OBJECTIVEType III glycogen storage disease (GSD-III, McKusick 232400), is a rare autosomal recessive disorder, also known as Cori's or Forbe's disease. The affected enzyme is amylo-1,6-glucosidase, 4-alpha-glucanotransferase (glycogen debrancher enzyme, GDE or amylogluco-sidase, AGL), which is responsible for the debranching of the glycogen molecule during catabolism. The AGL gene is located on chromosome 1p21 and contains 35 exons translated in a monomeric protein product. The clinical manifestations of GSD-III are represented by hepatomegaly, recurrent hypoglycemia, seizures, growth failure, dysmorphism, hyperlipidemia, raised transaminases and creatine kinase concentrations and, in a number of subjects, myopathy and cardiomyopathy. The hepatocellular adenoma, hepatocellular carcinoma, diabetes mellitus and liver fibrosis remain rare events. The diagnosis of debrancher deficiency was established by laboratory tests, electromyography (EMG), and muscle and liver biopsy.

METHODSWe studied six GSD-III families after patients or parental consent and the clinical characteristics were documented. Analysis of 33 exons and part exon-intron boundaries of the AGL gene in patients and their parents were carried out by PCR and direct DNA sequencing.

RESULTSThe clinical features included hepatomegaly, splenomegaly, recurrent hypoglycemia, hyperlipidemia, growth failure, raised transaminases and acidosis. Administration of epinephrine 2 hours after a carbohydrate meal could provoke normal rise of blood glucose in the affected individuals, but could not evoke any response after overnight fasting. Administration of raw-corn-starch could maintain normoglycemia and improve the disease condition. Mutation analysis for patient 1 was normal. Patient 2 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (come from father, 1294C > T, L 298 L) in exon 8 and a G-to-T transition at nucleotide 4747 (from mother, 4747G > T, E1450X) in exon 34. Patient 3 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (from father, 1294C > T, L 298 L) in exon 8 and a G-to-A transition at nucleotide -10 (from mother, -10G > A) in exon 3. Patient 4 was a homozygote: an insertion of a nucleotide CT into position +65 in exon 35 (4664 ins CT). Patient 5 had a compound heterozygote: a 8 bp deletion at nucleotide 2341 (from father, 2341delGCCATAGA, frameshift mutation) in exon 16 and a G-to-A transition at nucleotide 1559 (from mother, 1559G > A, R 387 Q) in exon 10. Patient 6 had a compound heterozygote: a T-to-G transition at nucleotide 1686 (from mother, 1686T > G, Y429 X) in exon 12 and a G-to-A transition at nucleotide 3742 (from father, 3742G > A, G 1115 R) in exon 26.

CONCLUSIONGSD-III patients have variable phenotypic characteristics. Administration of raw-corn-starch can effectively improve the disease outcome. We identified 8 new mutations on AGL gene through nucleotide sequence analysis.

Child ; Child, Preschool ; Female ; Glycogen Debranching Enzyme System ; genetics ; Glycogen Storage Disease Type III ; genetics ; therapy ; Humans ; Male ; Mutation

OBJECTIVEGlycogen debranching enzyme (AGL) plays an important role in complete degradation of the glycogen, and has two independent catalytic activities, i.e., those of alpha-1, 4-glucanotransferase (EC 2.4. 1.25) and amylo-1,6-glucosidase (EC 3.2. 1.33). A deficiency in activities of AGL causes excessive accumulation of glycogen with short branched outer chains and results in glycogen storage disease type III (GSD III; MIM #232 400), an autosomal recessive inborn disorder of glycogen metabolism. The present study aimed to investigate the mutation of AGL in 10 Chinese patients with GSD III.

METHODClinical and laboratory data of 10 patients with typical clinical manifestations of GSD III suggesting hypoglycemia, hyperlipidemia, increased creatine-phosphokinase and its isozyme were collected. The coding regions and their flanking introns of AGL gene of the 10 patients were amplified by PCR and analyzed by direct DNA sequencing. All the mutated alleles were confirmed by bidirectional DNA sequencing. The 3 novel splicing mutations were analyzed by restriction fragment length polymorphism (RFLP) in 50 healthy children (control). The 2 small deletions (c.408-411delTTTG, c.2717-2721delAGATC) were analyzed by fluorescent polymerase chain reaction and gene scan analysis to confirm the number of deleted bases.

RESULTThirteen different mutations were identified, including 4 splicing mutations (IVS6 + 1G > A, IVS6-1G > A, IVS14 + 1G > T, IVS26-2A > C), 5 nonsense mutations (R469X, R864X, S929X, R977X, Y1428X), 3 small deletions (c.408-411delTTTG, c.2717-2721delAGATC, c.2823delT) and 1 insert mutation (c.4234insT). Except for IVS14 + 1G > T, R864X, and R977X, the other 10 mutations are novel; 18 mutated alleles were identified in the 20 alleles (90%). IVS14 + 1G > T was the most frequently seen mutation, accounting for 5 of 20 (25%) alleles examined. None of homozygote and heterozygote of the 3 novel splicing mutations was found in the 50 healthy controls by RFLP analysis. With the fluorescent polymerase chain reaction and gene scan analysis, c.408411deTTTG mutation and c.2717-2721delAGATC mutation were confirmed to have 4 and 5 bases deletion respectively.

CONCLUSIONThirteen mutations were identified in the 10 cases with GSD III, with 10 novel mutations. IVS14 + 1G > T was a relatively common mutation. This study revealed the heterozygosity of AGL gene in Chinese patients with GSD III.

Adolescent ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; DNA Mutational Analysis ; Glycogen Debranching Enzyme System ; genetics ; Glycogen Storage Disease Type III ; genetics ; Humans

OBJECTIVETo detect potential mutation of the AGL gene in two siblings affected with glycogen storage disease type IIIa.

METHODSClinical data of the two siblings was collected and analyzed. Genomic DNA was extracted from peripheral venous blood samples from the patients and their parents. All exons and their flanking sequences of the AGL gene were subjected to PCR amplification and Sanger sequencing. Suspected mutation was verified in 75 healthy controls.

RESULTSThe main clinical features of the two siblings included hypoglycemia and hepatomegaly, along with markedly elevated liver and myocardial enzymes. Genetic analysis revealed that both siblings harbored compound heterozygous mutations c.1735+1G>T and c.959-1G>C of the AGL gene. Among these, the splicing mutation c.959-1G>C was a novel one with an allele frequency of <1%.

CONCLUSIONBased on their clinical features and genetic analysis, the siblings were diagnosed with glycogen storage disease type IIIa. The c.959-1G>C has enriched the spectrum of AGL gene mutations.

Adolescent ; Amino Acid Sequence ; Female ; Glycogen Debranching Enzyme System ; genetics ; Glycogen Storage Disease Type III ; genetics ; Humans ; Infant ; Male ; Mutation ; genetics ; Siblings

OBJECTIVEGlycogen storage disease type III (GSD III) is an autosomal recessive disease caused by glycogen debranching enzyme (GDE) gene (AGL gene) mutation resulting in hepatomegaly, hypoglycemia, short stature and hyperlipidemia. GSD IIIA, involves both liver and muscle, and accounts for up to 80% of GSD III. The definitive diagnosis depends on either mutation analysis or liver and muscle glycogen debranching enzyme activity tests. This study aimed to establish enzymologic diagnostic method for GSD IIIA firstly in China by detecting muscular GDE activity, glycogen content and structure and to determine the normal range of muscular GDE activity, glycogen content and structure in Chinese children.

METHODMuscle samples were collected from normal controls (male 15, female 20; 12-78 years old), molecularly confirmed GSD III A patients (male 8, female 4, 2-27 years old) and other myopathy patients (male 9, 2-19 years old). Glycogen in the muscle homogenate was degraded into glucose by amyloglucosidase and phosphorylase respectively. The glycogen content and structure were identified by glucose yield determination. The debranching enzyme activity was determined using limit dextrin as substrate. Independent samples Kruskal-Wallis H test, Nemenyi-Wilcoxson-Wilcox test, and Chi-square test were used for statistical analyses by SPSS 11.5.

RESULT(1) GSD III A patients' glycogen content were higher, but G1P/G ratio and GDE activity were lower than those of the other two groups (P < 0.01). In all of the three parameters, there were no significant difference between normal controls and other myopathy patients. (2) The range of normal values: glycogen content 0.31%-0.43%, G1P/G ratio 22.37%- 26.43%, GDE activity 0.234-0.284 micromol/(g. min). (3) Enzymologic diagnostic method had a power similar to that of gene analysis in diagnosis of GSD-IIIA patients. The sensitivity and specificity of enzymologic diagnostic method and mutation detection were 91.7% and 100% respectively.

CONCLUSIONEnzymologic diagnostic method of GSD IIIA was firstly established in China. The range of normal values was determined. This method could be used in diagnosing suspected GSD IIIA patients in the clinic.

Adolescent ; Adult ; Aged ; Biopsy ; Case-Control Studies ; Child ; Child, Preschool ; China ; Female ; Glycogen ; analysis ; Glycogen Debranching Enzyme System ; analysis ; Glycogen Storage Disease Type III ; diagnosis ; enzymology ; pathology ; Humans ; Male ; Middle Aged ; Muscles ; chemistry ; pathology ; Young Adult

OBJECTIVETo investigate the clinical features and AGL gene mutations in a family with glycogen storage disease type IIIa (GSD IIIa).

METHODSClinical data for diagnosis, treatment and follow-up of a sick child with GSD III was collected and analyzed. Genomic DNA was extracted from the peripheral blood samples from the patient and his parents. Polymerase chain reaction and direct DNA sequencing were utilized to analyze all of the exons of the AGL gene.

RESULTSThe genotype of the child was found to be c.3710_3711delTA/IVS14+1G>T. The former was a maternally-inherited mutation, which has not been reported previously. The latter was an abnormal splice-site mutation inherited from the father.

CONCLUSIONBased on its clinical and molecular evidences, the patient was diagnosed as GSD IIIa in conjunction with retrobular optic neuritis.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child, Preschool ; China ; Female ; Glycogen Debranching Enzyme System ; genetics ; metabolism ; Glycogen Storage Disease Type III ; enzymology ; genetics ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Point Mutation

OBJECTIVETo reveal the molecular genetic pathogenesis of the glycogen storage disease type III (GSDIII) and to provide a prerequisite for prenatal gene diagnosis in future.

METHODAll the coding regions as well as the border areas between exons and introns of the AGL gene and the parental relevant mutation sites were directly sequenced, so that to affirm the origin of the mutation. Then, detected novel heterozygous mutation was confirmed by cloning sequencing. Finally, definite diagnoses of the novel mutation were performed by a series of identification methods, including screening for the 100 normal controls by DHPLC in order to count the mutational frequency, analyze the conservative of the mutant amino acid sequence from 11 kinds of species and comprise the difference of the tertiary structure between the mutant protein and the normal one.

RESULTThe patient had compound heterozygous mutations, the c.100C>T (p.R34X) nonsense mutation and c. 1176_1178 del TCA deletion mutation. The p.R34X has been reported abroad, but the 1176_1178 del TCA/p.His392fs mutation is a novel one. The proband's father is heterozygous with the p.R34X mutation while his mother carries the c.1176_1178 del TCA mutation. The result from searching the dbSNP database, HGMD database and papers published in recent years showed that the c.1176_1178 del TCA is a novel mutation, but not an SNP. Conservative analysis results in 11 species indicate that the amino acid of the mutation site is highly conserved in the stage of evolution. Comparison results between the mutant protein and the normal one demonstrate that the deletion mutation results in the obvious variation of the spatial conformation of AGL protein.

CONCLUSIONThe "c.1176_1178 del TCA (p.392delHis)" mutation is a novel pathogenic mutation. This mutation and the c.100C>T (p.R34X) is the cause that the proband suffer from the GSDIIIa disease. These two mutations are inherited from mother and father respectively. The methods from this paper can be used for further prenatal gene diagnosis.

Adult ; Amino Acid Sequence ; Base Sequence ; Case-Control Studies ; Child, Preschool ; DNA Mutational Analysis ; Exons ; Female ; Glycogen Debranching Enzyme System ; chemistry ; genetics ; Glycogen Storage Disease Type III ; diagnosis ; genetics ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Protein Conformation ; Sequence Alignment