DELETIONS OF MITOCHONDRIAL DNA IN NEUROMUSCULAR DISEASE

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We recently discovered that essentially all patients with progressive external ophthalmoplegia (PEO) presenting with ragged red fibers in muscle - either with the less-severe form, called Ocular Myopathy (OM), or with the fatal form, called Kearns-Sayre Syndrome (KSS) - harbor large deletions of mitochondrial DNA (mtDNA) in their skeletal muscle. This is the first time a mtDNA, and it opens up new avenues for study of both disease process itself and the basic biology of mitochondria. We have mapped and sequenced the deletion breakpoints; studied heteroplasmy, both horizontally (i.e. among cells and tissue) and vertically deleted mtDNAs; analyzed KSS muscle sections by immunofluorescence, cytochemistry, and in-situ hybridization; and obtained evidence of segregation of normal and deleted mtDNAs. We now propose to analyze these deletions further at the molecular level, with an aim of understanding the etiology and pathogenesis of PEO, and of demonstrate definitively that there is segregation of normal and deleted mtDNAs in individual mitochondria. Second, we will measure the relative rates of replication of normal and deleted mtDNAs, to see whether the proliferation of deleted mtDNAs in PEO is due to replicative advantage. Third, we will create an in-vitro model of myopathies due to PEO, by taking advantage of a novel tissue culture system whereby isolated, intact mitochondria are introduced by microinjection or cell fusion into the cytoplasm of recipient human cells that have been depleted of all their mitochondrial DNA (rhoo cells). Finally, we will explore the possibility of creating a mouse model of PEO. In both the in-vitro and mouse models, we will attempt to eliminate selectively mitochondria containing deleted mtDNA molecules, with the ultimate goal of developing criteria for a rational therapy to treat these currently incurable, and in the care of Kearns-Sayne Syndrome, fatal diseases.