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.

Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes.

Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.

[Molecular analysis of a patient with carnitine palmitoyltransferase II deficiency].

Carnitine palmitoyltransferase (CPT), one of the key enzymes of beta-oxidation, translocates long-chain fatty acids from the cytosolic compartment into the mitochondrial matrix to undergo beta-oxidation. Recently, the CPT system has been characterized to consist of two distinct mitochondrial membrane-bound enzymes, CPT I, located on the inner side of the outer mitochondrial membrane, and CPT II, located on the inner mitochondrial membrane. We have investigated a Japanese patient with muscular manifestations who was previously reported as CPT deficiency. Enzymatic analysis of her cultured lymphoblasts revealed that CPT II activity was reduced to 5.8%, indicating that the patient suffered from CPT II deficiency. Molecular analysis identified a missense mutation, a glutamate (174)-to-lysine substitution (E174K), in the CPT II cDNA. The patient was homozygous for the mutation. The presence of the mutation was confirmed by PCR-RFLP with a mismatched primer to generate Mbo II recognition sequence at the mutation site. It has been reported that CPT II deficiency manifests as two different clinical phenotypes: a muscular form and a hepatocardiomuscular form. To our knowledge, this is the first case of CPT II deficiency with muscular symptoms to be characterized by molecular analysis in Japan.

[Identification of missense mutations and haplotyping of carnitine palmitoyltransferase II gene].

Carnitine palmitoyltransferase II(CPTII) deficiency manifests as two different clinical phenotypes: an adult form associated with muscular symptoms and an infantile form presenting with hepatocardiomuscular manifestations. We have investigated three Japanese patients with CPT II deficiency. Molecular analysis revealed two novel missense mutations, a glutamate (174)-to-lyine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPTII cDNA. Transfection experiments demonstrated that the two mutations reduced CPTII catalytic activity. We also identified a novel polymorphism in the CPTII gene, a phenylalanine (352)-to-cysteine substitution (F352C). According to an expression analysis this mutation did not alter CPTII activity. It was present in 21 out of 100 normal alleles in the Japanese population, but was not observed among Caucasians. Genotyping with the F352C polymorphism and the previously reported polymorphisms V368I and M647V allowed normal alleles to be classified into five haplotypes. In all three families, the E174K mutation resided only on F1V1M1 allele, while the F383Y mutation was observed on F2V2M1 allele, suggesting a single origin of each mutation.