We are investigating the role of mitochondrial DNA (mtDNA) mutations in adult-onset hereditary neuromuscular disease. During the previous grant period, we identified a mtDNA point mutation that causes Leber's Hereditary Optic Neuropathy (LHON), obtained evidence that the MERRF and MELAS syndromes are the product of mtDNA point mutations, characterized mtDNA deletions of ocular myopathy patients and explored the therapeutic potential of coenzyme Q and succinate. These studies have raised three questions. What are other mtDNA mutations that cause maternally inherited diseases? What is the relationship between clinical phenotypes and mtDNA mutations? What is the relationship between the proportion of mutant mtDNAs and the variable disease manifestations in pedigrees harboring a mixture of mutant and wild type mtDNAs (heteroplasmy)? We propose to address these questions through three specific aims. First, we will identify additional mtDNA point mutations associated with LHON, MERRF, MELAS and maternally inherited ocular myopathy by analysis of mtDNA polymerase chain reaction (PCR) products. Heteroplasmic disease-causing mutations will be identified within individuals and between individuals of the same pedigree by mapping mismatches using DNA:DNA and RNA:RNA heteroduplexes and denaturing gradient gel electrophoresis. Homoplasmic mutations will be located by direct PCR sequencing. The mtDNA origin of the severe mitochondrial defects of smaller pedigrees will be confirmed by cybrid transfer and possibly mapped by complementation with deleted mtDNAs derived from ocular myopathy patients. Second, we will correlate the gene functions affected by mtDNA mutations with their associated phenotypes. This should reveal if similar diseases are the result of mutations in the same enzyme or are simply the consequence of comparable reductions in mitochondrial ATP production. Third, we will characterize the kinetics of heteroplasmic mtDNA segregation, and correlate the proportion of mutant mtDNAs with the severity of the clinical manifestations. These studies should generate new molecular diagnostic tests for mitochondrial diseases, lead to more effective metabolic therapy and provide more accurate prognosis of the disease course.
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