Expression Of mtDNA Mutations Research Articles

Primary and secondary defects of the mitochondrial respiratory chain.

Over 100 mutations of mitochondrial DNA (mtDNA) have been associated with human disease. The phenotypic manifestation of mtDNA mutations is extremely broad, from oligosymptomatic patients with isolated deafness, diabetes, ophthalmoplegia, etc., to complex encephalomyopathic disorders that may include dementia, seizures, ataxia, stroke-like episodes, etc. The genotype variants are also wide, with rearrangements (deletions, duplications) and point mutations affecting protein coding genes, tRNAs and rRNAs. There are some broad genotype/phenotype correlations but also substantial overlap. The pathogenetic mechanisms involved in the expression of mtDNA mutations are still not yet fully understood. More recently, mutations of nuclear genes encoding subunits of the respiratory chain, particularly those of complex I, have been identified. These predominantly, but not exclusively, involve infant onset disease with early death. Recently it has become clear that the function of the respiratory chain may be impaired by mutations affecting other mitochondrial proteins or as a secondary phenomenon to other intracellular biochemical derangements. Examples include Friedreich ataxia where a mutation of a nuclear encoded protein (frataxin), probably involved in iron homeostasis in mitochondria, results in severe deficiency of the respiratory chain in a pattern indicative of free radical mediated damage. Mutations of nuclear encoded proteins involved in cytochrome oxidase assembly and maintenance have been characterised and, as predicted, are associated with severe deficiency of cytochrome oxidase and, most frequently, Leigh syndrome. Defects of intracellular metabolism, with particularly excess-free radical generation including nitric oxide or peroxynitrite, may cause secondary damage to the respiratory chain. This is probably of relevance in Huntington disease, motor neuron disease (amyotrophic lateral sclerosis) and Wilson disease. These disorders seem to have defective oxidative phosphorylation as a common pathway in their pathogenesis and it may be that treatments designed to improve respiratory chain function may ameliorate the progression of these disorders.

40] Use of myoblast cultures to study mitochondrial myopathies

This chapter describes the procedures used for (1) direct cloning of primary human muscle satellite cells, (2) immunofluorescent staining of myoblasts for fluorescence-activated cell sorting (FACS), and (3) analysis of mitochondrial translation products in myoblasts and myotubes. Mutations in mitochondrial DNA (mtDNA) that affect mitochondrial protein translation (large-scale deletions, tRNA point mutations) are associated by many clinical entities classified as mitochondrial encephalomyopathies. Skeletal muscle fibers in patients with these diseases contain heteroplasmic mixtures of wild-type and mutant mtDNAs, inhomogeneously distributed along their length. Skeletal muscle fibers are postmitotic, multinuclear syncytia that cannot be grown in tissue culture. They are surrounded by mononuclear, undifferentiated myoblasts called “satellite cells” that may proliferate in culture. Although a number of different cell types are often available to study the expression of pathogenic mtDNA mutations, two important advantages are gained by using primary myoblast cultures. First, myoblasts can be induced to fuse in culture to form multinucleate myotubes that terminally differentiate and contract, thus, making it possible to study the effects of growth versus terminal differentiation on the expression of mtDNA mutations. Second, because satellite cells are dormant most of the time, the turnover of mitochondria and mtDNA is extremely slow as compared to that in differentiated muscle fibers. Thus, the relative proportions of mutant and wild-type mtDNAs in the satellite cell population reflects the degree of heteroplasmy that existed in the myoblast precursor population during embryological development.