| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS) is a major type of disease in mitochondrial myopathies. To identify the defective gene in MELAS, mitochondrial DNA from a patient with MELAS was sequenced by using amplified DNA fragments as sequencing templates. At least 30 nucleotides were different from those of a control human mitochondrial DNA. One of the substitutions was a transition of A to G in tRNA-Leu (UUR) gene at Cambridge nucleotide number 3,243. For the sequencing analyses, myogenic cells which were clones from a patient with MELAS were used. Myogenic clone with normal mitochondrial respiratory enzymes had A at this region, while a clone without respiratory enzyme activity had G at this site. These indicate that this substitution was directly linked with the molecular pathology of MELAS. n ApaI restriction site was gained by the substitution of this nucleotide. The ApaI digestion of the amplified DNA fragment revealed that all indepen
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dent 6 patients had G at nucleotide number 3,243 in their mitochondrial DNAs, but none of ll control subjects had G at this site. The ApaI digestion of autopsied tissues of a patient with MELAS revealed that all tissues examined showed heteroplasmy with he wild-type and mutant mitochondrial DNA. Among the tissues examined, cerebrum contained approximately 90 % mutant DNA, while spleen had 35 %. These suggest that the amount of the mutant mitochondrial DNA may, at least partially, have relation wih clinical phenotype of patients with MELAS. Mitochondrial DNA analyses on lymphocytes of patient and thier family members revealed that patients had approximately 60 % mutant DNA and mothers of these patients had smaller amount of mutant mitochondrial DNA in their lymphocytes. Some of the siblings also had mutant mitochondrial DNA, amount of which were between their mother and patients. These results suggest that MELAS is caused by a point mutation in mitochondrial tRNA-Leu (UUR) gene and the mutant gene is inherited from their mother. We also revealed that the analyses of lymphocyte mitochondrial DNA can be used for the diagnosis of MELAS as well as the diagnosis of carriers. Less
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