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.

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.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

A novel GBE1 gene variant in a child with glycogen storage disease type IV.

Glycogen storage disease type IV is an autosomal recessive disorder of carbohydrates caused by deficiency of amylo-1-4-glycanoglycosyltransferase, which leads to accumulation of amylopectin-like polysaccharides in tissues including liver, heart and neuromuscular system. More than 40 different mutations in the glycogen branching enzyme gene (GBE1) have been described. In this study, we report a 2-year-old boy who presented with developmental delay and muscle weakness. He subsequently was diagnosed with glycogen storage disease type IV based on a liver biopsy histology and electron microscopy. Glycogen branching enzyme activity was in the low range. Genetic analysis demonstrated a novel heterozygous variant (c.760A>G; p.Thr254Ala) in exon 6 of the GBE1 gene, which is believed to be pathogenic. This variant was inherited from the patient's mother who was asymptomatic with normal glycogen branching enzyme activity. Whole-exome sequencing failed to reveal additional variations in the GBE1 gene.

Role in tumor growth of a glycogen debranching enzyme lost in glycogen storage disease.

BACKGROUND: Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS: Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS: We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS: Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.

Mouse model of glycogen storage disease type III.

Glycogen storage disease type IIIa (GSD IIIa) is caused by a deficiency of the glycogen debranching enzyme (GDE), which is encoded by the Agl gene. GDE deficiency leads to the pathogenic accumulation of phosphorylase limit dextrin (PLD), an abnormal glycogen, in the liver, heart, and skeletal muscle. To further investigate the pathological mechanisms behind this disease and develop novel therapies to treat this disease, we generated a GDE-deficient mouse model by removing exons after exon 5 in the Agl gene. GDE reduction was confirmed by western blot and enzymatic activity assay. Histology revealed massive glycogen accumulation in the liver, muscle, and heart of the homozygous affected mice. Interestingly, we did not find any differences in the general appearance, growth rate, and life span between the wild-type, heterozygous, and homozygous affected mice with ad libitum feeding, except reduced motor activity after 50 weeks of age, and muscle weakness in both the forelimb and hind legs of homozygous affected mice by using the grip strength test at 62 weeks of age. However, repeated fasting resulted in decreased survival of the knockout mice. Hepatomegaly and progressive liver fibrosis were also found in the homozygous affected mice. Blood chemistry revealed that alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities were significantly higher in the homozygous affected mice than in both wild-type and heterozygous mice and the activity of these enzymes further increased with fasting. Creatine phosphokinase (CPK) activity was normal in young and adult homozygous affected mice. However, the activity was significantly elevated after fasting. Hypoglycemia appeared only at a young age (3 weeks) and hyperlipidemia was not observed in our model. In conclusion, with the exception of normal lipidemia, these mice recapitulate human GSD IIIa; moreover, we found that repeated fasting was detrimental to these mice. This mouse model will be useful for future investigation regarding the pathophysiology and treatment strategy of human GSD III.

High frequency of W1327X mutation in glycogen storage disease type III patients from central Tunisia.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by the deficiency of glycogen debranching enzyme (AGL). It is characterized by hepatomegaly, progressive myopathy, cardiomyopathy and fasting hypoglycemia. Several mutations in AGL gene have been described in different populations. The W1327X mutation was reported in one Tunisian patient resident in Italy. We looked in this report to determine the frequency of W1327X mutation among Tunisian patients. The W1327X mutation was screening in 26 GSD III patients originated from various geographic locations in Tunisia. The sequence analysis revealed that among nine patients carried the W1327X mutation. Eight of them were from six unrelated families and they were originated from Mahdia (centre of Tunisia) suggesting the existence of a founder effect in this region. Taking into account historical migratory waves, screening for this mutation should be performed in priority for molecular diagnosis confirmation of GSD III in North African populations.

Bulbar muscle weakness and fatty lingual infiltration in glycogen storage disorder type IIIa.

Glycogen storage disorder type III (GSD III) is a rare autosomal recessive disorder resulting from a deficiency of glycogen debranching enzyme, critical in cytosolic glycogen degradation. GSD IIIa, the most common form of GSD III, primarily affects the liver, cardiac muscle, and skeletal muscle. Although skeletal muscle weakness occurs commonly in GSD IIIa, bulbar muscle involvement has not been previously reported. Here we present three GSD IIIa patients with clinical evidence of bulbar weakness based on instrumental assessment of lingual strength. Dysarthria and/or dysphagia, generally mild in severity, were evident in all three individuals. One patient also underwent correlative magnetic resonance imaging (MRI) which was remarkable for fatty infiltration at the base of the intrinsic tongue musculature, as well as abnormal expansion of the fibro-fatty lingual septum. Additionally, we provide supportive evidence of diffuse glycogen infiltration of the tongue at necropsy in a naturally occurring canine model of GSD IIIa. While further investigation in a larger group of patients with GSD III is needed to determine the incidence of bulbar muscle involvement in this condition and whether it occurs in GSD IIIb, clinical surveillance of lingual strength is recommended.

Glycogen storage disease type III in the Irish population.

Glycogen storage disease type III (GSD III) results from mutations of the AGL gene encoding the glycogen debrancher enzyme. The disease has clinical and biochemical heterogeneity reflecting the severity of the AGL mutations. We sought to characterise the molecular defects in our cohort of Irish patients with GSD III. Fifteen patients from eight unrelated Irish families were identified: six males and nine females. The age ranged from 2-39 years old, and all presented in the first 3 years of life. Four patients (of three families) had mild disease with hepatomegaly, mild hypoglycaemia and normal creatine kinase (CK) levels. Five families had more severe disease, with liver and skeletal muscle involvement and elevated CK. Eleven different mutations were identified amongst the eight families. Of the 11, six were novel: p.T512fs, p.S736fs, p.A1400fs, p.K1407fs, p.Y519X and p.D627Y. The family homozygous for p.A1400fs had the most severe phenotype (early-onset hypoglycaemia, massive hepatomegaly, myopathy and hypertrophic cardiomyopathy before age 2 years), which was not halted by aggressive carbohydrate and protein supplementation. Conversely, the only missense mutation identified in the cohort, p.D627Y, was associated with a mild phenotype. The phenotypic diversity in our GSD III cohort is mirrored by the allelic heterogeneity. We describe two novel null mutations in exon 32 in two families with severe GSD III resistant to current treatment modalities. Knowledge of the specific mutations segregating in this cohort may allow for the development of new therapeutic interventions.

Neuropathological study of skeletal muscle, heart, liver, and brain in a neonatal form of glycogen storage disease type IV associated with a new mutation in GBE1 gene.

Glycogen storage disease type IV (GSD IV, or Andersen disease) is an autosomal recessive disorder due to the deficiency of 1,4-alpha-glucan branching enzyme (or glycogen branching enzyme, GBE1), resulting in an accumulation of amylopectin-like polysaccharide in muscle, liver, heart and central and peripheral nervous system. Typically, the presentation is in childhood with liver involvement up to cirrhosis. The neuromuscular form varies in onset (congenital, perinatal, juvenile and adult) and in severity. Congenital cases are rare, and fewer than 20 cases have been described and genetically determined so far. This form is characterized by polyhydramnios, neonatal hypotonia, and neuronal involvement; hepatopathy is uncommon, and the babies usually die between 4 weeks and 4 months of age. We report the case of an infant who presented severe hypotonia, dilatative cardiomyopathy, mild hepatopathy, and brain lateral ventricle haemorrhage, features consistent with the congenital form of GSD IV. He died at one month of life of cardiorespiratory failure. Muscle biopsy and heart and liver autoptic specimens showed many vacuoles filled with PAS-positive diastase-resistant materials. Electron-microscopic analysis showed mainly polyglucosan accumulations in all the tissues examined. Postmortem examination showed the presence of vacuolated neurons containing this abnormal polysaccharide. GBE1 biochemical activity was virtually absent in muscle and fibroblasts, and totally lacking in liver and heart as well as glycogen synthase activity. GBE1 gene sequence analysis revealed a novel homozygous nonsense mutation, p.E152X, in exon 4, correlating with the lack of enzyme activity and with the severe neonatal involvement. Our findings contribute to increasing the spectrum of mutation associated with congenital GSD IV.

Clinicopathological analysis of the homozygous p.W1327X AGL mutation in glycogen storage disease type 3.

We report on clinicopathological and whole body MRI analyses of the index patient of a large nonconsanguineous German-Ukraine family with homozygous and heterozygous AGL gene mutations at position p.W1327X (c.3980G > A). There are only limited reports on this phenotype with a homozygous genotype. The index patient, a 49-year-old woman presented with hepatomegaly, cardiomyopathy and moderate progressive proximal limb myopathy. Skeletal muscle showed severe vacuolar myopathy with storage of PAS-positive non-membrane-limited glycogen. An increase in glycogen content and completely decrease of debranching enzyme activity was measured in erythrocytes. Mutational analysis of the AGL gene showed a homozygous p.W1327X mutation. In the family, two brothers had been affected by severe infantile onset hepatomegaly and died within their first years of life by fatal liver cirrhosis. Furthermore, another sister severely affected by hepatomegaly, cardiomyopathy and proximal skeletal myopathy died at age 33. Three younger heterozygous sisters and a brother noticed exercise-induced myalgia and weakness since their teens. In sum, a homozygous p.W1327X mutation leads to a severe generalized glycogenosis types 3a and 3b within the same family. Even heterozygous p.W1327X mutation carriers may present with mild non-progressive neuromuscular symptoms, such as exercise-induced myalgia and fatigue.

[Polyglycosan body myopathy]. / Spätmanifestation einer Polyglykosankörpermyopathie.

We report two patients with polyglycosan body disease manifesting in adulthood. Clinical, electrophysiological, and histopathological characteristics of their disorders are summarized, and diagnostic classification is discussed.

Compound heterozygous patient with glycogen storage disease type III: identification of two novel AGL mutations, a donor splice site mutation of Chinese origin and a 1-bp deletion of Japanese origin.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency of glycogen-debranching enzyme (AGL). We studied a 2-year-old GSD III patient whose parents were from different ethnic groups. Nucleotide sequence analysis of the patient showed two novel mutations: a single cytosine deletion at nucleotide 2399 (2399delC) in exon 16, and a G-to-A transition at the +5 position at the donor splice site of intron 33 (IVS33+5G>A). Analysis of the mRNA produced by IVS33+5G>A showed aberrant splicing: skipping of exon 33 and activation of a cryptic splice site in exon 34. Mutational analysis of the family revealed that the 2399delC was inherited from her father, who is of Japanese origin, and the IVS33+5G>A from her mother, who is of Chinese descent, establishing that the patient was a compound heterozygote. To our knowledge, this is the first report of a mutation identified in a GSD III patient from the Chinese population.

Glycogen storage diseases. Phenotypic, genetic, and biochemical characteristics, and therapy.

The glycogen storage diseases are caused by inherited deficiencies of enzymes that regulate the synthesis or degradation of glycogen. In the past decade, considerable progress has been made in identifying the precise genetic abnormalities that cause the specific impairments of enzyme function. Likewise, improved understanding of the pathophysiologic derangements resulting from individual enzyme defects has led to the development of effective nutritional therapies for each of these disorders. Meticulous adherence to dietary therapy prevents hypoglycemia, ameliorates the biochemical abnormalities, decreases the size of the liver, and results in normal or nearly normal physical growth and development. Nevertheless, serious long-term complications, including nephropathy that can cause renal failure and hepatic adenomata that can become malignant, are a major concern in GSD-I. In GSD-III, the risk for hypoglycemia diminishes with age, and the liver decreases in size during puberty. Cirrhosis develops in some adult patients, and progressive myopathy and cardiomyopathy occur in patients with absent GDE activity in muscle. It remains unclear whether these complications of glycogen storage disease can be prevented by dietary therapy. Glycogen storage diseases caused by lack of phosphorylase activity are milder disorders with a good prognosis. The liver decreases in size, and biochemical abnormalities disappear by puberty.

Cardiomyopathy of glycogen storage disease type III.

To identify the severity of cardiac involvement in glycogen storage disease type III (GSDIII), and its relation to skeletal muscle involvement and age, 23 patients were studied. The median age was 10 years. Echocardiography, electrocardiography, and creatine phosphokinase (CK) levels were used to assess cardiac and skeletal muscle involvement. Septal and left ventricular posterior wall measurements were compared with normal data. Shortening fraction was derived from left ventricular cavity dimensions. In some patients the echocardiogram resembled that of hypertrophic cardiomyopathy. Thirteen of 20 electrocardiograms (ECG) were abnormal. Eleven patients had septal and/or posterior wall thickness > 95% confidence limits (CL). Despite this, cardiac symptoms were uncommon. The CK levels were not directly associated with cardiac abnormalities. Older patients (> 20 years) had more abnormal measurements of posterior wall thickness than did younger ones (< 20 years). This finding, albeit in a cross-sectional series, suggests progressive myocardial involvement with age despite the absence of symptoms.

Glycogen debranching enzyme deficiency: long-term study of serum enzyme activities and clinical features.

In glycogen storage disease type III (glycogen debranching enzyme (DE) deficiency), the activities of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase may be strikingly elevated during childhood but are low during adult life. To determine the pattern of the elevated serum enzyme activities in relationship to diet, the biochemical subtype and clinical symptoms, 13 patients with DE deficiency were studied. Activities of serum aspartate and alanine transaminases, lactate dehydrogenase, and alkaline phosphatase were markedly elevated during infancy. Continued elevation of enzyme activities during childhood appeared to be related to DE deficiency in liver, but unrelated to DE deficiency in muscle. Activity elevations correlated inconsistently with diet and poorly with childhood growth rate or the presence of hypoglycaemia. The serum enzyme activities declined around puberty concomitantly with a decrease in liver size. Although periportal fibrosis and micronodular cirrhosis indicated the presence of hepatocellular damage during childhood, the decline in serum enzyme activities with age and the absence of overt hepatic dysfunction suggest that the fibrotic process may not always progress.

Diet therapy in severe clinical expression of debrancher deficiency.

An eleven year old boy was referred because of sudden loss of consciousness, muscular weakness, poor general health, severe hypoglycemia with seizures and hepatomegaly. Response to oral glucose and galactose increased blood lactic acid and glucose at different times. Fasting values of blood lactic was normal, but glucose was found at 33 mg/dl. Similar test made up two hours after feeding revealed hyperlactatemia (35-50 mg/dL) and hyperglycemia (129 mg/dL). Glucagon did not result in a rise of glucose at fasting or feeding. Hepatic glycogen content was found 15 gm/100 mg of tissue. The enzyme activities revealed a deficiency of the liver debranching enzyme while leukocytes had normal enzyme activity. Hepatic biopsy showed liver fibrosis. The present case had the clinical characteristics of severe form of glycogen storage disease. A low carbohydrate and high protein diet was indicated in order to increase the gluconeogenic precursors. Although debranching enzyme deficiency is almost always benign a high carbohydrate diet induced a more severe expression of the disease.