glycogen and Glycogen-Storage-Disease-Type-II

Cipaglucosidase alfa (Pombiliti

Topics: 1-Deoxynojirimycin; Adult; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans

Pompe disease is a rare, inherited, devastating condition that causes progressive weakness, cardiomyopathy and neuromotor disease due to the accumulation of glycogen in striated and smooth muscle, as well as neurons. While enzyme replacement therapy has dramatically changed the outcome of patients with the disease, this strategy has several limitations. Gene therapy in Pompe disease constitutes an attractive approach due to the multisystem aspects of the disease and need to address the central nervous system manifestations. This review highlights the recent work in this field, including methods, progress, shortcomings, and future directions.. Recombinant adeno-associated virus (rAAV) and lentiviral vectors (LV) are well studied platforms for gene therapy in Pompe disease. These products can be further adapted for safe and efficient administration with concomitant immunosuppression, with the modification of specific receptors or codon optimization. rAAV has been studied in multiple clinical trials demonstrating safety and tolerability.. Gene therapy for the treatment of patients with Pompe disease is feasible and offers an opportunity to fully correct the principal pathology leading to cellular glycogen accumulation. Further work is needed to overcome the limitations related to vector production, immunologic reactions and redosing.

Topics: Central Nervous System; Dependovirus; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid-α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. A lack of GAA leads to accumulation of glycogen in the lysosomes of cardiac, skeletal, and smooth muscle cells, as well as in the central and peripheral nervous system. Enzyme replacement therapy has been the standard of care for 15 years and slows disease progression, particularly in the heart, and improves survival. However, there are limitations of ERT success, which gene therapy can overcome.. Gene therapy offers several advantages including prolonged and consistent GAA expression and correction of skeletal muscle as well as the critical CNS pathology. We provide a systematic review of the preclinical and clinical outcomes of adeno-associated viral mediated gene therapy and alternative gene therapy strategies, highlighting what has been successful.. Although the preclinical and clinical studies so far have been promising, barriers exist that need to be addressed in gene therapy for Pompe disease. New strategies including novel capsids for better targeting, optimized DNA vectors, and adjuctive therapies will allow for a lower dose, and ameliorate the immune response.

Topics: alpha-Glucosidases; Animals; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Muscle, Skeletal

Metabolic myopathies are a heterogenous group of muscle diseases typically characterized by exercise intolerance, myalgia and progressive muscle weakness. Effective treatments for some of these diseases are available, but while our understanding of the pathogenesis of metabolic myopathies related to glycogen storage, lipid metabolism and β-oxidation is well established, evidence linking treatments with the precise causative genetic defect is lacking.. The objective of this study was to collate all published evidence on pharmacological therapies for the aforementioned metabolic myopathies and link this to the genetic mutation in a format amenable to databasing for further computational use in line with the principles of the "treatabolome" project.. A systematic literature review was conducted to retrieve all levels of evidence examining the therapeutic efficacy of pharmacological treatments on metabolic myopathies related to glycogen storage and lipid metabolism. A key inclusion criterion was the availability of the genetic variant of the treated patients in order to link treatment outcome with the genetic defect.. Of the 1,085 articles initially identified, 268 full-text articles were assessed for eligibility, of which 87 were carried over into the final data extraction. The most studied metabolic myopathies were Pompe disease (45 articles), multiple acyl-CoA dehydrogenase deficiency related to mutations in the ETFDH gene (15 articles) and systemic primary carnitine deficiency (8 articles). The most studied therapeutic management strategies for these diseases were enzyme replacement therapy, riboflavin, and carnitine supplementation, respectively.. This systematic review provides evidence for treatments of metabolic myopathies linked with the genetic defect in a computationally accessible format suitable for databasing in the treatabolome system, which will enable clinicians to acquire evidence on appropriate therapeutic options for their patient at the time of diagnosis.

Topics: Glycogen; Glycogen Storage Disease Type II; Humans; Lipid Metabolism; Metabolism, Inborn Errors; Multiple Acyl Coenzyme A Dehydrogenase Deficiency; Muscle Weakness; Mutation

Glycogen storage disease type II (GSDII, Pompe disease) is a rare metabolic disorder caused by a deficiency of acid alpha-glucosidase (GAA), an enzyme localized within lysosomes that is solely responsible for glycogen degradation in this compartment. The manifestations of GSDII are heterogeneous but are classified as early or late onset. The natural course of early-onset Pompe disease (EOPD) is severe and rapidly fatal if left untreated. Currently, one therapeutic approach, namely, enzyme replacement therapy, is available, but advances in molecular medicine approaches hold promise for even more effective therapeutic strategies. These approaches, which we review here, comprise splicing modification by antisense oligonucleotides, chaperone therapy, stop codon readthrough therapy, and the use of viral vectors to introduce wild-type genes. Considering the high rate at which innovations are translated from bench to bedside, it is reasonable to expect substantial improvements in the treatment of this illness in the foreseeable future.

Topics: alpha-Glucosidases; Animals; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Mutation; Oligonucleotides, Antisense; Phenotype

The glycogen storage diseases are a group of inherited metabolic disorders that are characterized by specific enzymatic defects involving the synthesis or degradation of glycogen. Each disorder presents with a set of symptoms that are due to the underlying enzyme deficiency and the particular tissues that are affected. Autophagy is a process by which cells degrade and recycle unneeded or damaged intracellular components such as lipids, glycogen, and damaged mitochondria. Recent studies showed that several of the glycogen storage disorders have abnormal autophagy which can disturb normal cellular metabolism and/or mitochondrial function. Here, we provide a clinical overview of the glycogen storage disorders, a brief description of autophagy, and the known links between specific glycogen storage disorders and autophagy.

Topics: Animals; Autophagy; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogenolysis; Humans; Muscle, Skeletal

Pompe disease, also known as glycogen storage disease type II, is caused by the lack or deficiency of a single enzyme, lysosomal acid alpha-glucosidase, leading to severe cardiac and skeletal muscle myopathy due to progressive accumulation of glycogen. The discovery that acid alpha-glucosidase resides in the lysosome gave rise to the concept of lysosomal storage diseases, and Pompe disease became the first among many monogenic diseases caused by loss of lysosomal enzyme activities. The only disease-specific treatment available for Pompe disease patients is enzyme replacement therapy (ERT) which aims to halt the natural course of the illness. Both the success and limitations of ERT provided novel insights in the pathophysiology of the disease and motivated the scientific community to develop the next generation of therapies that have already progressed to the clinic.

Topics: alpha-Glucosidases; Autophagy; Enzyme Replacement Therapy; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomal Storage Diseases; Lysosomes; Muscle, Skeletal

Autophagy, a highly conserved self-digestion process, is initially regarded as non-selectively sequestering and degradation cytoplasmic contents. Nowadays, many kinds of selective autophagy have been found in response to various physiological cues such as mitophagy, reticulophagy and glycophagy. Glycophagy, as a selective autophagy, plays a crucial role in maintaining glucose homeostasis in many tissues including heart, liver and skeletal muscles. Moreover, glycophagy is highly regulated by many signal pathways like the cyclic AMP protein kinase A/protein kinase A, PI3K-Akt/PKB-mTOR and Calcium. Latest studies have demonstrated that glycophagy is triggered by STBD1, which tethers glycogen to membranes via binding itself to the cognate autophagy protein GABARAPL1. More importantly, glycophagy might act as a protective role in coping with the accumulation of glycogen-rich lysosomes in infant patients with Pompe disease. However, glycophagy might aggravate diabetic cardiomyopathy via FoxO1 signal pathway. In this review, we focus on some findings about the occurrence and development, as well as the regulatory mechanism of glycophagy. We also analyze the role of glycophagy in Pompe disease and diabetic cardiomyopathy. Targeting glycophagy may open a new avenue of therapeutic intervention to these diseases.

Topics: Autophagy; Diabetic Cardiomyopathies; Glycogen; Glycogen Storage Disease Type II; Humans

Glycogenosis II (GSD II) is an autosomal recessive lysosomal storage disorder resulting from acid alpha-glucosidase deficiency, subsequent accumulation of glycogen in tissues, impairment of autophagic processes and progressive cardiac, motor and respiratory failure. The late-onset form is characterized by wide variability in residual enzyme activity, age of onset, rate of disease progression and phenotypical spectrum. Although the pathological process mainly affects the skeletal muscle, several other tissues may be involved in the course of the disease; therefore GSD II should be regarded as a multisystem disorder in which glycogen accumulation is present in skeletal and smooth muscle, heart, brain, liver, spleen, salivary glands, kidney and blood vessels. In this review, we briefly summarize the main non-muscle targets of the pathological process in late-onset GSD II. Further studies aimed at evaluating the extra-muscle involvement in this group of patients will help to better define clinical features and prognostic factors and to delineate the natural history of the disease.

Topics: Age of Onset; alpha-Glucosidases; Bone Diseases; Disease Progression; Glycogen; Glycogen Storage Disease Type II; Humans; Nervous System Diseases; Phenotype; Prognosis; Vascular Diseases

Late-onset glycogenosis type II (glycogen storage disease type II [GSDII]) is a rare autosomal disorder caused by deficiency of acid maltase, a lysosomal enzyme that hydrolyzes glycogen to glucose. Recently, both infantile and adult GSDII patients have been treated with enzyme replacement therapy (ERT), and a number of studies including large cohorts of GSDII patients have recently demonstrated that ERT is effective in modifying the natural course of the disease. The opportunity of this new treatment gave new hope to patients, but also an important impulse to the research on every feature of the disease, leading to a deeper knowledge on the response to treatment, on clinical manifestations, and on pathophysiologic aspects such as the role of autophagy and immune status.

Topics: alpha-Glucosidases; Animals; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle, Skeletal

Macroautophagy (often referred to as autophagy) is an evolutionarily conserved intracellular system by which macromolecules and organelles are delivered to lysosomes for degradation and recycling. Autophagy is robustly induced in response to starvation in order to generate nutrients and energy through the lysosomal degradation of cytoplasmic components. Constitutive, basal autophagy serves as a quality control mechanism for the elimination of aggregated proteins and worn-out or damaged organelles, such as mitochondria. Research during the last decade has made it clear that malfunctioning or failure of this system is associated with a wide range of human pathologies and age-related diseases. Our recent data provide strong evidence for the role of autophagy in the pathogenesis of Pompe disease, a lysosomal glycogen storage disease caused by deficiency of acid alpha-glucosidase (GAA). Large pools of autophagic debris in skeletal muscle cells can be seen in both our GAA knockout model and patients with Pompe disease. In this review, we will focus on these recent data, and comment on the not so recent observations pointing to the involvement of autophagy in skeletal muscle damage in Pompe disease.

Topics: alpha-Glucosidases; Animals; Autophagy; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Mice; Mice, Knockout; Mitochondria; Muscle, Skeletal

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder that is caused by acid α-glucosidase (GAA) deficiency and is due to pathogenic sequence variations in the corresponding GAA gene. The correlation between genotypes and phenotypes is strict, in that patients with the most severe phenotype, classic infantile Pompe disease, have two pathogenic mutations, one in each GAA allele, that prevent the formation of GAA or totally obliterates its function. All patients with less progressive phenotypes have at least one sequence variation that allows normal or low level synthesis of GAA leading to the formation of analytically measurable, low level GAA activity in most cases. There is an overall trend of finding higher GAA enzyme levels in patients with onset of symptoms in adulthood when compared to patients who show clinical manifestations in early childhood, aged 0-5 years, with a rapidly progressive course, but who lack the severe characteristics of classic infantile Pompe disease. However, several cases have been reported of adult-onset disease with very low GAA activity, which in all those cases corresponds with the GAA genotype. The clinical diversity observed within a large group of patients with functionally the same GAA genotype and the same c.-32-13C > T haplotype demonstrates that modifying factors can have a substantial effect on the clinical course of Pompe disease, disturbing the GAA genotype-phenotype correlation. The present day challenge is to identify these factors and explore them as therapeutic targets.

Topics: Age of Onset; alpha-Glucosidases; Genetic Association Studies; Glycogen; Glycogen Storage Disease Type II; Haplotypes; Humans; Mutation; Structure-Activity Relationship

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.

Topics: Clinical Trials as Topic; Dependovirus; Enzyme Replacement Therapy; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Treatment Outcome

Topics: Carbohydrate Metabolism; Carbohydrate Metabolism, Inborn Errors; Fructose-Bisphosphate Aldolase; Glycogen; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VII; Glycogen Storage Disease Type VIII; Humans; L-Lactate Dehydrogenase; Mitochondrial Myopathies; Phosphoglycerate Kinase; Phosphoglycerate Mutase; Phosphorylase b

Topics: alpha-Glucosidases; Diagnosis, Differential; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Intellectual Disability; Lysosomes; Muscle, Skeletal; Myocardium

Metabolic cardiomyopathy of babies and children accounts for approximately 15% of all cardiomyopathies presenting at these ages. The confirmation of the aetiology is essential for treatment, which is rarely curative. For establishing a prognosis which is often poor, and, above all, for family counselling in cases of mendelian transmission or mitochondrial disease. Cardiomyopathy due to glycogen (Pompe's disease) or mucopolysaccharide (Hurler's disease) disorders are easy to diagnose because of obvious extracardiac manifestations. The diagnosis of the enzyme deficiency only requires a blood and/or urine test. Cardiomyopathies due to a deficit of oxidative metabolism are usually associated with multi-system abnormalities but may be isolated or the presenting sign of the deficit. The diagnosis should be suspected in cases of a positive family history of cardiomyopathy or sudden death, of co-sanguinity, of unusual or unexplained extracardiac disease, of atypical ECG changes or of hypoglycaemia. Chromatography of organic acids, analysis of acylcarnitines and -oxidation of the fatty acid oxidation. Of these conditions, only primary carnitine deficits are curable. The diagnosis of mitochondrial cardiomyopathy is based on the ratios of oxidoreduction and, above all, on spectrophotometric analysis of the respiratory chain complexes in skeletal or cardiac muscle (when the heart is the only organ involved). Genetic counselling is difficult and punctual mutations or deletions of mitochondrial DNA are rarely observed, and also few nuclear genes coding for the proteins of the respiratory chain have been identified to this day.

Topics: Cardiomyopathies; Child; Child, Preschool; Diagnosis, Differential; Electrocardiography; Family Health; Female; Genetic Counseling; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Male; Mitochondrial Myopathies; Mucopolysaccharidosis I

Clinical, neurophysiological, morphological and biochemical investigations were performed in 2 patients with the adult form of glycogenosis II and related to the findings of 58 well-documented cases published in the literature. According to these findings three types can be distinguished from each other. The first one is characterized by an involvement of the limb-girdle muscles only. The second type shows the same pattern with additional progressive insufficiency of the respiratory muscles. The third type presents with weakness of the respiratory muscles without any other severe muscle involvement. Our case 1 can be related to the first, our case 2 to the second type. EMG-studies in case 1 showed myopathic changes and myotonic discharges without clinical signs of myotonia. A myotonic pattern was described in one third of the published cases. In case 2 neurogenic changes as well as in 4 cases in the literature were found. The muscle biopsy is the diagnostic clue in the differential diagnosis of progressive myopathy in the adult. Patients with glycogenosis II show glycogen storage specially in type I-fibres. The enzyme defect can be confirmed biochemically in muscle tissue or cultured fibroblasts. Various therapeutic concepts have been tried in patients with glycogenosis II but most of them remain disappointing. A diet with a low carbohydrate and a high protein proportion was observed to be of some benefit. In patients with respiratory muscle involvement artificial ventilation support showed a positive effect on the general condition for some time.

Topics: Adult; Biopsy; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Microscopy, Electron; Muscles; Muscular Atrophy; Neuromuscular Diseases; Reaction Time; Synaptic Transmission; Tibial Nerve

Topics: alpha-Glucosidases; AMP Deaminase; Carnitine; Carnitine O-Palmitoyltransferase; Creatine Kinase; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VII; Humans; Lipid Metabolism, Inborn Errors; Male; Metabolism, Inborn Errors; Muscular Diseases; Phosphofructokinase-1; Phosphorylase a

Topics: Animals; Cats; Cattle; Disease Models, Animal; Dogs; G(M2) Ganglioside; Gangliosidoses; Gaucher Disease; Glycogen; Glycogen Storage Disease Type II; Glycopeptides; Humans; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipidoses; Lysosomes; Mannosidases; Metabolism, Inborn Errors; Mice; Niemann-Pick Diseases; Rabbits; Sphingolipids

Topics: 1,4-alpha-Glucan Branching Enzyme; Animals; Bacteria; Biodegradation, Environmental; Chick Embryo; Chickens; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VI; Glycogen Storage Disease Type VII; Goats; Humans; Liver; Mice; Mutation; Rabbits; Sugar Phosphates

Topics: Adult; alpha-Glucosidases; Enzyme Replacement Therapy; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Middle Aged; Treatment Outcome

This 52-week, phase I/II double-blind, randomized, placebo-controlled study investigated the novel use of clenbuterol in late-onset Pompe disease (LOPD) stably treated with ERT. Eleven of thirteen participants completed the study. No serious adverse events were related to clenbuterol, and transient minor adverse events included mild elevations of creatine kinase, muscle spasms, and tremors. At week 52, the 6-min walk test distance increased by a mean of 16 m (p = 0.08), or a mean of 3% of predicted performance (p = 0.03), and the maximum inspiratory pressure increased 8% (p = 0.003) for the clenbuterol group. The quick motor function test score improved by a mean of seven points (p = 0.007); and the gait, stairs, gower, chair test improved by a mean of two points (p = 0.004). Clenbuterol decreased glycogen content in the vastus lateralis by 50% at week 52. Transcriptome analysis revealed more normal muscle gene expression for 38 of 44 genes related to Pompe disease following clenbuterol. The placebo group demonstrated no significant changes over the course of the study. This study provides initial evidence for safety and efficacy of adjunctive clenbuterol in patients with LOPD (NCT01942590).

Topics: Adult; Aged; Clenbuterol; Double-Blind Method; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Middle Aged; Muscle, Skeletal; Quadriceps Muscle

Late-onset Pompe disease is characterized by progressive skeletal myopathy followed by respiratory muscle weakness, typically leading to loss of ambulation and respiratory failure. In this population, enzyme replacement therapy (ERT) with alglucosidase alfa has been shown to stabilize respiratory function and improve mobility and muscle strength. Muscle pathology and glycogen clearance from skeletal muscle in treatment-naïve adults after ERT have not been extensively examined.. This exploratory, open-label, multicenter study evaluated glycogen clearance in muscle tissue samples collected pre- and post- alglucosidase alfa treatment in treatment-naïve adults with late-onset Pompe disease. The primary endpoint was the quantitative reduction in percent tissue area occupied by glycogen in muscle biopsies from baseline to 6months. Secondary endpoints included qualitative histologic assessment of tissue glycogen distribution, secondary pathology changes, assessment of magnetic resonance images (MRIs) for intact muscle and fatty replacement, and functional assessments.. Sixteen patients completed the study. After 6months of ERT, the percent tissue area occupied by glycogen in quadriceps and deltoid muscles decreased in 10 and 8 patients, respectively. No changes were detected on MRI from baseline to 6months. A majority of patients showed improvements on functional assessments after 6months of treatment. All treatment-related adverse events were mild or moderate.. This exploratory study provides novel insights into the histopathologic effects of ERT in late-onset Pompe disease patients. Ultrastructural examination of muscle biopsies demonstrated reduced lysosomal glycogen after ERT. Findings are consistent with stabilization of disease by ERT in treatment-naïve patients with late-onset Pompe disease.

Topics: Adult; Age of Onset; Aged; alpha-Glucosidases; Biopsy; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Magnetic Resonance Imaging; Male; Middle Aged; Muscle, Skeletal; Physical Therapy Modalities; Treatment Outcome

Pompe disease is a disorder originating from an acid alpha-glycosidase (AAG) enzyme deficiency. This disease produces an accumulation of lysosomal glycogen in different tissues, whereby the skeletal and heart muscles are especially involved. The established diagnosis is achieved through the identification of the AAG deficiency. There are also other secondary diagnostic biomarkers, such as tetra-saccharide glucose (Glc4), which shows high levels in the urine of these patients. In this study it is highlighted the usefulness of Glc4 as a diagnostic biomarker for Pompe disease in its different forms of presentation, using a high-performance liquid chromatography with ultraviolet detection (HPLC/UV) adapted to the study.. A total of 75 individuals have been analyzed: 40 healthy controls and 35 patients diagnosed with Pompe disease. Twenty-four hour samples of urine were collected from all of the patients and their Glc4 levels were determined by means of HPLC/UV.. The evaluation of the urinary Glc4 shows a high discrimination ability between healthy/sick individuals. In addition, the results obtained have allowed to establish the most appropriate level of decision or cut-off point for the identification of sick people.. Glc4 urinary levels are found to be high in patients suffering from Pompe disease and even though increased levels are also found in other conditions, the existence of a AAG deficiency together with a compatible clinical symptoms, prove very helpful for a correct diagnosis of this serious disease.

Topics: Adolescent; Adult; Age of Onset; Area Under Curve; Biomarkers; Child; Child, Preschool; Chromatography, High Pressure Liquid; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Male; Middle Aged; Oligosaccharides; ROC Curve; Young Adult

This report describes the cognitive development of 17 children with infantile Pompe disease who participated in a 52-week clinical trial of enzyme replacement therapy (ERT) via biweekly infusion of Myozyme® (alglucosidase alfa). Subjects were six months of age or younger (adjusted for gestational age) upon initiation of ERT. The Mental Scale of the Bayley Scales of Infant Development-Second Edition (BSID-II) was administered to obtain a Mental Development Index (MDI) at baseline and weeks 12, 26, 38, and 52 of ERT to assess cognitive development in this treated cohort. Data regarding motor development were also obtained at the same visits and these were used to determine correlations between cognitive and motor development. Over the course of the study, two subgroups of subjects emerged: high responders who were sitting independently and/or ambulating by week 52 (n=13) and limited responders who showed minimal motor gains throughout the first year of ERT (n=4). In the high responder group, MDI scores on the BSID-II remained stable throughout the study and were within normal limits. Positive correlations between cognitive and motor development were also present. These data suggest that the cognitive function of infants up to 18 months of age with Pompe disease is unaffected by the possible presence of glycogen in the central nervous system. Continued investigation of the cognitive development of older survivors is warranted.

Topics: alpha-Glucosidases; Cognition; Cohort Studies; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Male; Motor Skills

Screening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51 controls. Lymphocyte vacuolization was quantified using three methods: percentage vacuolated lymphocytes, percentage PAS-positive lymphocytes, and a PAS score depending on staining intensity. Diagnostic accuracy of the tests was assessed using receiver operating characteristic (ROC) curves. All three methods fully discerned classic infantile patients from controls. The mean values of patients with milder forms of Pompe disease were significantly higher than those of controls, but full separation was not obtained. The area under the ROC curve was 0.98 for the percentage vacuolated lymphocytes (optimal cutoff value 3; sensitivity 91%, specificity 96%) and 0.99 for the percentage PAS-positive lymphocytes and PAS score (optimal cutoff value 9; sensitivity 100%, specificity 98%). Our data indicate that PAS-stained blood films can be used as a reliable screening tool to support a diagnosis of Pompe disease. The percentage of PAS-positive lymphocytes is convenient for use in clinical practice but should always be interpreted in combination with other clinical and laboratory parameters.

Topics: Adult; Aged; Child; Child, Preschool; Enzyme Replacement Therapy; Eosine Yellowish-(YS); Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Lymphocytes; Male; Mass Screening; Methylene Blue; Middle Aged; Periodic Acid-Schiff Reaction; ROC Curve; Sensitivity and Specificity; Vacuoles; Young Adult

A clinical trial was conducted to evaluate the safety and efficacy of alglucosidase alfa in infants and children with advanced Pompe disease.. Open-label, multicenter study of IV alglucosidase alfa treatment in 21 infants 3-43 months old (median 13 months) with minimal acid alpha-glucosidase activity and abnormal left ventricular mass index by echocardiography. Patients received IV alglucosidase alfa every 2 weeks for up to 168 weeks (median 120 weeks). Survival results were compared with an untreated reference cohort.. At study end, 71% (15/21) of patients were alive and 44% (7/16) of invasive-ventilator free patients remained so. Compared with the untreated reference cohort, alglucosidase alfa reduced the risk of death by 79% (P < 0.001) and the risk of invasive ventilation by 58% (P = 0.02). Left ventricular mass index improved or remained normal in all patients evaluated beyond 12 weeks; 62% (13/21) achieved new motor milestones. Five patients were walking independently at the end of the study and 86% (18/21) gained functional independence skills. Overall, 52% (11/21) of patients experienced infusion-associated reactions; 95% (19/20) developed IgG antibodies to recombinant human lysosomal acid alpha-glucosidase; no patients withdrew from the study because of safety concerns.. In this population of infants with advanced disease, biweekly infusions with alglucosidase alfa prolonged survival and invasive ventilation-free survival. Treatment also improved indices of cardiomyopathy, motor skills, and functional independence.

Topics: alpha-Glucosidases; Body Height; Body Weight; Child, Preschool; Cough; Echocardiography; Enzyme-Linked Immunosorbent Assay; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Immunoglobulin G; Infant; Kaplan-Meier Estimate; Male; Muscle, Skeletal; Skin Diseases; Time Factors; Treatment Outcome

To investigate the correlation of the urinary glucose tetrasaccharide, Glcalpha1-6Glcalpha1-4Glcalpha1-4Glc, (Glc4) with skeletal muscle glycogen content and the long-term clinical response to enzyme replacement therapy with recombinant human acid alpha glucosidase in infantile Pompe disease.. Eighteen patients, < or =6 months old, were enrolled in a clinical trial of enzyme replacement therapy for up to 142 weeks. Urinary Glc4, skeletal muscle glycogen, and other clinical and laboratory assessments were made at baseline and at regular intervals. Urinary Glc4 was determined using an isotope-dilution tandem mass spectrometric assay. The clinical response to treatment was defined according to the motor function response. Trends in urinary Glc4 were correlated with the clinical response and compared with serum enzyme markers of skeletal muscle damage, creatine kinase, aspartate aminotransferase, and alanine aminotransferase.. Urinary Glc4, in contrast to the serum markers, correlated closely with skeletal muscle glycogen content and with the clinical response. Patients with the best response to treatment maintained the lowest levels of Glc4 throughout the trial.. The results from this study support the use of urinary Glc4 for monitoring patients with infantile-onset Pompe disease on therapy.

Topics: Alanine Transaminase; alpha-Glucosidases; Aspartate Aminotransferases; Biomarkers; Creatine Kinase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Monitoring, Physiologic; Muscle, Skeletal; Oligosaccharides; Tandem Mass Spectrometry

To conduct an open-label, multinational, multicenter study examining the safety and efficacy of recombinant human acid alpha-glucosidase (rhGAA) in treatment of infantile-onset Pompe disease.. We enrolled 8 infant patients who had Pompe disease with GAA activity <1% of normal, cardiomyopathy, and hypotonia. In the 52-week initial phase, rhGAA was infused intravenously at 10 mg/kg weekly; an extension phase continued survivors' treatment with 10 to 20 mg/kg of rhGAA weekly or 20 mg/kg every 2 weeks for as long as 153 weeks. Safety measurements included adverse events, laboratory tests, and anti-rhGAA antibody titers. Efficacy evaluations included survival, ventilator use, echocardiograms, growth, and motor and cognitive function.. After 52 weeks of treatment, 6 of 8 patients were alive, and 5 patients were free of invasive ventilator support. Clinical improvements included ameliorated cardiomyopathy and improved growth and cognition. Five patients acquired new motor milestones; 3 patients walked independently. Four patients died after the initial study phase; the median age at death or treatment withdrawal for all patients was 21.7 months, significantly later than expected for patients who were not treated. Treatment was safe and well tolerated; no death was drug-related.. rhGAA improved ventilator-free survival, cardiomyopathy, growth, and motor function in patients with infantile-onset Pompe disease compared with outcomes expected for patients without treatment.

Topics: alpha-Glucosidases; Body Height; Body Weight; Cardiomyopathy, Hypertrophic; Child Development; Europe; Female; Glycogen; Glycogen Storage Disease Type II; Hearing Disorders; Humans; Infant; Infant, Newborn; Infusions, Intravenous; Male; Muscle Hypotonia; Muscle, Skeletal; Respiration, Artificial; Treatment Outcome; United States

Pompe disease is an autosomal recessive muscle-wasting disorder caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase. Due to virtual absence of acid alpha-glucosidase, patients with classical infantile Pompe disease develop progressive cardiomyopathy, skeletal muscle weakness and respiratory insufficiency leading to death in early infancy. We report on the results of a phase II clinical trial including two patients with classical infantile Pompe disease receiving enzyme replacement therapy over a period of 48 weeks by weekly infusions. Recombinant acid alpha-glucosidase was derived from the milk of transgenic rabbits. Safety was evaluated by recording adverse events while clinical efficacy was evaluated by ventilator-free survival, left ventricular mass index, motor development as well as histologic and biochemical analysis of muscle biopsies. This therapy was in general well-tolerated. There was an overall improvement in left ventricular mass, cardiac function, skeletal muscle function and histological appearance of skeletal muscle.

Topics: alpha-Glucosidases; Drug Administration Schedule; Drug Evaluation; Electrocardiography; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Male; Motor Activity; Muscles; Myocardium; Recombinant Proteins; Time Factors; Treatment Outcome

Infantile Pompe disease (IPD) is a fatal, autosomal recessive muscle-wasting disorder. Due to a deficiency of the lysosomal enzyme acid alpha-glucosidase patients develop a generalized myopathy, diaphragmatic weakness, and cardiomyopathy leading to death usually within the first year of life. So far there is no therapy available. We report on the safety and efficacy of transgenically derived recombinant human precursor acid alpha-glucosidase (rhGAA) in a 10-month follow-up study in two children with IPD who previously completed a 48-week course of enzyme replacement therapy (ERT) with the same medication at the same dose in a phase II clinical trial. Under this therapy cardiac status and muscle strength had improved, leading to survival beyond the age of one year. These results, together with data from two other phase II clinical trials encouraged further evaluation of the long-term safety and efficacy of enzyme replacement therapy in patients with infantile-onset Pompe disease. During the 10-month follow-up period, ERT was well-tolerated and neither patient experienced a single infusion-associated reaction. The initial improvements in cardiac size and function, as measured by left ventricular mass index and the fractional shortening, were maintained in both patients, and a continued improvement of motor function, as measured by the Alberta infant motor scale, was observed.

Topics: Adolescent; alpha-Glucosidases; Animals; Animals, Genetically Modified; Drug Administration Schedule; Evaluation Studies as Topic; Female; Follow-Up Studies; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Motor Activity; Muscle, Skeletal; Recombinant Proteins; Time Factors; Treatment Outcome; Ventricular Function, Left

Pompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease onset and severity, although other factors, including dysregulation of cytoplasmic glycogen metabolism, are suspected to modulate the disease course. In this study, performed in mice and patient biopsies, we found elevated protein levels of enzymes involved in glucose uptake and cytoplasmic glycogen synthesis in skeletal muscle from mice with Pompe disease, including glycogenin (GYG1), glycogen synthase (GYS1), glucose transporter 4 (GLUT4), glycogen branching enzyme 1 (GBE1), and UDP-glucose pyrophosphorylase (UGP2). Expression levels were elevated before the loss of muscle mass and function. For first time, quantitative mass spectrometry in skeletal muscle biopsies from five adult patients with Pompe disease showed increased expression of GBE1 protein relative to healthy controls at the group level. Paired analysis of individual patients who responded well to treatment with enzyme replacement therapy (ERT) showed reduction of GYS1, GYG1, and GBE1 in all patients after start of ERT compared to baseline. These results indicate that metabolic changes precede muscle wasting in Pompe disease, and imply a positive feedforward loop in Pompe disease, in which lysosomal glycogen accumulation promotes cytoplasmic glycogen synthesis and glucose uptake, resulting in aggravation of the disease phenotype.

Topics: alpha-Glucosidases; Animals; Glucose; Glycogen; Glycogen Storage Disease Type II; Lysosomes; Mice; Muscle, Skeletal

Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in the gene that encodes acid alpha-glucosidase (GAA)-an enzyme responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Glycogen accumulation in skeletal muscles, motor neurons, and airway smooth muscle cells is known to contribute to respiratory insufficiency in Pompe disease. However, the impact of GAA deficiency on the distal alveolar type 1 and type 2 cells (AT1 and AT2) has not been evaluated. AT1 cells rely on lysosomes for cellular homeostasis so that they can maintain a thin barrier for gas exchange, whereas AT2 cells depend on lysosome-like structures (lamellar bodies) for surfactant production. Using a mouse model of Pompe disease, the

Topics: alpha-Glucosidases; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle, Skeletal; Pulmonary Surfactant-Associated Protein D; Respiratory Insufficiency

Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness affecting infants, children, and adults with different severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid α-glucosidase (GAA). Here, we demonstrated that adeno-associated virus-mediated (AAV-mediated) systemic gene transfer reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system - in both young and severely affected old Gaa-knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as those in autophagy and mTORC1/AMPK signaling. We used an AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for a future clinical development strategy in Pompe disease.

Topics: alpha-Glucosidases; Animals; Child; Dependovirus; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout

Pompe disease is a rare glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to glycogen deposition in multiple tissues. Infantile-onset Pompe disease (IOPD) patients present within the first year of life with profound hypotonia and hypertrophic cardiomyopathy. Treatment with enzyme replacement therapy (ERT) has significantly improved survival for this otherwise lethal disorder. This study aims to describe the clinical and molecular spectrum of Malaysian IOPD patients, and to analyze their long term treatment outcomes.. Seventeen patients diagnosed with IOPD between 2000 and 2020 were included in this retrospective cohort study. Clinical and biochemical data were collated and analyzed using descriptive statistics. GAA enzyme levels were performed on dried blood spots. Molecular analysis of the GAA gene was performed by polymerase chain reaction and Sanger sequencing. Structural modelling was used to predict the effect of the novel mutations on enzyme structure.. Our cohort had a median age of presentation of 3 months and median age of diagnosis of 6 months. Presenting features were hypertrophic cardiomyopathy (100%), respiratory insufficiency (94%), hypotonia (88%), failure to thrive (82%), feeding difficulties (76%), and hepatomegaly (76%). Fourteen different mutations in the GAA gene were identified, with three novel mutations, c.1552-14_1552-1del, exons 2-3 deletion and exons 6-10 deletion. The most common mutation identified was c.1935C > A p.(D645E), with an allele frequency of 33%. Sixteen patients received ERT at the median age of 7 months. Overall survival was 29%. Mean age of death was 17.5 months. Our longest surviving patient has atypical IOPD and is currently 20 years old.. This is the first study to analyze the genotype and phenotype of Malaysian IOPD patients, and has identified the c.1935C > A p.(D645E) as the most common mutation. The three novel mutations reported in this study expands the mutation spectrum for IOPD. Our low survival rate underscores the importance of early diagnosis and treatment in achieving better treatment outcomes.

Topics: alpha-Glucosidases; Cardiomyopathy, Hypertrophic; Genotype; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle Hypotonia; Phenotype; Retrospective Studies; Treatment Outcome

Pompe disease (PD), also defined as acid maltase deficiency, is a rare autosomal recessive disease that causes glycogen accumulation due to a deficiency of the lysosomal enzyme acid α-glucosidase. An excessive amount of undisposed glycogen causes progressive muscle weakness throughout the body. It particularly affects skeletal muscles and the nervous system, especially in the late-onset phase. Here, we present a clinical case of late-onset PD (LOPD) with normal CK (creatinine kinase) values treated after a misdiagnosis of demyelinating motor polyneuropathy and chronic inflammatory neuropathy. The suspicion of possible fibromyalgia induced the patient to seek a rheumatology consultation, and the investigations performed led to the diagnosis of PD. The patient was investigated for genetic and enzymatic studies. PD was diagnosed using the α-glucosidase assay on DBS. In LOPD, clinical manifestations, such as muscle weakness, exercise intolerance, myalgia, or even high hyperCKemia, often appear as nonspecific and may mimic a wide variety of other muscle disorders, such as limb muscle dystrophies, congenital, metabolic, or inflammatory myopathies. In our case, the patient had CK values in the normal range but with continued complaints typical of PD. An analysis of enzyme activity revealed a pathologic value, and genetic analysis identified the c.-32-13T>G mutation in homozygosis. The association of the pathological enzyme value and mutation in homozygosity with LOPD led to a familial segregation study. Our results contribute to the characterization of PD in Italy and support the importance of rheumatologic attention. This suggests further studies are needed to define the broad clinical and pathological spectrum observed in this disease.

Topics: alpha-Glucosidases; Creatine Kinase; Fibromyalgia; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle Weakness

Glycogenosis type II (GSDII), or Pompe disease (MIM 232300), is an inherited autosomal recessive disorder caused by deficiency of the lysosomal acid-α-glucosidase. Mutations in the GAA gene alter normal enzyme production and lead to progressive buildup of intralysosomal glycogen, which plays an essential role in the severity and progression of the disease. We report here the study of 76 patients from Spain with either infantile or late onset form of Pompe disease. The analysis consisted in the molecular study of exons and intron flanking fragments of GAA gene. We have identified 55 different molecular pathogenic variants, 12 of them not previously described. In addition, we have determined a frequency of 84.37% for the c.-32-13T>G mutation in patients with the late-onset form of the disease. Functional characterization of some splice mutations showed deleterious mechanisms on the processing of mRNA.

Topics: Alleles; alpha-Glucosidases; Exons; Female; Gene Frequency; Genetic Predisposition to Disease; Genetic Testing; Genotype; Glycogen; Glycogen Storage Disease Type II; Humans; Introns; Male; Mutation; Polymorphism, Single Nucleotide; RNA Splicing; Spain

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy that progresses despite currently available therapy in some patients. The development of gene therapy with adeno-associated virus (AAV) vectors revealed a sex-dependent decrease in efficacy in female mice with Pompe disease. This study evaluated the effect of testosterone on gene therapy with an AAV2/8 vector containing a liver-specific promoter to drive expression of GAA (AAV2/8-LSPhGAA) in female GAA-knockout (KO) mice that were implanted with pellets containing testosterone propionate before vector administration. Six weeks after treatment, neuromuscular function and muscle strength were improved as demonstrated by increased Rotarod and wirehang latency for female mice treated with testosterone and vector, in comparison with vector alone. Biochemical correction improved after the addition of testosterone as demonstrated by increased GAA activity and decreased glycogen content in the skeletal muscles of female mice treated with testosterone and vector, in comparison with vector alone. An alternative androgen, oxandrolone, was evaluated similarly to reveal increased GAA in the diaphragm and extensor digitorum longus of female GAA-KO mice after oxandrolone administration; however, glycogen content was unchanged by oxandrolone treatment. The efficacy of androgen hormone treatment in females correlated with increased mannose-6-phosphate receptor in skeletal muscle. These data confirmed the benefits of brief treatment with an androgen hormone in mice with Pompe disease during gene therapy.

Topics: alpha-Glucosidases; Androgens; Animals; Dependovirus; Female; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Muscle, Skeletal; Oxandrolone; Testosterone

Electrical impedance methods, including electrical impedance myography, are increasingly being used as biomarkers of muscle health since they measure passive electrical properties of muscle that alter in disease. One disorder, Pompe Disease (Glycogen storage disease type II (GSDII)), remains relatively unstudied. This disease is marked by dramatic accumulation of intracellular myofiber glycogen. Here we assessed the electrical properties of skeletal muscle in a model of GSDII, the Pompe

Topics: Animals; Electric Conductivity; Electric Impedance; Female; Glycogen; Glycogen Storage Disease Type II; Mice; Muscle, Skeletal

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

Topics: Adult; Diagnosis, Differential; Genetic Testing; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycogen; Glycogen Storage Disease Type II; High-Throughput Nucleotide Sequencing; Humans; Muscle Weakness; Myasthenic Syndromes, Congenital

α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6-

Topics: alpha-Glucosidases; Animals; Cyclohexanols; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Glycoside Hydrolase Inhibitors; Humans; Zebrafish

Pompe disease results from GAA mutations that leads to lysosomal glycogen accumulation and cardiac and skeletal muscle pathology. We have previously generated an infantile-onset Pompe disease patient-derived human-induced pluripotent stem cells (iPSCs) line carrying compound GAA mutations (R608X and E888X). Using his parents' peripheral blood mononuclear cells (PBMCs), we here generated two iPSCs lines which carry mutations of R608X E888X respectively. Both lines show typical cell morphology, high expressed pluripotent and self-renewal markers, normal karyotype, and trilineage differentiation potential. These two lines are valuable re-sources for studying the pathological mechanisms of GAA mutation-caused Pompe disease.

Topics: alpha-Glucosidases; Glycogen; Glycogen Storage Disease Type II; Humans; Induced Pluripotent Stem Cells; Infant; Leukocytes, Mononuclear; Mutation

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaa

Topics: alpha-Glucosidases; Animals; Cardiomyopathy, Hypertrophic; Disease Models, Animal; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Mice; Muscle, Skeletal

Pompe disease is a metabolic myopathy due to acid alpha-glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients' cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N-acetylcysteine, idebenone, resveratrol, edaravone) improved alpha-glucosidase activity in rhGAA-treated cells, enhanced enzyme processing, and improved mannose-6-phosphate receptor localization. When co-administered with rhGAA, antioxidants improved alpha-glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder.

Topics: alpha-Glucosidases; Animals; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Oxidative Stress

Pompe disease is a lysosomal storage disease caused by mutations within the GAA gene, which encodes acid α-glucosidase (GAA)-an enzyme necessary for lysosomal glycogen degradation. A lack of GAA results in an accumulation of glycogen in cardiac and skeletal muscle, as well as in motor neurons. The only FDA approved treatment for Pompe disease-an enzyme replacement therapy (ERT)-increases survival of patients, but has unmasked previously unrecognized clinical manifestations of Pompe disease. These clinical signs and symptoms include tracheo-bronchomalacia, vascular aneurysms, and gastro-intestinal discomfort. Together, these previously unrecognized pathologies indicate that GAA-deficiency impacts smooth muscle in addition to skeletal and cardiac muscle. Thus, we sought to characterize smooth muscle pathology in the airway, vascular, gastrointestinal, and genitourinary in the Gaa

Topics: alpha-Glucosidases; Animals; Disease Models, Animal; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Muscle, Smooth

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA

Topics: alpha-Glucosidases; Animals; Dependovirus; Disease Models, Animal; Glycogen; Glycogen Storage Disease Type II; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Contraction; Muscle Development; Muscle, Skeletal; Myocardium; Tissue Engineering

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.

Topics: alpha-Glucosidases; Animals; Animals, Genetically Modified; Autophagy; Drug Evaluation, Preclinical; Gene Knockdown Techniques; Glycogen; Glycogen Storage Disease Type II; Glycolysis; Hexokinase; Humans; Lysosomes; Microscopy, Electron; Morpholinos; Motor Activity; Muscle, Skeletal; Pyruvates; Zebrafish; Zebrafish Proteins

In late-onset Pompe disease (LOPD), a lysosomal storage disorder with glycogen accumulation in several tissues, patients suffer from progressive skeletal muscle weakness. Lower urinary tract symptoms (LUTS) have rarely been reported. The aim of this study is to objectively assess LUTS in patients with LOPD for the first time using urodynamic studies and to determine differences between LOPD patients with and without LUTS.. Eighteen patients with LOPD were recruited, of whom seven patients (38.9%) reported LUTS (both voiding and storage symptoms). Six of these patients underwent urodynamic studies. Medical histories and motor function tests were compared between the 7 patients with LUTS and the 11 patients without LUTS. The Student t test was used to determine an association between the two cohorts.. In the seven LOPD patients with LUTS urodynamics revealed neurogenic dysfunction, underactive detrusor, and bladder outlet obstruction. These patients had suffered from clinical symptoms for a longer period of time before starting enzyme replacement therapy (P = .017) than patients without LUTS. They also scored more poorly on muscle function tests. Urodynamic results point to neurogenic causes for LUTS in LOPD, that is, neurogenic reflex bladder or impaired filling sensation. This could be due to glycogen accumulation in the urothelium and central nervous system. Patients with LUTS also seem to be more severely affected by LOPD than patients without LUTS.. LUTS in LOPD requires early and specific treatment to limit the development of severe health problems. Urodynamic studies should be considered in assessing LUTS.

Topics: Aged; Cohort Studies; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Lower Urinary Tract Symptoms; Male; Middle Aged; Muscle, Skeletal; Neurologic Examination; Surveys and Questionnaires; Urinary Bladder, Neurogenic; Urinary Bladder, Underactive; Urodynamics; Urothelium

Pompe disease (PD) is a rare autosomal recessive multi-systemic lysosomal storage disorder, caused by mutations in the acid alpha-glucosidase (GAA) gene located on 17q25.2-q25.3. It is one of about 50 rare genetic diseases categorized as lysosomal storage disorders. This disease is characterized by a range of different symptoms related to acid alpha-glucosidase deficiency. Mutation recognition in the GAA gene can be very significant for purposes such as therapeutic interference, early diagnosis and genotype-phenotype relationship. In the current study, peripheral blood samples were gathered from patients with PD and healthy members of three families. Enzymatic activity of GAA was checked. Then, mutation detection was performed by polymerase chain reaction followed by direct sequencing of all exons in samples with decreased enzyme activity. The identified mutations were investigated using bioinformatics tools to predict possible effects on the protein product and also to compare the mutated sequence with near species. Three novel mutations (c.1966-1968delGAG, c.2011-2012delAT and c.1475-1481dupACCCCAC) were identified in the GAA gene. Assessment of the effects of these mutations on protein structure and function showed the possibility of harmful effects and their significant alterations in the protein structure. The three novel GAA gene mutations detected in this study expand the information about the molecular genetics of PD and can be used to helpdiagnosis and genetic counseling of affected families.

Topics: Adolescent; alpha-Glucosidases; Child; Child, Preschool; DNA Mutational Analysis; Female; Genetic Predisposition to Disease; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Male; Mutation; Turkey

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa

Topics: alpha-Glucosidases; Animals; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Liver; Lysosomes; Male; Mice; Mice, Knockout; Muscle, Skeletal; Phenotype; Secretory Pathway; Signal Transduction; Transcriptome; Transfection; Treatment Outcome

Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaa

Topics: Age of Onset; alpha-Glucosidases; Animals; Cardiomyopathy, Hypertrophic; CRISPR-Cas Systems; Disease Models, Animal; Female; Gene Knock-In Techniques; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Male; Mice; Mice, Transgenic; Muscle Weakness; Muscle, Skeletal; Myocardium; RNA, Guide, Kinetoplastida

Pompe disease (PD) is caused by lysosomal glycogen accumulation in tissues, including muscles and the central nervous system (CNS). The intravenous infusion of recombinant human acid alpha-glucosidase (rhGAA) rescues the muscle pathologies in PD but does not treat the CNS because rhGAA does not cross the blood-brain barrier (BBB). To understand the CNS pathologies in PD, control and PD mice were followed and analyzed at 9 and 18 months with brain structural and ultrastructural studies. T2-weighted brain magnetic resonance imaging studies revealed the progressive dilatation of the lateral ventricles and thinning of the corpus callosum in PD mice. Electron microscopy (EM) studies at the genu of the corpus callosum revealed glycogen accumulation, an increase in nerve fiber size variation, a decrease in the g-ratio (axon diameter/total fiber diameter), and myelin sheath decompaction. The morphology of oligodendrocytes was normal. Diffusion tensor imaging (DTI) studies at the corpus callosum revealed an increase in axial diffusivity (AD) and mean diffusivity (MD) more significantly in 9-month-old PD mice. The current study suggests that axon degeneration and axon loss occur in aged PD mice and are probably caused by glycogen accumulation in neurons. A drug crossing the BBB or a treatment for directly targeting the brain might be necessary in PD.

Topics: Animals; Axons; Case-Control Studies; Corpus Callosum; Diffusion Magnetic Resonance Imaging; Diffusion Tensor Imaging; Disease Models, Animal; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Mice; Microscopy, Electron; Oligodendroglia

In studies on the degradation of glycogen by rhGAA, a glycosylated protein core material was found which consists of about 5-6% of the total starting glycogen. There was an additional 25% of the glycogen unaccounted for based on glucose released. After incubation of glycogen with rhGAA until no more glucose was released, no other carbohydrate was detected on HPAEC-PAD. Several oligosaccharides are then detectable if the medium is first boiled in 0.1 N HCl or incubated with trypsin. It is present in serum either in an HCl extract or in a trypsin digest. The characteristics of the in vivo serum material are identical to the material in the in vitro incubation medium. One oligosaccharide cannot be further degraded by rhGAA, from the incubation medium as well as from serum co-elute on HPAEC-PAD. Several masked oligosaccharides in serum contain

Topics: alpha-Glucosidases; Female; Glycogen; Glycogen Storage Disease Type II; Glycosylation; Humans; Infant; Lysosomes; Recombinant Proteins; Solubility

Glycogen storage disease type II, or Pompe disease, is an autosomal recessive disorder caused by deficiency of lysosomal acid alpha-glucosidase (GAA). We performed genetic analysis to confirm the diagnosis of Pompe disease in a 61-year-old patient with progressive weakness in extremities, severe Sleep Apnea-Hypopnea Syndrome, a significant reduction of alpha-glucosidase in liquid sample of peripheral blood and muscular biopsy diagnosis. GAA gene sequencing showed the patient is homozygous for the splice-site mutation c.1194+5G>A, considered as nonpathogenic in Pompe Center mutation database. Further molecular RNA characterization of GAA transcripts allowed us to identify abnormal processing of pre-mRNA, leading to aberrant transcripts and a significant reduction of GAA mRNA levels. Our results indicate that c.1194+5G>A is a pathogenic splice-site mutation and should be considered as such for diagnostic purposes. This study emphasizes the potential role of functional studies to determine the consequences of mutations with no evident pathogenicity.

Topics: alpha-Glucosidases; Biopsy; Female; Genetic Testing; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Homozygote; Humans; Middle Aged; Mutation; Phenotype; Virulence

Pompe disease is a rare inherited disorder of lysosomal glycogen metabolism due to acid α-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA (rhGAA), is the only approved treatment for Pompe disease. Although alglucosidase alfa has provided clinical benefits, its poor targeting to key disease-relevant skeletal muscles results in suboptimal efficacy. We are developing an rhGAA, ATB200 (Amicus proprietary rhGAA), with high levels of mannose-6-phosphate that are required for efficient cellular uptake and lysosomal trafficking. When administered in combination with the pharmacological chaperone AT2221 (miglustat), which stabilizes the enzyme and improves its pharmacokinetic properties, ATB200/AT2221 was substantially more potent than alglucosidase alfa in a mouse model of Pompe disease. The new investigational therapy is more effective at reversing the primary abnormality - intralysosomal glycogen accumulation - in multiple muscles. Furthermore, unlike the current standard of care, ATB200/AT2221 dramatically reduces autophagic buildup, a major secondary defect in the diseased muscles. The reversal of lysosomal and autophagic pathologies leads to improved muscle function. These data demonstrate the superiority of ATB200/AT2221 over the currently approved ERT in the murine model.

Topics: 1-Deoxynojirimycin; alpha-Glucosidases; Animals; Disease Models, Animal; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Male; Mannosephosphates; Mice; Mice, Knockout; Muscle, Skeletal; Rats; Rats, Sprague-Dawley

In Pompe disease, deficient lysosomal acid α-glucosidase (GAA) activity causes glycogen accumulation in the muscles, which leads to weakness, cardiomyopathy, and respiratory failure. Although glycogen accumulation also occurs in the nervous system, the burden of neurological deficits in Pompe disease remains obscure. In this study, a neuron-specific gene therapy was administered to Pompe mice through intracerebroventricular injection of a viral vector carrying a neuron-specific promoter. The results revealed that gene therapy increased GAA activity and decreased glycogen content in the brain and spinal cord but not in the muscles of Pompe mice. Gene therapy only slightly increased the muscle strength of Pompe mice but substantially improved their performance on the rotarod, a test measuring motor coordination. Gene therapy also decreased astrogliosis and increased myelination in the brain and spinal cord of Pompe mice. Therefore, a neuron-specific treatment improved the motor coordination of Pompe mice by lowering glycogen accumulation, decreasing astrogliosis, and increasing myelination. These findings indicate that neurological deficits are responsible for a significant burden in Pompe disease.

Topics: alpha-Glucosidases; Animals; Brain; Genetic Therapy; Gliosis; Glycogen; Glycogen Storage Disease Type II; Mice; Motor Activity; Muscle Strength; Myelin Sheath; Neurons; Respiration; Rotarod Performance Test; Spinal Cord; Tissue Distribution

Pompe disease is a rare disorder due to deficiency of the acid α-glucosidase (GAA) treated by enzyme replacement therapy. The present authorized treatment with rhGAA, the recombinant human enzyme, provides an important benefit in the infantile onset; however, the juvenile and adult forms of the disease corresponding to >80% of the patients are less responsive to this treatment. This resistance has been mainly attributed to an insufficiency of mannose 6-phosphate residues in rhGAA to address lysosomes through the cation-independent mannose 6-phosphate receptor (CI-M6PR). As yet, several attempts to improve the enzyme delivery by increasing the number of mannose 6-phosphate on the enzyme were poorly effective on the late onset form of the disease. Here, we show that chemical conjugation of a synthetic analogue of the mannose 6-phosphate, named AMFA, onto rhGAA improves the affinity for CI-M6PR and the uptake of the enzyme in fibroblasts and myoblasts of adult Pompe patients. More importantly, only the conjugated rhGAA-AMFA was effective in aged Pompe mice when compared to rhGAA. Weekly treatment with 5-20mg·kg

Topics: Adult; alpha-Glucosidases; Animals; Cells, Cultured; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Mannosephosphates; Mice, Knockout; Muscle, Skeletal; Myoblasts

The complexity of the pathogenic cascade in lysosomal storage disorders suggests that combination therapy will be needed to target various aspects of pathogenesis. The standard of care for Pompe disease (glycogen storage disease type II), a deficiency of lysosomal acid alpha glucosidase, is enzyme replacement therapy (ERT). Many patients have poor outcomes due to limited efficacy of the drug in clearing muscle glycogen stores. The resistance to therapy is linked to massive autophagic buildup in the diseased muscle. We have explored two strategies to address the problem. Genetic suppression of autophagy in muscle of knockout mice resulted in the removal of autophagic buildup, increase in muscle force, decrease in glycogen level, and near-complete clearance of lysosomal glycogen following ERT. However, this approach leads to accumulation of ubiquitinated proteins, oxidative stress, and exacerbation of muscle atrophy. Another approach involves AAV-mediated TSC knockdown in knockout muscle leading to upregulation of mTOR, inhibition of autophagy, reversal of atrophy, and efficient cellular clearance on ERT. Importantly, this approach reveals the possibility of reversing already established autophagic buildup, rather than preventing its development.

Topics: alpha-Glucosidases; Animals; Autophagy; Disease Models, Animal; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Lysosomes; Male; Mice; Mice, Knockout; Muscle, Skeletal; TOR Serine-Threonine Kinases; Up-Regulation

Type 1 polysaccharide storage myopathy (PSSM1) is a glycogen storage disorder of known cause whereas the basis for type 2 PSSM (PSSM2) is unknown. The same diet and exercise regime prescribed for PSSM1 is recommended for PSSM2; however, the benefit of these recommendations for PSSM2 is undocumented. The objectives of this study were to determine traits of PSSM2 Warmblood horses (WB), determine the changes in exercise responses that occur with a recommended low-starch/fat-supplemented diet and exercise regime, and determine if glycogen concentrations correspond to the severity of signs. Owners of PSSM2 WB (2008-2016), completed a retrospective questionnaire regarding their horse. Glycogen concentrations were analyzed in skeletal muscle of PSSM2 WB (n = 36) obtained prior to recommendations and in control WB with no evident myopathy (n = 23). Chi-square, Fisher's exact, McNemar's tests with Bonferroni correction and Mann Whitney testing were utilized. Abnormal exercise responses reported by owners, began at approximately 6 years of age and included a decline in performance, a reluctance to collect and reluctance to go forward in over 50% of horses. With the recommended diet and exercise regime, 80% of PSSM2 WB owners reported an overall improvement with significant decreases in the proportion of horses showing a decline in performance and rhabdomyolysis. However, 53% of PSSM2 WB were still not advancing as expected with reluctance to go forward and collect persisting in approximately one third of horses. Median muscle glycogen concentrations did not differ between PSSM2 WB and WB with no evident myopathy. PSSM2 WB with the highest glycogen concentrations were significantly more likely to show a decline in performance than those with lower glycogen concentrations. In conclusion, diet and exercise recommendations ideal for PSSM1 improve but do not eliminate the decline in performance and reluctance to go forward under saddle characteristic of PSSM2.

Topics: Animal Feed; Animals; Glycogen; Glycogen Storage Disease Type II; Horse Diseases; Horses; Muscle, Skeletal; Muscular Diseases; Physical Conditioning, Animal; Starch

Pompe disease, which is due to acid alpha-glucosidase deficiency, is characterized by skeletal muscle dysfunction attributed to the accumulation of glycogen-filled lysosomes and autophagic buildup. Despite the extensive tissue damages, a failure of satellite cell (SC) activation and lack of muscle regeneration have been reported in patients. However, the origin of this defective program is unknown. Additionally, whether these deficits occur gradually over the disease course is unclear. Using a longitudinal pathophysiological study of two muscles in a Pompe mouse model, here, we report that the enzymatic defect results in a premature saturating glycogen overload and a high number of enlarged lysosomes. The muscles gradually display profound remodeling as the number of autophagic vesicles, centronucleated fibers, and split fibers increases and larger fibers are lost. Only a few regenerated fibers were observed regardless of age, although the SC pool was preserved. Except for the early age, during which higher numbers of activated SCs and myoblasts were observed, no myogenic commitment was observed in response to the damage. Following in vivo injury, we established that muscle retains regenerative potential, demonstrating that the failure of SC participation in repair is related to an activation signal defect. Altogether, our findings provide new insight into the pathophysiology of Pompe disease and highlight that the activation signal defect of SCs compromises muscle repair, which could be related to the abnormal energetic supply following autophagic flux impairment.

Topics: Age Factors; Animals; Autophagy; Cardiotoxins; Collagen; Disease Models, Animal; Dystrophin; Gene Expression Regulation; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Ki-67 Antigen; Laminin; Longitudinal Studies; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Muscle, Skeletal; Regeneration; Satellite Cells, Skeletal Muscle

Pompe disease is a metabolic myopathy that is caused by glycogen accumulation as a result of deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Previously, we showed that adult muscle stem cells termed satellite cells are present at normal levels in muscle from patients with Pompe disease, but that these are insufficiently activated to repair the severe muscle pathology. Here we characterized the muscle regenerative response during disease progression in a mouse model of Pompe disease and investigated the intrinsic capacity of Gaa

Topics: Age Factors; alpha-Glucosidases; Animals; Barium Compounds; Cardiotoxins; Chlorides; Disease Models, Animal; Female; Glycogen; Glycogen Storage Disease Type II; Ki-67 Antigen; Laminin; Lysosomal-Associated Membrane Protein 1; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; PAX7 Transcription Factor; Regeneration; Satellite Cells, Skeletal Muscle

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the

Topics: Albuterol; alpha-Glucosidases; Animals; Bronchi; Calcium Signaling; Extracellular Space; Glycogen; Glycogen Storage Disease Type II; Lung; Methacholine Chloride; Mice; Muscle Contraction; Muscle, Skeletal; Potassium Chloride; Trachea

Topics: Dermatomyositis; Glycogen; Glycogen Storage Disease Type II; Humans; Liver

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII.

Topics: Alleles; alpha-Glucosidases; Cell Line; Exons; Gene Order; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle Fibers, Skeletal; Mutation; Oligonucleotides, Antisense; Protein Binding; RNA Splicing; RNA Splicing Factors; Silencer Elements, Transcriptional; Targeted Gene Repair

Pompe disease is a lysosomal storage disorder caused by acid-α-glucosidase (GAA) deficiency, leading to glycogen storage. The disease manifests as a fatal cardiomyopathy in infantile form. Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The neurologic phenotype and the persistence of selective muscular weakness in some patients could be attributed to the central nervous system (CNS) storage uncorrected by ERT. GAA-KO 6neo/6neo mice were treated with a single intrathecal administration of adeno-associated recombinant vector (AAV) mediated gene transfer of human GAA at 1 month and their neurologic, neuromuscular, and cardiac function was assessed for 1 year. We demonstrate a significant functional neurologic correction in treated animals from 4 months onward, a neuromuscular improvement from 9 months onward, and a correction of the hypertrophic cardiomyopathy at 12 months. The regions most affected by the disease i.e. the brainstem, spinal cord, and the left cardiac ventricular wall all show enzymatic, biochemical and histological correction. Muscle glycogen storage is not affected by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction. This unprecedented global and long-term CNS and cardiac cure offer new perspectives for the management of patients.

Topics: alpha-Glucosidases; Animals; Brain; Cardiomyopathy, Hypertrophic; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; HEK293 Cells; Humans; Injections, Spinal; Male; Muscle Strength; Random Allocation; Single-Blind Method; Spinal Cord

Pompe disease is an extra-rare metabolic storage disease with deficiency of acid-alpha-glucosidase (GAA) enzyme activity, which leads to the pathologic accumulation of glycogen in target tissues (skeletal muscles, heart, brain). Clinical features and severity vary by the age of onset, rate of extent of organ involvement. In the late-onset Pompe disease (LOPD) form, essential cardiomyopathy seems to be uncommon. Muscles weakness and respiratory failure are the main symptoms of adult patient with Pompe disease. In presented case LOPD coupled with patient's regular sporting activity and healthy diet, which may explain the low intensity of the symptoms and the slow progress of the disease, lack of skeletal muscles weakness and lack of brain manifestation. Myocardial storage deposits are the only abnormalities found.

Topics: Glycogen; Glycogen Storage Disease Type II; Humans; Middle Aged; Muscle, Skeletal; Myocardium; Respiratory Insufficiency; Syncope

Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.

Topics: alpha-Glucosidases; Cell Differentiation; Cell Line; Energy Metabolism; Gene Expression; Glucose; Glycogen; Glycogen Storage Disease Type II; Humans; Induced Pluripotent Stem Cells; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Muscle Cells; Muscle Development; Muscle, Skeletal; MyoD Protein; Phenotype; Transduction, Genetic

OBJECTIVE To characterize clinical findings for polysaccharide storage myopathy (PSSM) in warmblood horses with type 1 PSSM (PSSM1; caused by mutation of the glycogen synthase 1 gene) and type 2 PSSM (PSSM2; unknown etiology). SAMPLE Database with 3,615 clinical muscle biopsy submissions. PROCEDURES Reported clinical signs and serum creatine kinase (CK) and aspartate aminotransferase (AST) activities were retrospectively analyzed for horses with PSSM1 (16 warmblood and 430 nonwarmblood), horses with PSSM2 (188 warmblood and 646 nonwarmblood), and warmblood horses without PSSM (278). Lameness examinations were reviewed for 9 warmblood horses with PSSM2. Muscle glycogen concentrations were evaluated for horses with PSSM1 (14 warmblood and 6 nonwarmblood), warmblood horses with PSSM2 (13), and horses without PSSM (10 warmblood and 6 nonwarmblood). RESULTS Rhabdomyolysis was more common for horses with PSSM1 (12/16 [75%] warmblood and 223/303 [74%] nonwarmblood) and nonwarmblood horses with PSSM2 (221/436 [51%]) than for warmblood horses with PSSM2 (39/147 [27%]). Gait abnormality was more common in warmblood horses with PSSM2 (97/147 [66%]) than in warmblood horses with PSSM1 (1/16 [7%]), nonwarmblood horses with PSSM2 (176/436 [40%]), and warmblood horses without PSSM (106/200 [53%]). Activities of CK and AST were similar in warmblood horses with and without PSSM2. Muscle glycogen concentrations in warmblood and nonwarmblood horses with PSSM1 were significantly higher than concentrations in warmblood horses with PSSM2. CONCLUSIONS AND CLINICIAL RELEVANCE Rhabdomyolysis and elevated muscle glycogen concentration were detected in horses with PSSM1 regardless of breed. Most warmblood horses with PSSM2 had stiffness and gait abnormalities with CK and AST activities and muscle glycogen concentrations within reference limits.

Topics: Animals; Biopsy; Female; Glycogen; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogen Synthase; Horse Diseases; Horses; Male; Muscular Diseases; Mutation; Polysaccharides; Retrospective Studies; Rhabdomyolysis

The underlying pathogenic lesions of glycogen storage disease type II (GSD II) and the diversity of this disease among different species are still under exploration. Thus, we created an acid alpha-glucosidase (gaa) gene-mutated zebrafish model of GSD II and examined the sequential pathogenic changes. gaa mRNA and protein expression, enzymatic activity, and lysosomal glycogen accumulation were assessed, and the phenotypic changes were compared between wild-type (WT) and gaa-mutated zebrafish. The presence of a Δ13 frameshift mutation in the gaa gene was confirmed at both the DNA and transcribed mRNA levels by Sanger sequencing. The relative amount of gaa mRNA was decreased before 2 days postfertilization (dpf), after which it unexpectedly increased in the mutant compared with the WT zebrafish. Consistent with the mRNA expression, the Gaa enzymatic activity in the mutant was downregulated before 3 dpf, while the Gaa protein level was slightly decreased at 4 dpf and was maintained at a consistent level in the adult gaa mutant muscle tissue. However, more than half of the adult mutant zebrafish exhibited excessive glycogen accumulation in the liver and muscles, along with the presence of autophagosomes, as determined by transmission electron microscopy. Thus, we have successfully generated a frameshift mutation in the gaa gene in zebrafish. The unique gaa gene expression changes and mild GSD II features during the adult stage strongly indicate the existence of species-specific differences, as well as an underlying compensatory network, which may warrant further examination.

Topics: alpha-Glucosidases; Animals; Base Sequence; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Glycogen; Glycogen Storage Disease Type II; Mutation; Phenotype; RNA, Messenger; Zebrafish

Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease.. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy for Pompe disease.

Topics: alpha-Glucosidases; Animals; Antibodies; Cytoplasm; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Mice; Mice, Knockout

Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) fails to completely reverse muscle weakness in Pompe disease. β2-agonists enhanced ERT by increasing receptor-mediated uptake of rhGAA in skeletal muscles.. To test the hypothesis that a β-blocker might reduce the efficacy of ERT, because the action of β-blockers opposes those of β2-agonists.. Mice with Pompe disease were treated with propranolol (a β-blocker) or clenbuterol in combination with ERT, or with ERT alone.. Propranolol-treated mice had decreased weight gain (p<0.01), in comparison with clenbuterol-treated mice. Left ventricular mass was decreased (and comparable to wild-type) in ERT only and clenbuterol-treated groups of mice, and unchanged in propranolol-treated mice. GAA activity increased following either clenbuterol or propranolol in skeletal muscles. However, muscle glycogen was reduced only in clenbuterol-treated mice, not in propranolol-treated mice. Cell-based experiments confirmed that propranolol reduces uptake of rhGAA into Pompe fibroblasts and also demonstrated that the drug induces intracellular accumulation of glycoproteins at higher doses.. Propranolol, a commonly prescribed β-blocker, reduced weight, increased left ventricular mass and decreased glycogen clearance in skeletal muscle following ERT. β-Blockers might therefore decrease the efficacy from ERT in patients with Pompe disease.

Topics: Adrenergic beta-Antagonists; alpha-Glucosidases; Animals; Cells, Cultured; Drug Antagonism; Drug Evaluation, Preclinical; Enzyme Replacement Therapy; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Heart Ventricles; Humans; Mice, Knockout; Propranolol

Respiratory and/or lingual dysfunction are among the first motor symptoms in Pompe disease, a disorder resulting from absence or dysfunction of the lysosomal enzyme acid α-glucosidase (GAA). Here, we histologically evaluated the medulla, cervical and thoracic spinal cords in 6 weeks old asymptomatic Pompe (Gaa(-/-)) mice to determine if neuropathology in respiratory motor regions has an early onset. Periodic acid-Schiff (PAS) staining indicated glycogen accumulation was exclusively occurring in Gaa(-/-) hypoglossal, mid-cervical and upper thoracic motoneurons. Markers of DNA damage (Tunel) and ongoing apoptosis (Cleaved Caspase 3) did not co-localize with PAS staining, but were prominent in a medullary region which included the nucleus tractus solitarius, and also in the thoracic spinal dorsal horn. We conclude that respiratory-related motoneurons are particularly susceptible to GAA deficiency and that neuronal glycogen accumulation and neurodegeneration may occur independently in early stage disease. The data support early therapeutic intervention in Pompe disease.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Caspase 3; Cervical Vertebrae; Cohort Studies; Disease Models, Animal; DNA Damage; Glial Fibrillary Acidic Protein; Glycogen; Glycogen Storage Disease Type II; Medulla Oblongata; Mice, 129 Strain; Mice, Knockout; Microfilament Proteins; Motor Neurons; Neuroimmunomodulation; Spinal Cord; Thoracic Vertebrae

Enzyme replacement therapy (ERT) with recombinant human (rh) acid α-glucosidase (GAA) has prolonged the survival of patients. However, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up rhGAA, correlated with a poor response to ERT by muscle in Pompe disease. Clenbuterol, a selective β2 receptor agonist, enhanced the CI-MPR expression in striated muscle through Igf-1 mediated muscle hypertrophy, which correlated with increased CI-MPR (also the Igf-2 receptor) expression. In this study we have evaluated 4 new drugs in GAA knockout (KO) mice in combination with an adeno-associated virus (AAV) vector encoding human GAA, 3 alternative β2 agonists and dehydroepiandrosterone (DHEA). Mice were injected with AAV2/9-CBhGAA (1E+11 vector particles) at a dose that was not effective at clearing glycogen storage from the heart. Heart GAA activity was significantly increased by either salmeterol (p<0.01) or DHEA (p<0.05), in comparison with untreated mice. Furthermore, glycogen content was reduced in the heart by treatment with DHEA (p<0.001), salmeterol (p<0.05), formoterol (p<0.01), or clenbuterol (p<0.01) in combination with the AAV vector, in comparison with untreated GAA-KO mice. Wirehang testing revealed that salmeterol and the AAV vector significantly increased performance, in comparison with the AAV vector alone (p<0.001). Similarly, salmeterol with the vector increased performance significantly more than any of the other drugs. The most effective individual drugs had no significant effect in absence of vector, in comparison with untreated mice. Thus, salmeterol should be further developed as adjunctive therapy in combination with either ERT or gene therapy for Pompe disease.

Topics: alpha-Glucosidases; Animals; Clenbuterol; Dehydroepiandrosterone; Dependovirus; Disease Models, Animal; Enzyme Replacement Therapy; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Myocardium; Salmeterol Xinafoate

Pompe disease, caused by deficiency of acid alpha-glucosidase (GAA), leads to widespread glycogen accumulation and profound neuromuscular impairments. There has been controversy, however, regarding the role of central nervous system pathology in Pompe motor dysfunction. We hypothesized that absence of GAA protein causes progressive activation of neuropathological signaling, including pathways associated with cell death. To test this hypothesis, genomic data (Affymetrix Mouse Gene Array 2.0ST) from the midcervical spinal cord in 6 and 16 mo old Pompe (Gaa

Topics: alpha-Glucosidases; Animals; Cell Death; Cervical Vertebrae; Gene Expression Profiling; Glycogen; Glycogen Storage Disease Type II; Inflammation; Mice; Nerve Degeneration; Neurons; RNA, Messenger; Signal Transduction; Spinal Cord; Transcriptome

Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca

Topics: Calcimycin; Cells, Cultured; Drug Evaluation, Preclinical; Exocytosis; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Iduronidase; Lysophosphatidylcholines; Lysosomes; Phagosomes; Pharmaceutical Vehicles

Pompe disease is an autosomal recessive disorder linked to GAA gene that leads to a multi-system intralysosomal accumulation of glycogen. Mutation identification in the GAA gene can be very important for early diagnosis, correlation between genotype-phenotype and therapeutic intervention. For this purpose, peripheral blood from 57 individuals susceptible to Pompe disease was collected and all exons of GAA gene were amplified; the sequences and the mutations were analyzed in silico to predict possible impact on the structure and function of the human protein. In this study, 46 individuals presented 33 alterations in the GAA gene sequence, among which five (c.547-67C>G, c.547-39T>G, p.R437H, p.L641V and p.L705P) have not been previously described in the literature. The alterations in the coding region included 15 missense mutations, three nonsense mutations and one deletion. One insertion and other 13 single base changes were found in the non-coding region. The mutation p.G611D was found in homozygosis in a one-year-old child, who presented low levels of GAA activity, hypotonia and hypertrophic cardiomyopathy. Two patients presented the new mutation p.L705P in association with c.-32-13T>G. They had low levels of GAA activity and developed late onset Pompe disease. In our study, we observed alterations in the GAA gene originating from Asians, African-Americans and Caucasians, highlighting the high heterogeneity of the Brazilian population. Considering that Pompe disease studies are not very common in Brazil, this study will help to better understand the potential pathogenic role of each change in the GAA gene. Furthermore, a precise and early molecular analysis improves genetic counseling besides allowing for a more efficient treatment in potential candidates.

Topics: Adolescent; Adult; alpha-Glucosidases; Asian People; Base Sequence; Black or African American; Brazil; Cardiomyopathy, Hypertrophic; Child; Child, Preschool; Codon, Nonsense; Early Diagnosis; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Testing; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Male; Middle Aged; Muscle Hypotonia; Mutation, Missense; Sequence Analysis, DNA; Sequence Deletion; White People; Young Adult

We have recently reported on the pathology of the neuromuscular junction (NMJ) in Pompe disease, reflecting disruption of neuronal and muscle homeostasis as a result of glycogen accumulation. The aim of this study was to examine how the alteration of NMJ physiology contributes to Pompe disease pathology; we performed molecular, physiological, and histochemical analyses of NMJ-related measures of the tibialis anterior muscles of young-, mid-, and late-stage alpha-glucosidase (GAA)-deficient mice.. We performed intramuscular injection of an adeno-associated virus (AAV)9 vector expressing GAA (AAV9-hGAA) into the tibialis anterior muscle of Gaa(-/-) mice at early, mid, and severe pathological time points. We analyzed expression of NMJ-related genes, in situ muscle force production, and clearance of glycogen in conjunction with histological assessment of the NMJ.. Our data demonstrate that AAV9-hGAA is able to replace GAA to the affected tissue and modify AChR mRNA expression, muscle force production, motor endplate area, and innervation status. Importantly, the degree of restoration for these outcomes is limited by severity of disease. Early restoration of GAA activity was most effective, whereas late correction of GAA expression was not effective in modifying parameters reflecting NMJ structure and function nor in force restoration despite resolution of glycogen storage in muscle.. Our data provide new mechanistic insight into the pathology of Pompe disease and suggest that early systemic correction to both neural and muscle tissues may be essential for successful correction of neuromuscular function in Pompe disease. Ann Neurol 2015;78:222-234.

Topics: alpha-Glucosidases; Animals; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Hindlimb; Injections, Intramuscular; Isometric Contraction; Mice; Mice, Knockout; Muscle Strength; Muscle, Skeletal; Neuromuscular Junction; Receptors, Cholinergic; RNA, Messenger; Time Factors

Pompe disease, or glycogen storage disease type II (GSD2), an autosomal recessive disease first described by Joannes Cassianus Pompe (1901-1945), causes deficient activity of acid α-glucosidase (GAA) enzyme. GAA catalyses α 1,4 and α 1,6 glucosidic linkages in lysosomes; destruction of these linkages permits glycogen to be separated into glucose and later used for energy. Without proper function of this enzyme, glycogen accumulates in lysosome, causing muscle hypotonia. We report a previously undescribed association of c.1437G>A intron 9 substitution on the GAA gene with severe infantile-onset Pompe disease in a deceased proband and carrier status in four of five surviving family members. Previous authors have found late-onset or moderate severity infantile-onset Pompe disease associated with this allelic variation. Our proband's family's village was suspicious for locally endemic disease. While our proband developed all features of classic infantile onset GSD2, socioeconomic and geographic factors initially suggested an infectious aetiology.

Topics: Alleles; alpha-Glucosidases; Family; Genetic Carrier Screening; Genotype; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Introns; Lysosomes; Male; Muscle Hypotonia; Phenotype; Point Mutation; Polymorphism, Single Nucleotide

Pompe disease (glycogen storage disease type II; acid maltase deficiency) is a devastating myopathy resulting from acid α-glucosidase (GAA) deficiency in striated and smooth muscle. Despite the availability of enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA), the limitations of ERT have prompted the preclinical development of gene therapy. Gene therapy has the advantage of continuously producing GAA, in contrast to ERT, which requires frequent injections of rhGAA. An adeno-associated viral (AAV) vector containing a muscle-specific promoter, AAV-MHCK7hGAApA, achieved high GAA expression in heart and skeletal muscle in mice with Pompe disease. However, elevated GAA activity was not sufficient to completely clear accumulated glycogen in skeletal muscle. The process of glycogen clearance from lysosomes might require improved trafficking of GAA to the lysosomes in skeletal muscle, previously achieved with the β(2)-agonist clenbuterol that enhanced glycogen clearance in skeletal muscle without increasing GAA activity. Glycogen clearance was clearly enhanced by treatment with a nondepleting anti-CD4 monoclonal antibody (anti-CD4 mAb) along with muscle-specific GAA expression in cardiac muscle, but that treatment was not effective in skeletal muscle. Furthermore, anti-CD4 mAb treatment along with clenbuterol achieved synergistic therapeutic efficacy in both cardiac and skeletal muscle. This triple therapy increased both muscle strength and weight gain. Overall, triple therapy to enhance GAA trafficking and to suppress immune responses significantly improved the efficacy of muscle-targeted gene therapy in murine Pompe disease.

Topics: Adrenergic beta-Agonists; alpha-Glucosidases; Animals; Antibodies, Monoclonal; CD4 Antigens; Clenbuterol; Combined Modality Therapy; Dependovirus; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Immune Tolerance; Mice; Mice, Knockout; Muscle, Skeletal; Myocytes, Cardiac; Recombinant Proteins

Pompe disease is an autosomal recessive disorder caused by mutations in the acid-α glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy (ERT). Using Pompe (Gaa(-/-)) mice, we tested the hypothesis that intralingual delivery of viral vectors encoding GAA results in GAA expression and glycogen clearance in both tongue myofibers and hypoglossal (XII) motoneurons. An intralingual injection of an adeno-associated virus (AAV) vector encoding GAA (serotypes 1 or 9; 1 × 10(11) vector genomes, CMV promoter) was performed in 2-month-old Gaa(-/-) mice, and tissues were harvested 4 months later. Both serotypes robustly transduced tongue myofibers with histological confirmation of GAA expression (immunochemistry) and glycogen clearance (Period acid-Schiff stain). Both vectors also led to medullary transgene expression. GAA-positive motoneurons did not show the histopathologic features which are typical in Pompe disease and animal models. Intralingual injection with the AAV9 vector resulted in approximately threefold more GAA-positive XII motoneurons (P < 0.02 versus AAV1); the AAV9 group also gained more body weight over the course of the study (P < 0.05 versus AAV1 and sham). We conclude that intralingual injection of AAV1 or AAV9 drives persistent GAA expression in tongue myofibers and motoneurons, but AAV9 may more effectively target motoneurons.

Topics: alpha-Glucosidases; Animals; Dependovirus; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Injections, Intramuscular; Mice; Motor Neurons; Muscle, Skeletal; Myofibrils; Promoter Regions, Genetic

Vascular abnormalities and glycogen accumulation in vascular smooth muscle fibres have been described in Pompe disease. Using carotid-femoral pulse wave velocity (cfPWV), the gold standard methodology for determining aortic stiffness, we studied whether aortic stiffness is increased in patients with Pompe disease. Eighty-four adult Pompe patients and 179 age- and gender-matched volunteers participated in this cross-sectional case-controlled study. Intima media thickness and the distensibility of the right common carotid artery were measured using a Duplex scanner. Aortic augmentation index, central pulse pressure, aortic reflexion time and cfPWV were assessed using the SphygmoCor® system. CfPWV was higher in patients than in volunteers (8.8 versus 7.4 m/s, p < 0.001). This difference was still present after adjustment for age, gender, mean arterial blood pressure (MAP), heart rate and diabetes mellitus (p = 0.001), and was shown by subgroup analysis to apply to the 40-59 years age group (p = 0.004) and 60+ years age group (p = 0.01), but not to younger age groups (p = 0.99). Except for a shorter aortic reflexion time (p = 0.02), indirect indicators of arterial stiffness did not differ between patients and volunteers. Relative to volunteers (20%), more Pompe patients had a history of hypertension (36%, p = 0.005), and the MAP was higher than in volunteers (100 versus 92 mmHg, p < 0.001). This study shows that patients with non-classic Pompe disease have increased aortic stiffness and blood pressure. Whether this is due to glycogen accumulation requires further investigation. To reduce the potential risk of cardiovascular diseases, we recommend that blood pressure and other common cardiovascular risk factors are monitored regularly.

Topics: Adult; Blood Pressure; Case-Control Studies; Cross-Sectional Studies; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Hypertension; Male; Middle Aged; Muscle, Smooth, Vascular; Pulse Wave Analysis; Vascular Stiffness

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Topics: Adrenergic beta-2 Receptor Agonists; alpha-Glucosidases; Animals; Cations; Clenbuterol; Densitometry; Dependovirus; Extremities; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; HEK293 Cells; Humans; Lysosomes; Mice; Mice, Knockout; Muscle, Skeletal; Receptor, IGF Type 2

Pompe disease (PD), which is also called glycogen storage disease type II (GSDII), is one of the lysosomal storage diseases (LSDs) caused by a deficiency in acid-α-glucosidase (GAA) in the lysosome and is characterized by the accumulation of glycogen in various cells. PD has been treated by enzyme replacement therapy (ERT). We generated induced pluripotent stem cells (iPSCs) from the cells of patients with infantile-type and late-onset-type PD using a retrovirus vector to deliver transgenes encoding four reprogramming factors, namely, OCT4, SOX2, c-MYC, and KLF4. We confirmed that the two types of PD-iPSCs exhibited an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. The PD-iPSCs exhibited strong positive staining with Periodic acid-Schiff (PAS). Moreover, ultrastructural features of these iPSCs exhibited massive glycogen granules in the cytoplasm, particularly in the infantile-type but to a lesser degree in the late-onset type. Glycogen granules of the infantile-type iPSCs treated with rhGAA were markedly decreased in a dose-dependent manner. Human induced pluripotent stem cell provides an opportunity to build up glycogen storage of Pompe disease in vitro. It represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies for Pompe disease.

Topics: alpha-Glucosidases; Cell Line; Dose-Response Relationship, Drug; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Models, Biological; Skin

Pompe disease or glycogen storage disease type II is a glycogen storage disorder associated with malfunction of the acid α-glucosidase enzyme (GAA; EC.3.2.1.3) leading to intracellular aggregations of glycogenin muscles. The infantile-onset type is the most life-threatening form of this disease, in which most of patients suffer from cardiomyopathy and hypotonia in early infancy. In this study, a typical case of Pompe disease was reported in an Iranian patient using molecular analysis of the GAA gene. Our results revealed a new c.1824_1828dupATACG mutation in exon 13 of the GAA gene. In conclusion, with the finding of this novel mutation, the genotypic spectrum of Iranian patients with Pompe disease has been extended, facilitating the definition of disease-related mutations.

Topics: alpha-Glucosidases; Cardiomyopathies; Consanguinity; Exons; Genetic Predisposition to Disease; Genome-Wide Association Study; Genome, Human; Genotype; Genotyping Techniques; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Iran; Male; Mutation; Oligosaccharides; Sequence Analysis, DNA

Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.

Topics: 1-Deoxynojirimycin; Administration, Oral; Animals; Biocatalysis; Biological Availability; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Stability; Gene Knockout Techniques; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Isoenzymes; Lysosomes; Mice; Mice, Transgenic; Mutant Proteins; Mutation; Protein Transport; Proteolysis

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

Topics: alpha-Glucosidases; Animals; Cell Line; Dietary Supplements; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Insulin; Kyphosis; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Multiprotein Complexes; Muscle, Skeletal; Muscular Atrophy; Myoblasts; RNA Interference; TOR Serine-Threonine Kinases; Transfection

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA(-/-) mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA(-/-) mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA(-/-) mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA(-/-) mice, phosphorylase activity is suppressed in the GAA(-/-) mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

Topics: alpha-Glucosidases; Animals; Disease Models, Animal; Gene Deletion; Glucosyltransferases; Glycogen; Glycogen Phosphorylase; Glycogen Storage Disease Type II; Glycogen Synthase; Glycoproteins; Hexokinase; Humans; Liver; Mice; Mice, Inbred C57BL; Myocardium; Quadriceps Muscle; Recombinant Proteins

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB-mediated cellular clearance.

Topics: Adenoviridae; alpha-Glucosidases; Animals; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cells, Cultured; Disease Models, Animal; Exocytosis; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Lysosomes; Mice; Mice, Knockout; Muscle, Skeletal

The physiological relevance of acid maltase (acid α-glucosidase, an enzyme that degrades lysosomal glycogen) is well recognized in liver and muscle. In late (adult)-onset acid maltase deficiency (glycogen storage disease type II [GSD II]), glycogen accumulates inside muscular lysosomes in the context of reduced enzymatic activity present not only in muscle, but also throughout the organism. Yet, disease manifestations are commonly attributed to lysosomal disruption and autophagic vesicle buildup inside the myofiber due to a lack of obvious hepatic or broader metabolic dysfunction. However, current therapies primarily focused on reducing glycogen deposition by dietary or enzyme replacement have not been consistently beneficial, providing the motivation for a better understanding of disease mechanisms.. To provide a systematic overview of metabolism and methylation capacity using widely available analytical methods by evaluating secondary compromise of (1) the citric acid cycle, (2) methylation capacity, and (3) nutrient sensor interaction in as many as 33 patients with GSD II (ie, not all patients were available for all assessments) treated with only a low-carbohydrate/high-protein, calorie-balanced diet.. Case series including clinical and analytical characterization in an academic setting involving 33 enzymatically proved adults with GSD II treated only with a low-carbohydrate/high-protein, calorie-balanced diet.. Biochemical analysis of blood and urine samples.. Patients exhibited evidence for disturbed energy metabolism contributing to a chronic catabolic state and those who were studied further also displayed diminished plasma methylation capacity and elevated levels of insulin-like growth factor type 1 and its carrier protein insulin-like growth factor binding protein 3 (IGFBP-3).. The simplest unifying interpretation of these abnormalities is nutrient sensor disturbance with secondary energy failure leading to a chronic catabolic state. Data also provide the framework for the investigation of potentially beneficial interventions, including methylation supplementation, as adjuncts specifically targeted to ameliorate the systemic metabolic abnormalities of this disorder.

Topics: Adolescent; Adult; Aged; Biomarkers; Diet, Carbohydrate-Restricted; Dietary Proteins; Energy Metabolism; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Middle Aged; Muscle, Skeletal; Young Adult

Previous studies strongly suggest that starch binding domain containing protein 1 (Stbd1) plays an important role in intracellular glycogen trafficking into lysosomes. We report here that Stbd1 expression is markedly increased in skeletal muscles but not in heart and liver of GAA-KO mice. An AAV2/9 vector expressing a Stbd1-specific shRNA effectively suppressed Stbd1 expression but did not alter lysosomal glycogen accumulation in the affected tissues of GAA-KO mice. Our results indicate that inhibition of Stbd1 does not appear to be an effective therapeutic approach for Pompe disease.

Topics: Animals; Cell Line; Disease Models, Animal; Gene Expression Regulation; Gene Knockdown Techniques; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Membrane Proteins; Mice; Mice, Knockout; Muscle Proteins; Muscle, Skeletal; RNA Interference

Infantile Pompe disease is a rare, autosomal recessive disorder due to deficiency of the enzyme acid α-glucosidase that degrades lysosomal glycogen. Clinical features of diffuse hypotonia, cardiomyopathy, and weakness are present within the first days to months of life in patients with classic infantile Pompe disease. Progression of the disease often leads to respiratory failure. Although sleep apnea is reported in late-onset Pompe disease, sleep pathology is not well characterized in infantile disease. In this retrospective study, we analyzed nocturnal polysomnography results from 17 patients with infantile-onset Pompe disease. Obstructive sleep apnea and hypoventilation were common among this cohort, even in those that did not have symptoms of sleep-disordered breathing. All patients with infantile-onset Pompe disease should undergo polysomnography as a routine part of their care.

Topics: Female; Glycogen; Glycogen Storage Disease Type II; Humans; Hypoventilation; Infant; Infant, Newborn; Lysosomes; Male; Polysomnography; Proteolysis; Sleep Apnea Syndromes

Glycogen storage disease type II - also called Pompe disease or acid maltase deficiency - is an autosomal recessive metabolic disorder, caused by an accumulation of glycogen in the lysosome due to deficiency of the lysosomal acid alpha-glucosidase enzyme. Pompe disease is transmitted as an autosomal recessive trait and is caused by mutations in the gene encoding the acid α-glucosidase (GAA), located on chromosome 17q25.2-q25.3. The different disease phenotypes are related to the levels of residual GAA activity in muscles. The clinical spectrum ranging from the classical form with early onset and severe phenotype to not-classical form with later onset and milder phenotype is described.

Topics: Age of Onset; alpha-Glucosidases; Diagnosis, Differential; Genetic Predisposition to Disease; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Muscle Weakness; Mutation; Prognosis

We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.

Topics: Animals; Biological Transport; Disease Models, Animal; Drug Delivery Systems; Enzyme Replacement Therapy; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Glycosylation; Half-Life; HEK293 Cells; Humans; Insulin-Like Growth Factor II; Kinetics; Lysosomes; Mice; Muscle, Skeletal; Mutant Chimeric Proteins; Myoblasts; Plasmids; Receptor, IGF Type 2; Transfection

Topics: alpha-Glucosidases; Central Nervous System; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans

Enzyme replacement therapies for lysosomal storage disorders are often hindered by suboptimal biodistribution of recombinant enzymes after systemic injection. This is the case for Pompe disease caused by acid α-glucosidase (GAA) deficiency, leading to excess glycogen storage throughout the body, mainly the liver and striated muscle. Targeting intercellular adhesion molecule-1 (ICAM-1), a protein involved in inflammation and overexpressed on most cells under pathological conditions, provides broad biodistribution and lysosomal transport of therapeutic cargoes. To improve its delivery, we coupled GAA to polymer nanocarriers (NCs; ∼180 nm) coated with an antibody specific to ICAM-1. Fluorescence microscopy showed specific targeting of anti-ICAM/GAA NCs to cells, with efficient internalization and lysosomal transport, enhancing glycogen degradation over nontargeted GAA. Radioisotope tracing in mice demonstrated enhanced GAA accumulation in all organs, including Pompe targets. Along with improved delivery of Niemann-Pick and Fabry enzymes, previously described, these results indicate that ICAM-1 targeting holds promise as a broad platform for lysosomal enzyme delivery.. In this study, ICAM-1 targeted nanocarriers were used to deliver GAA (acid alpha glucosidase) into cells to address the specific enzyme deficiency in Pompe's disease. The results unequivocally demonstrate enhanced enzyme delivery over nontargeted GAA in a mice model.

Topics: alpha-Glucosidases; Animals; Antibodies, Monoclonal; Disaccharides; Drug Carriers; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Lysosomal Storage Diseases; Lysosomes; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Muscle, Skeletal; Nanoparticles; Polymers; Saccharomyces cerevisiae; Tissue Distribution

Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.

Topics: alpha-Glucosidases; Animals; Dependovirus; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Injections, Spinal; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Ventilation; Spinal Cord

Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.

Topics: alpha-Glucosidases; Animals; Chromatography, Liquid; Disease Models, Animal; Glycogen; Glycogen Storage Disease Type II; Mice; Mice, Knockout; Muscle, Skeletal; Spectrometry, Mass, Electrospray Ionization

Defining disease severity in patients with Pompe disease is important for prognosis and monitoring the response to therapies. Current approaches include qualitative and quantitative assessments of the disease burden, and clinical measures of the impact of the disease on affected systems. The aims of this manuscript were to review a noninvasive urinary glucose tetrasaccharide biomarker of glycogen storage, and to discuss advances in imaging techniques for determining the disease burden in Pompe disease. The glucose tetrasaccharide, Glcα1-6Glcα1-4Glcα1-4Glc (Glc(4) ), is a glycogen-derived limit dextrin that correlates with the extent of glycogen accumulation in skeletal muscle. As such, it is more useful than traditional biomarkers of tissue damage, such as CK and AST, for monitoring the response to enzyme replacement therapy in patients with Pompe disease. Glc(4) is also useful as an adjunctive diagnostic test for Pompe disease when performed in conjunction with acid alpha-glucosidase activity measurements. Review of clinical records of 208 patients evaluated for Pompe disease by this approach showed Glc(4) had 94% sensitivity and 84% specificity for Pompe disease. We propose Glc(4) is useful as an overall measure of disease burden, but does not provide information on the location and distribution of excess glycogen accumulation. In this manuscript we also review magnetic resonance spectroscopy and imaging techniques as alternative, noninvasive tools for quantifying glycogen and detailing changes, such as fibrofatty muscle degeneration, in specific muscle groups in Pompe disease. These techniques show promise as a means of monitoring disease progression and the response to treatment in Pompe disease. © 2012 Wiley Periodicals, Inc.

Topics: Adult; Age Factors; Aged; alpha-Glucosidases; Biomarkers; Child; Child, Preschool; Diagnostic Imaging; Disease Progression; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Middle Aged; Muscle, Skeletal; Mutation; Oligosaccharides; Prognosis; Sequence Analysis, DNA; Treatment Outcome; Young Adult

Pompe disease (glycogen storage disease type 2 or acid maltase deficiency) is a rare autosomal recessive lysosomal storage disorder. Since the advent of ERT a lot has been learned about the phenotypic spectrum especially in the late onset patients. We describe in detail 44 patients diagnosed with late-onset Pompe disease (LOPD) at our neuromuscular department from 1985 to 2011 and compare them to patients with LOPD in the literature of the past 40 years. Study of the Munich LOPD group revealed varying musculoskeletal and cardio-cerebrovascular manifestation patterns. Several of these symptom patterns commonly appeared in conjunction with one another, highlighting the multisystem involvement of this condition. Common symptom patterns include: (i) Classic limb girdle and diaphragmatic weakness, (ii) rigid spine syndrome (RSS), scoliosis, and low body mass, and (iii) several cardio-cerebrovascular manifestation patterns. The most common presentation, limb girdle and diaphragmatic weakness, appeared in 78% (34/44) of our patients and over 80% of those in the literature. Sixteen percent (7/44) of our patients presented with rigid spine, scoliosis, and low body mass. Although scoliosis had a reported frequency of 33% in the general LOPD patient population, the literature only occasionally reported low body mass and RSS. Importantly, a multisystem extramuscular finding accompanied by cardio-cerebrovascular manifestations was found in 29% (13/44) of our LOPD patients; the literature showed an increasing prevalence of this latter finding. By examining the phenotype of patients with confirmed LOPD, we found a more subtle clinical multisystem involvement in LOPD. Whether patients presenting with the different symptom patterns respond differently to enzyme replacement therapy remains a key question for future research. © 2012 Wiley Periodicals, Inc.

Topics: Adolescent; Adult; Age of Onset; Aged; Cardiovascular Abnormalities; Cerebrovascular Disorders; Child; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Mallory Bodies; Middle Aged; Muscular Dystrophies; Muscular Dystrophies, Limb-Girdle; Musculoskeletal Abnormalities; Phenotype; Scoliosis

Pompe disease is an autosomal recessive neuromuscular disorder marked by progressive muscle weakness due to lysosomal buildup of glycogen. Presentation is described as a spectrum, varying by age of onset, organ involvement, and degree of myopathy. Given the phenotypic variability, Pompe disease is broadly classified into an infantile form and a late onset (juvenile, childhood, adult onset) form. Prior to the advent of enzyme replacement therapy (ERT) with alglucosidase alfa and approval for human use in 2006, the natural history was limited due to death before age 2 years for infantile onset cases and significant morbidity and early mortality for late onset Pompe disease (LOPD). ERT with alglucosidase alfa redefined the once fatal outcome in infantile Pompe, establishing an emergent phenotype. Treatment in late onset patients resulted in improved outcomes, enhancing understanding of the phenotype, presentation, and extent of organ involvement. This Issue of the Seminars seeks to enumerate the recent advancements in the field of Pompe disease, including newborn screening, novel therapeutic targets, new insights in the pathophysiology including role of autophagy, and impacts of long-term disease burden and CNS glycogen accumulation on cognition in infantile survivors. It also addresses immunological challenges and the critical role of immunomodulation in ERT treatment outcome. Other topics discussed include the role of biomarkers in monitoring disease progression and treatment responses, the role of genotype in defining phenotype and treatment response, better insights into the clinical presentations in LOPD and finally the importance of a multidisciplinary approach to care with the role of physical therapy as an example. Many gaps in our scientific understanding of this disease still remain; however, we hope the next decade will bring new knowledge and therapies to the horizon.

Topics: alpha-Glucosidases; Autophagy; Biomarkers; Central Nervous System; Disease Progression; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Treatment Outcome

Pompe disease is a very rare disorder of glycogen metabolism. Due to a deficiency of the enzyme glucosidase glycogen accumulates inside the lysosomes. The clinical picture varies widely as a consequence of varying participation of skeletal and heart muscle. In adults respiratory insufficiency can occur which must be taken into consideration during anesthesiology procedures for affected patients. This case report describes a 60-year-old patient scheduled for punch biopsy of the prostate.

Topics: Anesthesia; Anesthesia, Intravenous; Biopsy; Glucosidases; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Male; Middle Aged; Muscle, Skeletal; Myocardium; Plethysmography, Whole Body; Prostate; Respiratory Function Tests; Spirometry

Topics: Age of Onset; alpha-Glucosidases; Biopsy; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle, Skeletal; Periodic Acid-Schiff Reaction; Tissue Embedding; Tissue Fixation

Pompe disease is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme, acid alpha-glucosidase (GAA). To the best of our knowledge, no studies have reported the results of systematic and sequential CT analyses before and during ERT. In this study we have treated three patients with late onset Pompe disease by ERT, and investigated the efficacy of treatment by computed tomography number.. We measured the serial changes in the computed tomography (CT) number of multiple organs in three patients with late onset of Pompe disease during 24 months of enzyme replacement therapy (ERT).. Before treatment, the liver and muscle CT numbers were higher in these patients than in the controls. The liver CT number decreased after performing ERT. Furthermore, the urinary glucose tetrasaccharide levels, a biomarker of glycogen accumulation, were elevated before ERT and reduced thereafter.. The findings in these cases suggest that the elevation of the liver CT number represents glycogen accumulation in the liver and that the analysis of the liver CT number is therefore a useful tool for assessing the efficacy of ERT.

Topics: alpha-Glucosidases; Child, Preschool; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Muscle, Skeletal; Oligosaccharides; Tomography, X-Ray Computed

Urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc₄) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc₄ a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc₄ in urine without interference of the Glc₄ isomer maltotetraose (M₄).. Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS.. We separated and quantified acarbose, M₄, and Glc₄ using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc₄ was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc₄), and 10.5% and 16.2% (200 μmol/L Glc₄). Glc₄ in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M₄ was present in 5% of urine samples. Mean Glc₄ concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001-0.005). The diagnostic sensitivity of Glc₄ for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc₄ was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients.. The UPLC-MS/MS assay of Glc₄ in urine was discriminative between Glc₄ and M₄ and confirmed the diagnosis in >98% of GSD-II cases.

Topics: Adolescent; Adult; Age Factors; Aged; Child; Child, Preschool; Chromatography, Liquid; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Humans; Infant; Infant, Newborn; Maltose; Middle Aged; Oligosaccharides; Reference Values; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Young Adult

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.

Topics: 1-Deoxynojirimycin; alpha-Glucosidases; Animals; Buffers; Disease Models, Animal; Enzyme Activation; Enzyme Stability; Glycogen; Glycogen Storage Disease Type II; Half-Life; Humans; Mice; Mice, Knockout; Protein Denaturation; Rats; Recombinant Proteins

Glycogen storage disease type II (GSD II), also known as Pompe disease, is an autosomal recessive inherited disorder caused by a reduced activity of acid alpha glucosidase (GAA). Two different clinical entities have been described: rapidly fatal infantile and late onset forms. Hearing loss has been described in classic infantile Pompe patients but rarely in late onset cases. The main purpose of this study was to investigate the involvement of the auditory system in a cohort of Italian patients with late onset GSD II. We have enrolled 20 patients, 12 males and 8 females. The auditory system assessment included speech and pure tone audiometry, impedance audiometry and auditory brainstem responses (ABR). A combined interpretation of those tests allowed us to define the origin of the hearing impairment (sensorineural, conductive or mixed). Clinically, all patients but one denied subjective hearing disturbances. On the other hand, audiological evaluation revealed that 21/40 patient ears (52.5%) had a hearing impairment: 57% had a sensorineural deficit, 33% showed a conductive hearing loss whereas 10% presented with a mixed pattern. Our study revealed that, in this group of GSDII late onset patients, the auditory system impairment was more frequently present than thought with a prominent cochlear involvement. Our results emphasize the importance of a routinely auditory function evaluation in all forms of Pompe disease.

Topics: Acoustic Impedance Tests; Adolescent; Adult; Age of Onset; Aged; alpha-Glucosidases; Audiometry, Pure-Tone; Child; Cochlea; Evoked Potentials, Auditory, Brain Stem; Female; Glycogen; Glycogen Storage Disease Type II; Hearing; Hearing Loss, Conductive; Hearing Loss, Sensorineural; Humans; Male; Middle Aged

Pompe disease is caused by absence of the lysosomal enzyme acid alpha-glucosidase. It is generally assumed that intra-lysosomal hydrolysis of glycogen does not contribute to skeletal muscle energy production during exercise. However, this hypothesis has never been tested in vivo during exercise. We examined the metabolic response to exercise in patients with late-onset Pompe disease, in order to determine if a defect in energy metabolism may play a role in the pathogenesis of Pompe disease. We studied six adult patients with Pompe disease and 10 healthy subjects. The participants underwent ischemic forearm exercise testing, and peak work capacity was determined. Fat and carbohydrate metabolism during cycle exercise was examined with a combination of indirect calorimetry and stable isotope methodology. Finally, the effects of an IV glucose infusion on heart rate, ratings of perceived exertion, and work capacity during exercise were determined. We found that peak oxidative capacity was reduced in the patients to 17.6 vs. 38.8 ml kg(-1) min(-1) in healthy subjects (p = 0.002). There were no differences in the rate of appearance and rate of oxidation of palmitate, or total fat and carbohydrate oxidation, between the patients and the healthy subjects. None of the subjects improved exercise tolerance by IV glucose infusion. In conclusion, peak oxidative capacity is reduced in Pompe disease. However, skeletal muscle fat and carbohydrate use during exercise was normal. The results indicate that a reduced exercise capacity is caused by muscle weakness and wasting, rather than by an impaired skeletal muscle glycogenolytic capacity. Thus, it appears that acid alpha-glucosidase does not play a significant role in the production of energy in skeletal muscle during exercise.

Topics: Age of Onset; alpha-Glucosidases; Calorimetry, Indirect; Case-Control Studies; Exercise; Fatty Acids; Female; Glucose; Glycogen; Glycogen Storage Disease Type II; Glycogenolysis; Humans; Infusions, Intravenous; Isotope Labeling; Male; Muscle Weakness; Muscle, Skeletal; Oxygen Consumption; Physical Exertion

Most adults with Pompe disease are compound heterozygotes in which one acid α-glucosidase (GAA) allele harbors the c.-32-13T>G mutation, causing partial loss of GAA, and the other allele harbors a fully deleterious mutation. The fibroblast GAA activity in these patients is usually between 5% and 25% of the average in healthy individuals. In some adult patients, however, the fibroblast GAA activity is much lower and is in the range that is normally observed in classic-infantile Pompe disease. We investigated the genotype-phenotype correlation in three such adult patients and measured the GAA activity as well as the glycogen content in muscle and fibroblasts in order to better understand the clinical course.. DNA was sequenced and GAA activity and glycogen content were measured in leukocytes, fibroblasts and muscle. Muscle biopsies were microscopically analyzed and the biosynthesis of GAA in fibroblasts was analyzed by immunoblotting. GAA activity and glycogen content in fibroblasts and muscle tissue in healthy controls, adult patients with Pompe disease and classic-infantile patients were compared with those of the three index patients.. One patient had genotype c.525delT/c.671G>A (r.0/p.Arg224Gln). Two affected brothers had genotype c.569G>A/c.1447G>A (p.Arg190His/p.Gly483Arg). In all three cases the GAA activity and the glycogen content in fibroblasts were within the same range as in classic-infantile Pompe disease, but the activity and glycogen content in muscle were both within the adult range. In fibroblasts, the first step of GAA synthesis appeared unaffected but lysosomal forms of GAA were not detectable with immunoblotting.. Some adult patients with mutations other than c.-32-13T>G can have very low GAA activity in fibroblasts but express higher activity in muscle and store less glycogen in muscle than patients with classic-infantile Pompe disease. This might explain why these patients have a slowly progressive course of Pompe disease.

Topics: Adult; Alleles; alpha-Glucosidases; Fibroblasts; Genetic Association Studies; Genotype; Glycogen; Glycogen Storage Disease Type II; Heterozygote; Humans; Infant, Newborn; Male; Middle Aged; Muscle, Skeletal; Mutation; Phenotype

Aerobic exercise may be used in conjunction with enzyme replacement therapy (ERT) to attenuate cardiovascular deconditioning, skeletal muscle wasting, and loss of motor function in Pompe disease (glycogen storage disease type II; GSDII), but the effects on lysosomal glycogen content and macroautophagy have not been defined to date.. The main objectives of this study were to determine if acute aerobic exercise enhances 24-h uptake of recombinant human enzyme (rhGAA; Myozyme® [aim 1]) and if endurance training improves disease pathology when combined with ERT [aim 2] in Pompe mice.. For the first aim in our study, Pompe mutant mice (6(neo)/6(neo)) were grouped into ERT (Myozyme® injection only [40 mg/kg]) and ERT+EX (Myozyme® injection followed by 90 min treadmill exercise) cohorts, and enzyme uptake was assessed in the heart and quadriceps 24h post injection. For the second aim of our study, mutant mice were randomized into control, endurance-trained, enzyme-treated, or combination therapy groups. Exercised animals underwent 14 weeks of progressive treadmill training with or without biweekly Myozyme® injections (40 mg/kg) and tissues were harvested 1 week post last treatment.. Myozyme® uptake (GAA activity) was not improved in ERT+EX over ERT alone at 24-h post injection. Endurance exercise training, with or without ERT, improved aerobic capacity and normalized grip strength, motor function, and lean mass (P<0.05), but did not reduce glycogen content or normalize macroautophagy beyond traditional enzyme replacement therapy.. Endurance training is beneficial as an adjunctive therapy to ERT in Pompe disease, although it works by mechanisms independent of a reduction in glycogen content.

Topics: alpha-Glucosidases; Animals; Enzyme Replacement Therapy; Exercise; Exercise Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Heart; Humans; Infusions, Intravenous; Male; Mice; Mice, Transgenic; Muscle, Skeletal; Physical Conditioning, Animal

Pompe disease (glycogen storage disease type II) is an autosomal recessive neuromuscular disorder arising from a deficiency of lysosomal acid α-glucosidase (GAA). Accumulation of autophagosomes is a key pathological change in skeletal muscle fibers and fibroblasts from patients with Pompe disease and is implicated in the poor response to enzyme replacement therapy (ERT). We previously found that mutant GAA-induced endoplasmic reticulum (ER) stress initiated autophagy in patient fibroblasts. However, the mechanism of induction of autophagy in fibroblasts from Pompe disease patients lacking ER stress remains unclear. In this study, we show that inactivated Akt induces ER stress-independent autophagy via mTOR suppression in patient fibroblasts. Activated autophagy as evidenced by increased levels of LC3-II and autophagic vesicles was observed in patient fibroblasts, whereas PERK phosphorylation reflecting the presence of ER stress was not observed in them. These patient fibroblasts showed decreased levels of not only phosphorylated Akt, but also phosphorylated p70 S6 kinase. Treatment with insulin, which acts as an activator of the Akt signaling pathway, resulted in increased phosphorylation of both Akt and p70 S6 kinase and suppression of autophagy in patient fibroblasts. In addition, following combination treatment with recombinant human GAA plus insulin, enhanced localization of the enzymes with lysosomes was observed in patient fibroblasts. These findings define a critical role of Akt suppression in the induction of autophagy in fibroblasts from patients with Pompe disease carrying an ER stress non-inducible mutation, and they provide evidence that insulin may potentiate the effect of ERT.

Topics: alpha-Glucosidases; Autophagy; Cells, Cultured; Endoplasmic Reticulum; Fibroblasts; Gene Expression; Glucose; Glycogen; Glycogen Storage Disease Type II; Humans; Infant, Newborn; Insulin; Lysosomes; Muscle, Skeletal; Phagosomes; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases

Pompe disease is caused by the deficiency of acid α-glucosidase (GAA), which degrades glycogen into glucose. Its manifestation is characterized by a broad and continuous spectrum of clinical severity ranging from severe infantile to relatively benign adult form. We describe a 12-year-old girl diagnosed at a presymptomatic stage of late form Pompe disease due to fortuitous detection of an elevated level of serum creatine kinase (CK) at the age of 4. Biopsies were taken from the quadriceps muscle and studied with histological and histochemical techniques, as well as in electron microscope. Sporadic muscle cells showed the accumulation of lysosomal glycogen, suggesting Pompe disease. Interestingly, we found lysosomal bound glycogen, located in the axons of intramuscular nerves. The diagnosis was confirmed by deficient GAA activity in leukocytes. Mutation analysis revealed changes IVS1-13T>G and p.C103G in the GAA gene. The patient was able to obtain enzyme replacement therapy in the early asymptomatic stage of the disease.

Topics: Age of Onset; alpha-Glucosidases; Asymptomatic Diseases; Axons; Child; Enzyme Replacement Therapy; Female; Glycogen; Glycogen Storage Disease Type II; Heterozygote; Humans; Leukocytes; Lysosomes; Microscopy, Electron; Muscle, Skeletal; Mutation, Missense; Peripheral Nerves; Staining and Labeling; Vacuoles

RNA interference has emerged as a powerful technique to down-regulate gene expression. The lentiviral vector-mediated expression of small hairpin RNAs (shRNAs) from polymerase III promoters allows permanent down-regulation of a specific gene in a wide range of cell types both in vitro and in vivo. In this chapter, we describe a method permitting the expression of shRNA from lentiviral vectors in primary murine myogenic cells. We designed shRNAs targeted to the muscular glycogen synthase isoform (shGYS1), a highly regulated enzyme responsible for glycogen synthesis, in order to modulate the muscle glycogen biosynthetic pathway and to improve the phenotype in primary myogenic cells from a murine model of glycogen storage disease type II (GSDII). This method based on shRNA-mediated down-regulation could be applied to other muscular disorders to evaluate new therapeutic options.

Topics: Animals; Cells, Cultured; Gene Expression; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Glycogen Synthase; Lentivirus; Membrane Glycoproteins; Mice; Muscular Diseases; Myoblasts; RNA Interference; RNA, Small Interfering; Viral Envelope Proteins

To determine whether newborn screening facilitates early detection and thereby early treatment initiation for later-onset Pompe disease.. We have conducted a newborn screening program since 2005. Newborns with deficient skin fibroblast acid α-glucosidase activity and two acid α-glucosidase gene mutations but no cardiomyopathy were defined as having later-onset Pompe disease, and their motor development and serum creatine kinase levels were monitored every 3 to 6 months.. Among 344 056 newborns, 13 (1 in 26 466) were found to have later-onset Pompe disease. During a follow-up period of up to 4 years, four patients were treated because of hypotonia, muscle weakness, delayed developmental milestones/motor skills, or elevated creatine kinase levels starting at the ages of 1.5, 14, 34, and 36 months, respectively. Muscle biopsy specimens obtained from the treated patients revealed increased storage of glycogen and lipids.. Newborn screening was found to facilitate the early detection of later-onset Pompe disease. A subsequent symptomatic approach then identifies patients who need early treatment initiation.

Topics: Adolescent; Adult; alpha-Glucosidases; Biopsy; Cardiomyopathies; Creatine Kinase; Enzyme Replacement Therapy; Female; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Infant, Newborn; Lipids; Male; Middle Aged; Mutation; Neonatal Screening; Skin

Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with the administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β(2)-agonist, enhanced the CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA.

Topics: Adrenergic beta-Agonists; alpha-Glucosidases; Animals; Clenbuterol; Disease Models, Animal; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Male; Mice; Mice, Knockout; Motor Activity; Muscle, Skeletal; Receptor, IGF Type 2

Topics: Aging; alpha-Glucosidases; Biopsy; Disease Progression; Early Diagnosis; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Male; Muscle Strength; Muscle, Skeletal; Young Adult

Pompe disease, or glycogen storage disease type 2, is a rare inheritable metabolic disease caused by a deficiency of the lysosomal enzyme acid α-glucosidase. Patients with the classic infantile form of Pompe disease present with symptoms during the first 3 months after birth, and most will die within their first year. Recently, enzyme replacement therapy (ERT) with recombinant human α-glucosidase became commercially available for Pompe disease. This is a case report of an 8-year-old girl with the infantile form of Pompe disease who is one of the longest survivors through ERT. The patient was tetraplegic when she started ERT. At age 3 years, she developed massive gingival overgrowth and could not close her mouth, prompting a reduction of the gingival overgrowth surgically. We expected that massive accumulation of glycogen would explain the gingival overgrowth. However, histopathology of the gingiva tissue showed marked glycogen accumulation in smooth muscle cells of the arteries, but the glycogen content in fibroblasts did not exceed that of control individuals. Further, there was an increase of immature collagen in the connective tissue, and signs of a mild chronic inflammation. We concluded that glycogen storage is not a direct causative factor of gingival overgrowth in our patient. Chronic inflammation, dryness of the gingiva, or even the minimal glycogen accumulation in the fibroblasts may have played a role.

Topics: Child; Connective Tissue; Enzyme Replacement Therapy; Epithelium; Female; Fibroblasts; Follow-Up Studies; Gingiva; Gingival Overgrowth; Gingivectomy; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Recombinant Proteins

Topics: Adult; Diagnosis, Differential; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Pyomyositis; Young Adult

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.

Topics: alpha-Glucosidases; Animals; Disease Models, Animal; Female; Glucose; Glycogen; Glycogen Storage Disease Type II; Glycogen Synthase; Humans; Lysosomes; Male; Mice; Mice, Knockout; Muscle, Skeletal

The authors describe a 50-year-old man who was evaluated for a rigid spine syndrome with onset at age 15, and subsequent walking difficulties. Cardiac and pulmonary functions were normal. Deltoid biopsy revealed the presence of small vacuoles and increased glycogen with Periodic Acid Schiff staining in a limited number of fibers. Acid alpha-glucosidase staining was decreased in leucocytes, and genetic analysis identified the presence of two mutations in that gene. This observation suggests that Pompe disease should be considered in the differential diagnosis of rigid spine syndrome, even in patients without respiratory involvement or with a muscle biopsy showing only mild histopathological changes.

Topics: Age Factors; Age of Onset; alpha-Glucosidases; Biopsy; DNA Mutational Analysis; Gait Disorders, Neurologic; Glycogen; Glycogen Storage Disease Type II; Humans; Leukocytes; Male; Middle Aged; Mobility Limitation; Muscle, Skeletal; Muscular Diseases; Mutation; Periodic Acid-Schiff Reaction; Spinal Diseases; Spine

Pompe disease results from the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.

Topics: alpha-Glucosidases; Animals; Animals, Newborn; Enzyme-Linked Immunosorbent Assay; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Lentivirus; Mice; Mice, Knockout; Polymerase Chain Reaction; Transgenes

To perform the ultrastructural examination of a chorionic villi biopsy as a predictor of foetal involvement in the infantile form of glycogenosis type II (Pompe disease).. Ultrastructural, biochemical and genetic analyses were performed on chorionic villi biopsies of three consecutive pregnancies in a woman with a previous child affected by Pompe disease.. In the only affected foetus, glycogen storage was observed in fibrocytes and endothelial cells of a chorionic villi sample at 11 week's gestation. Severe multi-organ involvement was demonstrated in the tissues of the aborted foetus. No abnormal material was found in the chorionic samples of two subsequent pregnancies, and a healthy boy and girl were born at term and remain unaffected. Both exhibited a partial reduction in acid maltase and were carriers of the maternal mutation.. Ultrastructural findings correlated with biochemical and genetic results, providing a clear and early indicator of the definite diagnosis for future pregnancy management or an early therapeutic approach.

Topics: alpha-Glucosidases; Cells, Cultured; Chorionic Villi; Chorionic Villi Sampling; DNA Mutational Analysis; Endothelial Cells; Fatal Outcome; Female; Fibroblasts; Genetic Predisposition to Disease; Genetic Testing; Gestational Age; Glycogen; Glycogen Storage Disease Type II; Heterozygote; Humans; Infant, Newborn; Male; Microscopy, Electron, Transmission; Mutation; Pedigree; Phenotype; Predictive Value of Tests; Pregnancy; Prenatal Diagnosis

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Topics: alpha-Glucosidases; Animals; Cells, Cultured; Chimerism; Gene Expression; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Hematopoietic System; Humans; Lentivirus; Mice; Mice, Knockout; Motor Activity; Transduction, Genetic

Topics: Adolescent; alpha-Glucosidases; Blepharoptosis; Disease Progression; Dose-Response Relationship, Drug; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Muscle Weakness; Muscle, Skeletal; Oculomotor Muscles; Recombinant Proteins; Recovery of Function; Treatment Outcome

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

Topics: alpha-Glucosidases; Animals; Body Mass Index; Dependovirus; Disease Models, Animal; Follistatin; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Inbred C57BL; Muscle, Skeletal

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1-2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle.

Topics: Aging; alpha-Glucosidases; Animals; Dose-Response Relationship, Drug; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Glycogen Synthase; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Muscle, Skeletal; Myocardium; Phosphorylation; Proteins; Recombinant Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.

Topics: alpha-Glucosidases; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Disease Models, Animal; Enzyme Replacement Therapy; Glycogen; Glycogen Storage Disease Type II; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Integrases; Mice; Muscle Fibers, Fast-Twitch; Myosin Light Chains; Phosphorylation; Ubiquitin

Lysosomal storage disorders such as Pompe disease can be more effectively treated, if immune tolerance to enzyme or gene replacement therapy can be achieved. Alternatively, immune responses against acid α-glucosidase (GAA) might be evaded in Pompe disease through muscle-specific expression of GAA with adeno-associated virus (AAV) vectors.. An AAV vector containing the MHCK7 regulatory cassette to drive muscle-specific GAA expression was administered to GAA knockout (KO) mice, immune tolerant GAA-KO mice and mannose-6-phosphate deficient GAA-KO mice. GAA activity and glycogen content were analyzed in striated muscle to determine biochemical efficacy.. The biochemical efficacy from GAA expression was slightly reduced in GAA-KO mice, as demonstrated by higher residual glycogen content in skeletal muscles. Next, immune tolerance to GAA was induced in GAA-KO mice by co-administration of a second AAV vector encoding liver-specific GAA along with the AAV vector encoding muscle-specific GAA. Antibody formation was prevented by liver-specific GAA, and the biochemical efficacy of GAA expression was improved in the absence of antibodies, as demonstrated by significantly reduced glycogen content in the diaphragm. Efficacy was reduced in old GAA-KO mice despite the absence of antibodies. The greatest impact upon gene therapy was observed in GAA-KO mice lacking the mannose-6-phosphate receptor in muscle. The clearance of stored glycogen was markedly impaired despite high GAA expression in receptor-deficient Pompe disease mice.. Overall, antibody formation had a subtle effect upon efficacy, whereas the absence of mannose-6-phosphate receptors markedly impaired muscle-targeted gene therapy in murine Pompe disease.

Topics: alpha-Glucosidases; Animals; Antibody Formation; Dependovirus; Disease Models, Animal; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Mice; Mice, Knockout; Muscle, Skeletal; Receptor, IGF Type 2; Transgenes

PGC-1α is a transcriptional co-activator that plays a central role in the regulation of energy metabolism. Our interest in this protein was driven by its ability to promote muscle remodeling. Conversion from fast glycolytic to slow oxidative fibers seemed a promising therapeutic approach in Pompe disease, a severe myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) which is responsible for the degradation of glycogen. The recently approved enzyme replacement therapy (ERT) has only a partial effect in skeletal muscle. In our Pompe mouse model (KO), the poor muscle response is seen in fast but not in slow muscle and is associated with massive accumulation of autophagic debris and ineffective autophagy. In an attempt to turn the therapy-resistant fibers into fibers amenable to therapy, we made transgenic KO mice expressing PGC-1α in muscle (tgKO). The successful switch from fast to slow fibers prevented the formation of autophagic buildup in the converted fibers, but PGC-1α failed to improve the clearance of glycogen by ERT. This outcome is likely explained by an unexpected dramatic increase in muscle glycogen load to levels much closer to those observed in patients, in particular infants, with the disease. We have also found a remarkable rise in the number of lysosomes and autophagosomes in the tgKO compared to the KO. These data point to the role of PGC-1α in muscle glucose metabolism and its possible role as a master regulator for organelle biogenesis - not only for mitochondria but also for lysosomes and autophagosomes. These findings may have implications for therapy of lysosomal diseases and other disorders with altered autophagy.

Topics: Animals; Autophagy; Disease Models, Animal; Glucose; Glycogen; Glycogen Storage Disease Type II; Golgi Apparatus; Humans; Lysosomes; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Skeletal; Muscles; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Trans-Activators; Transcription Factors

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

Topics: alpha-Glucosidases; Animals; Disease Models, Animal; Glycogen; Glycogen Storage Disease Type II; Humans; Mannosephosphates; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Oligosaccharides; Protein Binding; Protein Engineering; Receptor, IGF Type 2

Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required.

Topics: Acarbose; Diagnostic Techniques, Neurological; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant, Newborn; Leukocytes; Substrate Specificity

Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in the absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy.. AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12- and 17-month-old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through acid alpha-glucosidase (GAA) activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, urinary biomarker, and Rotarod performance were evaluated after vector administration.. The AAV vector containing the liver-specific regulatory cassette secreted high-level human GAA into the blood and corrected glycogen storage in the heart and diaphragm. The biochemical correction of the heart and diaphragm was associated with efficacy, as reflected by increased Rotarod performance; however, the clearance of glycogen from skeletal muscles was relatively impaired compared to in younger Pompe disease mice. An alternative vector containing a muscle-specific regulatory cassette transduced skeletal muscle with high efficiency, but also failed to achieve complete clearance of accumulated glycogen. Decreased transduction of the heart and liver in older mice, especially in females, was implicated as a cause for reduced efficacy in advanced Pompe disease.. The impaired efficacy of AAV vector-mediated gene therapy in old Pompe disease mice emphasizes the need for early treatment to achieve full efficacy.

Topics: Age Factors; alpha-Glucosidases; Animals; Dependovirus; Enzyme Replacement Therapy; Female; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Lysosomes; Male; Mice; Muscles; Sex Factors; Transgenes

Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada, and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO cell-derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO cell-derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.

Topics: alpha-Glucosidases; Animals; Antibodies; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Drug Evaluation, Preclinical; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Lectins, C-Type; Mannose Receptor; Mannose-Binding Lectins; Mice; Oligosaccharides; Protein Binding; Rabbits; Receptor, IGF Type 2; Receptors, Cell Surface; Recombinant Proteins

Glycogen storage disease type II (Pompe disease; MIM 232300) stems from the deficiency of acid alpha-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. An adeno-associated virus 2/8 (AAV2/8) vector containing the muscle creatine kinase (MCK) (CK1) reduced glycogen content by approximately 50% in the heart and quadriceps in GAA-knockout (GAA-KO) mice; furthermore, an AAV2/8 vector containing the hybrid alpha-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) cassette reduced glycogen content by >95% in heart and >75% in the diaphragm and quadriceps. Transduction with an AAV2/8 vector was higher in the quadriceps than in the gastrocnemius. An AAV2/9 vector containing the MHCK7 cassette corrected GAA deficiency in the distal hindlimb, and glycogen accumulations were substantially cleared by human GAA (hGAA) expression therein; however, the analogous AAV2/7 vector achieved much lower efficacy. Administration of the MHCK7-containing vectors significantly increased striated muscle function as assessed by increased Rotarod times at 18 weeks after injection, whereas the CK1-containing vector did not increase Rotarod performance. Importantly, type IIb myofibers in the extensor digitalis longus (EDL) were transduced, thereby correcting a myofiber type that is unresponsive to enzyme replacement therapy. In summary, AAV8 and AAV9-pseudotyped vectors containing the MHCK7 regulatory cassette achieved enhanced efficacy in Pompe disease mice.

Topics: alpha-Glucosidases; Animals; Creatine Kinase, MM Form; Dependovirus; Enhancer Elements, Genetic; Female; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Hindlimb; Mice; Mice, Knockout; Muscle, Skeletal; Muscle, Striated; Myocardium; Myosin Heavy Chains; Promoter Regions, Genetic; Quadriceps Muscle; Transduction, Genetic

Ultrastructural analysis of the skeletal muscle in adult-onset Pompe disease revealed lysosomal and cytoplasmic glycogen storage, autophagic vacuoles and abnormal mitochondria. Significant glycogen accumulation within lysosomes causes their rupture and release of glycogen into the cytoplasm. Excess cytoplasmic glycogen could lead to damage of the structure of muscle cells including myofibrils. In consequence, parts of the sarcoplasm and damaged organelles were sequestered within membrane-limited vacuoles. We suppose that massive accumulation of autophagic vacuoles results from the inability of destroyed lysosomes to fuse with vacuoles. Autophagic vacuoles may be a prominent feature of muscle cells in adult glycogenosis type II.

Topics: Adolescent; Adult; Age of Onset; Diagnosis, Differential; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Muscle Weakness; Muscle, Skeletal; Severity of Illness Index

Pompe disease (glycogen storage disease II) is caused by mutations in the acid alpha-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, which leads to profound weakness, cardiac failure, and death by the age of 2 years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathologic and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of 1 month; accumulations then spread progressively within many CNS cell types. "Silver degeneration" and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at 3 to 9 months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging, and foot fault tests. The extensive neuropathologic alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.

Topics: Age Factors; alpha-Glucosidases; Animals; Behavior, Animal; Central Nervous System; Disease Models, Animal; Disease Progression; Glial Fibrillary Acidic Protein; Glycogen; Glycogen Storage Disease Type II; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Motor Activity; Muscle Strength; Muscle, Skeletal; Phenotype; Psychomotor Performance; Reaction Time

Topics: Age Factors; Disease Progression; Fatal Outcome; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant, Newborn; Lysosomes; Male; Muscle Weakness; Respiration, Artificial; Respiratory Insufficiency; Respiratory Muscles; Treatment Failure; Ventilator Weaning

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.

Topics: Animals; Animals, Newborn; Cell Line; Dependovirus; Genetic Therapy; Genetic Vectors; Glucosyltransferases; Glycogen; Glycogen Storage Disease Type II; Glycogen Synthase; Glycoproteins; Humans; Mice; Mice, Knockout; RNA Interference

We discuss four cases of acid alpha-glucosidase deficiency (EC, 3.2.1.3/20) without evident symptoms of Pompe disease (OMIM No 232300) in individuals of Asian descent. In three cases, the deficiency was associated with homozygosity for the sequence variant c.[1726G>A; 2065G>A] in the acid alpha-glucosidase gene (GAA) translating into p.[G576S; E689K]. One of these cases was a patient with profound muscular atrophy, another had cardio-myopathy and the third had no symptoms. The fourth case, the mother of a child with Pompe disease, was compound heterozygote for the GAA sequence variants c.[1726G>A; 2065G>A]/c.2338G>A (p.W746X) and had no symptoms either. Further investigations revealed that c.[1726A; 2065A] is a common GAA allele in the Japanese and Chinese populations. Our limited study predicts that approximately 4% of individuals in these populations are homozygote c.[1726A; 2065A]. The height of this figure in contrast to the rarity of Pompe disease in Asian populations and the clinical history of the cases described in this paper virtually exclude that homozygosity for c.[1726A; 2065A] causes Pompe disease. As c.[1726A; 2065A] homozygotes have been observed with similarly low acid alpha-glucosidase activity as some patients with Pompe disease, we caution they may present as false positives in newborn screening programs especially in Asian populations.

Topics: Adolescent; Adult; alpha-Glucosidases; Cells, Cultured; Child; Female; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Homozygote; Humans; Leukocytes; Lymphocytes; Male; Muscles; Polymorphism, Genetic

Topics: Adult; Creatine Kinase; Diagnosis, Differential; Electromyography; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Male; Muscle Weakness; Muscular Atrophy; Respiratory Insufficiency; Respiratory Muscles; Respiratory Paralysis; Thorax; Tomography, X-Ray Computed

Pompe disease is caused by a lack of functional lysosomal acid alpha-glucosidase (GAA) and can ultimately lead to fatal hypertrophic cardiomyopathy and respiratory insufficiency. Previously, we demonstrated the ability of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to restore the therapeutic levels of cardiac and diaphragmatic GAA enzymatic activity in vivo in a mouse model of Pompe disease. We have further characterized cardiac and respiratory function in rAAV2/1-treated animals 1 year post-treatment. Similar to the patient population, electrocardiogram measurements (P-R interval) are significantly shortened in the Pompe mouse model. In rAAV2/1-treated mice, we show a significant improvement in cardiac conductance with prolonged P-R intervals of 39.34+/-1.6 ms, as compared to untreated controls (35.58+/-0.57 ms) (P

Topics: alpha-Glucosidases; Animals; Dependovirus; Electrophysiology; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Heart; Mice; Mice, Knockout; Myocardium; Organ Size; Phenotype; Transgenes

Pompe disease (glycogenosis type II) is a rare lysosomal disorder caused by a mutational deficiency of acid alpha-glucosidase (GAA). This deficiency leads to glycogen accumulation in multiple tissues: heart, skeletal muscles, and the central nervous system. A knockout mouse model mimicking the human condition has been used for histological evaluation. Currently, the best method for preserving glycogen in Pompe samples uses epon-araldite resin. Although the preservation by this method is excellent, the size of the tissue is limited to 1 mm(3). To accurately evaluate brain pathology in the Pompe mouse model, a modified glycol methacrylate (JB-4 Plus) method was developed. This approach allowed the production of larger tissue sections encompassing an entire mouse hemisphere (8 x 15 mm) while also providing a high level of morphological detail and preservation of glycogen. Application of the JB-4 Plus method is appropriate when a high level of cellular detail is desired. A modified paraffin method was also developed for use when rapid processing of multiple samples is a priority. Traditional paraffin processing results in glycogen loss. The modified paraffin method with periodic acid postfixation resulted in improved tissue morphology and glycogen preservation. Both techniques provide accurate anatomic evaluation of the glycogen distribution in Pompe mouse brain.

Topics: Animals; Brain; Buffers; Cerebellum; Disease Models, Animal; Fixatives; Formaldehyde; Glycogen; Glycogen Storage Disease Type II; Mice; Mice, Inbred C57BL; Mice, Knockout; Paraffin Embedding; Periodic Acid; Tissue Fixation

Pompe's disease or glycogen storage disease type II is a genetic disorder affecting skeletal and cardiac muscle. The infantile form is associated with gross hypertrophic cardiomegaly and death in the early years. General anesthesia is associated with potential major morbidity in these patients. We present our experience of regional anesthetic blocks used in five patients with the infantile form of glycogen storage disease type II with and without sedation for 11 surgical procedures during a clinical trial of replacement therapy for this condition. Both femoral nerve blockade and caudal epidural blockade were used with good result. The relative merits of the type of block are discussed in addition to the choice of sedation and risks of general anesthesia. The avoidance of general anesthesia in the newly presenting patient with Pompe's disease may reduce potential morbidity until enzyme replacement has been established.

Topics: Anesthesia, Conduction; Anesthesia, General; Anesthetics, Combined; Biopsy; Catheters, Indwelling; Child, Preschool; Conscious Sedation; Female; Femoral Nerve; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Lidocaine; Lidocaine, Prilocaine Drug Combination; Male; Muscle, Skeletal; Nerve Block; Prilocaine; Treatment Outcome

Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.

Topics: alpha-Glucosidases; Animals; Antibodies; Blotting, Western; Cell Line; Dependovirus; Endpoint Determination; Enzyme-Linked Immunosorbent Assay; Glycogen; Glycogen Storage Disease Type II; Humans; Immune Tolerance; Mice; Muscle, Skeletal; Myocardium; Recombinant Proteins

An elevated serum biotinidase activity in patients with glycogen storage disease (GSD) type Ia has been reported previously. The aim of this work was to investigate the specificity of the phenomenon and thus we expanded the study to other types of hepatic GSDs. Serum biotinidase activity was measured in a total of 68 GSD patients and was compared with that of healthy controls (8.7 +/- 1.0; range 7.0-10.6 mU/ml; n = 26). We found an increased biotinidase activity in patients with GSD Ia (17.7 +/- 3.9; range: 11.4-24.8; n = 21), GSD I non-a (20.9 +/- 5.6; range 14.6-26.0; n = 4), GSD III (12.5 +/- 3.6; range 7.8-19.1; n = 13), GSD VI (15.4 +/- 2.0; range 14.1-17.7; n = 3) and GSD IX (14.0 +/- 3.8; range: 7.5-21.6; n = 22). The sensitivity of this test was 100% for patients with GSD Ia, GSD I non-a and GSD VI, 62% for GSD III, and 77% for GSD IX, indicating reduced sensitivity for GSD III and GSD IX, respectively. In addition, we found elevated biotinidase activity in all sera from 5 patients with Fanconi-Bickel Syndrome (15.3 +/- 3.7; range 11.0-19.4). Taken together, we propose serum biotinidase as a diagnostic biomarker for hepatic glycogen storage disorders.

Topics: Biomarkers; Biotinidase; DNA Mutational Analysis; Glycogen; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type VI; Humans; Liver; Liver Diseases; Sensitivity and Specificity; Specimen Handling

Glycogen-storage disease type II (GSD II, Pompe's disease) is an autosomal recessive disorder caused by a functional deficiency of acid alpha-glucosidase (GAA) that leads to glycogen accumulation within lysosomes in most tissues. The GAA gene is located to human chromosome 17q25 and contains 20 exons, 19 of which are coding. Clinically, patients with the severe infantile form of GSD II have muscle weakness and cardiomyopathy eventually leading to death before the age of two years. Patients with the juvenile or the adult form of GSD II present with myopathy with a slow progression over several years or decades. A broad genetic heterogeneity has been described in GSD II in Europe, South Africa, USA, Japan and Korea, however, the investigation has not been performed in the patients from the mainland of China. In this study, clinical analysis and mutation detection were done on Chinese patients.. Two unrelated juvenile patients with late onset GSD II (one boy, 3 years old and one girl, 9 years old) were included in the study with the informed consents. The diagnosis was confirmed by alpha-glucosidase determination in cultured fibroblasts. In addition, their clinical presentation, laboratory findings, electrophysiologic studies and muscle biopsy findings were analyzed in detail. Genomic DNA samples were extracted from fibroblasts of the probands, from peripheral blood of their parents and 50 unrelated, normal individuals. All the coding 19 exons and exon-intron boundaries of GAA were detected in the proband by polymerase chain reaction (PCR) and direct sequencing.. One patient presented decrease of muscle strength, limb-girdle hypotonia, the other patient presented reduced muscle volumes and respiratory problems. Both had increased CPK value, myopathic pattern on EMG; vacuoles on muscle biopsy, and deficiency of 1, 4-alpha-glucosidase activity. After 1 year follow up, the girl died after pneumonia at 10 years of age. One patient was found to be compound heretozygote for the novel mutation Arg702His, and the previously reported mutation Pro266Ser, which was reported in Korean population, with the late-onset phenotype. Two novel missense mutations Thr711Arg, Val723Met were found on the other patients.. Three mutations identified in the patient were new missense mutations causing late onset GSD II, which had not been reported elsewhere before.

Topics: Child; China; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Mutation; Mutation, Missense; Phenotype; Young Adult

Glycogen storage disease type II (GSD-II) patients manifest symptoms of muscular dystrophy secondary to abnormal glycogen storage in cardiac and skeletal muscles. For GSD-II, we hypothesized that a fully deleted adenovirus (FDAd) vector expressing hGAA via nonviral regulatory elements (PEPCK promoter/ApoE enhancer) would facilitate long-term efficacy and decrease propensity to generate anti-hGAA antibody responses against hepatically secreted hGAA. Intravenous delivery of FDAdhGAA into GAA-tolerant or nontolerant GAA-KO mice resulted in long-term hepatic secretion of hGAA. Specifically, nontolerant mice achieved complete reversal of cardiac glycogen storage and near-complete skeletal glycogen correction for at least 180 days and tolerant mice for minimally 300 days coupled with the preservation of muscle strength. Anti-hGAA antibody levels in both mouse strains were significantly less relative to those previously generated by CMV-driven hGAA expression in nontolerant GAA-KO mice. However, plasma GAA levels decreased in nontolerant GAA-KO mice despite long-term intrahepatic GAA expression from the persistent vector. This intriguing result is discussed in light of other examples of "tolerance" induction by gene-transfer-based approaches.

Topics: Adenoviridae; alpha-Glucosidases; Animals; Enhancer Elements, Genetic; Gene Transfer Techniques; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Immune Tolerance; Liver; Mice; Mice, Knockout; Muscle, Skeletal; Myocardium; Promoter Regions, Genetic

Infantile Pompe disease is caused by deficiency of lysosomal acid alpha-glucosidase. Trials with recombinant human acid alpha-glucosidase enzyme replacement therapy (ERT) show a decrease in left ventricular mass and improved function. We evaluated 24-hour ambulatory electrocardiograms (ECGs) at baseline and during ERT in patients with infantile Pompe disease.. Thirty-two ambulatory ECGs were evaluated for 12 patients with infantile Pompe disease from 2003 to 2005. Patients had a median age of 7.4 months (2.9-37.8 months) at initiation of ERT. Ambulatory ECGs were obtained at determined intervals and analyzed.. Significant ectopy was present in 2 of 12 patients. Patient 1 had 211 and 229 premature ventricular contractions (0.2% of heart beats) at baseline and at 11.5 weeks of ERT, respectively. Patient 2 had 10,445 premature ventricular contractions (6.7% of heart beats) at 11 weeks of therapy.. Infantile Pompe disease may have preexisting ectopy; it may also develop during the course of ERT. Therefore, routinely monitoring patients using 24-hour ambulatory ECGs is useful. Periods of highest risk may be early in the course of ERT when there is a substantial decrease in left ventricular mass and an initial decrease in ejection fraction.

Topics: alpha-Glucosidases; Cardiomegaly; Child, Preschool; Electrocardiography, Ambulatory; Female; Glycogen; Glycogen Storage Disease Type II; Heart Conduction System; Humans; Infant; Male; Recombinant Proteins; Ventricular Premature Complexes

Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice.

Topics: Animals; Blotting, Western; Cell Line; Cells, Cultured; Dependovirus; Fibroblasts; Genetic Therapy; Genetic Vectors; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Protein Sorting Signals; Recombinant Fusion Proteins; Transfection; Treatment Outcome

Enzyme replacement therapy (ERT) became a reality for patients with Pompe disease, a fatal cardiomyopathy and skeletal muscle myopathy caused by a deficiency of glycogen-degrading lysosomal enzyme acid alpha-glucosidase (GAA). The therapy, which relies on receptor-mediated endocytosis of recombinant human GAA (rhGAA), appears to be effective in cardiac muscle, but less so in skeletal muscle. We have previously shown a profound disturbance of the lysosomal degradative pathway (autophagy) in therapy-resistant muscle of GAA knockout mice (KO). Our findings here demonstrate a progressive age-dependent autophagic buildup in addition to enlargement of glycogen-filled lysosomes in multiple muscle groups in the KO. Trafficking and processing of the therapeutic enzyme along the endocytic pathway appear to be affected by the autophagy. Confocal microscopy of live single muscle fibers exposed to fluorescently labeled rhGAA indicates that a significant portion of the endocytosed enzyme in the KO was trapped as a partially processed form in the autophagic areas instead of reaching its target--the lysosomes. A fluid-phase endocytic marker was similarly mistargeted and accumulated in vesicular structures within the autophagic areas. These findings may explain why ERT often falls short of reversing the disease process and point toward new avenues for the development of pharmacological intervention.

Topics: Age Factors; Animals; Autophagy; CHO Cells; Cricetinae; Cricetulus; Endocytosis; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomal-Associated Membrane Protein 1; Lysosomes; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Muscle, Skeletal; Receptor, IGF Type 2; Recombinant Proteins

Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice.. An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated.. After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles.. Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.

Topics: Adenoviridae; Age Factors; alpha-Glucosidases; Animals; Blotting, Western; Genetic Therapy; Genetic Vectors; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Histological Techniques; Mice; Mice, Transgenic; Muscles; Time Factors

Pompe disease (type II glycogen storage disease) is an autosomal recessive disorder caused by a deficiency of lysosomal acid alpha-glucosidase (GAA) leading to the accumulation of glycogen in the lysosomes primarily in cardiac and skeletal muscle. The recombinant human GAA (rhGAA) is currently in clinical trials for enzyme replacement therapy of Pompe disease. Both clinical data and the results of preclinical studies in our knockout model of this disease show that rhGAA is much more effective in resolving the cardiomyopathy than the skeletal muscle myopathy. By contrast, another form of human GAA--transgenic enzyme constitutively produced in liver and secreted into the bloodstream of knockout mice (Gaa-/-)--completely prevented both cardiac and skeletal muscle glycogen accumulation. In the experiments reported here, the transgenic enzyme was much less efficient when delivered to skeletal muscle after significant amounts of glycogen had already accumulated. Furthermore, the transgenic enzyme and the rhGAA have similar therapeutic effects, and both efficiently clear glycogen from cardiac muscle and type I muscle fibers, but not type II fibers. Low abundance of proteins involved in endocytosis and trafficking of lysosomal enzymes combined with increased autophagy in type II fibers may explain the resistance to therapy.

Topics: alpha-Glucosidases; Animals; Autophagy; Cell Line; Cricetinae; Endocytosis; Genetic Therapy; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Lysosomes; Mice; Microscopy, Electron; Muscle Fibers, Fast-Twitch; Muscle, Skeletal; Myocardium; Recombinant Proteins

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid alpha-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.

Topics: alpha-Glucosidases; Animals; Glycogen; Glycogen Storage Disease Type II; Humans; Image Processing, Computer-Assisted; Mice; Mice, Knockout; Microscopy; Muscle, Skeletal; Myocardium; Reproducibility of Results; Tissue Embedding; Tissue Fixation

Unexplained left ventricular hypertrophy often prompts the diagnosis of hypertrophic cardiomyopathy, a sarcomere-protein gene disorder. Because mutations in the gene for AMP-activated protein kinase gamma2 (PRKAG2) cause an accumulation of cardiac glycogen and left ventricular hypertrophy that mimics hypertrophic cardiomyopathy, we hypothesized that hypertrophic cardiomyopathy might also be clinically misdiagnosed in patients with other mutations in genes regulating glycogen metabolism.. Genetic analyses performed in 75 consecutive unrelated patients with hypertrophic cardiomyopathy detected 40 sarcomere-protein mutations. In the remaining 35 patients, PRKAG2, lysosome-associated membrane protein 2 (LAMP2), alpha-galactosidase (GLA), and acid alpha-1,4-glucosidase (GAA) genes were studied.. Gene defects causing Fabry's disease (GLA) and Pompe's disease (GAA) were not found, but two LAMP2 and one PRKAG2 mutations were identified in probands with prominent hypertrophy and electrophysiological abnormalities. These results prompted the study of two additional, independent series of patients. Genetic analyses of 20 subjects with massive hypertrophy (left ventricular wall thickness, > or =30 mm) but without electrophysiological abnormalities revealed mutations in neither LAMP2 nor PRKAG2. Genetic analyses of 24 subjects with increased left ventricular wall thickness and electrocardiograms suggesting ventricular preexcitation revealed four LAMP2 and seven PRKAG2 mutations. Clinical features associated with defects in LAMP2 included male sex, severe hypertrophy, early onset (at 8 to 17 years of age), ventricular preexcitation, and asymptomatic elevations of two serum proteins.. LAMP2 mutations typically cause multisystem glycogen-storage disease (Danon's disease) but can also present as a primary cardiomyopathy. The glycogen-storage cardiomyopathy produced by LAMP2 or PRKAG2 mutations resembles hypertrophic cardiomyopathy but is distinguished by electrophysiological abnormalities, particularly ventricular preexcitation.

Topics: Adolescent; Adult; Aged; Algorithms; AMP-Activated Protein Kinases; Antigens, CD; Cardiomyopathy, Hypertrophic; Child; Diagnosis, Differential; Electrocardiography; Fabry Disease; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hypertrophy, Left Ventricular; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Male; Middle Aged; Multienzyme Complexes; Mutation; Myocardium; Pedigree; Protein Serine-Threonine Kinases

Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.

Topics: alpha-Glucosidases; Animals; Antibodies, Monoclonal; Dependovirus; Female; Genetic Therapy; Genetic Vectors; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Muscle, Skeletal; Myocardium

Glycogen storage disease II is an inherited progressive muscular disease in which the lack of functional acid 1-4 alpha-glucosidase results in the accumulation of lysosomal glycogen. In the present study, we examine the effect of these non-contractile inclusions on the mechanical performance of skeletal muscle. To this end, force developed in an isometrically contracting slice of a muscle was calculated with a finite element model. Force was calculated at several inclusion densities and distributions and compared to muscle lacking inclusions. Furthermore, ankle dorsal flexor torque was measured in situ of alpha-glucosidase null mice of 6 months of age and unaffected litter mates as was inclusion density in the dorsal flexor muscles. The calculated force loss was shown to be almost exclusively dependent on the inclusion density and less on the type of inclusion distribution. The force loss predicted by the model (6%) on the basis of measured inclusion density (3.3%) corresponded to the loss in mass-normalized strength in these mice measured in situ (7%). Therefore, we conclude that the mechanical interaction between the non-contractile inclusions and the nearby myofibrils is a key factor in the loss of force per unit muscle mass during early stages of GSD II in mice. As glycogen accumulation reaches higher levels in humans, it is highly probable that the impact of this mechanical interaction is even more severe in human skeletal muscle.

Topics: alpha-Glucosidases; Animals; Biomechanical Phenomena; Computer Simulation; Finite Element Analysis; Glycogen; Glycogen Storage Disease Type II; Humans; Inclusion Bodies; Isometric Contraction; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Muscle Fibers, Skeletal; Muscle, Skeletal; Stress, Mechanical

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid alpha-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

Topics: Aging; alpha-Glucosidases; Animals; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Mice; Molecular Structure; Muscle, Skeletal; Myocardium; Oligosaccharides; Protein Binding; Receptor, IGF Type 2; Recombinant Proteins

A tetraglucose oligomer, Glcalpha1-6Glcalpha1-4Glcalpha1-4Glc, designated Glc4, has been shown to be a putative biomarker for the diagnosis of Pompe disease. The purpose of this study was to assess whether Glc4 could be used to monitor the therapeutic response to recombinant human acid alpha glucosidase (rhGAA) enzyme replacement therapy (ERT) in patients with Pompe disease. Urinary Glc4 levels in 11 patients receiving rhGAA therapy was determined by both HPLC-UV and stable isotope dilution ESI-MS/MS. Combined Glc4 and maltotetraose, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glc, (M4) concentrations, designated Hex4, in plasma from these patients were measured by HPLC-UV only. Baseline urinary Glc4 and plasma Hex4 in these patients (mean+/-SD: 34.2+/-11.3 mmol/mol creatinine and 1.7+/-0.8 microM, respectively) were higher than age-matched control values (mean+/-SD, 6.1+/-5.1 mmol/mol creatinine and 0.22+/-0.15 microM, respectively). Both urinary Glc4 and plasma Hex4 levels decreased after initiation of ERT for all patients. In the four patients with the best overall clinical response in both skeletal and cardiac muscle, levels decreased to within, or near, normal levels during the first year of treatment. In contrast, levels fluctuated and were persistently elevated above the control ranges in those patients with a less favorable clinical response (good cardiac response but limited motor improvement). These results suggest that urinary Glc4 and plasma Hex4 could serve as a valuable adjunct to clinical endpoints for monitoring the efficacy of therapeutic interventions such as rhGAA ERT in Pompe disease.

Topics: alpha-Amylases; alpha-Glucosidases; Biomarkers; Case-Control Studies; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Monitoring, Physiologic; Oligosaccharides

Glycogen storage disease type II (Pompe disease) causes death in infancy from cardiorespiratory failure due to acid alpha-glucosidase (GAA; acid maltase) deficiency. An AAV2 vector pseudotyped as AAV6 (AAV2/6 vector) transiently expressed high-level human GAA in GAA-knockout (GAA-KO) mice without reducing glycogen storage; however, in immunodeficient GAA-KO/SCID mice the AAV2/6 vector expressed high-level GAA and reduced the glycogen content of the injected muscle for 24 weeks. A CD4+/CD8+ lymphocytic infiltrate was observed in response to the AAV2/6 vector in immunocompetent GAA-KO mice. When a muscle-specific creatine kinase promoter was substituted for the CB promoter (AAV-MCKhGAApA), that AAV2/6 vector expressed high-level GAA and reduced glycogen content in immunocompetent GAA-KO mice. Muscle-restricted expression of hGAA provoked only a humoral (not cellular) immune response. Intravenous administration of a high number of particles of AAV-MCKhGAApA as AAV2/7 reduced the glycogen content of the heart and skeletal muscle and corrected individual myofibers in immunocompetent GAA-KO mice 24 weeks postinjection. In summary, persistent correction of muscle glycogen content was achieved with an AAV vector containing a muscle-specific promoter in GAA-KO mice, and this approach should be considered for muscle-targeted gene therapy in Pompe disease.

Topics: alpha-Glucosidases; Animals; Antibodies; Antibody Formation; Creatine Kinase; Creatine Kinase, MM Form; Dependovirus; DNA, Viral; Enhancer Elements, Genetic; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Injections, Intramuscular; Isoenzymes; Mice; Mice, Knockout; Muscle, Skeletal; Myocardium; Promoter Regions, Genetic

Three unrelated patients, one girl, one boy, and an adult female, aged 14, 11 and 41 years, respectively, at the time of biopsy, revealed lysosomal glycogen storage, autophagic vacuoles and peculiar globular inclusions of distinct ultrastructure, which were reducing but did not appear like true "reducing bodies" as described in the congenital myopathy "reducing body myopathy". All three patients had residual activity of acid alpha-glucosidase in their muscle biopsy samples. Leukocytes in the girl showed normal acid alpha-glucosidase activity, but in the boy activity was reduced. Molecular genetic analysis of the GAA gene revealed disease-causing mutations in each patient: H568L/R672W, IVS1-13T>G/G615F, and IVS1-13T>G/IVS1-13T>G. Although only one patient with such globular inclusions has been reported up to now, the three patients described here indicate that in the late-onset type of GSD II such inclusions may not be rare.

Topics: Adult; alpha-Glucosidases; Child; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Inclusion Bodies; Male; Microscopy, Electron, Transmission; Middle Aged; Muscle, Skeletal; Mutation; Polymerase Chain Reaction

Pompe disease is an autosomal recessive disorder of glycogen metabolism resulting from a deficiency of acid alpha-glucosidase. Pompe disease can present within a broad clinical spectrum, from the severe infantile to the attenuated adult onset phenotypes. Early diagnosis, in the form of newborn screening has been proposed. However, in the absence of clinical symptoms, prediction of disease severity and progression will be critical to provide appropriate management and treatment of affected individuals.. We have used sensitive immune-assays to measure levels of acid alpha-glucosidase protein and activity in cultured skin fibroblasts and a new glycogen assay to specifically determine the lysosomal accumulation of glycogen in the same cells. These markers were assessed for their ability to predict age of onset.. Acid alpha-glucosidase activity and specific activity as well as lysosomal glycogen showed significant correlations with age of onset, with acid alpha-glucosidase activity having the highest Spearman correlation coefficient (0.887, p<0.001). Lysosomal glycogen accumulated only in cells from infantile and juvenile patients but not from adult-onset patients. However, cells from adult-onset patients had relatively low cytoplasmic glycogen compared to control individuals and other forms of the disease.. Acid-alpha-glucosidase activity and specific activity, and lysosomal glycogen content are useful predictors of age of onset in Pompe disease.

Topics: Acids; Age of Onset; alpha-Glucosidases; Cell Line; Culture Media; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Skin

Glycogen storage disease type II (GSD-II; Pompe disease) is caused by a deficiency of acid alpha-glucosidase (GAA; acid maltase) and manifests as muscle weakness, hypertrophic cardiomyopathy, and respiratory failure. Adeno-associated virus vectors containing either a liver-specific promoter (LSP) (AAV-LSPhGAApA) or a hybrid CB promoter (AAV-CBhGAApA) to drive human GAA expression were pseudotyped as AAV8 and administered to immunocompetent GAA-knockout mice. Secreted hGAA was detectable in plasma between 1 day and 12 weeks postadministration with AAV-LSPhGAApA and only from 1 to 8 days postadministration for AAV-CBGAApA. No anti-GAA antibodies were detected in response to AAV-LSPhGAApA (<1:200), whereas AAV-CBhGAApA provoked an escalating antibody response starting 2 weeks postadministration. The LSP drove approximately 60-fold higher GAA expression than the CB promoter in the liver by 12 weeks following vector administration. Furthermore, the detected cellular immunity was provoked by AAV-CBhGAApA, as detected by ELISpot and CD4+/CD8+ lymphocyte immunodetection. GAA activity was increased to higher than normal and glycogen content was reduced to essentially normal levels in the heart and skeletal muscle following administration of AAV-LSPhGAApA. Therefore, liver-restricted GAA expression with an AAV vector evaded immunity and enhanced efficacy in GSD-II mice.

Topics: alpha-Glucosidases; Animals; Antibody Formation; Creatine Kinase; Creatine Kinase, MM Form; Dependovirus; DNA, Viral; Enhancer Elements, Genetic; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Mice; Mice, Knockout; Muscle, Skeletal; Plasmids; Promoter Regions, Genetic

Topics: Adult; alpha-Glucosidases; Aspartic Acid; Female; Glutamic Acid; Glycogen; Glycogen Storage Disease Type II; Homozygote; Humans; Lysosomes; Mutation; Pregnancy; Pregnancy Trimester, Second

Clinical studies of enzyme replacement therapy for Pompe disease have indicated that relatively high doses of recombinant human acid alpha-glucosidase (rhGAA) may be required to reduce the abnormal glycogen storage in cardiac and skeletal muscles. This may be because of inefficient cation-independent mannose 6-phosphate receptor (CI-MPR)-mediated endocytosis of the enzyme by the affected target cells. To address this possibility, we examined whether the addition of a high affinity ligand to rhGAA would improve its delivery to these cells. Chemical conjugation of high mannose oligosaccharides harboring mono- and bisphosphorylated mannose 6-phosphates onto rhGAA (neo-rhGAA) significantly improved its uptake characteristics by muscle cells in vitro. Infusion of neo-rhGAA into Pompe mice also resulted in greater delivery of the enzyme to muscle tissues when compared with the unmodified enzyme. Importantly, this increase in enzyme levels was associated with significantly improved clearance of glycogen ( approximately 5-fold) from the affected tissues. These results suggest that CI-MPR-mediated endocytosis of rhGAA is an important pathway by which the enzyme is delivered to the affected lysosomes of Pompe muscle cells. Hence, the generation of rhGAA containing high affinity ligands for the CI-MPR represents a strategy by which the potency of rhGAA and therefore the clinical efficacy of enzyme replacement therapy for Pompe disease may be improved.

Topics: alpha-Glucosidases; Animals; Disease Models, Animal; Glycogen; Glycogen Storage Disease Type II; Mannosephosphates; Mice; Muscles; Myoblasts; Oligosaccharides; Protein Transport

We administered an adenovirus-adeno-associated virus (Ad-AAV) vector encoding human acid alpha-glucosidase (hGAA) to acid alpha-glucosidase-knockout (GAA-KO) mice on day 3 of life by gastrocnemius injection. In contrast to previous results for muscle-targeted Ad vector in adult GAA-KO mice, the muscles of the hindlimb showed reduced glycogen content and persistent hGAA for as long as 6 months after neonatal Ad-AAV vector administration. Not only the injected gastrocnemius muscles, but also the hamstrings and quadriceps muscles produced therapeutic levels of hGAA as a result of widespread transduction with the Ad-AAV vector; moreover, hGAA activity was 50-fold elevated as compared to normal mice. Vector RNA was detected in the hindlimb muscles, the hearts, and the livers by northern blot analysis and/or by RT-PCR for as long as 6 months. The low levels of hGAA detected in the heart were attributable to transduction with the Ad-AAV vector, not to secretion of hGAA by the injected muscle and uptake by the heart. Finally, although an antibody response to hGAA was present, it did not prevent the correction of glycogen storage in the skeletal muscle of GAA-KO mice.

Topics: Adenoviridae; Animals; Blotting, Northern; Blotting, Western; Cell Line; Dependovirus; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; HeLa Cells; Humans; Liver; Mice; Mice, Knockout; Muscle, Skeletal; Myocardium; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tissue Distribution

Pompe's disease (glycogen storage disease type II) is an autosomal recessive myopathy caused by lysosomal alpha-glucosidase deficiency. Enzyme replacement therapy (ERT) is currently under development for this disease. We evaluated the morphological changes in muscle tissue of four children with infantile Pompe's disease who received recombinant human alpha-glucosidase from rabbit milk for 72 weeks. The patients were 2.5-8 months of age at entry. Prior to treatment, all patients showed lysosomal glycogen storage in skeletal and smooth muscle cells, vascular endothelium, Schwann cells, and perineurium. The first response to treatment was noticed in vascular endothelium and in peripheral nerves after 12 weeks of treatment at an enzyme dose of 15-20 mg/kg. Increasing the dose to 40 mg/kg led, after 72 weeks of treatment, to a reduction of glycogen storage and substantial improvement of muscle architecture in the least affected patient. Not all patients responded equally well, possibly due to differences in degree of glycogen storage and concomitant muscle pathology at the start of treatment. We conclude that intravenous administration of recombinant human alpha-glucosidase from rabbit milk can improve muscle morphology in classic infantile Pompe's disease when treatment is started before irreversible damage has occurred.

Topics: alpha-Glucosidases; Animals; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Lysosomes; Male; Microscopy, Electron; Muscle Fibers, Skeletal; Muscle, Skeletal; Myocytes, Smooth Muscle; Peripheral Nerves; Rabbits; Recombinant Fusion Proteins; Schwann Cells; Treatment Outcome

Muscle glycogen storage was measured by in vivo, natural abundance 13C nuclear magnetic resonance spectroscopy in distal and proximal lower limb segments of patients suffering from adult-onset acid maltase deficiency. Interleaved T1-weighted acquisitions of glycogen and creatine served to quantify glycogen excess. For acid maltase deficient patients (n=11), glycogen:creatine was higher than controls (n=12), (1.20+/-0.39 vs. 0.83+/-0.18, P=0.0005). Glycogen storage was above the normal 95% confidence limits in at least one site for 7/11 patients. The intra-individual coefficient of reproducibility was 12%. This totally atraumatic measurement of glycogen allows repeated measurement at different muscle sites of acid maltase deficient patients, despite selective fatty replacement of tissue. This could provide an additional parameter to follow the development of disease in individual patients, including in the perspective of forthcoming therapeutic trials. It may also offer an appropriate tool to study the role of glycogen accumulation in progression of the pathology.

Topics: Adolescent; Adult; alpha-Glucosidases; Carbon Isotopes; Child; Creatine; Evaluation Studies as Topic; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Magnetic Resonance Spectroscopy; Male; Middle Aged; Molecular Biology; Muscle, Skeletal; Phenotype; Reproducibility of Results

Danon disease (DD) is a rare lysosomal glycogen storage disease with normal acid maltase activity, which is characterised clinically by cardiomyopathy and myopathy, and a variable degree of mental retardation. The causative gene, LAMP2, has been mapped to chromosome Xq24-q25. LAMP2 encodes a lysosome-associated membrane glycoprotein. We identified a novel LAMP2 mutation of the exon 8 splice acceptor site (IVS7-1G --> A) in an affected male and female, which predicts abnormal splicing. Both affected individuals presented solely with hypertrophic cardiomyopathy. Muscle weakness and mental impairment were absent. Diagnosis of Danon disease was established by muscle biopsy, when the male index patient developed transient severe muscle weakness following heart transplantation. Typical biopsy findings were also found in a heart muscle specimen. Demonstration of the LAMP2 mutation in affected male and female siblings is compatible with X-linked dominant inheritance. Danon disease should be actively looked for in cardiomyopathy patients.

Topics: Adult; Antigens, CD; Cardiomyopathies; Chromosomes, Human, X; DNA Mutational Analysis; Exons; Female; Glycogen; Glycogen Storage Disease Type II; Heart Transplantation; Humans; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Male; Muscle, Skeletal; Muscle, Smooth; Point Mutation

Glycogen storage disease type II (GSD II) is an inherited progressive muscle disease in which lack of functional acid alpha-glucosidase (AGLU) results in lysosomal accumulation of glycogen. We report on the impact of a null mutation of the acid alpha-glucosidase gene (AGLU(-/-)) in mice on the force production capabilities, contractile mass, oxidative capacity, energy status, morphology, and desmin content of skeletal muscle. Muscle function was assessed in halothane-anesthetized animals, using a recently designed murine isometric dynamometer. Maximal torque production during single tetanic contraction was 50% lower in the knockout mice than in wild type. Loss of developed torque was found to be disproportionate to the 20% loss in muscle mass. During a series of supramaximal contraction, fatigue, expressed as percentile decline of developed torque, did not differ between AGLU(-/-) mice and age-matched controls. Muscle oxidative capacity, energy status, and protein content (normalized to either dry or wet weight) were not changed in knockout mice compared to control. Alterations in muscle cell morphology were clearly visible. Desmin content was increased, whereas alpha-actinin was not. As the decline in muscle mass is insufficient to explain the degree in decline of mechanical performance, we hypothesize that the large clusters of noncontractile material present in the cytoplasm hamper longitudinal force transmission, and hence muscle contractile function. The increase in muscular desmin content is most likely reflecting adaptations to altered intracellular force transmission.

Topics: Actinin; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; alpha-Glucosidases; Animals; Body Weight; Desmin; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Inosine Monophosphate; Mice; Mice, Knockout; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Weakness; Muscle, Skeletal; Phosphocreatine; Stress, Mechanical

Although many lysosomal disorders are corrected by a small amount of the missing enzyme, it has been generally accepted that 20-30% of normal acid alpha-glucosidase (GAA) activity, provided by gene or enzyme replacement therapy, would be required to reverse the myopathy and cardiomyopathy in Pompe disease. We have addressed the issue of reversibility of the disease in the Gaa(-/-) mouse model. We have made transgenic lines expressing human GAA in skeletal and cardiac muscle of Gaa(-/-) mice, and we turned the transgene on at different stages of disease progression by using a tetracycline-controllable system. We have demonstrated that levels of 20-30% of normal activity are indeed sufficient to clear glycogen in the heart of young Gaa(-/-) mice, but not in older mice with a considerably higher glycogen load. However, in skeletal muscle-a major organ affected in infantile and in milder, late-onset variants in humans-induction of GAA expression in young Gaa(-/-) mice to levels greatly exceeding wildtype values did not result in full phenotypic correction, and some muscle fibers showed little or no glycogen clearance. The results demonstrate that complete reversal of pathology in skeletal muscle or long-affected heart muscle will require much more enzyme than previously expected or a different approach.

Topics: alpha-Glucosidases; Animals; Blotting, Western; Cardiomyopathies; Female; Gene Transfer Techniques; Glycogen; Glycogen Storage Disease Type II; Humans; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Skeletal; Mutation; Myocardium; Phenotype; Transgenes

A 26-year-old woman presented with elevated liver enzymes, which were diagnosed two months ago. Examination revealed mild proximal muscle weakness, though the patient herself did not realise any impairment. The abdominal ultrasound and the histology of the liver remained unsuspicious. Muscle biopsy showed vacuolar degeneration, which could be ultrastructurally identified as large deposits of membrane-bound glycogen. The morphological findings prompted biochemical investigations which showed an excess of muscle glycogen. Acid maltase activity was reduced to < 10 % of normal, leading together with the clinical findings to the diagnosis of glycogenosis type II (Pompe's disease) of the adult type. Because of the modest impairment of the patient and the limited therapeutic possibilities, the patient remained thus untreated for.

Topics: Adult; Alanine Transaminase; alpha-Glucosidases; Aspartate Aminotransferases; Biopsy; Creatine Kinase; Diagnosis, Differential; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Liver Function Tests; Muscle, Skeletal

Glycogen storage disease type II (GSD II), Pompe's disease, is caused by the deficiency of acid alpha-D-glucosidase (GAA) in lysosome and is the most common form of GSD in Taiwan. Most cases are the infantile form. The disease is relentless and most patients die of cardiac failure and respiratory tract infection in the first year of life. At present, no treatment has been proved effective for this fatal disease. The applicability of enzyme replacement therapy is under investigation. However, high price and transient efficiency are the major problems to be solved. Accordingly, gene therapy by viral method has been conducted. In this study we constructed a plasmid that contained 5'-shortened BglII-NotI fragment human GAA cDNA, downstream of CMV promoter and bovine growth hormone polyadenylation signal, as well as AAV ITR region. When fibroblasts obtained from GSD II patients were cultured and infected with rAAV-GAA, the GAA activity of the fibroblasts increased four- to five-fold. Using acid maltase deficient (AMD) Japanese quail as the animal model, rcAAV-GAA 0.1 ml per site (1 x 10(9)-10) particles), totally 10 different sites to make 1 ml (1 x 10(1)0-11) particles), was injected into unilateral deep pectoral muscle of AMD quails. Medium (hepes) was only injected in the same way into the contralateral deep pectoral muscle to serve as control. Four days after injection, PAS staining showed disappearance of the glycogenosomes with regeneration of myocytes surrounding the intramuscular injected area as compared with the contralateral muscle of the same birds. Using anti-GAA monoclonal antibody, GAA was demonstrated on the regenerated myocytes by immunohistochemical staining and absent on the contralateral muscle of the same birds. Nevertheless, T lymphocytes infiltration was noted in both the rcAAV-GAA and hepes (medium) injected muscles and more prominent in the rcAAV-GAA-injected site. Functional evaluation demonstrated that wing flapping movement improved with wide flapping in the rAAV-GAA injected side, but not in the counterpart. Unfortunately, these histochemical and functional improvements faded away in 14 days, probably due to destruction of rcAAV by cell-mediated immunity of infiltrated T cells. Taken together, the present study suggests that rAAV can enter either human or quail cells and express and effectively reduce the glycogen accumulation in the skeletal muscle of AMD quails. These preliminary results are similar to these of low-dose rGAA repl

Topics: alpha-Glucosidases; Animals; Coturnix; Dependovirus; Genetic Therapy; Genetic Vectors; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Injections, Intramuscular; Injections, Intravenous; Male; Muscle, Skeletal; Transduction, Genetic; Wings, Animal

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Topics: alpha-Glucosidases; Animals; Cardiovascular Diseases; Dependovirus; Disease Models, Animal; Fibroblasts; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Homozygote; Humans; Immunoenzyme Techniques; Infant; Lysosomal Storage Diseases; Mice; Mice, Inbred BALB C; Mice, Knockout; Muscle, Skeletal; Myocardium; Transduction, Genetic

Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.

Topics: Adenoviridae; alpha-Glucosidases; Animals; Genetic Therapy; Genetic Vectors; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Injections, Intramuscular; Lysosomes; Mice; Mice, Knockout; Microscopy, Electron; Muscle, Skeletal

Both enzyme replacement and gene therapy of lysosomal storage disorders rely on the receptor-mediated uptake of lysosomal enzymes secreted by cells, and for each lysosomal disorder it is necessary to select the correct cell type for recombinant enzyme production or for targeting gene therapy. For example, for the therapy of Pompe disease, a severe metabolic myopathy and cardiomyopathy caused by deficiency of acid alpha-glucosidase (GAA), skeletal muscle seems an obvious choice as a depot organ for local therapy and for the delivery of the recombinant enzyme into the systemic circulation. Using knockout mice with this disease and transgenes containing cDNA for the human enzyme under muscle or liver specific promoters controlled by tetracycline, we have demonstrated that the liver provided enzyme far more efficiently. The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. Furthermore, using tetracycline regulation, we somatically induced human GAA in the knockout mice, and demonstrated that the skeletal and cardiac muscle pathology was completely reversible if the treatment was begun early.

Topics: alpha-Glucosidases; Animals; Blotting, Western; Cells, Cultured; Gene Expression; Gene Expression Regulation, Enzymologic; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Skeletal; Organ Specificity; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Mitochondrial oxidative metabolism was examined in two infants with Pompe's disease. The clinical diagnosis was confirmed by the demonstration of intralysosomal glycogen accumulation and a deficiency of acid alpha-D-glucosidase in muscle biopsies. Light and electron microscopy studies demonstrated a normal number of mitochondria with normal ultrastructure. Spectrophotometric measurements revealed that the specific activities of citrate synthase and the partial reactions of electron transport were markedly elevated in the skeletal muscle homogenates prepared from both infants with Pompe's disease when calculated as micromoles per minute per gram wet weight of tissue. However, when respiratory chain enzyme activities were expressed relative to citrate synthase as a marker mitochondrial enzyme, a different pattern emerged, in which all Pompe muscle respiratory enzymes, except complex IV, were decreased relative to control subjects. These observations demonstrate that caution should be exercised when analyzing and interpreting data obtained from tissue homogenates in general and, in particular, in those prepared from tissues in which the wet weight of tissue may be altered, for example, by pathologic accumulation of carbohydrate or lipid.

Topics: alpha-Glucosidases; Biopsy; Citrate (si)-Synthase; Diagnosis, Differential; Electron Transport; Female; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Mitochondria; Muscles; Oxidation-Reduction

Glycogen storage disease type II (Pompe's disease) is a rare inherited metabolic disorder, which often leads to infantile death from severe cardiomyopathy. This case of sudden death illustrates the features of the cardiac findings in the disorder, resulting from massive lysosomal accumulation of glycogen in the heart and other tissues. Pompe's disease should be considered in cases of unexplained infantile cardiomyopathy.

Topics: Biopsy; Cardiomyopathy, Hypertrophic; Death, Sudden, Cardiac; Echocardiography; Electrocardiography; Endothelium, Vascular; Fatal Outcome; Follow-Up Studies; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Lysosomes; Male; Radiography, Thoracic; Skin

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid alpha-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.

Topics: Adenoviridae; alpha-Glucosidases; Animals; Cytomegalovirus; Genes, pol; Genetic Therapy; Genetic Vectors; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Muscular Diseases; Promoter Regions, Genetic

Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid alpha-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid alpha-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa -/-) lack Gaa mRNA and have a virtually complete acid alpha-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions.

Topics: alpha-Glucosidases; Animals; Cardiomegaly; Disease Models, Animal; Female; Glycogen; Glycogen Storage Disease Type II; Male; Mice; Mice, Knockout

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.

Topics: alpha-Glucosidases; Animals; Bird Diseases; Body Weight; CHO Cells; Coturnix; Cricetinae; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Male; Muscles; Recombinant Fusion Proteins; Tissue Distribution

Acid alpha-glucosidase (GAA) deficiency causes Pompe disease, a lethal lysosomal glycogen storage disease for which no effective treatment currently exists. We investigated the endocytic process in deficient cells of human recombinant GAA produced in Chinese hamster ovary cells, and the potential of GAA-deficient Japanese acid maltase-deficient quail as a model for evaluating the enzyme replacement therapy for Pompe disease. After 24-h incubation with a single dose of recombinant enzyme, intracellular GAA and glycogen levels in deficient human fibroblasts were normalized, and this correction lasted for 7 d. The 110-kD precursor recombinant enzyme was processed to the 76-kD mature form within 24 h after uptake. Intracellular GAA levels in deficient quail fibroblasts and myoblasts were similarly corrected to their average normal levels within 24 h. Differences existed in the efficiency of endocytosis among subfractions of the enzyme, and among different cell types. Fractions with a larger proportion of precursor GAA were endocytosed more efficiently. Quail fibroblasts required a higher dose, 4200 nmol.h-1.mL-1 to normalize intracellular GAA levels than human fibroblasts, 1290 nmol.h-1.mL-1, whereas primary quail myoblasts required 2800 nmol.h-1.mL-1. In all three cell lines, the endocytosed enzyme localized to the lysosomes on immunofluorescence staining, and the endocytosis was inhibited by mannose 6-phosphate (Man-6-P) added to the culture medium. Despite structural differences in Man-6-P receptors between birds and mammals, these studies illustrate that Man-6-P receptor mediated endocytosis is present in quail muscle cells, and demonstrate the potential of acid maltase-deficient quail to test receptor mediated enzyme replacement therapy for Pompe disease.

Topics: alpha-Glucosidases; Animals; Biological Transport, Active; Cells, Cultured; CHO Cells; Cricetinae; Disease Models, Animal; Endocytosis; Fibroblasts; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Kinetics; Muscles; Quail; Receptor, IGF Type 2; Recombinant Proteins

Glycogen storage disease type II (GSD II) is an autosomal recessive disorder caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. We investigated the feasibility of using a recombinant adenovirus containing the human GAA gene under the control of the cytomegalovirus promoter (AdCMV-GAA) to correct the enzyme deficiency in different cultured cells from patients with the infantile form of GSD II. In GAA-deficient fibroblasts infected with AdCMV-GAA, transduction and transcription of the human transgene resulted in de novo synthesis of GAA protein. The GAA enzyme activity was corrected from the deficient level to 12 times the activity of normal cells. The transduced cells overexpressed the 110 kDa precursor form of GAA, which was secreted into the culture medium and was taken up by recipient cells. The recombinant GAA protein was correctly processed and was active on both an artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4MUG) and glycogen. In GAA-deficient muscle cells, a significant increase in cellular enzyme level, approximately 20-fold higher than in normal cells, was also observed after viral treatment. The transduced muscle cells were also able to efficiently secrete the recombinant GAA. Moreover, transfer of the human transgene resulted in normalization of cellular glycogen content with clearance of glycogen from lysosomes, as assessed by electron microscopy, in differentiated myotubes. These results demonstrate phenotypic correction of cultured skeletal muscle from a patient with infantile-onset GSD II using a recombinant adenovirus. We conclude that adenovirus-mediated gene transfer might be a suitable model system for further in vivo studies on delivering GAA to GSD II muscle, not only by direct cell targeting but also by a combination of secretion and uptake mechanisms.

Topics: Adenoviridae; alpha-Glucosidases; Blotting, Western; Cells, Cultured; Fibroblasts; Gene Transfer Techniques; Genetic Therapy; Glycogen; Glycogen Storage Disease Type II; Humans; Muscle, Skeletal; Recombinant Proteins; Transduction, Genetic

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Topics: alpha-Glucosidases; Animals; Base Sequence; Biological Transport; Cells, Cultured; CHO Cells; Cricetinae; DNA Primers; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Humans; Liver; Lysosomes; Molecular Sequence Data; Muscles; Myocardium; Recombinant Proteins; Transfection

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

Topics: alpha-Glucosidases; Amino Acid Sequence; Animals; Cells, Cultured; CHO Cells; Cricetinae; Endocytosis; Enzyme Precursors; Glucan 1,4-alpha-Glucosidase; Glucosides; Glycogen; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Hymecromone; Lysosomes; Mannosephosphates; Molecular Sequence Data; Muscle, Skeletal; Peptide Elongation Factor 1; Peptide Elongation Factors; Protein Processing, Post-Translational; Recombinant Proteins; Sequence Analysis

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.

Topics: alpha-Galactosidase; alpha-Glucosidases; alpha-Macroglobulins; Animals; Biological Transport; Carrier Proteins; Endocytosis; Fibroblasts; Glycogen; Glycogen Storage Disease Type II; Hydrolysis; Lysosomes; Subcellular Fractions; Swine

Glycogen-storage disease type II (GSDII) is caused by the deficiency of lysosomal alpha-glucosidase (acid maltase). This paper reports on the analysis of the mutant alleles in an American black patient with an adult form of GSDII (GM1935). The lysosomal alpha-glucosidase precursor of this patient has abnormal molecular features: (i) the molecular mass is decreased, (ii) the phosphorylation is deficient and (iii) the proteolytic processing is impaired. Sequence analysis revealed four mutations leading to amino acid alterations: Asp-645-->Glu, Val-816-->Ile, Arg-854-->Stop and Thr-927-->Ile. By using allele-specific oligonucleotide hybridization on PCR-amplified cDNA we have demonstrated that the Arg-854-->Stop mutation is located in one allele that is not expressed, and that the other allele contains the remaining three mutations. Each of the mutations was introduced in wild-type cDNA and expressed in COS cells to analyse the effect on biosynthesis, transport and phosphorylation of lysosomal alpha-glucosidase. The Val-816-->Ile substitution appeared to have no significant effect in contrast with results [Martiniuk, Mehler, Bodkin, Tzall, Hirshhorn, Zhong and Hirschhorn (1991) DNA Cell Biol. 10, 681-687] and was therefore defined as a polymorphism. The Thr-927-->Ile substitution deleting one of the seven glycosylation sites was found to be responsible for the decrease in molecular-mass, but not for the deficient proteolytic processing and phosphorylation. It did not cause the enzyme deficiency either. The third mutation leading to the Asp-645-->Glu substitution was proven to account in full for the observed defects in transport, phosphorylation and proteolytic processing of the newly synthesized alpha-glucosidase precursor of the patient.

Topics: Adult; Alleles; alpha-Glucosidases; Aspartic Acid; Base Sequence; beta-N-Acetylhexosaminidases; Biological Transport; Black People; Cells, Cultured; Codon; DNA Mutational Analysis; Glutamates; Glutamic Acid; Glycogen; Glycogen Storage Disease Type II; Humans; Lysosomes; Microscopy, Immunoelectron; Molecular Sequence Data; Phenotype; Phosphorylation; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Protein Processing, Post-Translational; Regulatory Sequences, Nucleic Acid; Sequence Analysis, DNA; Tunicamycin

Clinical, diagnostic and biochemical features of generalised glycogenosis are described in 96 Brahman-type calves. Typically the calves were presented when about 6 months of age, with ill-thrift and muscular weakness as the most common signs. Acidic alpha-glucosidase activity was reduced in peripheral blood lymphocytes and skeletal muscle. Muscle glycogen concentration was consistently higher in affected animals than in clinically normal cattle. Other observations in affected calves included elevation of serum aspartate aminotransferase and creatine kinase activities and excessive amounts of high molecular weight oligosaccharides in urine. Fine cytoplasmic vacuolation of neurones in the brain and spinal cord, skeletal muscle, myocardium and of Purkinje fibres were consistent histological observations. Periodic acid-Schiff staining revealed the presence of glycogen-like material in peripheral blood lymphocytes of all affected calves, indicating that this is a useful aid for the diagnosis of glycogenosis. While 3 of the 96 calves showed somewhat different clinical signs, the similarity of pathology and the biochemical and clinical evidence in the remainder suggested that, in these animals, the disease was expressed as a single syndrome.

Topics: alpha-Glucosidases; Animals; Aspartate Aminotransferases; Cattle; Cattle Diseases; Central Nervous System; Creatine Kinase; Eye; Female; Glycogen; Glycogen Storage Disease Type II; Male; Muscles; Myocardium; Neurons; Oligosaccharides; Purkinje Fibers

Muscular glycogenosis is a disease resulting from genetical abnormalities altering an enzyme which is involved in glycogen metabolism. In addition to disorders of glycogenosis and glycolysis, there are other pathological processes such as alpha-glycosidase deficiency and diseases associated with abnormal polysaccharide structure. A short review of the various diseases with their particular features is reported.

Topics: Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VII; Glycolysis; Humans; Muscles; Muscular Diseases

Adding acid alpha-glucosidase to cultures of Pompe's disease muscle has resulted in enzyme uptake and reduction in concentration of glycogen. However, bone marrow transplantation has been unsuccessful as a treatment. Immune rejection may have contributed to this failure. Twin calves share a placenta and carry lymphoreticular cells of each other's type, they become lymphoreticular chimeras in utero and immune rejection does not occur. One natural and three sets of twins produced by embryo transfer were studied in Pompe's disease cattle. Chimerism persisted throughout life and the situation was analogous to a transplant of histocompatible bone marrow stem cells. The activity of acid alpha-glucosidase in leucocytes and in biopsies of the semitendinosus muscle and the mean activity in diaphragm, spleen and lymph node obtained after death from affected twins were significantly higher than in single affected calves. Glycogen concentration was lowered in liver, spleen and lymph node but not in muscles. The affected twins showed clinical signs and changes in muscle similar to those seen in affected single calves. It is concluded that bone marrow transplantation is unlikely to be a successful treatment for Pompe's disease.

Topics: alpha-Glucosidases; Animals; Bone Marrow Transplantation; Cattle; Cattle Diseases; Chimera; Embryo Transfer; Female; Glycogen; Glycogen Storage Disease Type II; Karyotyping; Lymphocytes; Male; Organ Specificity

Topics: Diagnosis, Differential; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VII; Glycogen Storage Disease Type VIII; Humans

We compared acid alpha-glucosidase of acid maltase-deficient Japanese quails, an animal model of human late-onset glycogenosis type II, with that of normal controls. Antibody produced in a rabbit against acid alpha-glucosidase purified from chicken pectoral muscle cross-reacted with that of Japanese quails. The presence of a 110K and 98K form of acid alpha-glucosidase was confirmed in normal controls by immunoblotting. However the 98K form was absent in the affected quails. Subcellular distribution studies demonstrated that the 98K form, but not the 110K form, was localized in the lysosomes. This suggests that the 110K form is a precursor of the mature 98K form of acid alpha-glucosidase. In the affected quails, the 110K precursor is synthesized, but maturation to the 98K form does not occur or may be extremely deficient.

Topics: alpha-Glucosidases; Animals; Coturnix; Disease Models, Animal; Electrophoresis; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Immunoblotting; Lysosomes

Properties of the acid alpha-glucosidase, GAA2, the product of the GAA*2 allele have been compared with those of the common allele product GAA1, GAA2 has an altered affinity for glycogen but resembles GAA1 in its affinity for low molecular weight substrates, and also in its processing, as judged by immunoblot analysis of the denatured polypeptides. Starch gel electrophoretic analysis of fibroblasts from 15 patients with late onset glycogen storage disease type II (GSDII) failed to reveal either homozygotes or heterozygotes for the GAA*2 allele (GAA2-2 or GAA2-0) providing evidence that neither of these genotypes lead to late onset GSDII despite the impaired activity of the enzyme towards glycogen.

Topics: Alleles; alpha-Glucosidases; Cell Line; Female; Fibroblasts; Genotype; Glycogen; Glycogen Storage Disease Type II; Humans; Immunoblotting; Lysosomes; Male; Phenotype; Substrate Specificity

Attempts at treatment of glycogenosis type II and other lysosomal storage disorders by enzyme replacement have been reported. Parenteral enzyme administration has been ineffectual. Treatment by bone marrow transplantation is currently under investigation. We have used cultured skeletal muscle cells from a patient with infantile glycogenosis type II to study fundamental aspects of enzyme replacement therapy. Efficient uptake of acid alpha-glucosidase was achieved by using the mannose-6-phosphate receptor on the cell surface as a target for an enzyme precursor with phosphorylated high-mannose types carbohydrate chains purified from human urine. We found that the enzyme was channeled to the lysosomes and converted to mature acid alpha-glucosidase. Glycogen storage was reversed. The results are discussed in relation to treatment of glycogenosis type II.

Topics: alpha-Glucosidases; Carrier Proteins; Cells, Cultured; Endocytosis; Enzyme Precursors; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Lysosomes; Microscopy, Electron; Muscles; Receptor, IGF Type 2

In addition to the infantile lethal form of glycogen storage disease with cardiomyopathy (GSD Type IIa, Pompe disease) 1,4 glucosidase or acid maltase deficiency has been reported in a few children and adults (GSD Type IIb or IIc) erroneously thought to have muscular dystrophies. The clinical heterogeneity of the muscle involvement in these latter cases is illustrated in a 12-year-old boy presenting with a right lumbar mass, growth retardation, muscular weakness including difficulty in walking, and marked elevations of muscle and liver enzymes. Light- and electron-microscopic examination of specimens from the lumbar mass, apparently normal skeletal muscle and liver, showed typical changes consistent with the biochemical and enzymatic features of acid maltase deficiency. GSD Type IIb and IIc are more frequent than suspected, may present as local pseudohypertrophy and should be considered in patients with progressive muscle disease and abnormal serum enzymes.

Topics: alpha-Glucosidases; Back; Child; Diagnosis, Differential; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Liver Diseases; Male; Muscular Diseases

Topics: alpha-Glucosidases; Animals; Fibroblasts; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease Type II; Humans; Muscles; Skin

Fibroblast cultures from patients with different clinical subtypes of glycogenosis type II were compared with respect to residual acid alpha-glucosidase activity and lysosomal glycogen content. Lysosomal glycogen storage was most pronounced in fibroblasts from patients with the rapidly progressive infantile form of the disease, and the most severe enzyme deficiency. In fibroblasts from adult patients with more than 10% of the control activity storage did not occur, and 15% of the total cellular glycogen was found in the lysosomes as in control cells. The strict correlation between residual acid alpha-glucosidase activity and lysosomal glycogen accumulation was further illustrated in two adult Pompe patients with an unusually low enzyme activity. The mild clinical course is unexplained in these particular cases. The enzyme deficiency in all the different mutant cell lines was corrected by the uptake of bovine testis acid alpha-glucosidase from the culture medium. As a result of this, the lysosomal glycogen storage disappeared, and the balance between lysosomal and cytoplasmic glycogen was restored to normal. The implications of this study as a model for enzyme replacement therapy are discussed.

Topics: alpha-Glucosidases; Cell Fractionation; Cell Line; Fibroblasts; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Lysosomes; Proteins

Adult acid maltase deficiency (AMD, glycogen storage disease type II) may involve respiratory muscles leading to severe respiratory failure even before the affection of pelvic girdle muscles has turned the patient non-ambulatory. The case of a 29-year-old woman is presented to demonstrate that long-term survival is possible even after acute respiratory failure has occurred. The examination of the patient's family revealed the diagnosis of AMD in her 24-year-old sister, so far without clinical symptoms. The comparison between the two patients of serum enzyme elevations (CK, LDH, GOT, GPT, aldolase) suggested that both physical activity and the stage of the disease may be correlated with the degree of enzyme level elevation.

Topics: Adult; alpha-Glucosidases; Biopsy; Diagnosis, Differential; Electromyography; Female; Follow-Up Studies; Glucan 1,4-alpha-Glucosidase; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Muscles; Muscular Atrophy

Glycogen storage disease type II Pompe (GSD II) is a lysosomal storage disease caused by an inherited deficiency of acid alpha-glucosidase. In addition to the classical infantile form of GSD II, several clinical variants are known. We describe an infant with the classical course of the disease. Our patient differs from the classical variant by the lack of cardiomegalia and the high residual activity of acid alpha-glucosidase in cultivated skin fibroblasts and muscle tissue. In the present case, however, glycogen storing lysosomes were found in peripheral lymphocytes and skeletal muscle cells. This finding underlines the particular value of ultrastructural investigation in the diagnosis of GSD II.

Topics: alpha-Glucosidases; Biopsy; Cardiomegaly; Fibroblasts; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Lysosomes; Male; Microscopy, Electron; Muscles; Organ Size

Topics: Echocardiography; Female; Furosemide; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Heart Failure; Humans; Infant; Infant, Newborn; Muscles; Myocardium; Prognosis; Twins, Monozygotic

Topics: Adult; Age Factors; alpha-Glucosidases; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Lysosomes; Motor Neurons; Muscles; Muscular Diseases; Neuromuscular Diseases; Vacuoles

An adult patient with lysosomal acid alpha-glucosidase deficiency was fully investigated, and then placed on various forms of therapy with favourable response to a high protein, low carbohydrate diet. The rationale for the employment of this therapy, the problem of acid maltase deficiency and the relationship to weakness and glycogenosome formation with accumulation or otherwise of glycogen within the muscle fibres is discussed.

Topics: Adult; alpha-Glucosidases; Dietary Carbohydrates; Dietary Proteins; Electromyography; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Muscles

Topics: alpha-Glucosidases; Bone Marrow Transplantation; Follow-Up Studies; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Liver; Liver Glycogen; Male; Muscles; Myocardium

In lamellar bodies isolated from adult human lung at least two acid alpha-glucosidases are present: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme. In the present study we measured the activity of this lamellar body-specific alpha-glucosidase and of lysosomal alpha-glucosidase in a patient with an inherited deficiency of lysosomal alpha-glucosidase. The activity of the lamellar body-specific alpha-glucosidase was not affected in the patient, whereas the lysosomal alpha-glucosidase activity was strongly depressed. The results strongly suggest that the lysosomal alpha-glucosidase and the lamellar body-specific alpha-glucosidase are different gene products.

Topics: alpha-Glucosidases; Concanavalin A; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Lung; Lysosomes

Five placentas from infants with enzymatically diagnosed glycogen storage disease type II (three from midtrimester abortions, two from term deliveries) were studied by light and electron microscopy. On routine histologic examination with hematoxylin and eosin staining, storage cells were identifiable in the connective tissue of the amnion. These cells provide the means to diagnose this glycogen storage disease prior to the development of clinical symptoms. Electron microscopy, even in the midtrimester placenta, shows typical membrane-bound, glycogen-filled vacuoles in the villous endothelium and stromal cells. These vacuoles can provide confirmation of glycogen storage in cases of prenatal enzymatic diagnosis and therapeutic abortion.

Topics: alpha-Glucosidases; Amnion; Cytoplasm; Eosine Yellowish-(YS); Female; Gestational Age; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Hematoxylin; Humans; Infant; Microscopy, Electron; Mucolipidoses; Placenta; Pregnancy; Staining and Labeling

acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.

Topics: alpha-Glucosidases; Animals; Cattle; Cattle Diseases; Cells, Cultured; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Lysosomes; Muscles

The pathology of canine glycogen storage disease type II (acid alpha-glucosidase deficiency, GSD II) was studied in three genetically related Lapland dogs and compared to the pathology of human GSD II (McKusick 23230). Canine GSD II closely parallels the infantile form of the human disease, except for the presence of oesophageal dilatation. Generalized glycogen storage particularly affected muscular tissues (skeletal, oesophageal, cardiac and smooth muscle). The altered cells showed glycogen accumulation in the cytosol and in autophagic membrane-bound vacuoles (glycogenosomes). They also showed increased acid phosphatase activity consistent with the lysosomal nature of this storage disorder. The cytopathology in canine and human GSD II appears to evolve from segregation of glycogen during regular cellular autophagy, phagolysosomal accumulation of the undigested glycogen, and eventually rupture of distended glycogenosomes. This study indicates that the usefulness of canine GSD II as an animal model of human disease, extends to the area of pathogenesis.

Topics: Acid Phosphatase; Animals; Brain; Cytoplasm; Disease Models, Animal; Dogs; Esophagus; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Kidney; Liver; Male; Microscopy, Electron; Muscle, Smooth; Muscles; Myocardium; Neurons; Spinal Cord; Vacuoles

Two siblings who developed adult form acid maltase deficiency (AMD) are reported. The elder sister, a 30-year-old Japanese woman whose parents are cousins was admitted because of respiratory disturbance which she noticed two years previously. The muscle histology demonstrated numerous acid phosphatase positive vacuoles filled with PAS positive materials, and the muscle enzyme assay demonstrated a reduction of acid maltase activity, thus confirming a diagnosis of acid maltase deficiency of adult form. Her younger sister, a 25-year-old woman who had no obvious history of muscle weakness was admitted because of coma due to subarachnoideal hemorrhage and died two days later. Postmortem examination revealed the rupture of a fusiform aneurysm of the basilar artery whose wall showed vacuolar degeneration, and the histological and biochemical examination revealed that she had also AMD of the adult form. It is considered that the fragility of arterial wall, caused by vacuolar degeneration due to AMD, resulted as the rupture of aneurysm. Immunologically cross reactive material against acid maltase antibody was not detected. To our knowledge, this family is considered to be the third of AMD of the adult form reported in Japan.

Topics: Adult; alpha-Glucosidases; Basilar Artery; Consanguinity; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Intracranial Aneurysm; Muscles; Myocardium; Respiratory Insufficiency; Vacuoles

The loss of normal ultrastructure of skeletal muscle during the relentless course of infantile acid maltase deficiency (AMD) is re-examined in the light of the lysosomal rupture hypothesis. This hypothesis suggests that movement and increased myofibril rigidity during contraction cause lysosomes in muscle to rupture and release glycogen and other lysosomal contents to a much greater extent than do lysosomes in other cell types in cases of infantile AMD. Muscle fibers are destroyed, while macrophages and other cells mostly accumulate glycogen in storage lysosomes without being destroyed. When morphological stages of fiber destruction are placed in a sequential series, from fibers most like normal infant muscle to those with only remnants of muscle ultrastructure, the earliest stages seen contain intact storage lysosomes. Intermediate stages exhibit ruptured lysosomal membranes and free glycogen as well as glycogen in lysosomes. Loss of myofibrillar material and loss of glycogen occur in later stages of fiber destruction. Membrane-enclosed glycogen and mitochondria are relatively protected from the process of destruction. The electron-microscopic observations support the lysosomal rupture hypothesis and are compatible with the original proposal of Hers, that the disease results from a deficiency of a single lysosomal enzyme. Secondary changes other than muscle fiber destruction probably relate to disrupted control mechanisms and the nature of muscle as a specialized cell. At least two different mechanisms could contribute to the loss of contractile activity and myofibrillar structure.

Topics: alpha-Glucosidases; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Infant; Lysosomes; Microscopy, Electron; Mitochondria, Muscle; Muscles; Myofibrils; Staining and Labeling

Infantile acid maltase deficiency (Pompe's disease, glycogenosis II) is a progressive, severe lysosomal storage disease in which skeletal and cardiac muscle fibers accumulate membrane-bound and free glycogen and are destroyed. New information in this report concerns 1) early hypertrophy of skeletal muscle fibers, 2) absence of size change as glycogen is lost, and 3) the ultrastructure of end-stage fibers empty of glycogen. Muscle fibers enlarge as they accumulate glycogen and then stay large as glycogen is lost. They are so large that, if empty fibers did in fact contain glycogen, over 80% of the muscle would be glycogen instead of 6.3-11.5% (from 37 published determinations). Fibers that have reached "empty" end-stage are shown to be more numerous than all other stages combined in biopsies from infantile acid maltase deficiency. Ultrastructurally, end-stage fibers contain much "empty" space (liquid-filled without fine structure) and various remnants and masses of altered myofibrillar and sarcoplasmic material. Many broken membranes originally enclosing glycogen in storage lysosomes are seen. A single broken membrane can enclose an area larger than the cross section area of a muscle fiber from a normal infant. The results support the proposal of Hers that the disease is due to a deficiency of the single lysosomal enzyme acid maltase. The results also support the lysosomal rupture hypothesis of Griffin, which accounts for muscle fibers being more damaged than are other cells and for the release of glycogen to the sarcoplasm.

Topics: alpha-Glucosidases; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Hypertrophy; Infant; Lysosomes; Microscopy, Electron; Muscles; Staining and Labeling

In infantile acid maltase deficiency (AMD), masses of glycogen accumulate in muscle fibers and are then gradually digested. The metachromatic material found in some glycogen-filled fibers, not previously studied with the electron microscope, has two different fine structural appearances. Some is similar in shape and size to glycogen beta granules, but is more intensely stained, and some is in larger granules, irregular in shape, and has even higher stain affinity. Since acid maltase deficiency was identified by Hers, others have proposed that more than one genetic defect or additional extralysosomal factors are required to account for massive glycogen accumulation and metachromasia. There is no direct evidence of additional rare genetic defects. Presented herein are two simple proposals consistent with the primary deficiency. The first is that some partly digested glycogen is condensed and that this concentrates the sites that bind dye, producing metachromasia and other differences from normal glycogen. The second is that the massive accumulation of glycogen in muscle fibers involves, in addition to previously recognized lysosomal storage and lysosomal rupture, inactivation of sarcoplasmic phosphorylase caused by disruption of excitation-contraction linkages. These two proposals are physiologically plausible and potentially testable and do not invoke the coincidence of two or more rare genetic mutations.

Topics: alpha-Glucosidases; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Inclusion Bodies; Infant; Microscopy, Electron; Muscles; Staining and Labeling

Topics: Adult; Aged; alpha-Glucosidases; Biopsy; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Male; Microscopy, Electron; Middle Aged; Muscles

Topics: Adolescent; Child; Child, Preschool; Erythrocytes; Glycogen; Glycogen Debranching Enzyme System; Glycogen Storage Disease; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Glycogen Storage Disease Type IV; Humans; Infant; Leukocytes; Liver; Liver Diseases; Phosphorylase a

Topics: Acarbose; Adrenal Glands; Animals; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Kidney; Lysosomes; Microscopy, Electron; Muscles; Oligosaccharides; Rats; Spleen; Trisaccharides

An infant died at 8 months of age with a history of developmental regression, hypotonia, severe weakness, cardiomegaly, congestive heart failure, and hepatomegaly. A diagnosis of Pompe's disease (glycogenosis type II) was established by muscle biopsy at 5 months of age. Vacuolar myopathy involved muscle fibers of histochemical type I more than type II. Many vacuoles were filled with glycogen. In addition, increased amounts of neutral lipid were demonstrated by oil red O stain, electron microscopy, and quantitative analysis. Acid alpha-1,4-glucosidase activity was demonstrated to be deficient. Biochemical studies failed to determine the cause of the lipid accumulation, but demonstrated a low total concentration of carnitine in the muscle (6.37 nmole/mg of protein), associated with elevated activities of carnitine palmityl-transferase and palmityl-coenzyme A dehydrogenase. Palmityl-coenzyme A synthetase activity was in the normal range.

Topics: Carnitine O-Palmitoyltransferase; Coenzyme A Ligases; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Lipid Metabolism; Lipid Metabolism, Inborn Errors; Microscopy, Electron; Muscle Hypotonia; Muscles; Palmitic Acids; Repressor Proteins; Saccharomyces cerevisiae Proteins; Vacuoles

Topics: Adult; alpha-Glucosidases; Animals; Female; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Liver; Male; Muscles; Rats

The case of a 26 year old man with acute respiratory difficulty was reported. Morphological and biochemical analysis of biopsied gastrocnemius muscle indicated a diagnosis of adult form acid maltase deficiency. Clinically, the most interesting point of our case was the presence of a thickening of the posterior papillary muscles and chordae without any functional disturbance, which was detected by echocardiogram. Another interesting point of our case was the existence of a sibling who died of progressive muscular dystrophy at the age of 31 years. This may raise the possibility that we are dealing with a familial type of adult form acid maltase deficiency.

Topics: Adult; alpha-Glucosidases; Chordae Tendineae; Echocardiography; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Male; Muscles; Papillary Muscles

This report describes a female patient with childhood form of acid maltase deficiency who survived till fifteen years old. Although acid alpha-1,4-glucosidase was deficient in the liver, kidney, skeletal and cardiac muscles, neutral alpha-1,4-glucosidase was present in normal concentrations in those organs. On light microscopic examination, numerous intracytoplasmic vacuoles containing acid phosphatase positive granules and PAS positive materials were present in both type 1 and 2 A fibers, predominantly in the latter. The striking finding in the present case was a selective type 2 fiber atrophy with type 2 B fiber deficiency believed to result from type 2 motor neuron dysfunction in the spinal cord. Electron microscopic study revealed extensive glycogen particle accumulation, autophagic vacuoles and myelin figures in the muscle fibers.

Topics: Adolescent; alpha-Glucosidases; Electromyography; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Muscles; Muscular Atrophy; Organoids; Vacuoles

Type II glycogenosis is an autosomal recessive storage disease characterized by absence of the enzyme acid alpha-1,4-glucosidase. The eye of a 16 week fetus, aborted after diagnosis by amniocentesis, was studied by light and electron microscopy. Extensive deposits of lysosomal and cytoplasmic glycogen were present in virtually all ocular tissues examined, with the notable exception of pigment epithelia (iris and retina). The massive glycogen deposits present in this, the youngest case thus far examined histologically, emphasize the involvement of the fetus from its earliest stages and the importance of prenatal diagnosis.

Topics: Eye; Female; Fetus; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Pregnancy; Prenatal Diagnosis; Retina

We analyzed clinical, histological and biochemical findings in 10 patients with glycogen storage disease in skeletal muscle. Four patients were deficient in acid-alpha-glucosidase (Glycogenosis type II), three of them with late infantile onset and one patient adult form. Five patients, two of them siblings, were deficient in myophosphorylase (glycogenosis type V, McArdle's disease). One patient was a newborn with phosphofructokinase deficiency (glycogenosis type VII, Tarui's disease). Of the study of our cases we would like to outline the following features: in the glycogenosis type II the deposit is fundamentally intralysosomal in the late infantile form, storage of mucopolysaccharides and deposit in interstitial fibroblasts were found, while in the adult form glycogen storage is minimal. In the glycogenosis type V the storage of glycogen is free and of a small amount. In two patients we have observed enzymatic activity in regenerating fibres. In glycogenosis type VII the storage is free, of considerable quantity and the interstitial cells are also affected; no storage is observed in the satellite cells.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type V; Glycogen Storage Disease Type VII; Humans; Male; Muscles; Muscular Diseases

Topics: Adult; Alkaline Phosphatase; alpha-Glucosidases; Amniotic Fluid; Cells, Cultured; Enzyme Induction; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Pregnancy; Prenatal Diagnosis

A herd of cattle which produces calves with generalised glycogenosis type II has been established. Seven affected animals have been born and their disease status as indicated by a decreased acid alpha-glucosidase activity and excessive glycogen deposition in muscle, can be detected on the day of birth. Two animals have died of heart failure aged 3 and 5 months and have shown cardiomegally. Five animals were clinically normal until 9 months of age when they failed to maintain weight gain, showed muscle weakness and four were killed aged between 12 and 16 months after showing difficulty in rising. All affected animals had abnormal ECG tracings and had elevated levels of CK, LDH and HBDH in serum. Excessive amounts of glycogen were deposited in voluntary, cardiac and smooth muscle, and in cells of the nervous system. The muscles showed a vacuolar myopathy. Both the infantile and late onset forms of generalised glycogenosis type II are present in this herd of cattle. The condition appears to be controlled by a recessive allele at a single autosomal locus.

Topics: alpha-Glucosidases; Animals; Animals, Newborn; Cattle; Cattle Diseases; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Male; Microscopy, Electron; Muscles

We present a case of glycogen storage disease type II (Pompe's disease) with the classical clinical presentation and characteristic electrocardiographic changes of this disorder. An acid maltase (EC 3.2.1.20) determination in the peripheral leukocytes revealed normal activity; however, acid maltase activity was completely absent in a pre-mortem skeletal muscle biopsy. Post-mortem studies showed acid maltase activity to be absent in all tissues examined, including cultured skin fibroblasts. Massive glycogen deposition corresponded to the localization of the enzymic deficiency, except in the brain, where glycogen content was within the normal range. The acid maltase activity in mixed peripheral leukocytes was due to an isoenzyme of acid maltase in the granulocyte series. Antenatal diagnosis was accurate in a subsequent pregnancy, but discordance between enzyme activity in different cell lines in an individual with a genetic disease is a conceivable source of error in both prenatal and postnatal diagnoses.

Topics: alpha-Glucosidases; Female; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Leukocytes; Muscles

Topics: alpha-Glucosidases; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Lipoproteins, LDL; Liver; Lysosomes; Muscles; Myocardium

Topics: alpha-Glucosidases; Animals; Coturnix; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Poultry Diseases; Quail

The authors report an uncommon case of type II glycogenosis. An 8-year-old boy developed a slow progressive myopathy. Biopsy of skeletal muscle showed scarce lesions under the optic microscope but in 50% of the fibers the presence of vacuoles filled with glycogen under the electron microscope. Ultrastructural analysis of fibroblasts in culture showed numerous vacuoles filled with glycogen, characteristic of type II glycogenosis. Enzymatic analysis revealed that acid-alpha-glucosidase activity was normal in muscle tissues but deeply deficient in leukocytes and fibroblasts in culture. This is, as far as we know, the first case with such a discrepancy in the distribution of the enzymatic activity, and it underlines the necessity of investigating several tissues in atypical cases.

Topics: alpha-Glucosidases; Biopsy; Child; Fibroblasts; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Leukocytes; Male; Microscopy, Electron; Muscles; Vacuoles

The concurrence of the infantile and adult form of acid maltase deficiency in one family is presented. The muscle of both cases possesses residual activity. The glycogen amount in the infantile form is increased tremendously, while in the late-onset the amount of glycogen is normal.

Topics: alpha-Glucosidases; Female; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Infant, Newborn; Infant, Newborn, Diseases; Male; Middle Aged; Muscles

The retina of a 9-month-old boy afflicted with biochemically proven type II glycogenosis contained abundant lysosomal glycogen. This was present in almost every cell type and occasionally associated with lipofuscin in choroidal macrophages. Lysosomal glycogen was absent from melanocytes and pigment epithelial cells. No degeneration of any cell layer was noted. The ubiquitous accretion of lysosomal glycogen resembles the widespread distribution of lipopigments in canine neural ceroid lipofuscinosis, another lysosomal disorder.

Topics: Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Lysosomes; Male; Microscopy, Electron; Retina

Furazolidone (FZ) at 700 ppm was added to feed mixtures fed turkey poults 2--5 weeks after hatching to induce acute experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. From EKG data obtained at weekly intervals, poults were selected for sacrifice at 5 and 10 weeks of age. Poults were sacrificed by cervical dislocation and appropriate samples of tissue from the left ventricle, liver, pectoralis and tibialis cranialis muscles were removed for glycogen assays. Character of glycogen, as determined by percent of branching and number of glucose units per segment, was not significantly altered in poults with spontaneous round heart disease or FZ-induced cardiomyopathy. This suggests that the glycogen accumulation noted in these conditions most closely resembles type II glycogenosis.

Topics: Animals; Cardiomyopathies; Furazolidone; Glucose; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Heart Diseases; Liver; Liver Glycogen; Muscles; Myocardium; Pectoralis Muscles; Poultry Diseases; Turkeys

Topics: Atrioventricular Node; Bundle of His; Cardiomyopathies; Consanguinity; Electrocardiography; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Muscles; Myocardium; Wolff-Parkinson-White Syndrome

Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.

Topics: Adolescent; Adult; alpha-Glucosidases; Antigen-Antibody Reactions; Cells, Cultured; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Kinetics; Middle Aged; Skin

3 adult women with distinct clinical pictures of progressive myopathy were studied. The morphological findings of biopsied skeletal muscle suggested the diagnosis of type II glycogenosis. Biochemical analysis confirmed a profound deficiency of alpha-1,4-glucosidase activity. Electrophoresis of muscle acid maltase showed the presence of one band in normal individuals. A very faint band with normal electrophoretic mobility was present in the patients' muscles. Muscle neutral maltase is composed of four bands in normal adult individuals: two of the four bands were clearly reduced in the muscles of the patients. The acid and neutral maltases were not significantly reduced in the patients' leukocytes. Acid maltase determination in urine made it possible to identify the homozygous, but not to completely segregate the heterozygous, from unaffected adult subjects.

Topics: alpha-Glucosidases; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Middle Aged; Mitochondria, Muscle; Muscles; Myofibrils; Vacuoles

Using electron microscopy, glycogen-filled lysosomes were found in peripheral lymphocytes in 5 cases of the infantile form of glycogenosis type II. In two infants whose blood smears were available, the ultrastructural demonstration of this pathognomonic storage corresponded to well-delineated vacuoles detected by routine light microscopy. Detection of such vacuoles in peripheral lymphocytes by light microscopy and demonstration of glycogen-filled lysosomes by electron microscopy could be a simple and harmless tool for diagnosing the classical form of type II glycogenosis.

Topics: Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Lymphocytes; Lysosomes; Male; Microscopy, Electron; Organoids; Vacuoles

The mild, generalized myopathy (glycogenosis type II) of a 23-year-old male, previously thought to have progressive muscular dystrophy, was studied clinically, electro-myographically, biochemically and with light- and electron microscopes. However, the history and clinical aspects, as well as the registration of high frequency discharges in the electromyogram first made the diagnosis uncertain. This kind of spontaneous activity has been found in nearly all cases reported in the literature. Light microscopic and histochemical examinations show vacular degeneration and glycogen storage in muscle fibres. With the electron microscope we found free dispersed glycogen in the cytoplasm and membrane-bound glycogen, glycogen-filled lysosomes. Biochemical measurements of the muscle enzymes, involved in the glycogen breakdown, were normal except for acid alpha-1,4-glucosidase, which was deficient. The evidence of these findings in this abortive form of glycogenosis type II is discussed and compared with the few cases found in the literature.

Topics: Adult; Diagnosis, Differential; Electromyography; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Histocytochemistry; Humans; Male; Muscles; Neuromuscular Diseases

Acid maltase deficiency is described in non-identical adult twins. The onset of the disease can be traced into late infancy; the clinical picture is one of severe muscular dystrophy; respiratory insuficiency was the cause of death in one case. The autopsy showed the central nervous system, heart and liver to be spared. Glycogen filled vacuoles are found in skin, mesenchymal cells, small nerves and skeletal muscles. The light microscopic study of 9 different muscles showed extremely variable involvement ranging from normal appearance to overt vacuolization. A 6--20% residual acid alpha-glucosidase activity was found in visceral organs, cultured fibroblasts and in some skeletal muscles. No satisfactory explanation can be given why this generalized acid alpha-glucosidase deficiency produces a selective involvement of skeletal muscles. If compared with infantile AMD (Pompe's disease) our cases have a much higher residual acid alpha-glucosidase activity and show the presence of an antigenically detectable protein. From our study and from a similar report in the literature (de Barsy et al., 1975), it appears that a combined approach of light microscopy, electron microscopy and biochemical analysis (determination of acid alpha-glucosidase) is necessary to make a diagnosis of AMD in adults.

Topics: Adult; Diseases in Twins; Female; Fibroblasts; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycoside Hydrolases; Humans; Male; Muscles; Muscular Atrophy; Pregnancy; Skin; Twins, Dizygotic

Topics: Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Male

Topics: Female; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycoside Hydrolases; Humans; Infant; Liposomes; Liver Glycogen; Muscles; Myocardium

Topics: Child; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Lymphocytes; Lysosomes; Microscopy, Electron

Pathological and biochemical data are reported on a 4(4)/12-year-old male patient with a severe myopathic disorder, hepatomegaly, recurrent pulmonary infections ending fatally. Combined morphological and enzymatic studies on muscle biopsy led to the diagnosis of acid maltase deficiency (Type II glycogenosis). On post mortem examination, lysosomal glycogen storage is found in skeletal muscles and liver, while heart and central nervous sytem are spared. Both hydrolytic and transferase activities of acid maltase are absent in cultured fibroblasts, heart, liver and postmortem skeletal muscles. That in the biopsied skeletal muscle only, the transferase activity alone is deficient while the hydrolytic function is maintained at low normal levels correlates well with the abnormal structure of the glycogen stored in that muscle. However, these findings on biopsied muscle cannot be reconciled with the absence of both functions and the presence of normal glycogen in other biopsied tissues or in postmortem specimens from the same patient.

Topics: Cartilage; Child, Preschool; Glucosidases; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Male; Muscles; Phenotype; Skin; Spinal Cord; Vacuoles

An autopsy case of Type II glycogenosis was reported with detailed description of ultrastructural findings. In addition to two typical patterns of glycogen deposition, membrane-bound lysosomal glycogen and membrane-free cytoplasmic glycogen, we observed numerous vacuolar structures in liver cells and a large deposition of nomogeneous materials between fragmented myocardial fibrils. These findings were briefly discussed in this manuscript.

Topics: Autopsy; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Infant; Infant, Newborn; Liver; Male; Myocardium

Topics: Child; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Kidney; Lysosomes

The clinical, biochemical, morphological and electrophysiological findings in a 13-month-old child, who died of glycogenosis type II, is presented. In addition to the deficiency of alpha-1,4-glucosidase, which is typical for the disease, a deficiency in hyaluronidase could be detected for the first time in the skeletal and heart muscles and in the liver. On the other hand, the beta-glucoronidase and beta-acetylglucosaminidase activity was highly increased. Deposits of a substance, most probably an acid mucopolysaccharide, which could be differentiated from glycogen by chromography and electronmicroscopy, could be detected in the muscle. A pathogenetical connection with the hyaluronidase defect is imminent.

Topics: Glucosidases; Glucuronidase; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycosaminoglycans; Hexosaminidases; Humans; Hyaluronoglucosaminidase; Infant; Kidney; Liver; Muscles; Myofibrils; Spleen; Vacuoles

Muscle biopsies were obtained from three infants under the age of 12 mth, each of whom was diagnosed as having Pompe's disease. The biopsies revealed a severe vacuolar myopathy with accumulation of large amounts of PAS positive material within the muscle fibres, changes similar to those in adult cases of the disease. In addition large amounts of metachromatic material were found within the muscle fibres in all three cases and in two of them scattered, rather sparse perivascular lymphocytic infiltrates were seen in the interstitial tissue. Review of material previously obtained from two adult cases showed no accumulation of metachromatic material in the older case and only moderate amounts in the younger. However, dense interstitial lymphocytic infiltrates were seen in the former, some concentrated around small vessels. These observations suggest that the pathogenesis of the muscle disorder in acid maltase deficiency may not depend on abnormal glycogen storage only.

Topics: Adult; Alcian Blue; Azure Stains; Basement Membrane; Female; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycosaminoglycans; Histocytochemistry; Humans; Inclusion Bodies; Infant; Lipids; Macrophages; Male; Microscopy, Electron; Muscle Tonus; Muscles; Myocardium; Periodic Acid; Sarcolemma; Schiff Bases; Tolonium Chloride; Vacuoles

Topics: alpha-Glucosidases; Brain; Female; Glycogen; Glycogen Storage Disease Type II; Humans; Infant; Liver; Lysosomes; Muscle, Skeletal; Myocardium; Pancreas; Tissue Distribution

Topics: Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Glycogenolysis; Humans

Topics: Child; Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Heart Diseases; Humans; Infant

Topics: Glycogen; Glycogen Storage Disease; Glycogen Storage Disease Type II; Heart; Heart Diseases; Humans