; 1.89% merosinopathies; 1.42% caveolinopathies; and 0.47% emerinopathies. Deficiencies in lamin A/C and telethonin were not detected.. We have described the frequency of common muscular dystrophies in Mexico.

Topics: Adolescent; Adult; Calpain; Caveolin 3; Child; Child, Preschool; Creatine Kinase; Dysferlin; Dystrophin; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Infant; Laminin; Membrane Proteins; Mexico; Middle Aged; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Nuclear Proteins; Sarcoglycans; Severity of Illness Index; Young Adult

The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.

Topics: Animals; Animals, Genetically Modified; Caenorhabditis elegans; Calcium; Calpain; Disease Models, Animal; Dystrophin-Associated Protein Complex; EF Hand Motifs; Evolution, Molecular; Gene Expression Regulation; Humans; Muscle, Skeletal; Muscular Dystrophies; Nuclear Proteins; Paralysis; Phosphotransferases; Phylogeny; Sequence Homology, Amino Acid; Transcription Factors

Ullrich congenital muscular dystrophy (UCMD) is a common form of muscular dystrophy associated with defects in collagen VI. It is characterized by loss of individual muscle fibers and muscle mass and proliferation of connective and adipose tissues. We sought to investigate the mechanisms by which collagen VI regulates muscle cell survival, size, and regeneration and, in particular, the potential role of the ubiquitin-proteasome and calpain-proteolytic systems. We studied muscle biopsies of UCMD (n = 6), other myopathy (n = 12), and control patients (n = 10) and found reduced expression of atrogin-1, MURF1, and calpain-3 mRNAs in UCMD cases. Downregulation of calpain-3 was associated with changes in the nuclear immunolocalization of nuclear factor-κB. We also observed increased expression versus controls of regeneration markers at the protein and RNA levels. Satellite cell numbers did not differ in collagen VI-deficient muscle versus normal nonregenerating muscle, indicating that collagen VI does not play a key role in the maintenance of the satellite cell pool. Our results indicate that alterations in calpain-3 and nuclear factor-κB signaling pathways may contribute to muscle mass loss in UCMD muscle, whereas atrogin-1 and MURF1 are not likely to play a major role.

Topics: Calpain; Child; Child, Preschool; Collagen Type VI; Female; Humans; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; NF-kappa B; Regeneration; Signal Transduction; Young Adult

LGMD (limb-girdle muscular dystrophy) and CMD (congenital muscular dystrophy) are two common forms of neuromuscular disorders which are distinguishable by their age of onset but with probably a similar underlying pathway. In the present study, we report immunohistochemical, Western-blot and genetic analyses in a large consanguineous Tunisian family with two branches, including seven patients sharing similar LGMD2 phenotype in one branch and one CMD patient in the other branch. Linkage analyses were compatible with the LGMD2A locus in one branch and the MDC1A (muscular dystrophy congenital type 1A) locus in the other branch. This result was supported by deficiency in merosin and calpain3 in the CMD patient and LGMD patients respectively. Mutation analysis revealed two distinct mutations: a c.8005delT frameshift deletion in exon 56 of the LAMA2 (laminin-α2) gene (MDC1A) was found in the CMD patient and a new homozygous mutation c.1536+1G>T in the donor splice site of intron 12 of the CAPN3 (calpain3) gene (LGMD2A) was found in the LGMD patients. RT-PCR (reverse transcription-PCR) performed on total RNA from a LGMD2A patient's muscle biopsy showed complete retention of intron 12 in CAPN3 cDNA, generating a PTC (premature termination codon) that potentially elicits degradation of the nonsense mRNA by NMD (nonsense-mediated mRNA decay). Our results indicate that mRNA analysis is necessary to clarify the primary effect of genomic mutations on splicing efficiency that alters mRNA processing and expression level.

Topics: Base Sequence; Blotting, Western; Calpain; Chromosome Mapping; Codon, Nonsense; Consanguinity; DNA Mutational Analysis; Family; Female; Genetic Heterogeneity; Genetic Linkage; Humans; Laminin; Male; Muscle Proteins; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA, Messenger; Sequence Analysis, RNA; Tunisia

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.

Topics: Aneuploidy; Animals; Calpain; Cells, Cultured; Comparative Genomic Hybridization; Disease Models, Animal; DNA Damage; Dysferlin; Dystrophin; Female; Humans; Membrane Proteins; Mice; Mice, Inbred C57BL; Models, Animal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; N-Acetylglucosaminyltransferases; Sarcoma

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Topics: Calpain; Cell Differentiation; Cell Line; Down-Regulation; Humans; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Dystrophies; Myoblasts; MyoD Protein; Proteasome Endopeptidase Complex; Regeneration; Satellite Cells, Skeletal Muscle; Transcription, Genetic; Ubiquitin

The definite molecular diagnosis in patients with muscular dystrophies often requires the assessment of muscular expression of multiple proteins in small amounts of muscle tissue. The sample material obtained in muscle biopsies is limited and the measurement of multiple proteins is often restricted to conventional, non-quantitative assays, i.e. immunohistochemistry and immunoblotting. Here, we demonstrate that reverse protein arrays are a novel and excellent material-saving method for the measurement and quantification of changes in protein expression between healthy and diseased muscle tissue as well as cultured primary myotubes. We evaluated a set of antibodies and found reproducible differences between Duchenne muscular dystrophy/limb-girdle muscular dystrophy patients and control samples for dystrophin, the sarcoglycans and the dystroglycans. As little as 10 mg of tissue is sufficient for the analysis of all diagnostically relevant proteins. The average coefficient of variation calculated for the sample signals confirmed that the method is highly reproducible. Thus, our experiments provide strong evidence that quantitative protein detection from very small amounts of muscle tissue is possible using reverse protein arrays. This technology may not only be of interest for diagnostic purposes, but also for protein quantification of multiple, follow-up biopsies during clinical trials when protein expression in muscle is considered an important outcome measure or biomarker.

Topics: Calpain; Dystroglycans; Dystrophin; Humans; Membrane Proteins; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myoblasts; Protein Array Analysis; Sarcoglycans; Tissue Culture Techniques

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

Topics: Animals; Biomarkers; Calpain; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Profiling; Male; Mice; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; Nuclear Proteins; Proteasome Endopeptidase Complex; Repressor Proteins; Signal Transduction; Up-Regulation

Topics: Adult; Calpain; Diagnosis, Differential; Disease Progression; Humans; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myopathies, Structural, Congenital

Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 (CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

Topics: Animals; Calpain; Dependovirus; Genetic Engineering; Genetic Therapy; Genetic Vectors; Isotonic Contraction; Male; Mice; Mice, Knockout; Muscle, Skeletal; Muscular Dystrophies; Mutation; Myostatin; Sarcoglycans; Transduction, Genetic; Transforming Growth Factor beta

Human muscular dystrophies are a heterogeneous group of genetic disorders which cause decreased muscle strength and often result in premature death. There is no known cure for muscular dystrophy, nor have all causative genes been identified. Recent work in the small vertebrate zebrafish Danio rerio suggests that mutation or misregulation of zebrafish dystrophy orthologs can also cause muscular degeneration phenotypes in fish. To aid in the identification of new causative genes, this study identifies and maps zebrafish orthologs for all known human muscular dystrophy genes.. Zebrafish sequence databases were queried for transcripts orthologous to human dystrophy-causing genes, identifying transcripts for 28 out of 29 genes of interest. In addition, the genomic locations of all 29 genes have been found, allowing rapid candidate gene discovery during genetic mapping of zebrafish dystrophy mutants. 19 genes show conservation of syntenic relationships with humans and at least two genes appear to be duplicated in zebrafish. Significant sequence coverage on one or more BAC clone(s) was also identified for 24 of the genes to provide better local sequence information and easy updating of genomic locations as the zebrafish genome assembly continues to evolve.. This resource supports zebrafish as a dystrophy model, suggesting maintenance of all known dystrophy-associated genes in the zebrafish genome. Coupled with the ability to conduct genetic screens and small molecule screens, zebrafish are thus an attractive model organism for isolating new dystrophy-causing genes/pathways and for use in high-throughput therapeutic discovery.

Topics: Animals; Calpain; Databases, Genetic; Disease Models, Animal; Expressed Sequence Tags; Gene Duplication; Gene Expression Regulation; Genome; Humans; Muscle Proteins; Muscular Dystrophies; Mutation; Phenotype; Physical Chromosome Mapping; Software; Zebrafish

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

Topics: Animals; Calpain; Dysferlin; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry; Membrane Proteins; Mice; Mice, Transgenic; Models, Biological; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myosin Light Chains; Proteomics; Substrate Specificity

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

Topics: Animals; Blotting, Western; Calpain; Camelids, New World; DNA Mutational Analysis; Dysferlin; Humans; Immunoassay; Immunoglobulin Fragments; Immunoglobulin Heavy Chains; Immunoprecipitation; Isoenzymes; Membrane Proteins; Microscopy, Fluorescence; Muscle Proteins; Muscular Dystrophies; Peptide Library; Recombinant Fusion Proteins

Limb gird muscular dystrophies (LGMD2) are a clinically and genetically heterogeneous group of hereditary diseases with autosomal recessive trait, characterized by progressive atrophy and weakness predominantly in the proximal limb muscles. The authors present clinical, histological, immunohistochemical and immunoblot results of two sisters suffering from so far unclassified autosomal recessive limb girdle muscular dystrophy. Haplotype analysis for genes possibly involved in autosomal recessive limb girdle muscular dystrophies was performed in the genetically informative family. All of the results pointed to a molecular genetic defect of the calpain-3 (CAPN3) gene. Direct sequencing of the CAPN3 gene revealed compound heterozygous state for two mutations previously described in association with limb girdle muscular dystrophy, proving pathogenicity. The authors would like to emphasize the importance of the above described combined strategy in diagnosing limb girdle muscular dystrophies.

Topics: Adult; Blotting, Western; Calpain; Extremities; Female; Genetic Linkage; Genetic Markers; Haplotypes; Humans; Hungary; Muscular Dystrophies; Pedigree

To determine types and frequency of CAPN3 mutations in 29 unrelated Croatian families, analyzed during 6-year prospective and ongoing genetic and epidemiological study of muscular dystrophies in Croatia.. Mutation analysis included allele-specific polymerase chain reaction (PCR) or combination of PCR and restriction fragment length polymorphisms (RFLP) methods. Haplotype analysis was performed by PCR and DNA electrophoresis using 5 highly polymorphic markers flanking CAPN3 gene locus.. Mutation analysis revealed the presence of 6 different CAPN3 mutations (550delA, R541W, P82L, delFWSAL, R49H, Y537X), accounting for 94.8% of CAPN3 chromosomes in the studied population. 550delA was the most frequent mutation, found in 43/58 (74%) CAPN3 chromosomes, whereas the frequency of other five mutations ranged from 2-9%. Haplotype analysis of 38 chromosomes carrying 550delA mutation showed the presence of the same haplotype on 66% of analyzed chromosomes.. The present data, together with our previously published results, explain the frequency and the distribution of the 550delA mutation in Croatia by founder effect and genetic drift. Results of haplotype study are in favor of the hypothesis that 550delA is an old, rather than a recurrent mutation. The findings are important for effective diagnostic screening of CAPN3 gene in Croatia and neighboring countries, as well as for accurate genetic counseling.

Topics: Calpain; Croatia; Female; Haplotypes; Humans; Male; Molecular Epidemiology; Muscular Dystrophies; Pedigree; Prospective Studies

Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.

Topics: Animals; Binding Sites; Calpain; Connectin; Crosses, Genetic; Exercise Test; Locomotion; Mice; Mice, Knockout; Mice, Transgenic; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Myositis; Protein Kinases; Protein Structure, Tertiary; Transcriptional Activation

We report on two siblings with late-onset, limb-girdle muscular dystrophy (LGMD) inherited in an autosomal recessive manner. The LGMD was characterized by many rimmed vacuoles and reduced expression of the laminin beta1 chain in skeletal muscle. Both patients developed a progressive wasting and weakness of limb-girdle muscles in the late forties or early fifties; their facial, ocular, bulbar, and cardiac muscles were not involved. Histopathology of skeletal muscles biopsies showed typical dystrophic changes with many rimmed vacuoles. The immunoreactivity of the laminin beta1 chain was reduced in the muscle fibers, while dystrophin, sarcoglycans, beta-dystroglycan, dysferlin, and other laminin components were normally expressed. A mutation search revealed that no mutation existed in the coding region of the calpain 3, telethonin and UDP-N-acetylglucosamine 2-epimerase/N-acetylmanosamine kinase (GNE) genes. We conclude that this autosomal recessive LGMD is unknown and characterized by its late onset, rimmed vacuoles and reduction of the laminin beta1 chain in muscle fibers.

Topics: Actin Cytoskeleton; Biopsy; Calpain; Chromosome Aberrations; Connectin; Consanguinity; Creatine Kinase; Cytoplasm; DNA Mutational Analysis; Female; Gait Apraxia; Genes, Recessive; Genetic Markers; Humans; Inclusion Bodies; Isoenzymes; L-Lactate Dehydrogenase; Microscopy, Electron; Microscopy, Fluorescence; Multienzyme Complexes; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; Neurologic Examination; Pedigree; Reverse Transcriptase Polymerase Chain Reaction; Tomography, X-Ray Computed; Vacuoles

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain-3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS-PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain-3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain-3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype-protein-phenotype correlations and provide insights into a critical protein domain.

Topics: Adolescent; Adult; Calpain; Child; Chromatography, High Pressure Liquid; DNA Mutational Analysis; Exons; Female; Genotype; Humans; Isoenzymes; Loss of Heterozygosity; Male; Molecular Diagnostic Techniques; Muscle Proteins; Muscular Dystrophies; Mutation, Missense; Phenotype; Polymorphism, Single-Stranded Conformational; Protein Denaturation; Sensitivity and Specificity; Sex Distribution

Limb girdle muscular dystrophy type 2B and Miyoshi myopathy are clinically distinct forms of muscular dystrophy that arise from defects in the dysferlin gene. Here, we report two novel lines of dysferlin-deficient mice obtained by (a) gene targeting and (b) identification of an inbred strain, A/J, bearing a retrotransposon insertion in the dysferlin gene. The mutations in these mice were located at the 3' and 5' ends of the dysferlin gene. Both lines of mice lacked dysferlin and developed a progressive muscular dystrophy with histopathological and ultrastructural features that closely resemble the human disease. Vital staining with Evans blue dye revealed loss of sarcolemmal integrity in both lines of mice, similar to that seen in mdx and caveolin-3 deficient mice. However, in contrast to the latter group of animals, the dysferlin-deficient mice have an intact dystrophin glycoprotein complex and normal levels of caveolin-3. Our findings indicate that muscle membrane disruption and myofiber degeneration in dysferlinopathy were directly mediated by the loss of dysferlin via a new pathogenic mechanism in muscular dystrophies. We also show that the mutation in the A/J mice arose between the late 1970s and the early 1980s, and had become fixed in the production breeding stocks. Therefore, all studies involving the A/J mice or mice derived from A/J, including recombinant inbred, recombinant congenic and chromosome substitution strains, should take into account the dysferlin defect in these strains. These new dysferlin-deficient mice should be useful for elucidating the pathogenic pathway in dysferlinopathy and for developing therapeutic strategies.

Topics: Animals; Calpain; Caveolin 3; Caveolins; Disease Models, Animal; Dysferlin; Dystrophin; Gene Expression; Gene Targeting; Humans; Membrane Proteins; Mice; Mice, Mutant Strains; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Phenotype; RNA, Messenger; Sarcolemma

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by any of over 150 mutations in the calpain-3 (CAPN3) gene. Of those, 2362AG --> TCATCT is particularly prevalent in Basque patients, and this mutation was hypothesized to have arisen in the Basque Country. To explore the natural history of this mutation, we genotyped 65 Basque and non-Basque patients with LGMD2A who carry the 2362AG --> TCATCT mutation for four microsatellites within or flanking the gene. A particular haplotype was found in three-fourths of the patients and was assumed to be ancestral. From the average number of recombinations and mutations accumulated from this ancestral haplotype, the age of the 2362AG ----> TCATCT mutation was estimated to be 50 generations (i.e., 1,250 years), which is more recent than the Paleolithic Basque heritage. The subsequent spread of the 2362AG --> TCATCT mutation can be related to gene flow out of the Basque Country, even across a cultural border.

Topics: Calpain; Gene Frequency; Genetic Predisposition to Disease; Genetic Variation; Genetics, Population; Haplotypes; Humans; Muscle Proteins; Muscular Dystrophies; Mutation; Polymorphism, Single-Stranded Conformational; Spain

Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFkappaB/IkappaB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4-5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by approximately 47% (P<0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFkappaB precursor and/or a defect in specific substrate targeting.

Topics: Animals; Blotting, Northern; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Down-Regulation; Lysosomes; Mice; Mice, Knockout; Multienzyme Complexes; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Peptide Fragments; Peptide Hydrolases; Proteasome Endopeptidase Complex; Ubiquitin

Defects in human calpain 3 are responsible for limb-girdle muscular dystrophy type 2A, an autosomal-recessive disorder characterized mainly by late-onset proximal muscular atrophy. A corresponding murine model has previously been generated by gene targeting. In this report, muscular activity of calpain 3-deficient (capn3(-/-)) mice was evaluated at different ages. Growth curves showed a progressive global muscular atrophy. Histological examination throughout the lifespan of mice confirmed the dystrophic lesions. Whole animal tests showed only a mild significant impairment of the forelimbs. Studies of the mechanical properties of selected isolated fast- and slow-twitch muscles demonstrated that slow-twitch muscles were significantly weaker in capn3(-/-) mice than in wild-type mice. Three different tests showed that there was no membrane disruption, suggesting a nonmechanical etiology of capn3(-/-) mice dystrophy. These findings are consistent with a mechanism involving signaling systems.

Topics: Animals; Calpain; Disease Models, Animal; Electric Impedance; Female; Mice; Mice, Mutant Strains; Muscle Contraction; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Phenotype

Mutations in the dysferlin gene cause muscular dystrophies called dysferlinopathy, which include limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). To clarify the frequency, clinicopathological and genetic features of dysferlinopathy in Japan, we performed protein and gene analyses of dysferlin. We examined a total of 107 unrelated Japanese patients, including 53 unclassified LGMD, 28 MM and 26 other neuromuscular disorders (ONMD). Expression of dysferlin protein was observed using immunohistochemistry (IHC) and mini-multiplex Western blotting (MMW), and mutation analysis was performed. We found a deficiency of dysferlin protein by both IHC and MMW in 19% of LGMD and 75% of MM patients, and mutations in the dysferlin gene were identified in this group alone. 19% of dysferlin-deficient patients had 3370G-->T missense mutation and 16% had 1939C-->G nonsense mutation. The patients with homozygous 3370G-->T mutation showed milder clinical phenotypes. Twenty-five percent of MM muscles had normal dysferlin protein contents that suggested the genetic heterogeneity of this disease. Altered immunolocalization of dysferlin was observed in not only primary dysferlinopathy, but also in the several diseased muscles with normal protein contents. This result implies the necessity of other protein(s) for proper membrane localization of dysferlin, or some roles of dysferlin in the cytoplasmic region.

Topics: Age of Onset; Alanine; Blotting, Western; Calpain; Cysteine; Cytoskeletal Proteins; DNA Mutational Analysis; Dysferlin; Dystroglycans; Dystrophin; Glycine; Humans; Immunohistochemistry; Japan; Membrane Glycoproteins; Membrane Proteins; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Neuromuscular Diseases; Sarcoglycans; Sequence Analysis, Protein; Tyrosine

Calpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils [Sorimachi et al. (1995) J. Biol. Chem. 270, 31158-31162]. However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined. In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains. This antibody stained myofibrils with a unique repeated doublet-pattern. Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils. Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.

Topics: Calpain; Fluorescent Antibody Technique; Humans; Isoenzymes; Microscopy, Confocal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myofibrils; Subcellular Fractions

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 gene. In a large family affected by LGMD2A with four severely affected members, three additional asymptomatic relatives had very high serum creatine kinase concentrations. All were homozygous for the R110X mutation and showed a total absence of calpain 3 in the muscle. Histological analysis of muscle in these three rare preclinical cases showed a consistent but unusual pattern, with isolated fascicles of degenerating fibres in an almost normal muscle. This pattern was also seen in one patient with early stage LGMD2A who had a P82L missense mutation and a partial deficiency of calpain 3 in the muscle, but was not seen in early stage patients affected by other forms of LGMD. These findings suggest that a peculiar pattern of focal degeneration occurs in calpainopathy, independently of the type of mutation or the amount of calpain 3 in the muscle.

Topics: Calpain; Case-Control Studies; Histocytochemistry; Humans; Immunohistochemistry; Isoenzymes; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation

Topics: Adult; Calpain; Cytoskeletal Proteins; Dystrophin-Associated Proteins; Female; Humans; Immunohistochemistry; Male; Membrane Proteins; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies; Mutation; Patient Selection; Protein Isoforms; Sarcolemma

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.

Topics: Adolescent; Adult; Age of Onset; Blotting, Western; Calpain; Child; DNA Mutational Analysis; Female; Humans; Isoenzymes; Male; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation, Missense; Polymerase Chain Reaction

Limb girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders characterised by progressive weakness of the pelvic and shoulder girdle muscles and a great variability in clinical course. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the calpain-3 gene (CAPN-3). More than 100 pathogenic mutations have been identified to date, however few genotype : phenotype correlation studies, including both DNA and protein analysis, have been reported. In this study we screened 26 unrelated LGMD2A Brazilian families (75 patients) through Single-Stranded Conformation Polymorphism (SSCP), Denaturing high-performance liquid chromatography (DHPLC) and sequencing of abnormal fragments which allowed the identification of 47 mutated alleles (approximately 90%). We identified two recurrent mutations (R110X and 2362-2363AG > TCATCT) and seven novel pathogenic mutations. Interestingly, 41 of the identified mutations (approximately 80%) were concentrated in only 6 exons (1, 2, 4, 5, 11 and 22), which has important implications for diagnostic purposes. Protein analysis, performed in 28 patients from 25 unrelated families showed that with exception of one patient (with normal/slight borderline reduction of calpain) all others had total or partial calpain deficiency. The effects of type of mutation, amount of calpain in the muscle, gender and ethnicity of affected patients on clinical course (age of onset and ascertainment) were analysed. Interestingly, it was observed that, on average, African-Brazilian calpainopathy patients are more severely affected than Caucasians.

Topics: Adolescent; Adult; Base Sequence; Calpain; Child; Child, Preschool; Chromatography, High Pressure Liquid; Female; Humans; Isoenzymes; Male; Middle Aged; Muscle Proteins; Muscles; Muscular Dystrophies; Mutation; Polymorphism, Single-Stranded Conformational

The clinico-pathological features of 17 patients displaying a myopathy with lobulated (trabeculated) fibers are reported. All these patients had a limb girdle phenotype and at least 20% of lobulated fibers in their muscle biopsies. There were ten females and seven males. The onset of symptoms ranged from 2 to 55 years (mean 24). The average age at the time of muscle biopsy was 39 (range 3-63). Interestingly, in six patients, high prevalence of lobulated fibers was observed at the second biopsy only, performed on average 11 years after the first or in another muscle. Six patients had a suggestively positive family history. Facial weakness was noted in two patients (genetic study confirmed FSH dystrophy). The course and the severity of weakness varied from one patient to another. Immunohistochemistry and Western blot analyses revealed one Duchenne carrier, one alpha-sarcoglycanopathy, no dysferlinopathy and four calpain deficiencies (including one patient with FSH dystrophy), but SSCP revealed mutation in the calpain gene in only one of the patients. These results show that (1) myopathies with lobulated fibers are clinically and genetically heterogeneous, (2) lack of calpain expression by Western blot analysis is not always associated with null mutation, (3) a molecular diagnosis is made in less than 40% of myopathy with lobulated fibers, (4) when observed, lobulated fibers are most prominent in proximal muscles and require time to appear.

Topics: Adolescent; Adult; Biopsy; Calpain; Child, Preschool; Dysferlin; Dystrophin; Female; Follicle Stimulating Hormone; Gene Expression; Genetic Heterogeneity; Humans; Immunohistochemistry; Male; Membrane Proteins; Middle Aged; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Dystrophies; Mutation; Phenotype

We describe a strategy for molecular diagnosis in the autosomal recessive limb-girdle muscular dystrophies, a highly heterogeneous group of inherited muscle-wasting diseases. Genetic mutation analysis is directed by immunoanalysis of muscle biopsies using antibodies against a panel of muscular dystrophy-associated proteins. Performing the molecular analysis in this way greatly increases the chance that mutations will be found in the first gene examined. The use of this strategy can significantly decrease the time involved in determining the genetic fault in a patient with a clinical diagnosis of recessive limb-girdle muscular dystrophy, as well as having a feedback effect, which is useful in helping clinicians to identify subtle clinical differences between the subtypes of the disease. The use of this approach has so far helped us to identify mutations in ten sarcoglycanopathy (limb-girdle muscular dystrophy 2C-2F) patients, and seven calpainopathy (limb-girdle muscular dystrophy 2A) patients.

Topics: Calpain; DNA Mutational Analysis; Dysferlin; Dystrophin; Genes, Recessive; Humans; Immunohistochemistry; Membrane Proteins; Muscle Proteins; Muscular Dystrophies

Mutations in the genes encoding for calpain-3 and dysferlin are responsible for limb-girdle muscular dystrophy (LGMD) type 2A and 2B, the most common forms of autosomal recessive LGMD.. To identify calpain-3 or dysferlin deficiency in a large cohort of patients with as yet unclassified LGMD and myopathy through candidate protein analysis.. The authors' muscle biopsy database search identified 407 candidate muscle biopsies with normal dystrophin and sarcoglycan. Calpain-3 and dysferlin were studied by Western blotting and immunohistochemistry.. Combined calpain-3 and dysferlin Western blot analysis identified calpain-3 deficiency in 66 (16%) muscle biopsies. In 31 cases (47%), the protein was absent, and in 35 (53%), it was severely reduced in amount (3 to 50% of control). Dysferlin deficiency was found in 26 (6.5%) muscle biopsies. In 9, the protein was absent (35%), and in 17 (65%), it was severely reduced in amount (traces to 20% of control). Twenty-eight percent (53/191) of patients with LGMD phenotype had calpain-3 deficiency. Sixty percent (21/35) of patients with distal myopathy had dysferlin deficiency. Dysferlin immunohistochemistry showed, in the completely dysferlin-deficient patients, absent reaction at the sarcolemma but positive nuclear membrane labeling and, in the partially dysferlin-deficient patients, scattered granular positive cytoplasmic areas and diffuse reaction in regenerating fibers.. About 25% of previously unclassified dystrophy/myopathy cases are due to calpain-3 or dysferlin protein deficiency. These results suggest that immunoblot analysis may be used to define patients for calpain-3 and dysferlin gene mutation studies.

Topics: Biopsy; Calpain; Dysferlin; Fluorescent Antibody Technique, Direct; Humans; Immunoblotting; Isoenzymes; Membrane Proteins; Muscle Proteins; Muscles; Muscular Dystrophies

Topics: Adult; Calpain; Gene Expression; Humans; Male; Muscles; Muscular Dystrophies

Tibial muscular dystrophy (TMD), a late-onset dominant distal myopathy, is caused by yet unknown mutations on chromosome 2q, whereas MD with myositis (MDM) is a muscular dystrophy of the mouse, also progressing with age and linked to mouse chromosome 2. For both disorders, linkage studies have implicated titin as a potential candidate gene.. The authors analyzed major candidate regions in the titin gene by sequencing and Southern blot hybridization, and performed titin immunohistochemistry on TMD patient material to identify the underlying mutation. Western blot studies were performed on the known titin ligands in muscle samples of both disorders and controls, and analysis of apoptosis was also performed.. The authors identified almost complete loss of calpain3, a ligand of titin, in the patient with limb-girdle MD (LGMD) with a homozygous state of TMD haplotype when primary calpain3 gene defect was excluded. Apoptotic myonuclei with altered distribution of transcription factor NF-kB and its inhibitor IkBalpha were encountered in muscle samples of patients with either heterozygous or homozygous TMD haplotype. Similar findings were confirmed in the MDM mouse.. These results imply that titin mutations may be responsible for TMD, and that the pathophysiologic pathway following calpain3 deficiency may overlap with LGMD2A. The loss of calpain3 could be a downstream effect of the deficient TMD gene product. The significance of the secondary calpain3 defect for the pathogenesis of TMD was emphasized by similar calpain3 deficiency in the MDM mouse, which is suggested to be a mouse model for TMD. Homozygous mutation at the 2q locus may thus be capable of producing yet another LGMD.

Topics: Animals; Calpain; Chromosomes, Human, Pair 2; Connectin; Genetic Linkage; Haplotypes; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Isoenzymes; Mice; Muscle Proteins; Muscles; Muscular Dystrophies; Pedigree; Protein Kinases

Calpainopathy (LGMD2A) is the most common type of autosomal recessive limb-girdle muscular dystrophy. We performed a systematic clinical evaluation in 13 calpainopathy patients from 11 families, with particular attention to the pattern of muscle involvement. Eleven patients had a muscle biopsy with deficiency of calpain 3 on western blotting. The other two patients were not biopsied as they were siblings from the same families. Confirmatory CAPN3 mutations were detected in seven patients. The age at presentation was 2-45 years, wider than previously reported. We confirm the highly characteristic and recognisable phenotype of predominant muscular atrophy with early pelvic girdle involvement, relative sparing of the hip abductors, scapular winging and abdominal laxity. Early primary contractures were also a prominent feature in this group, expanding the breadth of the phenotype. Recognition of the clinical pattern of calpainopathy is of diagnostic significance. It is important, especially in sporadic cases, in targeting and interpreting laboratory investigations in order to provide accurate diagnostic and prognostic information.

Topics: Adolescent; Adult; Calpain; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Muscle, Skeletal; Muscular Dystrophies; Mutation; Phenotype

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.

Topics: Amino Acid Sequence; Animals; Calpain; Humans; Isoenzymes; Models, Molecular; Molecular Sequence Data; Muscle Proteins; Muscular Dystrophies; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Rats; Sequence Alignment; Structure-Activity Relationship

Mutations in the calpain 3 gene have been proven to be responsible for limb-girdle muscular dystrophy (LGMD) type 2A. To determine the incidence and genotypes of the calpain 3 (p94) gene mutations in Japanese LGMD patients, we sequenced the gene in 80 patients with clinical characteristics of autosomal recessive or sporadic LGMD. We identified 13 distinct pathogenic mutations in 21 patients (26%), including seven missense mutations, four splice-site mutations and two insertions in which six were novel mutations. Among the 21 patients, 15 (71%) had three types of the common missense (G233V, R461C, D707G) and one insertion (1795-1796insA) mutation. The patients had slowly progressive muscle weakness with age of onset of the disease varying from 6 to 52 years, averaging 20.9. The most striking pathologic findings were the presence of lobulated fibers in 14 patients, especially in the advanced stages. Differing from Duchenne and Becker muscular dystrophy, opaque (hypercontracted) fibers were very rarely seen. These findings may be helpful in establishing diagnostic screening strategies in Japanese LGMD patients.

Topics: Adolescent; Adult; Calpain; Child; DNA Mutational Analysis; Female; Genetic Testing; Humans; Isoenzymes; Japan; Male; Middle Aged; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation, Missense

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Topics: Adult; Age of Onset; Calcium-Binding Proteins; Calpain; Child; Connectin; Dysferlin; Dystrophin; Female; Genetic Linkage; Humans; Immunohistochemistry; Male; Membrane Proteins; Middle Aged; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Polysaccharides

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.

Topics: Adolescent; Adult; Age of Onset; Blotting, Western; Calpain; Child; Child, Preschool; Chromosomes, Human, Pair 19; Cytoskeletal Proteins; DNA Primers; Dystroglycans; Female; Genetic Linkage; Genotype; Haplotypes; Humans; Immunoenzyme Techniques; Infant; Laminin; Male; Membrane Glycoproteins; Microsatellite Repeats; Middle Aged; Muscular Dystrophies; Mutation; Pedigree; Pentosyltransferases; Phenotype; Polymerase Chain Reaction; Proteins

Autosomal recessive limb gird muscular dystrophy (LGMD2) is a clinically and genetically heterogeneous group of diseases that are characterized by progressive atrophy and weakness of the proximal limb muscles. At least eight genetic loci leading to LGMD2 are recognized. The proportion of particular gene involved in producing different forms of LGMD2 shows a marked geographical variation. We studied 19 LGMD2 patients from Russia (15 families) and found calpain 3 (CAPN3) gene mutations in most of the patients studied. Sequence analysis of the fourth exons revealed two sibs - heterozygous compound for a 15-bp deletion (nt598-612) and 550 adenine deletion, and two sibs homozygous for a 550delA. We developed assay based on allele specific amplification (ASA) for rapid screening of the 550delA. The ASA assay of the LGMD2 patients under study showed that 7 patients from 6 families were homozygous for 550delA and 7 patients from 4 families were heterozygous for 550delA. A linkage analysis employing four microsatellites flanking the LGMD2A locus was performed. We found complete haplotype identity in most cases what favors the possibility of a common founder. Heterozygous carriers of 550delA were found in general population. The crude estimate of the mutation frequency is 1/150. Hum Mutat 15:295, 2000.

Topics: Calpain; Haplotypes; Isoenzymes; Muscle Proteins; Muscular Dystrophies; Mutation; Peptide Fragments; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Russia

The sarcoglycan complex is composed of four membrane-spanning dystrophin-associated proteins (DAPs) and is essential for skeletal muscle survival, since the absence or markedly reduced expression of this complex due to mutation of any one of the sarcoglycan genes causes a group of muscular dystrophies, collectively termed sarcoglycanopathy. Although one of the putative functions of the sarcoglycan complex is its participation in signaling processes, detailed studies have been scarce. Very recently, it was shown that gene knockout mice for a DAP, alpha-dystrobrevin, exhibit a dystrophic phenotype, possibly due to defects in muscle cell signaling. To clarify the putative function of the sarcoglycan complex, it is essential to determine whether or not there is a link between it and the intracellular signaling molecules. To elucidate this, we developed new methods for preparing various DAP complexes containing the sarcoglycan complex from the purified dystrophin-DAP complex. It was suggested from one of the complexes prepared that the sarco-glycan-sarcospan complex (the sarcoglycan complex associated with sarcospan) is associated with syntrophin and/or dystrobrevin. Further analysis of this complex revealed that the N-terminal half of dystrobrevin participates in this association. It is thus considered that the sarcoglycan-sarcospan complex is linked to the signaling protein neuronal nitric oxide synthase via alpha-syntrophin associated with dystrobrevin.

Topics: Animals; Calpain; Carrier Proteins; Chromatography, Gel; Cytoskeletal Proteins; Dystroglycans; Dystrophin; Dystrophin-Associated Proteins; Electrophoresis, Polyacrylamide Gel; Immunohistochemistry; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Knockout; Models, Biological; Muscles; Muscular Dystrophies; Neoplasm Proteins; Precipitin Tests; Protein Binding

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.

Topics: Animals; Binding Sites; Body Weight; Calpain; Connectin; Cysteine; Female; Immunohistochemistry; Mice; Mice, Transgenic; Muscle Proteins; Muscle, Skeletal; Muscular Diseases; Muscular Dystrophies; Mutation; Phenotype; Plasmids; Polymerase Chain Reaction; Protein Kinases; Serine

Topics: Amino Acid Sequence; Animals; Autolysis; Base Sequence; Calpain; Cloning, Molecular; COS Cells; DNA Primers; DNA, Complementary; Gene Expression; Humans; Molecular Sequence Data; Muscular Dystrophies; Mutation; Recombinant Proteins; RNA; Transfection

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin alpha 2 chain (merosin) and dysferlin-deficiency.

Topics: Calpain; DNA Mutational Analysis; Dysferlin; Humans; Membrane Proteins; Muscle Proteins; Muscular Diseases; Muscular Dystrophies

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

Topics: Animals; Apoptosis; Calpain; Creatine Kinase; Crosses, Genetic; DNA-Binding Proteins; Evans Blue; Female; Fertility; Gene Deletion; Gene Targeting; Genotype; I-kappa B Proteins; Male; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophies; NF-kappa B; NF-KappaB Inhibitor alpha; Phenotype; RNA, Messenger; Sarcolemma; Signal Transduction

A 45-year-old housewife had proximal dominant limb muscle weakness from around 25 years of age. Her parents were cousins. None of family members was affected. Progressive muscle weakness and atrophy were prominent at the posterior compartments of legs and trunk. Serum CK was moderately elevated. Muscle pathology revealed variation in fiber size, moderate increase in numbers of internal nuclei and abundant lobulated fibers. On immunostaining using by monoclonal antibody against human calpain 3 (NCL-CALP-2 C4; Novocastra) to the biopsied muscle, calpain 3 was completely absent in the sarcoplasm, while granular debris and in part positive striation were noted in control muscle. By Western blot analysis, a band corresponding to 94 kDa of calpain 3 was not detected. A genetic analysis of calpain 3 revealed homozygous C-565-G mutation (Leu189Val). From the present study. Western blot analysis and immunostaining by using calpain 3 antibody were suggested to be useful to diagnose LGMD2A in LGMD patients.

Topics: Biomarkers; Blotting, Western; Calpain; Female; Humans; Immunohistochemistry; Isoenzymes; Middle Aged; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation

The autosomal recessive limb-girdle muscular dystrophies (AR-LGMDs) are a heterogeneous group of disorders of progressive weakness of the pelvic and shoulder girdle musculature. The clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression resembling clinically Xp21 Duchenne muscular dystrophy (DMD) to milder forms with later onset and slower course. Eight genes are mapped for the AR-LGMDs; they are: LGMD2A (CAPN3) at 15q, LGMD2B (dysferlin) at 2p, LGMD2C (gamma-SG) at 13q, LGMD2D (alpha-SG) at 17q, LGMD2E (beta-SG) at 4q, LGMD2F (6-SG) at 5q, LGMD2G at 17q, and more recently LGMD2H at 9q. The LGMD2F (delta-SG) and LGMD2G genes were mapped in Brazilian AR-LGMD families. Linkage analysis in two unlinked families excluded the eight AR-LGMD genes, indicating that there is at least one more gene responsible for AR-LGMD. We have analyzed 140 patients (from 40 families) affected with one of seven autosomal recessive LGMD loci, that is, from LGMD2A to LGMD2G. The main observations were: 1) all LGMD2E and LGMD2F patients had a severe condition, but considerable inter- and intra-familial clinical variability was observed among patients from all other groups; 2) serum CK activities showed the highest values in LGMD2D (alpha-SG) patients among sarcoglycanopathies and LGMD2B (dysferlin) patients among nonsarcoglycanopathies; 3) comparison between LGMD2A (CAPN3) and LGMD2B (dysferlin) showed that the first have on average a more severe course and have calf hypertrophy more frequently (86% versus 13%); and 4) inability to walk on toes was observed in approximately 70% of LGMD2B patients.

Topics: Adolescent; Adult; Age of Onset; Brazil; Calpain; Child; Child, Preschool; Connectin; Creatine Kinase; Cytoskeletal Proteins; Dysferlin; Dystroglycans; Female; Genotype; Humans; Isoenzymes; Male; Membrane Glycoproteins; Membrane Proteins; Middle Aged; Muscle Proteins; Muscular Dystrophies; Mutation; Mutation, Missense; Peptide Fragments; Phenotype; Sarcoglycans

To determine the frequency of calpain III mutations in a heterogeneous limb-girdle muscular dystrophy (LGMD) population.. Mutations of the calpain III gene have been shown to cause a subset of autosomal recessive LGMDs. Patient populations studied to date have been primarily of French and Spanish origin, in which calpain III may cause 30% of autosomal recessive MDs. The incidence of calpain III mutations in non-French/Spanish MD patients has not been studied thoroughly. No sensitive and specific biopsy screening methods for detecting patients with abnormal calpain III protein are available. Thus, detection of patients relies on direct detection of gene mutations.. The authors studied the calpain III gene in 107 MD patient muscle biopsies exhibiting normal dystrophin. Muscle biopsy RNA was produced for each patient, and the entire calpain III complementary DNA was screened for mutations by reverse-transcriptase PCR/single-strand conformation polymorphism using three different conditions.. The authors identified nine patients (eight unrelated) with causative mutations. Six of the seven distinct mutations identified are novel mutations and have not been described previously.. The results suggest that approximately 9.2% of patients in the heterogeneous population with an LGMD diagnosis will show mutations of the calpain III gene. Interestingly, two patients were heterozygous for a single mutation at the DNA level, whereas only the mutant allele was observed at the RNA level. This suggests that there are undetectable, nondeletion mutations that ablate expression of the calpain III gene.

Topics: Adolescent; Adult; Calpain; DNA; Female; Humans; Male; Muscular Dystrophies; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational

Topics: Adolescent; Adult; Apoptosis; Biological Transport; Calpain; Cell Compartmentation; Cell Nucleus; Child; DNA-Binding Proteins; Female; Fetus; Fluorescent Antibody Technique; Humans; I-kappa B Proteins; Male; Microscopy, Confocal; Muscle, Skeletal; Muscular Dystrophies; NF-kappa B; NF-KappaB Inhibitor alpha; Recombinant Proteins

A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers.

Topics: Biopsy; Blotting, Western; Calpain; Cytoskeletal Proteins; Diagnosis, Differential; Dysferlin; Dystroglycans; Dystrophin; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Isoenzymes; Laminin; Membrane Glycoproteins; Membrane Proteins; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Myosin Heavy Chains; Peptide Fragments

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized mainly by symmetrical and selective atrophy of the proximal limb muscles. It derives from defects in the human CAPN3 gene, which encodes the skeletal muscle-specific member of the calpain family. This report represents a compilation of the mutations and variants identified so far in this gene. To date, 97 distinct pathogenic calpain 3 mutations have been identified (4 nonsense mutations, 32 deletions/insertions, 8 splice-site mutations, and 53 missense mutations), 56 of which have not been described previously, together with 12 polymorphisms and 5 nonclassified variants. The mutations are distributed along the entire length of the CAPN3 gene. Thus far, most mutations identified represent private variants, although particular mutations have been found more frequently. Knowledge of the mutation spectrum occurring in the CAPN3 gene may contribute significantly to structure/function and pathogenesis studies. It may also help in the design of efficient mutation-screening strategies for calpainopathies.

Topics: Amino Acid Sequence; Base Sequence; Calpain; DNA Primers; Genetic Testing; Humans; Isoenzymes; Molecular Sequence Data; Muscle Proteins; Muscular Dystrophies; Mutation, Missense; Peptide Fragments; Phenotype; Polymorphism, Genetic

Mutations of the calpain 3 gene, an intracellular calcium-activated neutral protease, is one of the causes of limb-girdle muscular dystrophy (LGMD). We examined 14 Japanese patients with sporadic LGMD for calpain 3 mutations, and found four mutations in five patients. Three (R461C, D707G and R147P) were novel missense mutations, and one was a splice-site mutation (801+1g-->a) resulting in skipping of exons 4 and 5. Of the five patients, three patients with homozygous missense mutations showed later onset and slower progression than the other two patients with an exon skipping or mRNA loss of unknown cause. It would appear that the occurrence of calpain 3 gene mutations in sporadic LGMD in Japan may be quite high since all five patients with mutations in this gene were among the 14 patients without apparent family history, an incidence of 36%. These findings also suggest that calpain 3 deficiency occurs in both sporadic and familial LGMD and that direct analysis of the calpain 3 gene may be useful in the definitive diagnosis not only of the 15q-linked familial but also of sporadic cases of LGMD.

Topics: Adolescent; Adult; Alleles; Alternative Splicing; Amino Acid Substitution; Blotting, Southern; Blotting, Western; Calpain; Child; DNA Mutational Analysis; Dystrophin; Exons; Female; Humans; Immunohistochemistry; Isoenzymes; Japan; Male; Middle Aged; Muscle Proteins; Muscular Dystrophies; Mutation, Missense; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction

The structures of the gene for calpain (CANP-3) and of the DMD gene were analyzed in patients with primary myopathies [limb-girdle muscular distrophy (LGMD) and Duchenne-Becker myodystrophy (DBM)] from various regions of Russia. Via amplification of DNA isolated from the peripheral blood lymphocytes of 74 patients, extended deletions were found in 18 out of 55 patients with DBM. In none of the 19 patients with LGMD, were extended deletions in the CANP-3 gene found. In most patients with LGMD, the amplification of the promoter region and exons 1, 2, 3, 4, 5, and 6 of the CANP-3 gene yielded a single product of corresponding length, but in six patients (three sib pairs), amplification of exon 4 of the CANP-3 gene yielded two products of different size. The following single-strand conformation polymorphism (SSCP) analysis revealed a pronounced polymorphism of exon 4 of the CANP-3 gene in 14 out of 19 patients with LGMD. This structure of exon 4 of the CANP-3 gene was found neither in 16 patients with DBM who had deletions in the DMD gene nor in 16 patients with DBM who had no deletions in the DMD gene.

Topics: Calpain; Exons; Gene Deletion; Humans; Muscular Dystrophies; Polymorphism, Single-Stranded Conformational

Topics: Adolescent; Age of Onset; Alternative Splicing; Amino Acid Sequence; Binding Sites; Calpain; Child; DNA; DNA Mutational Analysis; Endopeptidases; Family Health; Female; Humans; Male; Muscular Dystrophies; Point Mutation; Reading Frames; Sequence Deletion

In order to measure the amount of m-calpain in platelets from Duchenne muscular dystrophy (DMD) patients, carriers and controls, an enzyme linked immunosorbent assay (ELISA) has been developed using polyclonal antibodies raised against m-calpain (Calcium dependent neutral protease), purified from human placenta. Compared to controls a nine-fold increase in the level of m-calpain was observed in all 25 DMD patients and eight obligate carrier mothers studied. Serum creatine kinase (SCK) values of four of these obligate carriers were found to be overlapping with that of normal values, limiting its usefulness. Since calpain levels were found to be raised in all DMD patients and obligate carriers, the estimation of calpain in all female relatives of the ones affected with DMD (with or without raised serum CK) would prove a facile index. Population screening for carrier females using this cost and labour effective index may help in assessing the carrier status and counselling the at risk families.

Topics: Adolescent; Adult; Blood Platelets; Calpain; Child; Enzyme-Linked Immunosorbent Assay; Female; Genetic Carrier Screening; Humans; Male; Middle Aged; Muscular Dystrophies; Phenotype; Sensitivity and Specificity

The developmental expression pattern of four human genes, three of which are involved in progressive muscular dystrophies, was investigated. The rationale for these experiments is that these patterns might provide useful information on the pathophysiology underlying these myopathies. Despite the presence of overlapping clinical signs, the spatiotemporal expression profiles of the corresponding genes differed widely. Transcripts of alpha-sarcoglycan (SGCA) were visible as soon as myotomes were formed, and constitute, together with titin transcripts, precocious muscular system landmarks. beta-sarcoglycan (SGCB) was initially transcribed in a ubiquitous manner, and, toward the second part of the embryonic period, became specific to striated muscle, heart, and the central nervous system. Whereas titin (TTN) transcription and translation seem to be coupled, for the sarcoglycans, translation seemed restricted to skeletal muscle. Calpain3 (CAPN3) RNA was found in only skeletal muscles during the fetal period. It was, however, present earlier in the whole heart, where it selectively disappeared. Finally, evidence for differentially spliced calpain3 variants in smooth muscles was also seen. The expression profiles of these genes is suggestive of their having a role during myogenesis, knowledge of which could be pertinent to the understanding of the pathophysiology of the associated diseases.

Topics: Adult; Base Sequence; Blotting, Northern; Calpain; Cell Differentiation; Connectin; Cytoskeletal Proteins; Dystroglycans; Embryonic and Fetal Development; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Isoenzymes; Membrane Glycoproteins; Molecular Sequence Data; Muscle Proteins; Muscles; Muscular Dystrophies; Polymerase Chain Reaction; Protein Kinases; Sarcoglycans

Calpain is thought to be involved in muscular degradation in progressive muscular dystrophy (PMD), especially Duchenne and Becker muscular dystrophies. To assess the expression of calpain genes in skeletal muscles of patients with myopathies, we examined mRNA levels of three calpain isoforms by the quantitative reverse transcriptase-polymerase chain reaction method in biopsied muscles from control, PMD and amyotrophic lateral sclerosis (ALS) patients. There was a statistically significant increase in calpain 1 and calpain 2 mRNA levels in PMD and ALS patients as compared to controls. In contrast, there was a decrease in expression of calpain 3 mRNA in PMD, but it was not statistically significant. Expression of calpain 1 and calpain 2 positively correlated with each other, but not with calpain 3. These results indicate that expression of calpain 1 and calpain 2, but not calpain 3, are upregulated in diseased human muscles, likely playing a regulatory role in the process of myofibrillar degradation at the transcriptional as well as posttranslational level.

Topics: Adolescent; Adult; Aged; Amyotrophic Lateral Sclerosis; Calpain; Child; DNA Primers; Female; Humans; Isoenzymes; Male; Middle Aged; Muscle, Skeletal; Muscular Dystrophies; Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

p94 (calpain3), a muscle-specific member of the calpain family, has been shown to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A), a form of autosomal recessive and progressive neuromuscular disorder. To elucidate the molecular mechanism of LGMD2A, we constructed nine p94 missense point mutants found in LGMD2A and analyzed their p94 unique properties. All mutants completely or almost completely lose the proteolytic activity against a potential substrate, fodrin. However, some of the mutants still possess autolytic activity and/or connectin/titin binding ability, indicating these properties are not necessary for the LGMD2A phenotypes. These results provide strong evidence that LGMD2A results from the loss of proteolysis of substrates by p94, suggesting a novel molecular mechanism leading to muscular dystrophies.

Topics: Animals; Calpain; Carrier Proteins; Connectin; COS Cells; Humans; Hydrolysis; Microfilament Proteins; Muscle Proteins; Muscular Dystrophies; Mutagenesis, Site-Directed; Point Mutation; Protein Kinases

Two siblings originating from Reunion Island were affected by a limb-girdle muscular dystrophy (LGMD) type 2A and carried the same two mutations in the calpain gene: 946-1 AG-->AA, affecting a splice site, and S744G. They demonstrated the clinical variability possible with calpain-3 mutations. Onset was around 20 years of age in each of them. The girl's symptoms mimicked a metabolic myopathy, while her brother, at the same age, presented a classical phenotype of LGMD in an advanced functional stage.

Topics: Adult; Atrophy; Calpain; Family Health; Female; Humans; Male; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophies; Nuclear Family; Phenotype; Reunion

The concept of limb-girdle muscular dystrophy (LGMD) is changing rapidly due to the advances in molecular genetics. Recently, seven different gene loci have been described, demonstrating that limb-girdle muscular dystrophy is a heterogeneous syndrome, which includes different diseases with a similar phenotype. In isolated populations which have little genetic exchange with neighbouring populations, an accumulation of cases may be found. We carried out an epidemiological study in Guipúzcoa, a small mountainous Basque province in northern Spain, and found the highest prevalence rate of LGMD described so far: 69 per million. Genetic studies demonstrated that 38 cases corresponded to the LGMD2A type, due to calpain-3 gene mutations. Only one patient with alpha-sarcoglycanopathy was found, and in 12 patients the genetic defect was not identified. Moreover, the particular calpain-3 mutation predominant in Basque chromosomes (exon 22, 2362AG-->TCATCT), has only been rarely found in the rest of the world. This observation strongly suggests a founder effect in the indigenous population of Guipúzcoa. The clinical characteristics of the patients with calpain-3 gene mutations were quite homogeneous and different from the other groups (sarcoglycanopathy and unknown gene defect), allowing for a precise clinical diagnostic. The disease onset was between the ages of 8 and 15 years, in most cases in the pelvic girdle, and the patients became wheelchair-bound between 11 and 28 years after onset. No pseudohypertrophy of calves or contractures were observed. No clear correlations were found between the nature and site of the mutation and the resulting phenotype.

Topics: Adult; Age of Onset; Aged; Base Sequence; Calpain; Chromosome Mapping; Chromosomes, Human, Pair 15; DNA Primers; Exons; Female; Genotype; Geography; Humans; Incidence; Isoenzymes; Male; Medical Records; Middle Aged; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Phenotype; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Prevalence; Retrospective Studies; Spain; Syndrome

We report on the clinical, pathological, and genetic features of 7 patients with limb-girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7+/-3.1 years (mean+/-SD), and loss of ambulance occurred at 38.5+/-2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+-binding domain.

Topics: Age of Onset; Biopsy; Calpain; Child; DNA Mutational Analysis; Exons; Family Health; Female; Haplotypes; Humans; Isoenzymes; Japan; Male; Microscopy, Electron; Middle Aged; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Mutation; Pedigree; Reverse Transcriptase Polymerase Chain Reaction

Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.

Topics: Adult; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Calpain; Chickens; Child; Child, Preschool; Cricetinae; Dogs; Humans; Isoenzymes; Mice; Molecular Sequence Data; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Peptide Fragments; Rabbits; Rats; Species Specificity; Swine

Eight females with a family history of Duchenne muscular dystrophy (DMD) were analysed for their carrier status by m-calpain test, which monitors the m-calpain (milli-calpain), a proteolytic enzyme in the platelets, using an ELISA technique. Four of the eight females were identified as carriers by virtue of their elevated enzyme levels as compared to control. DNA samples of these members were analysed to ascertain the carrier status, by PCR followed by dosage analysis by densitometry. DNA analysis confirmed the findings by calpain test, which underlines the reliability of this phenotypic test for carrier detection in DMD. Calpain test has been informative in a large group of patients and carriers tested so far. Since the calpain test is cost and labour effective, it is suited for routine and widespread screening purposes.

Topics: Blood Platelets; Calpain; Enzyme-Linked Immunosorbent Assay; Female; Heterozygote; Humans; Muscular Dystrophies; Pedigree; Polymerase Chain Reaction; Sequence Deletion

Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature alpha- and beta-dystroglycan proteins. The beta-dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processing of dystroglycan.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Calpain; Dystrophin; Humans; Immunoblotting; Muscle, Skeletal; Muscular Dystrophies; Rabbits

Limb-girdle muscular dystrophies (LGMDs) are a group of neuromuscular diseases presenting great clinical heterogeneity. Mutations in CANP3, the gene encoding muscle-specific calpain, were used to identify this gene as the genetic site responsible for autosomal recessive LGMD type 2A (LGMD2A; MIM 253600). Analyses of the segregation of markers flanking the LGMD2A locus and a search for CANP3 mutations were performed for 21 LGMD2 pedigrees from various origins. In addition to the 16 mutations described previously, we report 19 novel mutations. These data indicate that muscular dystrophy caused by mutations in CANP3 are found in patients from all countries examined so far and further support the wide heterogeneity of molecular defects in this rare disease.

Topics: Adolescent; Adult; Age of Onset; Calpain; Child; Child, Preschool; Chromosomes, Human, Pair 15; Europe; Female; Genetic Heterogeneity; Genetic Markers; Haplotypes; Humans; Isoenzymes; Lod Score; Male; Middle East; Muscle Proteins; Muscular Dystrophies; Mutation; Pedigree; United States

Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a clinically and genetically heterogenous group of diseases involving at least six different loci. Five genes have already been identified: calpain-3 at LGMD2A (15q15), and four members of the sarcoglycan (SG) complex, alpha-SG at LGMD2D (17q21), beta-SG at LGMD2E (4q12), gamma-SG at LGMD2C (13q12), and delta-SG at LGMD2F (5q33-q34). The gene product at LGMD2B (2p13-p16) is still unknown and at least one other gene is still unmapped. We investigated 20 Turkish families (18 consanguineous) diagnosed as having LGMD2. Most of our patients had onset of symptoms before age 10. The phenotypes varied from severe to benign. We analyzed the SG complex by immunofluorescence and/or western blot. Genotyping was performed using markers defining the six known loci and the suspected genes were screened for mutations. Six of 17 index cases showed deficiency of the SG complex, by immunofluorescence and/or western blot. Seven cases involved one of the known genes of the SG complex (alpha, 2; beta, 1; and gamma, 4 cases), and five mutations were documented in the alpha- and gamma-SG genes. After linkage analysis, 10 families were characterized as having LGMD2A (calpain-3 deficiency), and all mutations were eventually identified. One family was classified as having LGMD2B and 1 family that has normal SGs was linked to the chromosome 5q33-q34 locus (LGMD2F). In 1 family there was no linkage to any of the known LGMD2 loci. It appears that in Turkey, there is a broad spectrum of genes and defects involved in LGMD2. It may be possible to correlate genotype to phenotype in LGMD2. All severe cases belonged to the gamma-SG-deficiency group. Nine calpain-3-deficient cases had intermediate and 1 had moderate clinical courses. The LGMD2B patient had a moderate clinical expression, whereas the LGMD2F case was truly benign.

Topics: Adolescent; Age of Onset; Calpain; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 4; Chromosomes, Human, Pair 5; Cytoskeletal Proteins; Dystroglycans; Female; Genes, Recessive; Genetic Linkage; Genetic Markers; Genotype; Humans; Male; Membrane Glycoproteins; Muscular Dystrophies; Sarcoglycans; Turkey

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Topics: Calpain; Chromosome Mapping; Chromosomes, Artificial, Yeast; Cloning, Molecular; Cosmids; DNA Probes; Electrophoresis, Gel, Pulsed-Field; Genetic Techniques; Genome, Fungal; Genome, Human; Humans; In Situ Hybridization, Fluorescence; Isoenzymes; Muscle Proteins; Muscular Dystrophies; Mutation; Proteins; Repressor Proteins; Reproducibility of Results; Sequence Deletion; Silanes; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Among our 20 families with LGMD2, 10 were documented to have muscle-specific calcium-activated neutral protease 3 (calpain-3) deficiency. Consanguinity was present in all. The current ages of the index cases were between 12 and 23 years, and there were additional nine members affected. Clinically, the patients showed mild courses; none of the cases below age 30 lost autonomy so far. The dystrophy is mainly proximal and atrophic with calf enlargement and scapular wasting in some. In three cases walking was delayed. Creatine kinase levels were at least 10 times elevated. All obligate carriers had normal creatine kinase levels. Five families shared the same 551 delA frameshift mutation. In four of these families there was the same core haplotype, whereas one was distinct suggesting an independent origin. Calpain-3 deficiency in general is a mild muscular dystrophy during childhood.

Topics: Adolescent; Adult; Age of Onset; Calpain; Child; Cohort Studies; Consanguinity; Disease Progression; Female; Frameshift Mutation; Haplotypes; Humans; Male; Muscular Dystrophies; Pedigree; Severity of Illness Index

Topics: Amino Acid Sequence; Base Sequence; Calpain; Chromosomes, Human, Pair 15; Cloning, Molecular; Gene Expression Regulation, Enzymologic; Gene Targeting; Humans; Isoenzymes; Molecular Sequence Data; Muscle Proteins; Muscular Dystrophies; Open Reading Frames; Protein Conformation; RNA Splicing; Sequence Alignment; Sequence Homology, Amino Acid

A branch of a highly inbred family was referred for prenatal counseling with an initial misdiagnosis of Becker Muscular Dystrophy (BMD) due to the limited clinical and laboratory data obtained in pre-dystrophin era and hidden family information. In a second branch of the family with a diagnosis of limb-girdle muscular dystrophy type 2A (LGMD2A) molecular studies revealed a homozygous 550 delta A mutation in the calcium-activated neutral protease 3 (calpain 3, CANP3) gene in the affected members. Finally, in the third branch of the family, it turned out that both parents were heterozygous for the 550 delta A mutation and the 13-week-old fetus was homozygous. The same mutation subsequently also was found in the first branch of the family. The parents were informed that the risk of their child of developing the disease would be very high given that he was carrying the same homozygous mutation of the other affected members. They were informed also that in another population (in Reunion Island) the same disease does not necessarily follow such a simple pattern of inheritance. After counseling the parents decided to terminate the pregnancy.

Topics: Adult; Calpain; Chromosome Mapping; Chromosomes, Human, Pair 15; Consanguinity; Cysteine Proteinase Inhibitors; DNA; Exons; Female; Fetus; Genetic Counseling; Heterozygote; Homozygote; Humans; Male; Muscular Dystrophies; Pregnancy; Prenatal Diagnosis

Increased expression of critical components of the ubiquitin-dependent proteolytic pathway occurs in any muscle wasting condition so far studied in rodents where proteolysis rises. We have recently reported similar adaptations in head trauma patients [Mansoor et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2714-2718]. We demonstrate here that the increased muscle protein breakdown seen in mdx mice only correlated with enhanced expression of m-calpain, a Ca(2+)-activated proteinase. By contrast, no change in mRNA levels for components of the ubiquitin-proteasome proteolytic process was seen in muscles from both mdx mice and Duchenne muscular dystrophy patients. Thus, gene expression of components of this pathway is not regulated in the chronic wasting that characterizes muscular dystrophy.

Topics: Adolescent; Animals; Calpain; Cathepsin D; Cathepsin L; Cathepsins; Child; Cysteine Endopeptidases; Dystrophin; Endopeptidases; Female; Fibrosis; Gene Expression; Humans; Male; Mice; Mice, Inbred mdx; Multienzyme Complexes; Muscle, Skeletal; Muscular Dystrophies; Necrosis; Proteasome Endopeptidase Complex; Reference Values; RNA, Messenger; Transcription, Genetic; Ubiquitins

Autosomal recessive limb-girdle muscular dystrophies (AR LGMD) represent a group of muscle diseases with a wide spectrum of clinical signs, varying from very severe to mild. Four different loci that when mutated cause the AR LGMD phenotype have been mapped or cloned or both: in two of them the linked families seem to have a relatively mild phenotype (LGMD2a and LGMD2b), in the third one the reported linked families show a more severe clinical course (LGMD2c), while mutations in the fourth locus may cause severe or mild phenotypes (LGMD2d). The relative proportion of each of these genetic forms among the LGMD families and whether there are other genes that when mutated cause this phenotype is unknown. The closest available informative markers for each of the mapped AR LGMD genes have been tested in 13 Brazilian families with at least three affected patients. The findings from the present report confirm non-allelic heterogeneity for LGMD and suggest that in our population about 33% of the LGMD families are caused by mutations in the 15q gene, 33% in the 2p gene, 17% by mutations in the adhalin gene, and less than 10% may be by mutations at the 13q locus. They also suggest that there is at least one other gene responsible for this phenotype. In addition, the main clinical features of the different forms are discussed.

Topics: Adolescent; Adult; Brazil; Calpain; Child; Child, Preschool; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 2; Consanguinity; Cytoskeletal Proteins; Female; Genes, Recessive; Genetic Heterogeneity; Genetic Markers; Genotype; Humans; Lod Score; Male; Membrane Glycoproteins; Muscular Dystrophies; Pedigree; Phenotype; Sarcoglycans

The genes for six forms of recessive muscular dystrophy have so far been identified, although more are certain to be revealed. Differential diagnosis on clinical grounds alone can be very difficult, so a classification system based on the underlying molecular defect has been introduced. Muscle biopsies are taken for routine diagnostic histopathology, and the various proteins implicated in muscular dystrophy can be analysed immunologically and the results used to indicate where to start searching for gene mutations. A flow diagram is presented which demonstrates how such protein analysis could be optimized.

Topics: Biopsy; Calpain; Diagnosis, Differential; Dystrophin; Extremities; Genes, Recessive; Genetic Linkage; Humans; Laminin; Mass Screening; Muscle, Skeletal; Muscular Dystrophies; Proteins

Erb's type limb-girdle muscular dystrophy (LGMD) was identified and clinically studied in detail in a small community living in the Reunion Island (RI). It was linked to chromosome 15q and related to mutations in the muscle specific calpain 3 gene. A series of cases were afterwards clinically and genetically identified in the French metropolitan community. The phenotype was identical to the RI type in the great majority of cases, although clinical differences were noticed in a few cases. Six different mutations were identified in the RI families, whereas a series of 39 mutations were detected in the French metropolitan families, all different from those present in the RI patients. Phenotype-genotype correlations were attempted in both communities.

Topics: Adolescent; Adult; Age of Onset; Calpain; Child; Chromosomes, Human, Pair 15; Extremities; France; Genetic Linkage; Genotype; Humans; Indian Ocean Islands; Muscle, Skeletal; Muscular Dystrophies; Mutation; Phenotype; Urban Health

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

Topics: Animals; Calpain; Chromosome Mapping; Chromosomes, Human, Pair 15; Cloning, Molecular; Cytoskeletal Proteins; Disease Progression; Dystroglycans; Genes, Recessive; Genetic Heterogeneity; Humans; Membrane Glycoproteins; Mice; Muscles; Muscular Dystrophies; Organ Specificity

Topics: Calpain; Chromosomes, Human, Pair 15; Genetic Linkage; Haplotypes; Humans; Muscular Dystrophies; Mutation

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 x g) but the membrane skeleton requires 100,000 x g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin alpha IIb beta 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following alpha IIb beta 3-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate an approximately 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Topics: Amino Acid Sequence; Base Sequence; Blood Platelets; Calpain; Cell Membrane; Cytoskeletal Proteins; Cytoskeleton; Detergents; DNA Primers; Humans; In Vitro Techniques; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Muscular Dystrophies; Octoxynol; Oligopeptides; Peptide Fragments; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Solubility; Thrombasthenia; Utrophin

A calcium-dependent proteinase (calpain) has been suggested to play an important role in muscle degradation in Duchenne muscular dystrophy (DMD). In immunohistochemical studies, calpain and its endogenous inhibitor (calpastatin) were located exclusively in the cytoplasm in normal human muscles. The intensity of the staining was stronger in type 1 than in type 2 fibers. Quantitative immunohistochemical study showed an increase of calpain in biopsied muscles from the patients with DMD and Becker muscular dystrophy. Abnormal increases in calpain and calpastatin were demonstrated mainly in atrophic fibers, whereas necrotic fibers showed moderate or weak immunoreactions for the enzymes. Opaque fibers and hypertrophic fibers were negative. Not all dystrophin-deficient muscle fibers necessarily showed a strong reaction for calpain. We suggest that calpain may play an important role in muscle fiber degradation, especially in the early stage of muscle degradation in muscular dystrophy.

Topics: Adolescent; Adult; Calcium-Binding Proteins; Calpain; Child; Cysteine Proteinase Inhibitors; Humans; Immunohistochemistry; Male; Muscle, Skeletal; Muscular Dystrophies

Topics: Amino Acid Sequence; Base Sequence; Calpain; Codon; DNA; Humans; Molecular Sequence Data; Muscular Dystrophies; Mutation; RNA Splicing; Tay-Sachs Disease

Limb-girdle muscular dystrophies (LGMDs) are a group of inherited diseases whose genetic etiology has yet to be elucidated. The autosomal recessive forms (LGMD2) constitute a genetically heterogeneous group with LGMD2A mapping to chromosome 15q15.1-q21.1. The gene encoding the muscle-specific calcium-activated neutral protease 3 (CANP3) large subunit is located in this region. This cysteine protease belongs to the family of intracellular calpains. Fifteen nonsense, splice site, frameshift, or missense calpain mutations cosegregate with the disease in LGMD2A families, six of which were found within La Réunion island patients. A digenic inheritance model is proposed to account for the unexpected presence of multiple independent mutations in this small inbred population. Finally, these results demonstrate an enzymatic rather than a structural protein defect causing a muscular dystrophy, a defect that may have regulatory consequences, perhaps in signal transduction.

Topics: Amino Acid Sequence; Base Sequence; Calpain; Chromosomes, Human, Pair 15; DNA; DNA Mutational Analysis; Exons; Gene Expression; Genetic Testing; Humans; Models, Genetic; Molecular Sequence Data; Muscular Dystrophies; Mutation; Nucleic Acid Heteroduplexes; Polymerase Chain Reaction; Restriction Mapping; Sequence Alignment

p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.

Topics: Amino Acid Sequence; Animals; Binding Sites; Calpain; Cell Line; Connectin; Humans; Hydrolysis; Membrane Proteins; Molecular Sequence Data; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies; Protein Kinases; Rabbits; Rats

Calcium and calcium dependent enzymes viz., calcium ATPase, protein kinase C and calcium activated neutral protease (milli CANP mCANP) were studied in the erythrocytes, platelets and lymphocytes of obligate carriers, in order to assess the usefulness of these indices for detection of carriers for Duchenne muscular dystrophy (DMD). With the exception of mCANP and lymphocyte calcium ATPase, other calcium dependent enzyme activities showed considerable overlap between carriers and control. Since the increase in the level of platelet mCANP was found in all affected boys (no false negatives) and obligate carriers, and patients with other myopathic conditions and some neurogenic causes did not show high platelet mCANP activity, this parameter could be considered as a good phenotypic index. Unlike SCK, the platelet mCANP of carriers did not overlap that of controls, hence tests are to be carried out to verify its usefulness as an index of carrier state in mutations other than DNA deletion since testing of non-deletion is both costly and has practical limitations.

Topics: Adult; Calcium; Calcium-Transporting ATPases; Calpain; Erythrocytes; Heterozygote; Humans; Male; Muscular Dystrophies; Protein Kinase C

As blood cells such as platelets, lymphocytes and erythrocytes from patients with Duchenne muscular dystrophy show evidence of membrane alterations and elevation of intra-cellular calcium, one of the calcium related changes i.e., the activity of calcium activated neutral protease (CANP) was monitored and found to be elevated in erythrocytes, lymphocytes and platelets. As similar changes were observed in platelets of carriers of this disease, CANP in platelets may serve as a useful index for carrier detection.

Topics: Adult; Calpain; Child; Female; Heterozygote; Humans; Male; Muscular Dystrophies

Calcium activated neutral protease (milli- and micro-forms) and its endogenous inhibitor have been quantified in muscle from Duchenne muscular dystrophy (DMD) patients. The specific activities of both the enzymes are found to be significantly elevated. Some of the properties studied indicate that the enzymes from DMD and normal are not qualitatively different. The ratios of milli- to micro-enzyme in normal and disease state suggest that these enzymes have independent roles to play. The absence of a significant increase in the level of the endogenous inhibitor is probably indicative of its mode of regulation, in disease condition.

Topics: Adolescent; Calpain; Child; Child, Preschool; Glycoproteins; Humans; Isoenzymes; Muscles; Muscular Dystrophies

Calcium-activated neutral protease (CANP) has been purified from its endogenous inhibitor and its activity is monitored in the muscle of Duchenne Muscular Dystrophy (DMD) patients. The specific activity of the enzyme is found to be significantly higher as compared to normal. CANP from platelets of DMD patients is significantly higher as compared to activities in other myopathies and controls. Platelet CANP, therefore, may prove to be an index of pathological condition in DMD, since other myopathies do not show such a significant change.

Topics: Adolescent; Aging; Blood Platelets; Calpain; Child; Creatine Kinase; Endopeptidases; Humans; L-Lactate Dehydrogenase; Muscles; Muscular Dystrophies; Pyruvate Kinase

Topics: Animals; Calpain; Endopeptidases; Humans; Hydrogen-Ion Concentration; Kinetics; Muscle Proteins; Muscles; Muscular Dystrophies; Protease Inhibitors; Swine

Topics: Calpain; Endopeptidases; Humans; Muscular Dystrophies