Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.     

The variable presentations of glycogen storage disease type IV: a review of clinical, enzymatic and molecular studies

Glycogen storage disease type IV (GSD-IV), also known as Andersen disease or amylopectinosis (MIM 23250), is a rare autosomal recessive disorder caused by a deficiency of glycogen branching enzyme (GBE) leading to the accumulation of amylopectin-like structures in affected tissues. The disease is extremely heterogeneous in terms of tissue involvement, age of onset and clinical manifestations. The human GBE cDNA is approximately 3-kb in length and encodes a 702-amino acid protein. The GBE amino acid sequence shows a high degree of conservation throughout species. The human GBE gene is located on chromosome 3p14 and consists of 16 exons spanning at least 118 kb of chromosomal DNA. Clinically the classic Andersen disease is a rapidly progressive disorder leading to terminal liver failure unless liver transplantation is performed. Several mutations have been reported in the GBE gene in patients with classic phenotype. Mutations in the GBE gene have also been identified in patients with the milder non-progressive hepatic form of the disease. Several other variants of GSD-IV have been reported: a variant with multi-system involvement including skeletal and cardiac muscle, nerve and liver; a juvenile polysaccharidosis with multi-system involvement but normal GBE activity; and the fatal neonatal neuromuscular form associated with a splice site mutation in the GBE gene. Other presentations include cardiomyopathy, arthrogryposis and even hydrops fetalis. Polyglucosan body disease, characterized by widespread upper and lower motor neuron lesions, can present with or without GBE deficiency indicating that different biochemical defects could result in an identical phenotype. It is evident that this disease exists in multiple forms with enzymatic and molecular heterogeneity unparalleled in the other types of glycogen storage diseases.     

Glycogenosis Type III in the Dog

Enzyme and glycogen structure studies have been carried out on tissues of a glycogenotic dog, the clinical and pathological characteristics of which are reported in the accompanying paper. Liver glucose-6-phosphatase, leukocyte and liver acid maltase, and liver and skeletal muscle glycogen Phosphorylase all appeared largely unaffected. The activity of the muscle and liver debranching enzyme (amylo-l,6-glucosidase), determined by two independent assay methods, was, however, reduced to between 0 and 7 % of normal activity. Glycogen structure studies with Phosphorylase or iodine spectra revealed that the abnormally large amounts of glycogen found in liver and skeletal muscle had abnormally short branches, as would be expected for a deficiency of debranching enzyme. It is thus clear that the dog had suffered from the equivalent of Cori's disease (limit dextrinosis, type III glycogen storage disease). Preliminary data indicate that it may be possible to identify heterozygotes based on a study of the debranching enzyme of leukocytes.     

Assignment of the human glycogen debrancher gene to chromosome 1p21

Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but approximately 15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1p21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein.     

Fanconi-Bickel syndrome--a congenital defect of facilitative glucose transport

Fanconi-Bickel syndrome (FBS, OMIM 227810) is a rare type of glycogen storage disease (GSD). It is caused by homozygous or compound heterozygous mutations within GLUT2, the gene encoding the most important facilitative glucose transporter in hepatocytes, pancreatic beta-cells, enterocytes, and renal tubular cells. To date, 112 patients have been reported in the literature. Most patients have the typical combination of clinical symptoms: hepatomegaly secondary to glycogen accumulation, glucose and galactose intolerance, fasting hypoglycemia, a characteristic tubular nephropathy, and severely stunted growth. In 63 patients, mutation analysis has revealed a total of 34 different GLUT2 mutations with none of them being particularly frequent. No specific therapy is available for FBS patients. Symptomatic treatment is directed towards a stabilization of glucose homeostasis and compensation for renal losses of various solutes. In addition to the clinical and molecular genetic aspects of FBS, this review discusses the pathophysiology of the disease and compares it to recent findings in GLUT2 deficient transgenic animals. An overview is also provided on recently discovered members of the rapidly growing family of facilitative glucose transporters, which are novel candidates for congenital disorders of carbohydrate metabolism.     

Genetic heterogeneity and the diagnosis of hepatic glycogenoses

Glycogen storage diseases constitute a highly heterogeneous group of disorders, because of the many complex enzyme systems involved in glycogen metabolism, and also because of the diversity of molecular defects connected with gene mutations. To illustrate these features, the authors studied four types of liver glycogen storage diseases, respectively caused by deficiencies of glucose-6-phosphatase, debranching enzyme, phosphorylase and phosphorylase kinase. In each case, the role and functional characteristics of the enzyme system are described, as well as the bioclinical aspects of the deficiency. The only reliable way of diagnosing glycogen storage disease is by assaying the activity of the enzyme concerned. Assay procedure must take account of various factors, especially the progress made in understanding the nature and mechanism of action of enzyme systems, the possible tissular heterogeneity of the deficiency and the functional characteristics of certain enzymes.     

Glycogenoses--genetic disorders leading to disturbed glycogen metabolism

Glycogen storage diseases (GSD, glycogenoses) is a group of genetic disorders resulting from abnormal metabolism of glycogen--a polymeric molecule involved in intercellular glucose storage. Currently 13 different types of glycogenoses are known. They all result from mutations in genes for different enzymes, which directly or indirectly regulate glycogen synthesis and degradation. The clinical manifestation of GSDs encompasses primarily liver, striated muscle and brain tissue dysfunction. In those tissues glycogen plays a particularly important role. spectrum and severity of symptoms is very diverse, depending on both the type and subtype of the disease as well as on the individual features of the patient. The therapy is based mainly on application of an appropriate diet. Enzyme replacement therapy is currently available for GSD type II. For some of the other types the possibility for gene therapy is intensively investigated.     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

A founder AGL mutation causing glycogen storage disease type IIIa in Inuit identified through whole-exome sequencing: a case series

Background:

Glycogen storage disease type III is caused by mutations in both alleles of the AGL gene, which leads to reduced activity of glycogen-debranching enzyme. The clinical picture encompasses hypoglycemia, with glycogen accumulation leading to hepatomegaly and muscle involvement (skeletal and cardiac). We sought to identify the genetic cause of this disease within the Inuit community of Nunavik, in whom previous DNA sequencing had not identified such mutations.

Methods:

Five Inuit children with a clinical and biochemical diagnosis of glycogen storage disease type IIIa were recruited to undergo genetic testing: 2 underwent whole-exome sequencing and all 5 underwent Sanger sequencing to confirm the identified mutation. Selected DNA regions near the AGL gene were also sequenced to identify a potential founder effect in the community. In addition, control samples from 4 adults of European descent and 7 family members of the affected children were analyzed for the specific mutation by Sanger sequencing.

Results:

We identified a homozygous frame-shift deletion, c.4456delT, in exon 33 of the AGL gene in 2 children by whole-exome sequencing. Confirmation by Sanger sequencing showed the same mutation in all 5 patients, and 5 family members were found to be carriers. With the identification of this mutation in 5 probands, the estimated prevalence of genetically confirmed glycogen storage disease type IIIa in this region is among the highest worldwide (1:2500). Despite identical mutations, we saw variations in clinical features of the disease.

Interpretation:

Our detection of a homozygous frameshift mutation in 5 Inuit children determines the cause of glycogen storage disease type IIIa and confirms a founder effect.Glycogen storage disease type III is a rare autosomal recessive disease characterized by recurrent hypoglycemia in childhood, as well as hepatomegaly with elevated transaminases and hyperlipidemia.¹ The disease involves a defect in the key glycogen debranching enzyme, which has 2 enzymatic activities (amylo-1,6-glucosidase and 4-α-glucanotransferase), resulting in reduced glycogen degradation, accumulation of limit dextrin in affected organs (primarily skeletal muscle, cardiac muscle and liver), organomegaly and dysfunction. Glycogen storage disease type IIIa involves the liver and cardiac and skeletal muscles, whereas glycogen storage disease type IIIb involves only the liver.Mutations in the AGL gene encoding glycogen debranching enzyme have been described in many populations, including Northern European,² Egyptian,³ Hispanic² and Asian;² a high prevalence of the disease was also found in the North African Jewish community (1/5400) and in the Faroe Islands (1/3600).^4,5 We previously described the presenting clinical characteristics of 4 Inuit children with putative glycogen storage disease type III and suspected the presence of a founder effect.⁶ However, targeted genetic analysis had failed to identify a mutation in the AGL gene. The aim of our present study was to identify the genetic cause of glycogen storage disease type III in the Inuit population of Nunavik on the eastern coast of Hudson Bay. By using exome sequencing, which examines all protein-encoding DNA sequences (exons), we hoped to facilitate early diagnosis, prenatal and neonatal screening and screening of family members.     

A newly identified c.1824_1828dupATACG mutation in exon 13 of the GAA gene in infantile-onset glycogen storage disease type II (Pompe disease)

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.     

Laforin–Malin Complex Degrades Polyglucosan Bodies in Concert with Glycogen Debranching Enzyme and Brain Isoform Glycogen Phosphorylase

In Lafora disease (LD), the deficiency of either EPM2A or NHLRC1, the genes encoding the phosphatase laforin and E3 ligase, respectively, causes massive accumulation of less-branched glycogen inclusions, known as Lafora bodies, also called polyglucosan bodies (PBs), in several types of cells including neurons. The biochemical mechanism underlying the PB accumulation, however, remains undefined. We recently demonstrated that laforin is a phosphatase of muscle glycogen synthase (GS1) in PBs, and that laforin recruits malin, together reducing PBs. We show here that accomplishment of PB degradation requires a protein assembly consisting of at least four key enzymes: laforin and malin in a complex, and the glycogenolytic enzymes, glycogen debranching enzyme 1 (AGL1) and brain isoform glycogen phosphorylase (GPBB). Once GS1-synthesized polyglucosan accumulates into PBs, laforin recruits malin to the PBs where laforin dephosphorylates, and malin degrades the GS1 in concert with GPBB and AGL1, resulting in a breakdown of polyglucosan. Without fountional laforin–malin complex assembled on PBs, GPBB and AGL1 together are unable to efficiently breakdown polyglucosan. All these events take place on PBs and in cytoplasm. Deficiency of each of the four enzymes causes PB accumulation in the cytoplasm of affected cells. Demonstration of the molecular mechanisms underlying PB degradation lays a substantial biochemical foundation that may lead to understanding how PB metabolizes and why mutations of either EPM2A or NHLRC1 in humans cause LD. Mutations in AGL1 or GPBB may cause diseases related to PB accumulation.     

Phosphofructokinase deficiency; past, present and future

Phosphofructokinase deficiency (Tarui disease, glycogen storage disease VII, GSD VII) stands out among all the GSDs. PFK deficiency was the first recognized disorder that directly affects glycolysis. Ever since the discovery of the disease in 1965, a wide range of biochemical, physiological and molecular studies of the disorder have greatly expanded our understanding of the function of normal muscle, general control of glycolysis and glycogen metabolism. The studies of PFK deficiency vastly enriched the field of glycogen storage diseases, as well as the field of metabolic and neuromuscular disorders. This article cites a historical overview of this clinical entity and the progress that has been made in molecular genetic area. We will also present the results of a search in-silico, which allowed us to identify a previously unknown sequence of the human platelet PFK gene (PFK-P). In addition, we will describe phylogenetic analysis of evolution of PFK genes.     

Hexosamines, insulin resistance, and the complications of diabetes: current status

Glycogen is the storage form of carbohydrate for virtually every organism from yeast to primates. Most mammalian tissues store glucose as glycogen, with the major depots located in muscle and liver. The French physiologist Claude Bernard first identified a starch-like substance in liver and muscle and coined the term glycogen, or "sugar former," in the 1850s. During the 150 years since its identification, researchers in the field of glycogen metabolism have made numerous discoveries that are now recognized as significant milestones in biochemistry and cell signaling. Even so, more questions remain, and studies continue to demonstrate the complexity of the regulation of glycogen metabolism. Under classical definitions, the functions of glycogen seem clear: muscle glycogen is degraded to generate ATP during increased energy demand, whereas hepatic glycogen is broken down for release of glucose into the bloodstream to supply other tissues. However, recent findings demonstrate that the roles of glycogen metabolism in energy sensing, integration of metabolic pathways, and coordination of cellular responses to hormonal stimuli are far more complex.     

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.     

Nemaline myopathy caused by mutations in the muscle alpha-skeletal-actin gene

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder characterized by muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle. Disease-causing mutations have been reported in five genes, each encoding a protein component of the sarcomeric thin filament. Recently, we identified mutations in the muscle alpha-skeletal-actin gene (ACTA1) in a subset of patients with NM. In the present study, we evaluated a new series of 35 patients with NM. We identified five novel missense mutations in ACTA1, which suggested that mutations in muscle alpha-skeletal actin account for the disease in approximately 15% of patients with NM. The mutations appeared de novo and represent new dominant mutations. One proband subsequently had two affected children, a result consistent with autosomal dominant transmission. The seven patients exhibited marked clinical variability, ranging from severe congenital-onset weakness, with death from respiratory failure during the 1st year of life, to a mild childhood-onset myopathy, with survival into adulthood. There was marked variation in both age at onset and clinical severity in the three affected members of one family. Common pathological features included abnormal fiber type differentiation, glycogen accumulation, myofibrillar disruption, and "whorling" of actin thin filaments. The percentage of fibers with rods did not correlate with clinical severity; however, the severe, lethal phenotype was associated with both severe, generalized disorganization of sarcomeric structure and abnormal localization of sarcomeric actin. The marked variability, in clinical phenotype, among patients with different mutations in ACTA1 suggests that both the site of the mutation and the nature of the amino acid change have differential effects on thin-filament formation and protein-protein interactions. The intrafamilial variability suggests that alpha-actin genotype is not the sole determinant of phenotype.     

Mutation analysis in glycogen storage disease type 1 non-a

We report molecular and clinical findings in 13 patients with rare types of glycogen storage disease 1 (GSD1 non-a). Analysis of G6PT encoding a microsomal transporter protein has revealed mutations on both chromosomes in each case, four of which are novel. Diagnosis has been confirmed in three patients suspected of having GSD1 non-a without enzymatic studies involving liver biopsy, thus emphasising the advantage of G6PT mutation analysis for all GSD1 non-a patients.     

Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS 14+1G-->T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29-1G-->C), a 1-bp deletion (587delC), a 2-bp deletion (4216-4217delAG), a 1-bp insertion (2072-2073insA), and a 3-bp insertion (4735-4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G-->T, and 4216-4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan.     

Clinical and genetic characterization of Chanarin-Dorfman syndrome

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers.All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel.These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.