Glycolytic ATP fuels phosphoinositide 3-kinase signaling to support effector T helper 17 cell responses.

Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Proteomic profiling of polyglucosan bodies associated with glycogenin-1 deficiency in skeletal muscle.

AIMS: Polyglucosan storage disorders represent an emerging field within neurodegenerative and neuromuscular conditions, including Lafora disease (EPM2A, EPM2B), adult polyglucosan body disease (APBD, GBE1), polyglucosan body myopathies associated with RBCK1 deficiency (PGBM1, RBCK1) or glycogenin-1 deficiency (PGBM2, GYG1). While the storage material primarily comprises glycans, this study aimed to gain deeper insights into the protein components by proteomic profiling of the storage material in glycogenin-1 deficiency. METHODS: We employed molecular genetic analyses, quantitative mass spectrometry of laser micro-dissected polyglucosan bodies and muscle homogenate, immunohistochemistry and western blot analyses in muscle tissue from a 45-year-old patient with proximal muscle weakness from late teenage years due to polyglucosan storage myopathy. RESULTS: The muscle tissue exhibited a complete absence of glycogenin-1 due to a novel homozygous deep intronic variant in GYG1 (c.7+992T>G), introducing a pseudo-exon causing frameshift and a premature stop codon. Accumulated proteins in the polyglucosan bodies constituted components of glycogen metabolism, protein quality control pathways and desmin. Muscle fibres containing polyglucosan bodies frequently exhibited depletion of normal glycogen. CONCLUSIONS: The absence of glycogenin-1, a protein important for glycogen synthesis initiation, causes storage of polyglucosan that displays accumulation of several proteins, including those essential for glycogen synthesis, sequestosome 1/p62 and desmin, mirroring findings in RBCK1 deficiency. These results suggest shared pathogenic pathways across different diseases exhibiting polyglucosan storage. Such insights have implications for therapy in these rare yet devastating and presently untreatable disorders.

Endocrine involvement in hepatic glycogen storage diseases: pathophysiology and implications for care.

Hepatic glycogen storage diseases constitute a group of disorders due to defects in the enzymes and transporters involved in glycogen breakdown and synthesis in the liver. Although hypoglycemia and hepatomegaly are the primary manifestations of (most of) hepatic GSDs, involvement of the endocrine system has been reported at multiple levels in individuals with hepatic GSDs. While some endocrine abnormalities (e.g., hypothalamic­pituitary axis dysfunction in GSD I) can be direct consequence of the genetic defect itself, others (e.g., osteopenia in GSD Ib, insulin-resistance in GSD I and GSD III) may be triggered by the (dietary/medical) treatment. Being aware of the endocrine abnormalities occurring in hepatic GSDs is essential (1) to provide optimized medical care to this group of individuals and (2) to drive research aiming at understanding the disease pathophysiology. In this review, a thorough description of the endocrine manifestations in individuals with hepatic GSDs is presented, including pathophysiological and clinical implications.

Gene therapy for glycogen storage diseases.

Glycogen storage disorders (GSDs) are inherited disorders of metabolism resulting from the deficiency of individual enzymes involved in the synthesis, transport, and degradation of glycogen. This literature review summarizes the development of gene therapy for the GSDs. The abnormal accumulation of glycogen and deficiency of glucose production in GSDs lead to unique symptoms based upon the enzyme step and tissues involved, such as liver and kidney involvement associated with severe hypoglycemia during fasting and the risk of long-term complications including hepatic adenoma/carcinoma and end stage kidney disease in GSD Ia from glucose-6-phosphatase deficiency, and cardiac/skeletal/smooth muscle involvement associated with myopathy +/- cardiomyopathy and the risk for cardiorespiratory failure in Pompe disease. These symptoms are present to a variable degree in animal models for the GSDs, which have been utilized to evaluate new therapies including gene therapy and genome editing. Gene therapy for Pompe disease and GSD Ia has progressed to Phase I and Phase III clinical trials, respectively, and are evaluating the safety and bioactivity of adeno-associated virus vectors. Clinical research to understand the natural history and progression of the GSDs provides invaluable outcome measures that serve as endpoints to evaluate benefits in clinical trials. While promising, gene therapy and genome editing face challenges with regard to clinical implementation, including immune responses and toxicities that have been revealed during clinical trials of gene therapy that are underway. Gene therapy for the glycogen storage diseases is under development, addressing an unmet need for specific, stable therapy for these conditions.

Platelet glycogenolysis is important for energy production and function.

Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (i.e., activation, secretion, aggregation, and clot contraction) remains unclear. Patients with glycogen storage disease often present with increased bleeding and glycogen phosphorylase (GP) inhibitors, when used as treatments for diabetes, induce bleeding in preclinical studies suggesting some role for this form of glucose in hemostasis. In the present work, we examined how glycogen mobilization affects platelet function using GP inhibitors (CP316819 and CP91149) and a battery of ex vivo assays. Blocking GP activity increased glycogen levels in resting and thrombin-activated platelets and inhibited platelet secretion and clot contraction, with minimal effects on aggregation. Seahorse energy flux analysis and metabolite supplementation experiments suggested that glycogen is an important metabolic fuel whose role is affected by platelet activation and the availability of external glucose and other metabolic fuels. Our data shed light on the bleeding diathesis in glycogen storage disease patients and offer insights into the potential effects of hyperglycemia on platelets.

What did we know? Activated platelets transition from a low-energy-requiring, resting state to a high-energy-demanding state.Platelet glycogen is degraded upon activation.Glycogen storage disorders and glycogen phosphorylase inhibitors are associated with bleeding.What did we discover? Glycogen turnover occurs in resting platelets and its degradation is important for platelet functions.Glycogen phosphorylase inhibitors block secretion and clot contraction of which the latter can be reversed with alternative metabolic fuels.Glucose derived from glycogen may be routed through TCA/OxPhos versus aerobic glycolysis.What is the impact? Glycogen breakdown contributes to the high energy requirements of platelet function.Our work offers insights into potential energy sources in activated platelets.

The Liver and Glycogen: In Sickness and in Health.

The liver is a major store of glycogen and is essential in maintaining systemic glucose homeostasis. In healthy individuals, glycogen synthesis and breakdown in the liver are tightly regulated. Abnormal glycogen metabolism results in prominent pathological changes in the liver, often manifesting as hepatic glycogenosis or glycogen inclusions. This can occur in genetic glycogen storage disease or acquired conditions with insulin dysregulation such as diabetes mellitus and non-alcoholic fatty liver disease or medication effects. Some primary hepatic tumors such as clear cell hepatocellular carcinoma also demonstrate excessive glycogen accumulation. This review provides an overview of the pathological manifestations and molecular mechanisms of liver diseases associated with abnormal glycogen accumulation.

Proteomic characterisation of polyglucosan bodies in skeletal muscle in RBCK1 deficiency.

AIMS: Several neurodegenerative and neuromuscular disorders are characterised by storage of polyglucosan, consisting of proteins and amylopectin-like polysaccharides, which are less branched than in normal glycogen. Such diseases include Lafora disease, branching enzyme deficiency, glycogenin-1 deficiency, polyglucosan body myopathy type 1 (PGBM1) due to RBCK1 deficiency and others. The protein composition of polyglucosan bodies is largely unknown. METHODS: We combined quantitative mass spectrometry, immunohistochemical and western blot analyses to identify the principal protein components of polyglucosan bodies in PGBM1. Histologically stained tissue sections of skeletal muscle from four patients were used to isolate polyglucosan deposits and control regions by laser microdissection. Prior to mass spectrometry, samples were labelled with tandem mass tags that enable quantitative comparison and multiplexed analysis of dissected samples. To study the distribution and expression of the accumulated proteins, immunohistochemical and western blot analyses were performed. RESULTS: Accumulated proteins were mainly components of glycogen metabolism and protein quality control pathways. The majority of fibres showed depletion of glycogen and redistribution of key enzymes of glycogen metabolism to the polyglucosan bodies. The polyglucosan bodies also showed accumulation of proteins involved in the ubiquitin-proteasome and autophagocytosis systems and protein chaperones. CONCLUSIONS: The sequestration of key enzymes of glycogen metabolism to the polyglucosan bodies may explain the glycogen depletion in the fibres and muscle function impairment. The accumulation of components of the protein quality control systems and other proteins frequently found in protein aggregate disorders indicates that protein aggregation may be an essential part of the pathobiology of polyglucosan storage.

A Mouse Model of Glycogen Storage Disease Type IX-Beta: A Role for Phkb in Glycogenolysis.

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, ß, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (Phkb−/−) mouse model. In this study, we assessed fasting blood glucose and ketone levels, serum metabolite concentrations, glycogen phosphorylase activity, and gene expression of gluconeogenic genes and fibrotic genes. Phkb−/− mice displayed hepatomegaly with lower fasting blood glucose concentrations. Phkb−/− mice showed partial liver glycogen phosphorylase activity and increased sensitivity to pyruvate, indicative of partial glycogenolytic activity and upregulation of gluconeogenesis. Additionally, gene expression analysis demonstrated increased lipid metabolism in Phkb−/− mice. Gene expression analysis and liver histology in the livers of old Phkb−/− mice (>40 weeks) showed minimal profibrogenic features when analyzed with age-matched wild-type (WT) mice. Collectively, the Phkb−/− mouse recapitulates mild clinical features in patients with GSD-IX-beta. Metabolic and molecular analysis confirmed that Phkb−/− mice were capable of sustaining energy homeostasis during prolonged fasting by using partial glycogenolysis, increased gluconeogenesis, and potentially fatty acid oxidation in the liver.

Gene therapy for glycogen storage diseases.

The focus of this review is the development of gene therapy for glycogen storage diseases (GSDs). GSD results from the deficiency of specific enzymes involved in the storage and retrieval of glucose in the body. Broadly, GSDs can be divided into types that affect liver or muscle or both tissues. For example, glucose-6-phosphatase (G6Pase) deficiency in GSD type Ia (GSD Ia) affects primarily the liver and kidney, while acid α-glucosidase (GAA) deficiency in GSD II causes primarily muscle disease. The lack of specific therapy for the GSDs has driven efforts to develop new therapies for these conditions. Gene therapy needs to replace deficient enzymes in target tissues, which has guided the planning of gene therapy experiments. Gene therapy with adeno-associated virus (AAV) vectors has demonstrated appropriate tropism for target tissues, including the liver, heart and skeletal muscle in animal models for GSD. AAV vectors transduced liver and kidney in GSD Ia and striated muscle in GSD II mice to replace the deficient enzyme in each disease. Gene therapy has been advanced to early phase clinical trials for the replacement of G6Pase in GSD Ia and GAA in GSD II (Pompe disease). Other GSDs have been treated in proof-of-concept studies, including GSD III, IV and V. The future of gene therapy appears promising for the GSDs, promising to provide more efficacious therapy for these disorders in the foreseeable future.

Biomarkers in Glycogen Storage Diseases: An Update.

Glycogen storage diseases (GSDs) are a group of 19 hereditary diseases caused by a lack of one or more enzymes involved in the synthesis or degradation of glycogen and are characterized by deposits or abnormal types of glycogen in tissues. Their frequency is very low and they are considered rare diseases. Except for X-linked type IX, the different types are inherited in an autosomal recessive pattern. In this study we reviewed the literature from 1977 to 2020 concerning GSDs, biomarkers, and metabolic imbalances in the symptoms of some GSDs. Most of the reported studies were performed with very few patients. Classification of emerging biomarkers between different types of diseases (hepatics GSDs, McArdle and PDs and other possible biomarkers) was done for better understanding. Calprotectin for hepatics GSDs and urinary glucose tetrasaccharide for Pompe disease have been approved for clinical use, and most of the markers mentioned in this review only need clinical validation, as a final step for their routine use. Most of the possible biomarkers are implied in hepatocellular adenomas, cardiomyopathies, in malfunction of skeletal muscle, in growth retardation, neutropenia, osteopenia and bowel inflammation. However, a few markers have lost interest due to a great variability of results, which is the case of biotinidase, actin alpha 2, smooth muscle, aorta and fibroblast growth factor receptor 4. This is the first review published on emerging biomarkers with a potential application to GSDs.

Benign or not benign? Deep phenotyping of liver Glycogen Storage Disease IX.

INTRODUCTION: Liver Glycogen Storage Disease Type IX (GSD IX) is one of the most common forms of GSD. It is caused by a deficiency in enzyme phosphorylase kinase (PhK), a complex, hetero-tetrameric enzyme comprised of four subunits - α, ß, γ, and δ - each with tissue specific isoforms encoded by different genes. Until the recent availability of gene panels and exome sequencing, the diagnosis of liver GSD IX did not allow for differentiation of these subtypes. This study presents the first comprehensive literature review for liver GSD IX subtypes - GSD IX α2, ß, and γ2. We aim to better characterize the natural history of liver GSD IX and further investigate if there are subtype-specific differences in clinical presentation. METHODS: A comprehensive literature review was performed with the help of a medical librarian at Duke University Medical Center to gather all published patients of liver GSD IX. Our refined search yielded 74 articles total. Available patient data were compiled into an excel spreadsheet. Data were analyzed via descriptive statistics. The number of patients with specific symptoms were individually summed and reported as a percentage of the total number of patients for which data were available or were averaged and reported as a mean numerical value. Published pathology reports were scored using the International Association of the Study of the Liver Scale. RESULTS: There were a total of 183 GSD IX α2 patients, 17 GSD IX ß patients, and 30 GSD IX γ2 patients. Average age at diagnosis was 4 years for GSD IX α2 patients, 2.34 years for GSD IX ß patients, and 1.81 years for GSD IX γ2 patients. Hepatomegaly was reported in 164/176 (93.2%) of GSD IX α2 patients, 16/17 (94.1%) of GSD IX ß patients, and 30/30 (100%) of GSD IX γ2 patients. Fasting hypoglycemia was reported in 53/121 (43.8%) of GSD IX α2 patients, 8/16 (50%) of GSD IX ß patients, and 18/19 (94.7%) of GSD IX γ2 patients. Liver biopsy pathology reports were available and interpreted for 46 GSD IX α2 patients, 3 GSD IX ß patients, and 24 GSD IX γ2 patients. 22/46 (47.8%) GSD IX α2 patients, 1/3 (33.3%) GSD IX ß patients, and 23/24 (95.8%) GSD IX γ2 patients with available pathology reports documented either some degree of fibrosis or cirrhosis. CONCLUSION: Our comprehensive review demonstrates quantitatively that the clinical presentation of GSD IX γ2 patients is more severe than that of GSD IX α2 or ß patients. However, our study also shows the existence of a severe phenotype in GSD IX α2, evidenced by early onset liver pathology in conjunction with clinical symptoms. There is need for a more robust natural history study to better understand the variability in liver pathophysiology within liver GSD IX; in addition, further study of mutations and gene mapping could bring a better understanding of the relationship between genotype and clinical presentation.

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1.

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease-related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild-type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. SYNOPSIS: Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

RBCK1-related disease: A rare multisystem disorder with polyglucosan storage, auto-inflammation, recurrent infections, skeletal, and cardiac myopathy-Four additional patients and a review of the current literature.

In this article, we report four new patients, from three kindreds, with pathogenic variants in RBCK1 and a multisystem disorder characterised by widespread polyglucosan storage. We describe the clinical presentation of progressive skeletal and cardiac myopathy, combined immunodeficiencies and auto-inflammation, illustrate in detail the histopathological findings in multiple tissue types, and report muscle MRI findings.

From the seminal discovery of proteoglycogen and glycogenin to emerging knowledge and research on glycogen biology.

Although the discovery of glycogen in the liver, attributed to Claude Bernard, happened more than 160 years ago, the mechanism involved in the initiation of glucose polymerization remained unknown. The discovery of glycogenin at the core of glycogen's structure and the initiation of its glucopolymerization is among one of the most exciting and relatively recent findings in Biochemistry. This review focuses on the initial steps leading to the seminal discoveries of proteoglycogen and glycogenin at the beginning of the 1980s, which paved the way for subsequent foundational breakthroughs that propelled forward this new research field. We also explore the current, as well as potential, impact this research field is having on human health and disease from the perspective of glycogen storage diseases. Important new questions arising from recent studies, their links to basic mechanisms involved in the de novo glycogen biogenesis, and the pervading presence of glycogenin across the evolutionary scale, fueled by high throughput -omics technologies, are also addressed.

A novel PHKA2 mutation in a Chinese child with glycogen storage disease type IXa: a case report and literature review.

BACKGROUND: PHKA2 gene mutations can cause liver phosphorylase kinase (PhK) deficiency, resulting in glycogen storage disease type IXa (GSD IXa). Elevated liver transaminase levels and liver enlargement are the most frequent phenotypes of GSD IXa. However, whether the phenotypes are applicable to Chinese patients remains unclear. CASE REPORT: A boy aged 2 years and 8 months with a history of episodic fatigue and weakness since he was 2 years old was referred to our endocrinology clinic. Apart from symptomatic ketotic hypoglycemic episodes (palpitation, hand shaking, sweating, etc.), no abnormalities of liver transaminase levels or liver size were found. To identify the aetiology of his clinically diagnosed hypoglycaemia, the proband and his parents were screened for PHKA2 gene mutations by next-generation sequencing. A heterozygous mutation (c.2972C > G, p.G991A) in PHKA2 was found in the proband and his mother. Twenty-one Chinese cases with GSD IXa have been reported in the literature to date, and elevated liver transaminase levels (95%) and liver enlargement (91%) are the most frequent phenotypes of GSD IXa in Chinese patients. Hypoglycaemia may be one of the early onset symptoms in infants with GSD IXa. CONCLUSIONS: This study enriches our knowledge of the PHKA2 gene mutation spectrum and provides further information about the phenotypic characteristics of Chinese GSD IXa patients.

G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a.

Glycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase-/- mouse model (L-G6PC-/-) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.

Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

Novel method for detection of glycogen in cells.

y: Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.

A novel mouse model that recapitulates adult-onset glycogenosis type 4.

Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen-branching enzyme (GBE). The diagnostic hallmark of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age at onset. Complete loss of enzyme activity is lethal in utero or in infancy and affects primarily the muscle and the liver. However, residual enzyme activity as low as 5-20% leads to juvenile or adult onset of a disorder that primarily affects the central and peripheral nervous system and muscles and in the latter is termed adult polyglucosan body disease (APBD). Here, we describe a mouse model of GSD IV that reflects this spectrum of disease. Homologous recombination was used to knock in the most common GBE1 mutation p.Y329S c.986A > C found in APBD patients of Ashkenazi Jewish decent. Mice homozygous for this allele (Gbe1(ys/ys)) exhibit a phenotype similar to APBD, with widespread accumulation of PG. Adult mice exhibit progressive neuromuscular dysfunction and die prematurely. While the onset of symptoms is limited to adult mice, PG accumulates in tissues of newborn mice but is initially absent from the cerebral cortex and heart muscle. Thus, PG is well tolerated in most tissues, but the eventual accumulation in neurons and their axons causes neuropathy that leads to hind limb spasticity and premature death. This mouse model mimics the pathology and pathophysiologic features of human adult-onset branching enzyme deficiency.

Multiple phenotypes in phosphoglucomutase 1 deficiency.

BACKGROUND: Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS: Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS: Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS: Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).