Article

Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, CA 92697, USA.
Biological Chemistry (Impact Factor: 3.27). 10/2010; 391(10):1115-30. DOI: 10.1515/BC.2010.125
Source: PubMed

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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    • "As the transmission of mtDNA is uniparental due to the lack of recombination, pathogenic, functional and neutral variants can interact and are often linked with one another [58]. Thus, a multitude of SNPs can accumulate along branches of a haplogroup, which may alter the significance of other sub-haplogroups; hence, the test for individual SNPs reveals an inadequate image of the evolutionary and functional role for mtDNA in an individual's haplotype [58]. "
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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. "
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