The mitochondrial myopathies are a genetically, biochemically, and clinically heterogenous group of disorders which have been difficult to classify and study. They are currently classified on the basis of a clinical constellation of symptoms and histochemical properties of skeletal muscle biopsies. Their molecular basis remains obscure, but there is recent evidence for deletions in the mitochondrial DNA of some of these patients. We propose to apply the powerful techniques of molecular biology to these disorders and to test the hypothesis that mutations mitochondrial DNA play a major pathogenetic role. The long term objective of this project is the characterization of the molecular genetic basis of the mitochondrial myopathies, beginning with a detailed analysis of a well-characterized patient.
The specific aims of this proposal are: (1) the preparation of a set of molecular DNA clones that span the entire human mitochondria genome; (2) the production of a complete set of molecular mitochondrial DNA clones from the proband patient with mitochondrial encephalomyopathy, lactic acidosis, and stroke (MELAS) syndromes; (3) the nucleotide sequence determination of the three mitochrondrially-encoded cytochrome oxidase genes and comparison to the published normal sequence; (4) the screening of a population of mitochondrial myopathy patients for large mitochondrial DNA deletions by Southern hybridization probe analysis, molecular cloning, and polymerase chain reaction (PCR) amplificance of any observed mutations in highly conserved regions by assessing mitochondrial protein synthesis, by focused DNA analysis of first degree relatives of affected patients, and by in situ hybridization studies of skeletal muscle. These studies should further our understanding of the molecular genetic basis of the mitochondrial myopathies. Such information is a necessary prerequisite of the proper diagnosis, genetic counselling, and eventual treatment of these patients.
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