Mutations in human mitochondrial genome may result in formation of local
lesions in vascular wall and atherosclerosis development. A novel method based on pyrosequencing technology was developed to estimate frequency of mutant allele in mitochondrial genome, and used for the measurement of frequencies of mutations in the specimens of lipofibrous plaques in comparison normal intima from human aorta.
Assayed fragments of DNA were amplified with PCR. Relative quantity of
mutant allele was determined with pyrosequencing method. Pyrosequencing was initially designed to analyze single nucleotide polymorphisms (SNP) presence in genome. We adopted it for conditions where both mutant and normal allele were present in the same specimen. So it became possible to determine a critical quantity of mutant allele, which may initiate development of atherosclerosis lesions.
To find mutations associated with atherosclerotic lesions of aorta we analyzed 42 mitochondrial mutations previosly revealed at such pathologies as coronary vessel stenosis, diabetes, deafness, cardiac infarction, cardiomyopathy, and stroke. Tissue samples from intima of 7 aortas were used taken at autopsy. It was found that the heteroplasmy level of eight mitochondrial mutations localized in 7 genes (rRNA 12S, tRNA-Leu (codon recognized UUR ), tRNA-Leu (codon recognized CUN), cytochrome B, and subunits 1, 5, and 6 NADH dehydrogenase) have higher frequency in plaques as compared to normal intima. We suppose that this mutations may be potentially associated with atherosclerosis development.
Meanwhile the heteroplasmy level of three mutations localized in two mitochondrial genes (rRNA 12S and subunit 2
NADH dehydrogenase) turned out to be significantly higher in normal intima than in atherosclerotic plaques. That may indicate their antiatherogenic effect.
