Shigeaki Miyabayashi

Identification of a common mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common recessively inherited metabolic diseases in man. We have studied fibroblast cultures obtained from three patients with MCAD deficiency by sequencing the entire coding region of MCAD mRNA. A single A to G nucleotide replacement which resulted in lysine329-to-glutamic acid329 substitution of the MCAD protein was identified in all cultures. Furthermore, this point mutation was present in 91% (31 of 34) of mutant MCAD alleles, indicating that the majority of cases with MCAD deficiency are caused by this type of mutation.

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.

Pancreatic beta-cell secretory defect associated with mitochondrial point mutation of the tRNALEU(UUR) gene: a study in seven families with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS)

SummaryRecent evidence suggests possible linkage between diabetes mellitus and mitochondrial gene mutation. We surveyed mitochondrial tRNALEU(UUR) (3243) mutation in 7 mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode (MELAS) families and identified 24 mutated subjects (7 MELAS probands and 17 non-MELAS relatives) as well as 11 non-mutant family members. An OGTT in the 24 mutant relatives revealed 14 diabetic subjects, 3 with impaired glucose tolerance and 7 with normal glucose tolerance and all non-mutant family members as having normal glucose tolerance. Insulinogenic index was significantly reduced in the mutant diabetic subjects and those with impaired and normal glucose tolerance in comparison with the normal control subjects and the non-mutant members. Urinary 24-h C-peptide immunoreactivity excretion was markedly reduced in the mutant diabetic subjects and those with normal and impaired glucose tolerance, compared with the control subjects and the non-mutant family members. Plasma C-peptide immunoreactivity 6 min after glucagon injection was markedly reduced in the mutant diabetic subjects and those with normal and impaired glucose tolerance compared with the control subjects and the non-mutant family members. Si, an index of insulin sensitivity of the four mutant subjects was within normal range. Islet cell antibodies were negative in sera of eight mutated diabetic subjects, 2 and 6 with impaired and normal glucose tolerance, respectively. Diabetic retinopathy and nephropathy were demonstrated in 7 (50%) and 12 (85.7%) of 14 mutant diabetic subjects, respectively. Neurosensory deafness was demonstrated in 12 (85.7%) of 14 mutated diabetic subjects, (66.7%) of 3 mutated impaired glucose tolerant subjects, but not detected in 6 mutated normal glucose tolerant subjects and 11 non-mutant family members. These findings suggest that the tRNALEU(UUR) mutation is associated with pancreatic beta-cell secretory defect of insulin.

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.

The GSTP1 Gene Is a Susceptibility Gene for Childhood Asthma and the GSTM1 Gene Is a Modifier of the GSTP1 Gene

Background: Bronchial asthma is a chronic airway disorder characterized by bronchial inflammation. Oxidative stress is a key component of inflammation. Glutathione S-transferase P1 (GSTP1), the abundant isoform of glutathione S-transferases (GSTs) in lung epithelium, plays a key role in cellular protection against oxidative stress. Several studies have shown that the GSTP1 geneis involved in the pathogenesis of asthma and a gene-gene interaction may occur within the GST gene superfamily. Methods: We screened single-nucleotide polymorphisms (SNPs) at the GSTP1 locus and performed an association study in the Japanese population using two independent case-control groups (group 1: 391 pediatric patients with asthma, 462 adult patients with asthma, and 639 controls, and group 2: 115 pediatric patients with asthma and 184 controls). The effect of GSTM1 null/present genotype on the association between GSTP1 Ile105Val and asthma was also investigated. Results: We identified 20 SNPs at this locus and found this region consisted of one linkage disequilibrium block represented by four SNPs (tag SNPs). The association between the Ile105Val polymorphism in the GSTP1 gene and childhood asthma was significant in both groups (p = 0.047 in group 1, and p = 0.021 in group 2). This association was only significant in patients with GSTM1-positive genotype in both groups (group 1: GSTM1 present p = 0.013 and GSTM1 null p = 0.925, and group 2: GSTM1 present p = 0.015 and GSTM1 null p = 0.362). Conclusions: These findings suggest that the GSTP1 gene is a childhood asthma susceptible gene, and the GSTM1 gene is a modifier gene of GSTP1 for the risk of childhood asthma in the Japanese population.

Linkage and association of childhood asthma with the chromosome 12 genes.

AbstractSeveral studies have shown linkage of chromosome region 12q13-24 to bronchial asthma and related phenotypes in ethnically diverse populations. In the Japanese population, a genome-wide study failed to show strong evidence of linkage of this region. Chromosome 12 genes that showed association with the disease in at least one report include: the signal transducer and activator of transcription 6 gene (STAT6), the nitrogen oxide synthetase 1 gene (NOS1), the interferon γ gene (IFNG), and the activation-induced cytidine deaminase gene (AICDA). To evaluate the linkage between chromosome 12 and childhood asthma in the Japanese population, we performed sib-pair linkage analysis on childhood asthma families using 18 microsatellite markers on chromosome 12. To investigate association between chromosome 12 candidate genes and asthma, distributions of alleles and genotypes of repeat polymorphisms of STAT6, NOS1, and IFNG were compared between controls and patients. Single nucleotide polymorphism of AICDA was also investigated. Chromosome region 12q24.23-q24.33 showed suggestive linkage to asthma. The NOS1 intron 2 GT repeat and STAT6 exon 1 GT repeat were associated with asthma. Neither the IFNG intron 1 CA repeat nor 465C/T of AICDA showed any association with asthma. Our results suggest that NOS1 and STAT6 are asthma-susceptibility genes and that chromosome region 12q24.23-q24.33 contains other susceptibility gene(s).

Ultrastructural study of the childhood mitochondrial myopathic syndrome associated with lactic acidosis

The quadriceps femoris muscle and the muscularis mucosae of the rectum from two children with mitochondrial myopathic syndrome associated with lactic acidosis were studied by electron microscopy. Striking morphological abnormalities of mitochondria were noted not only in the skeletal but also in the smooth muscle cells. Endothelial cells of blood capillaries distributed in these affected muscles were so greatly swollen that the capillary lumen was almost occluded. In contrast, surface epithelial and glandular epithelial cells of the rectum contained normal mitochondria, and fenestrated capillaries in the propria mucosae remained intact. Long-term ischemia resulting from occlusive changes of the capillary wall may be responsible for the mitochondrial alterations of muscle cells.

Extensive defects of mitochondrial electron-transfer chain in muscular cytochrome c oxidase deficiency.

ABSTRACT: This study was undertaken to estimate the extent of molecular defects in the mitochondrial electron-transfer chain of a patient with mitochondrial myopathy. Biochemical and immunochemical studies were performed on the skeletal muscle mitochondria. Spectrophotometry and enzyme activity measurements localized a definite defect at the segment of cytochrome c oxidase (complex IV) of the electron-transfer chain. Immunoblotting and immunoprecipitation studies using the anti-complex IV antibody revealed that the contents of subunits 1, 4, 5, 6, and 7 of complex IV were markedly diminished and that subunit 2 was almost absent. Inummohistochemistry of the muscle tissue revealed a considerable accumulation of immunoreactive materials of complex IV in the ragged-red fibers. The immunoblots using the anti-NADH-ubiquinone oxidoreductase antibody demonstrated that the contents of NADH-ubiquinone oxidoreductase subunits were 47% of control and that the contents of three subunits were considerably decreased. The contents of ubiquinol-cytochrome c oxidoreductase subunits were also somewhat low (77% of control) and one of the minor contaminants detected in the control was completely absent. High-resolution one-dimensional sodium dodecyl sulfate-urea-gel electrophoresis disclosed that six additional unidentified polypeptides in the control were markedly diminished or completely missing. These results demonstrate that the molecular defects in the mitochondrial electron-transfer chain are more extensive than would be expected from either spectral analysis or enzyme activity measurements alone, and involve not only complex IV but also NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase and several unidentified mitochondrial proteins.

Immunochemical study in three patients with cytochromec oxidase deficiency presenting leigh's encephalomyelopathy

Cytochrome c oxidase deficiency has been characterized by remarkable clinical heterogeneity because of the complexity of the enzyme. This complex enzyme consists of thirteen subunits which are the product of two separate genomes. Three larger subunits (I-III) are encoded by mitochondrial DNA and synthesized on mitochondrial ribosomes, whereas smaller subunits (IV-VII) are nuclear gene products. The clinical heterogeneity may be caused by biochemical heterogeneity. In this paper, we made immunological studies of cytochrome c oxidase (CCO; EC 1.9.3.1.) in muscle biopsies, post-mortem tissues and cultured skin fibroblasts from three patients with CCO deficiency presenting with Leighs encephalomyelopathy (McKusick 25600) in order to understand the molecular defect in this disorder.

Cerebral glucose utilization in pediatric neurological disorders determined by positron emission tomography

We measured local cerebral glucose utilization in 19 patients with Lennox-Gastaut syndrome (LG), partial seizures (PS), atypical and classical phenylketonuria (PKU), Leigh disease, and subacute sclerosing panencephalitis (SSPE), using positron emission tomography (PET). The mean values of regional glucose utilization in interictal scans of LG were significantly reduced in all brain regions when compared with that of PS (P<0.005). PET studies of glucose utilization in LG revealed more widespread hypometabolism than in PS. Two siblings with dihydropteridine reductase deficiency, a patient with classical PKU, and a boy with cytochrome c oxidase deficiency showed reduced glucose utilization in the caudate and putamen. A marked decrease in glucose utilization was found in the cortical gray matter of a patient with rapidly progressive SSPE, despite relatively preserved utilization in the caudate and putamen. The PET study of a patient with slowly progressive SSPE revealed patterns and values of glucose utilization similar to those of the control. Thus, PET provided a useful clue toward understanding brain dysfunction in LG, PS, PKU, Leigh disease, and SSPE.