Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants
ABSTRACT The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.
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- "Following digestion, the relative levels of the fragments was determined by capillary electrophoresis , and the ratio of the two mtDNAs normalized using a standard curve. As an independent validation of this method, in some experiments the proportion of NZB and 129 mtDNAs was also determined using SNaPshot primer extension method with the latter also normalized by comparison with a standard curve (Poole et al., 2010). Supporting SNaPshot data are provided in Figures S1and S2. "
ABSTRACT: Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.Cell 10/2012; 151(2):333. DOI:10.1016/j.cell.2012.09.004 · 33.12 Impact Factor
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ABSTRACT: How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. This article is part of a special issue entitled: Mitochondrial Gene Expression.Biochimica et Biophysica Acta 06/2012; 1819(9-10):979-91. DOI:10.1016/j.bbagrm.2012.06.002 · 4.66 Impact Factor
Article: The Problem with Mixing Mitochondria[Show abstract] [Hide abstract]
ABSTRACT: Mixing of mitochondrial DNAs (heteroplasmy) is unfavorable for reasons unknown. Sharpley et al. show that heteroplasmy has surprising genetic and behavioral effects in mice, even when each haplotype alone produces a normal phenotype. This interference is bioenergetic and may have contributed to the evolution of sexes.Cell 10/2012; 151(2):246-8. DOI:10.1016/j.cell.2012.09.028 · 33.12 Impact Factor