Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Severe neuromuscular forms of glycogen storage disease type IV: Histological, clinical, biochemical, and molecular findings in a large French case series.

Glycogen storage disease type IV (GSD IV), also called Andersen disease, or amylopectinosis, is a highly heterogeneous autosomal recessive disorder caused by a glycogen branching enzyme (GBE, 1,4-alpha-glucan branching enzyme) deficiency secondary to pathogenic variants on GBE1 gene. The incidence is evaluated to 1:600 000 to 1:800 000 of live births. GBE deficiency leads to an excessive deposition of structurally abnormal, amylopectin-like glycogen in affected tissues (liver, skeletal muscle, heart, nervous system, etc.). Diagnosis is often guided by histological findings and confirmed by GBE activity deficiency and molecular studies. Severe neuromuscular forms of GSD IV are very rare and of disastrous prognosis. Identification and characterization of these forms are important for genetic counseling for further pregnancies. Here we describe clinical, histological, enzymatic, and molecular findings of 10 cases from 8 families, the largest case series reported so far, of severe neuromuscular forms of GSD IV along with a literature review. Main antenatal features are: fetal akinesia deformation sequence or arthrogryposis/joint contractures often associated with muscle atrophy, decreased fetal movement, cystic hygroma, and/or hydrops fetalis. If pregnancy is carried to term, the main clinical features observed at birth are severe hypotonia and/or muscle atrophy, with the need for mechanical ventilation, cardiomyopathy, retrognathism, and arthrogryposis. All our patients were stillborn or died within 1 month of life. In addition, we identified five novel GBE1 variants.

[Infant glycogen storage disease type â £: a clinicopathological and genetic characteristics analysis of five cases].

Objective: To investigate the clinical pathology and gene mutation characteristics of patients with glycogen storage disease type Ⅳ (GSD Ⅳ). Methods: The clinical data, liver histopathology and ultrastructural morphology, and gene sequencing results of 5 GSD Ⅳ cases diagnosed in the Children's Hospital Affiliated to Shanghai Jiaotong University School of Medicine and the Children's Hospital of Fudan University from January 2015 to February 2022 were collected and analyzed retrospectively. Results: Among the 5 cases, 3 were male and 2 were female, ranging in age from 4 months to 1 year and 9 months. The clinical manifestations included fever, hepatosplenomegaly, liver insufficiency, growth retardation and hypotonia. Four cases had liver biopsy showing ground-glass-like changes in hepatocytes with intracytoplasmic inclusion bodies and varying degrees of fibrosis. Liver electron microscopy in 2 cases showed that the level of glycogen increased to varying degrees, and the cytoplasm was filled with low electron density substances. Genetic testing revealed that 3 cases had compound heterozygous variants in GBE1 gene; 1 case had a single pathogenic variant in GBE1 gene; and 1 case was deceased with no genetic testing, but each parent was tested for a heterozygous variant in the GBE1 gene. A total of 9 GBE1 gene mutations were detected, 3 of which were reported mutations and 6 novel mutations. One case died of liver cirrhosis, and 1 case underwent autologous liver transplantation. After transplantation, the liver function basically returned to normal, and the growth and development improved; the other 3 cases were managed through diet control and symptomatic treatment. Conclusions: CSD Ⅳ is an extremely rare inherited metabolic disease caused by GBE1 gene mutation, often presenting with hepatic and neuromuscular disorders, with heterogeneous clinical manifestations. The diagnosis mainly depends on histopathology and a pedigree gene analysis.

Glycogen storage disease type IV without detectable polyglucosan bodies: importance of broad gene panels.

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme 1 (GBE1) gene and is characterized by accumulation of polyglucosan bodies in liver, muscle and other tissues. We report three cases with neuromuscular forms of GSD IV, none of whom had polyglucosan bodies on muscle biopsy. The first case had no neonatal problems and presented with delayed walking. The other cases presented at birth: one with arthrogryposis, hypotonia, and respiratory distress, the other with talipes and feeding problems. All developed a similar pattern of axial weakness, proximal upper limb weakness and scapular winging, and much milder proximal lower limb weakness. Our cases expand the phenotypic spectrum of neuromuscular GSD IV, highlight that congenital myopathy and limb girdle weakness can be caused by mutations in GBE1, and emphasize that GSD IV should be considered even in the absence of characteristic polyglucosan bodies on muscle biopsy.

Diagnosis and management of glycogen storage disease type IV, including adult polyglucosan body disease: A clinical practice resource.

Glycogen storage disease type IV (GSD IV) is an ultra-rare autosomal recessive disorder caused by pathogenic variants in GBE1 which results in reduced or deficient glycogen branching enzyme activity. Consequently, glycogen synthesis is impaired and leads to accumulation of poorly branched glycogen known as polyglucosan. GSD IV is characterized by a remarkable degree of phenotypic heterogeneity with presentations in utero, during infancy, early childhood, adolescence, or middle to late adulthood. The clinical continuum encompasses hepatic, cardiac, muscular, and neurologic manifestations that range in severity. The adult-onset form of GSD IV, referred to as adult polyglucosan body disease (APBD), is a neurodegenerative disease characterized by neurogenic bladder, spastic paraparesis, and peripheral neuropathy. There are currently no consensus guidelines for the diagnosis and management of these patients, resulting in high rates of misdiagnosis, delayed diagnosis, and lack of standardized clinical care. To address this, a group of experts from the United States developed a set of recommendations for the diagnosis and management of all clinical phenotypes of GSD IV, including APBD, to support clinicians and caregivers who provide long-term care for individuals with GSD IV. The educational resource includes practical steps to confirm a GSD IV diagnosis and best practices for medical management, including (a) imaging of the liver, heart, skeletal muscle, brain, and spine, (b) functional and neuromusculoskeletal assessments, (c) laboratory investigations, (d) liver and heart transplantation, and (e) long-term follow-up care. Remaining knowledge gaps are detailed to emphasize areas for improvement and future research.

Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency.

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

Alteration of mitochondrial function in the livers of mice with glycogen branching enzyme deficiency.

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1+/-) mice. Mitochondria isolated from the livers of Gbe1+/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates.

The potential of dietary treatment in patients with glycogen storage disease type IV.

There is paucity of literature on dietary treatment in glycogen storage disease (GSD) type IV and formal guidelines are not available. Traditionally, liver transplantation was considered the only treatment option for GSD IV. In light of the success of dietary treatment for the other hepatic forms of GSD, we have initiated this observational study to assess the outcomes of medical diets, which limit the accumulation of glycogen. Clinical, dietary, laboratory, and imaging data for 15 GSD IV patients from three centres are presented. Medical diets may have the potential to delay or prevent liver transplantation, improve growth and normalize serum aminotransferases. Individual care plans aim to avoid both hyperglycaemia, hypoglycaemia and/or hyperketosis, to minimize glycogen accumulation and catabolism, respectively. Multidisciplinary monitoring includes balancing between traditional markers of metabolic control (ie, growth, liver size, serum aminotransferases, glucose homeostasis, lactate, and ketones), liver function (ie, synthesis, bile flow and detoxification of protein), and symptoms and signs of portal hypertension.

Hallmarks of oxidative stress in the livers of aged mice with mild glycogen branching enzyme deficiency.

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1+/-), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1+/- mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1+/- livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress.

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.

Glycogen Storage Disease Type IV Diagnosed at Fetal Autopsy.

Glycogen storage disease type IV (GSD IV; Andersen's disease) is a rare autosomal recessive disorder that results from defects in the GBE1 gene (3p12.2) and subsequent deficiencies of glycogen branching. We report a case of GSD IV diagnosed at autopsy in a 35 4/7 weeks gestational age female neonate that died shortly after birth. Multisystem blue, ground glass inclusions initially presumed artefactual were periodic acid-Schiff positive, diastase resistant. Chromosomal microarray analysis identified a deletion of exons 2 through 16 of the GBE1 gene and whole exome sequencing identified a nonsense mutation within exon 14, confirming the diagnosis of GSD IV. A strong index of suspicion was required determine GSD IV as the ultimate cause of death, illustrating the need for critical evaluation of postmortem artifact in the setting of fetal demise of unknown etiology and highlighting the role of postmortem molecular diagnostics in a subset of cases.

Glycogen storage disease type IV: dilated cardiomyopathy as the isolated initial presentation in an adult patient.

Glycogen storage disease type IV (GSD IV, Andersen disease) is a rare autosomal recessive condition. The childhood neuromuscular subtype of GSD IV is characterised by a progressive skeletal myopathy with cardiomyopathy also reported in some individuals. We report a case of a 19-year-old man who presented with severe non-ischaemic dilated cardiomyopathy (NIDCM) necessitating heart transplantation, with biopsy showing aggregations of polyglucosan bodies in cardiac myocytes. He had no signs or symptoms of muscle weakness, liver dysfunction or neurologic involvement. A homozygous GBE1 c.607C>A (p.His203Asn) variant was identified. Our case is unusual in that our patient presented with an isolated NIDCM in the absence of other clinical manifestations of GSD IV. This case highlights the importance of considering storage disorders in young adults presenting with isolated NIDCM of unknown aetiology. It also emphasises the potential synergy between histopathological evaluation and genomic testing in enhancing diagnostic certainty.

Update on polyglucosan storage diseases.

An abnormal structural form of glycogen (with less branching points or amylopectin-like polysaccharide) called polyglucosan (PG) may accumulate in various tissues such as striated and smooth muscles, brain, nerve, liver and skin, and cause a group of nine different genetic disorders manifesting with a variety of clinical phenotypes that affect mainly the nervous system (Lafora disease, adult PG body disease), the heart (glycogen storage disease type XV, hypertrophic cardiomyopathy type 6, PG body myopathy type 1) and the skeletal muscle (glycogen storage disease type IV, glycogen storage disease type VII, PG body myopathy type 2), depending on the organs which are mostly affected by the PG aggregates. The pathological feature of PG storage in tissues is a hallmark of these disorders. Whole-genome sequencing has allowed to obtain a diagnosis in a large number of patients with a previously unrecognized disorder. We describe the clinical, pathological and molecular features of these genetic disorders, for many of which the pathological mechanisms underlying the corresponding mutant gene have been investigated and, at least in part, understood.

Novel pathogenic variants in GBE1 causing fetal akinesia deformation sequence and severe neuromuscular form of glycogen storage disease type IV.

Glycogen storage disease IV (GSD IV), caused by a defect in GBE1, is a clinically heterogeneous disorder. A classical hepatic form and a neuromuscular form have been described. The severe neuromuscular form presents as a fetal akinesia deformation sequence or a congenital subtype. We ascertained three unrelated families with fetuses/neonates who presented with fetal akinesia deformation sequence to our clinic for genetic counseling. We performed a detailed clinical evaluation, exome sequencing, and histopathology examination of two fetuses and two neonates from three unrelated families presenting with these perinatally lethal neuromuscular forms of GSD IV. Exome sequencing in the affected fetuses/neonates identified four novel pathogenic variants (c.1459G>T, c.144-1G>A, c.1680C>G, and c.1843G>C) in GBE1 (NM_000158). Histopathology examination of tissues from the affected fetuses/neonate was consistent with the diagnosis. Here, we add three more families with the severe perinatally lethal neuromuscular forms of GSD IV to the GBE1 mutation spectrum.

Polyglucosan Bodies in Placental Extravillious Trophoblast for the Diagnosis of Fatal Perinatal Neuromuscular-type Glycogen Storage Disease Type IV.

The fatal infantile neuromuscular type is the most severe form of glycogen storage disease type IV (GSD IV). We report a case of a 22-day-old female neonate born at 34 weeks gestation with polyhyramnios, fetal hydrops, and severe hypotonia. Placental examination revealed numerous periodic acid schiff-positive diastase-resistant polyglucosan bodies in the cytoplasm of extravillous trophoblast predominantly in the placental basal plate. Muscle biopsy and autopsy findings supported a diagnosis of neuromuscular-type glycogen storage disease type IV with extensive involvement of skeletal muscle, heart, and liver. The diagnosis was confirmed by molecular genetic testing. We could only find 1 prior report in the English literature that describes placental pathological changes. Our findings suggest that placental examination can be a useful adjunct for early diagnosis, as placentas are often received for pathological examination shortly after birth and usually before a diagnostic muscle biopsy can be performed. Pathologists need to be aware of characteristic placental features.

A Case of Glycogen Storage Disease IV with Rare Homozygous Mutations in the Glycogen Branching Enzyme Gene / 대한소아소화기영양학회지

Glycogen storage disease (GSD) IV is a rare autosomal recessive inherited disorder caused by mutations in the gene coding for glycogen branching enzyme leading to progressive liver disease. GSD IV is associated with mutations in GBE1, which encodes the glycogen branching enzyme. We report a case of GSD IV with rare homozygous mutations in the GBE1 gene (c.791G>A (p.Gly264Glu), which was successfully treated by liver transplantation.

Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy.

Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1ys/ys). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1ys/ys mice at a dose of 5 × 1011 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1ys/ys mice.