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.     

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Glycogen branching enzyme (<Emphasis Type="Italic">GBE1</Emphasis>) mutation causing equine glycogen storage disease IV

Comparative biochemical and histopathological evidence suggests that a deficiency in the glycogen branching enzyme, encoded by the GBE1 gene, is responsible for a recently identified recessive fatal fetal and neonatal glycogen storage disease (GSD) in American Quarter Horses termed GSD IV. We have now derived the complete GBE1 cDNA sequences for control horses and affected foals, and identified a C to A substitution at base 102 that results in a tyrosine (Y) to stop (X) mutation in codon 34 of exon 1. All 11 affected foals were homozygous for the X34 allele, their 11 available dams and sires were heterozygous, and all 16 control horses were homozygous for the Y34 allele. The previous findings of poorly branched glycogen, abnormal polysaccharide accumulation, lack of measurable GBE1 enzyme activity and immunodetectable GBE1 protein, coupled with the present observation of abundant GBE1 mRNA in affected foals, are all consistent with the nonsense mutation in the 699 amino acid GBE1 protein. The affected foal pedigrees have a common ancestor and contain prolific stallions that are likely carriers of the recessive X34 allele. Defining the molecular basis of equine GSD IV will allow for accurate DNA testing and the ability to prevent occurrence of this devastating disease affecting American Quarter Horses and related breeds.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AY505107–AY505110.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.     

Characterization of the branching patterns of glycogen branching enzyme truncated on the N-terminus

Truncation of 112 amino acids at the N-terminus (Nd(1-112)) changes the chain transfer pattern of the Escherichia coli glycogen branching enzyme (GBE) [Arch. Biochem. Biophys. 397 (2002) 279]. We investigated further the role of the N-terminus by engineering other truncated GBEs and analyzing the branching pattern by high-performance anion-exchange chromatography. The wild type GBE transfers mainly chains with a degree of polymerization (d.p.) of 8-14, the Nd(1-112) enzyme transfers a greater proportion of chains with higher d.p. 15-20, whereas the 63- and 83-amino acid deleted enzymes had an intermediate pattern of transferred chains (d.p. 10-20). These data showed that a progressive shortening of the N-terminus leads to a gradual increase in the length of the transferred chains, suggesting that the N-terminus provides a support for the glucan substrate during the processes of cleavage and transfer of the alpha-(1-4) glucan chains.     

Genetic mapping of GBE1 and its association with glycogen storage disease IV in American Quarter horses

Comparative biochemical and histopathological data suggest that a deficiency in the glycogen branching enzyme (GBE) is responsible for a fatal neonatal disease in Quarter Horse foals that closely resembles human glycogen storage disease type IV (GSD IV). Identification of DNA markers closely linked to the equine GBE1 gene would assist us in determining whether a mutation in this gene leads to the GSD IV-like condition. FISH using BAC clones as probes assigned the equine GBE1 gene to a marker deficient region of ECA26q12-->q13. Four other genes, ROBO2, ROBO1, POU1F1, and HTR1F, that flank GBE1 within a 10-Mb segment of HSA3p12-->p11, were tightly linked to equine GBE1 when analyzed on the Texas A&M University 5000 rad equine radiation hybrid panel, while the GLB1, MITF, RYBP, and PROS1 genes that flank this 10-Mb interval were not linked with markers in the GBE1 group. A polymorphic microsatellite (GBEms1) in a GBE1 BAC clone was then identified and genetically mapped to ECA26 on the Animal Health Trust full-sibling equine reference family. All Quarter Horse foals affected with GSD IV were homozygous for an allele of GBEms1, as well as an allele of the most closely linked microsatellite marker, while a control horse population showed significant allelic variation with these markers. This data provides strong molecular genetic support for the candidacy of the GBE1 locus in equine GSD IV.     

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene ( GBE1 ). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene ( GYS1 ) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.     

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation.     

Increased glycogen storage in yeast results in less branched glycogen

Glycogen is a branched polymer of glucose, synthesized as a reserve of both energy and carbon. The branched nature of glycogen is important for its function and polyglucosan bodies, particles that contain a glycogen-like polymer with reduced branching, are a feature of several disease states. The degree of glycogen branching is thought to be governed by the balance between glycogen synthesis and branching activities. However, there have been reports that the intrinsic properties of individual branching enzymes govern the degree of branching. To address the relationship between synthesis and branching more fully, we made use of the yeast Saccharomyces cerevisiae. The glycogen content of yeast cells was manipulated by using different growth conditions or by the introduction of specific mutations. Whenever glycogen storage was elevated, the polysaccharide formed was found to be less branched but normal branching could be restored by overexpression of branching enzyme.     

Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.     

Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia

Monogenic hypobetalipoproteinemias include three disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) with recessive transmission and familial hypobetalipoproteinemia (FHBL) with dominant transmission. We investigated three unrelated Tunisian children born from consanguineous marriages, presenting hypobetalipoproteinemia associated with chronic diarrhea and retarded growth. Proband HBL-108 had a moderate hypobetalipoproteinemia, apparently transmitted as dominant trait, suggesting the diagnosis of FHBL. However, she had no mutations in FHBL candidate genes (APOB, PCSK9 and ANGPTL3). The analysis of MTTP gene was also negative, whereas SAR1B gene resequencing showed that the patient was homozygous for a novel mutation (c.184G>A), resulting in an amino acid substitution (p.Glu62Lys), located in a conserved region of Sar1b protein. In the HBL-103 and HBL-148 probands, the severity of hypobetalipoproteinemia and its recessive transmission suggested the diagnosis of ABL. The MTTP gene resequencing showed that probands HBL-103 and HBL-148 were homozygous for a nucleotide substitution in the donor splice site of intron 9 (c.1236+2T>G) and intron 16 (c.2342+1G>A) respectively. Both mutations were predicted in silico to abolish the function of the splice site. In vitro functional assay with splicing mutation reporter MTTP minigenes showed that the intron 9 mutation caused the skipping of exon 9, while the intron 16 mutation caused a partial retention of this intron in the mature mRNA. The predicted translation products of these mRNAs are non-functional truncated proteins.     

A novel enzymatic process for glycogen production

Two well-established methods to prepare glycogen are available: (1) extraction from natural resources such as shellfish and animal tissues; (2) synthesis from glucose-1-phosphate using two enzymes, α-glucan phosphorylase (EC 2.4.1.1) and branching enzyme (EC 2.4.1.18). We have developed a novel enzymatic process for glycogen production, in which short-chain amylose is first prepared from starch or dextrin by using isoamylase (EC 3.2.1.68), and then branching enzyme and amylomaltase (EC 2.4.1.25) are added to synthesize glycogen. Our enzymatic process, using isoamylase, branching enzyme and amylomaltase, is currently the most efficient for glycogen production. Furthermore, the molecular weight of glycogen is controllable in a range of 3.0×10⁶ to 3.0×10⁷ by adjusting some parameters of the reaction.     

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and -glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or -glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.Electronic Supplementary Material Suplementary material is available for this article at .Electronic database information: Online Mendelian Inheritance in Man, (MIM 266130 for GS deficiency); GenBank, ( nos. AL133324.13 for genomic DNA and NM_000178.2 for mRNA were used for sequence referencing)     

Phenotype guided characterization and molecular analysis of Indian patients with long QT syndromes

BackgroundLong QT syndromes (LQTS) are characterized by prolonged QTc interval on electrocardiogram (ECG) and manifest with syncope, seizures or sudden cardiac death. Long QT 1–3 constitute about 75% of all inherited LQTS. We classified a cohort of Indian patients for the common LQTS based on T wave morphology and triggering factors to prioritize the gene to be tested. We sought to identify the causative mutations and mutation spectrum, perform genotype-phenotype correlation and screen family members.MethodsThirty patients who fulfilled the criteria were enrolled. The most probable candidate gene among KCNQ1, KCNH2 and SCN5A were sequenced.ResultsOf the 30 patients, 22 were classified at LQT1, two as LQT2 and six as LQT3. Mutations in KCNQ1 were identified in 17 (77%) of 22 LQT1 patients, KCNH2 mutation in one of two LQT2 and SCN5A mutations in two of six LQT3 patients. We correlated the presence of the specific ECG morphology in all mutation positive cases. Eight mutations in KCNQ1 and one in SCN5A were novel and predicted to be pathogenic by in-silico analysis. Of all parents with heterozygous mutations, 24 (92%) of 26 were asymptomatic. Ten available siblings of nine probands were screened and three were homozygous and symptomatic, five heterozygous and asymptomatic.ConclusionsThis study in a cohort of Asian Indian patients highlights the mutation spectrum of common Long QT syndromes. The clinical utility for prevention of unexplained sudden cardiac deaths is an important sequel to identification of the mutation in at-risk family members.     

Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria

d-2-hydroxyglutaric aciduria is a neurometabolic disorder with both a mild and a severe phenotype and with unknown etiology. Recently, a novel enzyme, d-2-hydroxyglutarate dehydrogenase, which converts d-2-hydroxyglutarate into 2-ketoglutarate, and its gene were identified. In the genes of two unrelated patients affected with d-2-hydroxyglutaric aciduria, we identified disease-causing mutations. One patient was homozygous for a missense mutation (c.1331T-->C; p.Val444Ala). The other patient was compound heterozygous for a missense mutation (c.440T-->G; p.Ile147Ser) and a splice-site mutation (IVS1-23A-->G) that resulted in a null allele. Overexpression studies in HEK-293 cells of proteins containing the missense mutations showed a marked reduction of d-2-hydroxyglutarate dehydrogenase activity, proving that mutations in the d-2-hydroxyglutarate dehydrogenase gene cause d-2-hydroxyglutaric aciduria.     

Mutation analyses in pedigrees and sporadic cases of ethnic Han Chinese Kallmann syndrome patients

Kallmann syndrome, a form of idiopathic hypogonadotropic hypogonadism, is characterized by developmental abnormalities of the reproductive system and abnormal olfaction. Despite association of certain genes with idiopathic hypogonadotropic hypogonadism, the genetic inheritance and expression are complex and incompletely known. In the present study, seven Kallmann syndrome pedigrees in an ethnic Han Chinese population were screened for genetic mutations. The exons and intron–exon boundaries of 19 idiopathic hypogonadotropic hypogonadism (idiopathic hypogonadotropic hypogonadism)-related genes in seven Chinese Kallmann syndrome pedigrees were sequenced. Detected mutations were also tested in 70 sporadic Kallmann syndrome cases and 200 Chinese healthy controls. In pedigrees 1, 2, and 7, the secondary sex characteristics were poorly developed and the patients’ sense of smell was severely or completely lost. We detected a genetic mutation in five of the seven pedigrees: homozygous KAL1 p.R191ter (pedigree 1); homozygous KAL1 p.C13ter (pedigree 2; a novel mutation); heterozygous FGFR1 p.R250W (pedigree 3); and homozygous PROKR2 p.Y113H (pedigrees 4 and 5). No genetic change of the assayed genes was detected in pedigrees 6 and 7. Among the 70 sporadic cases, we detected one homozygous and one heterozygous PROKR2 p.Y113H mutation. This mutation was also detected heterozygously in 2/200 normal controls and its pathogenicity is likely questionable. The genetics and genotype–phenotype relationships in Kallmann syndrome are complicated. Classical monogenic inheritance does not explain the full range of genetic inheritance of Kallmann syndrome patients. Because of stochastic nature of genetic mutations, exome analyses of Kallmann syndrome patients may provide novel insights.     

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997