Kuniaki Narisawa

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.

Glutamate triggers internucleosomal DNA cleavage in neuronal cells.

Glutamate neurotoxicity is responsible for neuronal loss associated with numerous obstinate disorders. In this report, the mechanism of glutamate neurotoxicity was investigated on a viewpoint of DNA degradation. We found that chromosomal DNA of cultured neurons was degraded into nucleosomal-sized DNA fragments by the addition of glutamate, prior to the glutamate-induced neuronal death. Both the neuronal death and DNA fragmentation were prevented by the inhibitors of endonucleases and mRNA synthesis. Furthermore, an injection of glutamate into the rat hippocampi resulted in DNA fragmentation with the similar time course observed in neuronal death in vitro. These results suggest that the glutamate neurotoxicity involves an active suicide process which leads to neuronal death through internucleosomal DNA cleavage.

Novel mutations in the connexin 26 gene (GJB2) responsible for childhood deafness in the Japanese population

Mutations in the connexin 26 gene (GJB2), which encodes a gap-junction protein and is expressed in the inner ear, have been shown to be responsible for a major part of nonsyndromic hereditary prelingual (early-childhood) deafness in Caucasians. We have sequenced the GJB2 gene in 39 Japanese patients with prelingual deafness (group 1), 39 Japanese patients with postlingual progressive sensorineural hearing loss (group 2), and 63 Japanese individuals with normal hearing (group 3). Three novel mutations were identified in group 1: a single nucleotide deletion (235delC), a 16-bp deletion (176-191 del (16)), and a nonsense mutation (Y136X) in five unrelated patients. The 235delC mutation was most frequently observed, accounting for seven alleles in 10 mutant alleles. Screening of 203 unrelated normal individuals for the three mutations indicated that the carrier frequency of the 235delC mutation was 2/203 in the Japanese population. No mutation was found in group-2 patients. We also identified two novel polymorphisms (E114G and I203T) as well as two previously reported polymorphisms (V27I andV37I). Genotyping with these four polymorphisms allowed normal Japanese alleles to be classified into seven haplotypes. All 235delC mutant alleles identified in four patients resided only on haplotype type 1. These findings indicate that GJB2 mutations are also responsible for prelingual deafness in Japan.

A new variant of glycogen storage disease Type I probably due to a defect in the glucose-6-phosphate transport system

A new variant of glycogen storage disease (GSD) Type 1, with clinical symptoms and laboratory findings consistent with those of glucose-6-phosphatase (G6Pase) deficiency, is described. Assay of G6Pase in liver from the patient immediately after biopsy by the method of Nordlie and Arion gave low activity (0.8µmol/min per g liver) in the absence of detergent, but was normal (10.2µmol/min per g liver) after addition of detergent. Liver stored for a day at −25°C had normal activity (3.4µmol/min per g liver) without detergent. In patients with GSD Type 1a, G6Pase activity was very low both with and without detergent. These findings suggest a defect in glucose-6-phosphate transport in the microsomal membrane of the patients liver. The integrity of microsomal membrane was destroyed by storage at −25°C, when activity of G6Pase in the patients liver could be demonstrated. This may be the first example of a disorder involving the transport system of an intracellular membrane.

Familial amyotrophic lateral sclerosis (ALS) in Japan associated with H46R mutation in Cu Zn superoxide dismutase gene: A possible new subtype of familial ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disorder that results in relentless damage to the motor neuron system. Although about 5-10% of cases are familial, the pathophysiologic process of ALS remains unknown. We identified a novel point mutation A to G in exon 2 of the Cu/Zn SOD gene, resulting in an amino acid substitution of histidine46 by arginine (H46R), in two Japanese familial ALS (FALS) families. The segregations of the mutation were evident. The enzymatic activities of Cu/Zn SOD of peripheral red blood cell lysate were reduced to about 80% in the affected members, compared with other non-affected family members. The patients in these families are clinically characterized by relative late onset, initial involvement in lower extremities, relative rare impairment of bulbar muscles and much slow progression of muscular weakness and atrophy, compared with other Japanese FALS cases who have no mutation in the Cu/Zn SOD gene. These findings suggest that the H46R mutation in Cu/Zn SOD gene is highly related to this unique subtype of FALS.

Biochemical study in 28 children with lactic acidosis, in relation to Leigh's encephalomyelopathy.

An enzymatic study of cultured skin fibroblasts was made in 28 patients with lactic acidosis. In three of these patients a diagnosis of Leighs encephalomyelopathy was established from autopsy findings. Pyruvate decarboxylase (PDC) deficiency was found in four patients. In two of them, in whom Leighs encephalomyelopathy was proved by autopsy, PDC activity was lower than 10% of the normal. The other two living patients, who showed 22%–25% of the normal activity, had clinical symptoms and courses different from Leighs disease. These findings suggest that the patients with severe PDC deficiency develop Leighs disease but those with mild deficiency may not. A deficiency of cytochrome c oxidase was found in two siblings. One of them, who was diagnosed as having Leighs encephalomyelopathy by postmortem examination, showed a reduction of cytochrome c oxidase in the liver and brain. In the other sibling, who is living, the reduction of cytochrome c oxidase was demonstrated in the cultured skin fibroblasts and biopsied muscle. In an electron-microscopic study of biopsied muscle, two patients with mitochondrial myopathy were found. Their fundamental enzymatic defects were unclear. In two patients, in whom Leighs disease was suspected following a brain CT, the production of 14CO2 from [3-14C] pyruvate was found to be low; suggesting a reduced activity of the TCA cycle. In another 18 patients, the fundamental defect was not clear.

Mutation detection by TaqMan‐allele specific amplification: Application to molecular diagnosis of glycogen storage disease type Ia and medium‐chain acyl‐CoA dehydrogenase deficiency

We have devised an allele‐specific amplification method with a TaqMan fluorogenic probe (TaqMan‐ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele‐specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by “threshold” cycles to differentiate mutant and normal alleles without post‐PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end‐point PCR products, therefore allowing greater flexibility in designing allele‐specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan‐ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium‐chain acyl‐CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases. Hum Mutat 15:189–196, 2000.

Cytochrome C oxidase deficiency in two siblings with leigh encephalomyelopathy

Two siblings with cytochrome c oxidase deficiency are described. One of them died of subacute necrotizing encephalomyelopathy which was proven by autopsy. The other was also suspected of having Leigh encephalomyelopathy by the findings on brain CT scans. The former, a younger brother, was in good health until the age of 10 months when progressive dysphagia, muscular hypotonia and abnormal eye movements became apparent. Six months later he suddenly died due to respiratory insufficiency. The latter, an elder brother, started to show nystagmus, abnormal eye movements and ataxia at the age of 5 years. A deficiency of cytochrome c oxidase in the younger brother was demonstrated in autopsied liver and brain, while such a deficiency in the elder brother was shown in biopsied peripheral muscle tissue and in cultured skin fibroblasts. Both patients showed a marked heat lability of cytochrome c oxidase. These results suggest that the biochemical defect observed in the siblings is due to a genetic defect. This seems to be the first case of a generalized defect in cytochrome c oxidase.

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. Hum Mutat 11:377–386, 1998.

Nonketotic hyperglycinemia: Biochemical, molecular, and neurological aspects

SummaryNonketotic hyperglycinemia (NKH) is a metabolic disorder with autosomal recessive inheritance, causing severe, frequently lethal, neurological symptoms in the neonatal period. The metabolic lesion of NKH is in the glycine cleavage system (GCS), a complex enzyme system with four enzyme components; P-, T-, H-, and L-protein. The enzymatic analysis revealed that 86% of the patients with NKH are deficient of P-protein activity. The cDNA clones encoding all four components were isolated and their primary structures were determined. Several mutations have been identified in P- and T-protein genes: One missense mutation, S564I, in P-protein gene accounts for 70% of the mutant alleles in Finland where the incidence of NKH is unusually high. The immunochemical and in situ hybridization analyses revealed that the strong GCS expression was observed in rat hippocampus, olfactory bulbus, and cerebellum. The distribution resembled that of N-methyl-d-aspartic acid (NMDA) receptor which has binding site for glycine. It is, therefore, suggested that the neurological disturbance in NKH may be caused by excitoneurotoxicity through the NMDA receptor allosterically activated by high concentration of glycine. Based on the hypothesis the NMDA antagonists such as ketamine and dextromethorphan were administered to the patients. We treated three neonatal case with dextromethorphan and it ameliorated their findings on electroencephalogram and behavior in two out of three patients. Thus the GCS is suggested to play a role in regulation of glycine level around the NMDA receptor.