Identification of three novel mutations (Q54P,W70X and T108I) in the glucose‐6‐phosphatase gene of patients with glycogen storage disease type Ia

Three novel mutations, Q54P, W70X and T108I, were identified in the gene encoding glucose‐6‐phosphatase in three patients with glycogen storage disease type Ia. Two sibs of Portuguese origin were homozygous for the Q54P mutation whereas the third patient, originating from both France and Lebanon, was a compound heterozygote for the W70X and T108I mutations. Glycogen storage disease type Ia is a heterogeneous autosomal recessive condition. Hum Mutat 14:91, 1999. © 1999 Wiley‐Liss, Inc.     

Mutations in the glucose-6-phosphatase-alpha (G6PC) gene that cause type Ia glycogen storage disease

Glucose-6-phosphatase-alpha (G6PC) is a key enzyme in glucose homeostasis that catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. Mutations in the G6PC gene, located on chromosome 17q21, result in glycogen storage disease type Ia (GSD-Ia), an autosomal recessive metabolic disorder. GSD-Ia patients manifest a disturbed glucose homeostasis, characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, lactic acidemia, and growth retardation. G6PC is a highly hydrophobic glycoprotein, anchored in the membrane of the endoplasmic reticulum with the active center facing into the lumen. To date, 54 missense, 10 nonsense, 17 insertion/deletion, and three splicing mutations in the G6PC gene have been identified in more than 550 patients. Of these, 50 missense, two nonsense, and two insertion/deletion mutations have been functionally characterized for their effects on enzymatic activity and stability. While GSD-Ia is not more prevalent in any ethnic group, mutations unique to Caucasian, Oriental, and Jewish populations have been described. Despite this, GSD-Ia patients exhibit phenotypic heterogeneity and a stringent genotype-phenotype relationship does not exist.     

Glucose-6-phosphatase gene mutations in Taiwan Chinese patients with glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD Ia) is caused by a deficiency of glucose-6-phosphatase (G6Pase) activity. Eighteen GSD Ia families were studied for G6Pase gene mutations. Thirty-two mutations were found in 36 GSD Ia chromosomes: 16 were 727 G→T (44.44%); 13 were R83H (327 G→T; 36.11%); 1 was 341delG; 1 was 933insAA; and 1 was 793 G→T. The 727 G→T and R83H mutations together accounted for 80.56% (29/36) of the GSD Ia chromosomes. These two mutations were easily examined by polymerase chain reaction-based methods, and the prenatal diagnosis of a non-affected fetus was successfully made. The 727 G→T mutation is the predominant mutation in Japanese GSD Ia patients, but is rarely seen in Western counties. The 727 G→T mutation is also the most prevalent mutation in Taiwan Chinese, although the incidence is not as high as in Japan. Received: January 4, 2000 / Accepted: February 28, 2000     

Mutation spectrum of the glucose-6-phosphatase gene and its implication in molecular diagnosis of Korean patients with glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD Ia; MIM 232200) is an autosomal recessive inherited metabolic disorder resulting from a deficiency of the microsomal glucose-6-phosphatase (G6Pase), the enzyme that catalyzes the terminal step in gluconeogenesis and glycogenolysis. Various mutations in the G6Pase gene (G6PC) have been found in patients with GSD Ia. To elucidate the spectrum of the G6PC gene mutations, 13 unrelated Korean patients with GSD Ia were analyzed. We were able to identify mutant alleles in all patients, including three known mutations (727G > T, G122D, and T255I) and two novel mutations (P178A and Y128X). The frequency of the 727G > T mutation in Korean patients with GSD Ia was 81% (21/26), which was slightly lower than that (86-92%) in Japanese but much higher than that (44.4%) in Taiwan Chinese. Except one, all patients were either homozygous (9/13) or compound heterozygous (3/13) for the 727G > T mutation; the only patient without the 727G > T mutation was a compound heterozygote for the G122D and Y128X mutations. Our findings suggest that a DNA-based test can be used as the initial diagnostic approach in Korean patients clinically suspected to have GSD Ia, thereby avoiding invasive liver biopsy.     

葡萄糖6磷酸酶基因热点突变检测结合1176多态位点连锁分析快速产前诊断Ia型糖原累积病

目的探讨中国人Ⅰa型糖原累积病简便、快速、准确的产前诊断方法。方法通过限制性内切酶图谱分析了葡萄糖6磷酸酶(glucose-6-phosphatase，G6Pase)基因727G→T和R83H的突变，并结合1176位点单核苷酸多态性连锁分析，对3个Ⅰa型糖原累积病家系进行了基因诊断和产前诊断。对发现的突变及1176位点多态性用DNA测序证实。结果3个家系先证者G6Pase基因的2个等位基因均携带727G→T突变，分别来自其父母。家系1和3胎儿为727G→T突变杂合子；家系2胎儿不携带该突变。1176位点单核苷酸多态性分析显示，3名胎儿1176位点单核苷酸多态性与3名先证者不同。DNA直接测序结果与限制性内切酶图谱分析结果相符。家系1和家系2胎儿已出生，井证实与产前诊断结果相符。结论通过限制性内切酶酶切法筛查727G→T和R83H突变结合1176位点单核苷酸多态性连锁分析可简便、快速、准确地诊断和产前诊断Ⅰa型糖原累积病。     

Glycogen storage disease type Ia: Four novel mutations (175delGG,R170X,G266V and V338F) identified

Deficient activity of glucose‐6‐phosphatase (G6Pase) causes glycogen storage disease type Ia (GSD Ia). We analysed the G6Pase gene of 16 GSD Ia patients using single strand conformation polymorphism (SSCP) analysis prior to automated sequencing of exon(s) revealing an aberrant SSCP pattern. In all GSD Ia patients we were able to identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure to identify mutations. Four novel mutations (175delGG, R170X, G266V and V338F) were identified. © 1998 Wiley‐Liss, Inc.     

Glycogen storage disease type Ia: molecular study in Brazilian patients

Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSDIa). This disease is characterized by growth retardation, hepatomegaly, hypoglycemia, hyperlipidemia, and lactic acidosis. In this study, we report mutations in the G6Pase gene in 8 of 25 Brazilian patients with clinical symptoms of GSDIa. Five previously described mutations (R83C, Q347X, V338F, D38V, and G68R) were detected. The two most common mutations identified were R83C and Q347X, accounting for 8 of 14 (57.14%) mutant alleles. A 1176 single-nucleotide polymorphism and two intronic mutations (IVS3-58T>A and IVS4+10G>A) were also analyzed. We used the minigene strategy in order to verify the effect of these intronic mutations on the splicing mechanism. This study emphasizes that molecular genetic analysis is a reliable and convenient alternative to the assay of enzyme activity in a fresh liver biopsy specimen for diagnosing GSDIa. Received: November 13, 2000 / Accepted: December 25, 2000     

Heterogeneous mutations in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type Ia

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. It is characterized by short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. Various mutations have been reported in the G6Pase gene (G6PC). However, in Japanese patients, a g727t substitution was found to be the major cause of GSD-Ia, accounting for 20 of 22 mutant alleles [Kajihara et al., 1995], and no other mutations have been found in this population. We analyzed four Japanese GSD-Ia patients and identified three other mutations in addition to the g727t. They included two missense mutations (R83H and P257L) and one nonsense mutation (R170X). Each of the three mutations exhibited markedly decreased G6Pase activity when expressed in COS7 cells. A patient homozygous for R170X showed multiple episodes of profound hypoglycemia associated with convulsions, while P257L was associated with a mild clinical phenotype. The presence of R170X in three unrelated families may implicate that it is another important mutation in the etiology of GSD-Ia in Japanese patients. Thus, the detection of non-g727t mutations is also important in establishing the DNA-based diagnosis of GSD-Ia in this population.     

Genetic analysis of the glucose-6-phosphatase mutation of type la glycogen storage disease in a Chinese family

Type la glycogen storage disease (GSD) is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase (GóPase). Polymerase chain reaction (PCR) and nucleotide sequence analysis were used to identify the location and nature of mutations at the GóPase locus in two siblings affected with type la GSD. Both patients are compound heterozygotes with two different single nucleotide substitutions in the two GóPase alleles. A guanine to adenine transition was identified at base position 327 in the exon 2, converting an arginine to a histidine at codon 83. The second substitution was a thymine to adenine transversion at base position 1101 in the exon 5, converting an isoleucine to an asparagine at codon 341. Family study reveals that both parents are heterozygous carriers: the father with a mutant GóPase allele at exon 2, the mother with another mutant GóPase allele at exon 5. This is the first family study in Taiwan on type la GSD identified by molecular analysis. The mutations identified herein are novel substitutions in the GóPase gene. In addition, an adenine to guanine substitution was observed at base position 653 in the exon 5 of GóPase gene in both sibling patients and their parents, as well as in 15 normal Chinese subjects and three normal Caucasian subjects.     

Identification of a point mutation (G727T) in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type 1a, and carrier screening in healthy volunteers

Glycogen storage disease type 1a (GSD 1a) is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase (G6Pase). We analyzed the G6Pase gene of two unrelated Japanese families with GSD 1a. DNA sequencing of all five exons and exon-intron junctions revealed a G-to-T transversion at nucleotide 727 (G727T) in exon 5, which has been previously reported to cause abnormal splicing. Family studies using mismatch PCR showed that three patients were homozygous for the G727T mutation, while the parents were heterozygous. To investigate allele frequencies, we screened 216 Japanese healthy volunteers and found one asymptomatic carrier. Our findings suggest that the G727T mutation may be prevalent in Japan.     

Glucose-6-phosphatase gene (727G→T) splicing mutation is prevalent in Hong Kong Chinese patients with glycogen storage disease type la

Glycogen storage disease type la (GSD1a) is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase (GóPase). We analyzed the GóPase genes of two unrelated Chinese families with GSD1a. DNA sequencing of all five exons and the exonintron boundaries revealed a G → T transversion at nucleotide 727 (727G→T) in exon 5, which has previously been reported to cause abnormal splicing. In one family, the subject and her affected sister were confirmed to be homozygous for this mutation and their parents to be heterozygotes. In the other family, the proband was identified to be heterozygous for this mutation, and a novel mutation, the 341delG in exon 2, was identified. This mutation alters the reading frame and creates a stop codon TAA 15 codons downstream from the mutation, resulting in a truncated protein. Family studies revealed that the father was heterozygous for the 727G → T mutation and that the mother was heterozygous for the 341delG mutation. This is the first time that the 727G→T mutation has been found in Chinese patients or outside Japan. Since we only tested two GSDla families and found 727G→T in both, we believe that this mutation may also be prevalent in our local Chinese population. To investigate allele frequencies, we screened 385 Chinese healthy volunteers and found two asymptomatic carriers. Our findings suggest that the 727G → T mutation is indeed prevalent in Hong Kong.     

Hepatocellular adenoma and metabolic balance in patients with type Ia glycogen storage disease

Glycogen storage disease type I (GSD I) is a metabolic disorder resulting from defects in the glucose-6-phosphatase system. Approximately 75% of adolescent and adult patients develop hepatocellular adenomas, which can lead to considerable morbidity and mortality. The pathogenesis of adenomas is unclear and the risk of developing adenomas in treated patients is uncertain. The objective of this study was to determine whether metabolic imbalance was related to the occurrence of adenomas in patients with GSD I, and to determine what specific biochemical pathways were involved. We performed a 1:1 case–control retrospective study; cases were GSD I patients with adenomas and controls were GSD I patients without adenomas. Controls and cases were matched according to age at diagnosis, age at adenoma detection, and gender. We investigated biochemical abnormalities indicative of metabolic balance and exogenous factors potentially related to the onset of adenomas in the two groups. We detected no significant differences in dietetic treatment, compliance to treatment, or biochemical parameters related to metabolic balance between the two groups. In conclusion, we were unable to identify any significant differences in metabolic balance between GSD I patients who developed adenomas and those who did not.     

一个糖原累积病Ⅱ型家系的酸性-α-葡萄糖苷酶及其产前基因诊断

目的 为1个糖原累积病Ⅱ型(glycogen storage disease typeⅡ,GSDⅡ)家系进行酶学和产前基因诊断.方法 用酸性-α-葡萄糖苷酶(acid-alpha-glucosidase,GAA)特异性水解荧光底物4-甲基伞型酮-α-D-吡喃葡萄糖苷(4-methylumbelliferyl-α-D-glucopyranoside,4-MUG)和阿卡波糖抑制其同工酶的方法检测外周血白细胞和羊水细胞GAA酶活性,聚合酶链反应扩增GAA基因外显子编码区序列,直接测序分析GAA基因突变情况.结果 先证者外周血白细胞与胎儿羊水细胞GAA酶活性均明显低于正常参考值范围,分别为正常对照平均值的12.3% 和1.1%.先证者和胎儿均携带新无义突变 p.W738X 和已报道的无义突变p.E888X;先证者、母亲和胎儿均携带假性缺陷等位基因[c.1726G>A; c.2065G>A].结论 通过GAA酶活性检测结合GAA基因分析对1个GSDⅡ家系进行了产前诊断.由于假性缺陷等位基因可引起GAA酶活性降低,故GAA基因分析应作为亚洲人群GSDⅡ产前诊断的常规手段.

Abstract：

Objective To carry out prenatal diagnosis for a glycogen storage disease typeⅡ(GSDⅡ) affected family. Methods The acid-α-glucosidase (GAA) activity was measured in whole leukocytes and cultured amniocytes with 4-methylumbelliferyl-α-D-glucopyranoside as substrate and with acarbose as inhibitor. The coding regions of GAA gene were amplified by polymerase chain reaction and analyzed by direct DNA sequencing. Results The proband and the fetus had low GAA activity (12.3% and 1.1% of the average normal range, respectively). Mutation analysis of the GAA gene revealed a novel nonsense mutation p.W738X and a reported nonsense mutation p.E888X in both the proband and the fetus; the reported pseudodeficiency allele c.[1726G>A;2065G>A] was found in the proband, the mother and the fetus. Conclusion The proband and the fetus were both GSDⅡaffected. A combination of GAA activity analysis and mutation analysis is efficient for the prenatal diagnosis of GSDⅡ. Mutation analysis should be a routine method in the prenatal diagnosis of GSDⅡ in Asian population, where pseudodeficiency allele can cause low GAA activity in normal individuals which is relatively common in Asian.     

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. Am. J. Med. Genet. 93:211–214, 2000. © 2000 Wiley‐Liss, Inc.     

Mutation profile of the GAA gene in 40 Italian patients with late onset glycogen storage disease type II

Glycogen storage disease type II (GSDII) is a recessively inherited disorder due to the deficiency of acid alpha-glucosidase (GAA) that results in impaired glycogen degradation and its accumulation in the lysosomes. We report here the complete molecular analysis of the GAA gene performed on 40 Italian patients with late onset GSDII. Twelve novel alleles have been identified: missense mutations were functionally characterized by enzyme activity and protein processing in a human GAA-deficient cell line while splicing mutations were studied by RT-PCR and in silico analysis. A complex allele was also identified carrying three different alterations in cis. The c.-32-13T > G was the most frequent mutation, present as compound heterozygote in 85% of the patients (allele frequency 42.3%), as described in other late onset GSDII Caucasian populations. Interestingly, the c.-32-13T > G was associated with the c.2237G > A (p.W746X) in nine of the 40 patients. Genotype-phenotype correlations are discussed with particular emphasis on the subgroup carrying the c.-32-13T > G/c.2237G > A genotype.     

Noncompaction myocardium in association with type Ib glycogen storage disease

Noncompaction myocardium is a rare disorder assumed to occur as an arrest of the compaction process during the normal development of the heart. Left ventricular noncompaction has been reported to be associated with a variety of cardiac and extracardiac, especially neuromuscular abnormalities. Moreover, it has been suggested that metabolic alterations could be responsible for the noncompaction. However, no association of noncompaction myocardium with type Ib glycogen storage disease (GSD) has been reported so far. Type Ib GSD is due to a defect of a transmembrane protein which results, similar to type Ia GSD, in hypoglycemia, a markedly enlarged liver and, additionally, in neutropenia, recurrent infections, and inflammatory bowel disease. Until now, no muscular or cardiac involvement has been described in type Ib GSD patients. The present case represents the first report of a noncompaction myocardium in a child with type Ib GSD who died of sudden clinical deterioration at the age of four.     

Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II

Patients with glycogen storage disease type II (GSDII, Pompe disease) suffer from progressive muscle weakness due to acid alpha-glucosidase deficiency. The disease is inherited as an autosomal recessive trait with a spectrum of clinical phenotypes. We have investigated 29 cases of GSDII and thereby identified 55 pathogenic mutations of the acid alpha-glucosidase gene (GAA) encoding acid maltase. There were 34 different mutations identified, 22 of which were novel. All of the missense mutations and two other mutations with an unpredictable effect on acid alpha-glucosidase synthesis and function were transiently expressed in COS cells. The effect of a novel splice-site mutation was investigated by real-time PCR analysis. The outcome of our analysis underscores the notion that the clinical phenotype of GSDII is largely dictated by the nature of the mutations in the GAA alleles. This genotype-phenotype correlation makes DNA analysis a valuable tool to help predict the clinical course of the disease.     

Functional analysis of mutations in the glucose-6-phosphate transporter that cause glycogen storage disease type Ib

The glucose-6-phosphate transporter (G6PT) deficient in glycogen storage disease type Ib is a phosphate (P_i)-linked antiporter capable of G6P: P_i and P_i:P_i exchanges. We previously characterized G6PT mutations by measuring G6P uptake activities in microsomes co-expressing G6PT and glucose-6-phosphatase-α. Here we report a new assay, based on reconstituted proteoliposomes carrying only G6PT, and characterize G6P and P_i uptake activities of 23 G6PT mutations. We show that co-expression and G6PT-only assays are equivalent in measuring G6PT activity. However, the p.Q133P mutation exhibits differential G6P and P_i transport activities, suggesting that characterizing G6P and P_i transport activities of G6PT mutations may yield insights to this genetic disorder.     

A nonsense mutation due to a single base insertion in the 3′-coding region of glycogen debranching enzyme gene associated with a severe phenotype in a patient with glycogen storage disease type IIIa

Glycogen storage disease type III (GSD-III) is an autosomal recessive disease resulting from deficient glycogen debranching enzyme (GDE) activity. A child with GDE deficient in both liver and muscle (GSD-IIIa) had recurrent hypoglycemia, seizures, severe cardiomegaly, and hepatomegaly and died at 4 years of age. Analysis of the GDE gene in this child by single-strand conformation polymorphism, followed by direct DNA sequencing and restriction analysis, revealed an insertion of a nucleotide A into position 4529 of the GDE cDNA (4529insA). This insertion resulted in substitution of a tyrosine to a stop codon at amino acid 1510 (Y1510X). The 4529insA mutation appeared to be homozygous in this patient and was not found in 20 unrelated controls or 18 other GSD-III patients (14 GSD-IIIa and 4 GSD-IIIb). This is the first identification of a disease mutation in this gene, and the data suggest that homozygous 4529insA may be associated with a severe phenotype in GSD-IIIa. © 1997 Wiley-Liss, Inc.     

A c.3216_3217delGA mutation in AGL gene in Tunisian patients with a glycogen storage disease type III: evidence of a founder effect

Mili A, Ben Charfeddine I, Amara A, MamaÏ O, Adala L, Ben Lazereg T, Bougulia J, Saad A, Limem K, Gribaa M. A c.3216_3217delGA mutation in AGL gene in Tunisian patients with a glycogen storage disease type III: evidence of a founder effect. Glycogen storage disease type III (GSD III) is an autosomal recessive disorder characterized by excessive accumulation of abnormal glycogen in the liver and muscles and caused by deficiency in the glycogen debranching enzyme, the amylo‐1,6‐glucosidase (AGL). In this study, we report the clinical, biochemical and genotyping features of five unrelated GSD III patients coming from the same region in Tunisia. The concentration of erythrocyte glycogen and AGL activity were measured by colorimetric and fluorimetric methods, respectively. Four CA/TG microsatellite markers flanking the AGL gene in chromosome 1 were amplified with fluoresceinated primers. The full coding exons and their relevant exon–intron boundaries of the AGL gene were directly sequenced for the patients and their parents. All patients showed a striking increase of erythrocytes glycogen content. No AGL activity was detected in peripheral leukocytes. Sequencing of the AGL gene identified a c.3216_3217delGA (p.Glu1072AspfsX36) mutation in the five patients which leads to a premature termination, abolishing the AGL activity. Haplotype analysis showed that the mutation was associated with a common homozygote haplotype. Our results suggested the existence of a founder effect responsible for GSD III in this region of Tunisia.