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Sazonova et al. Threshold heteroplasmy levels of atherogenic mutations

As a working hypothesis for the present study, we the threshold value of the heteroplasmic percentage for
used the monoclonal hypothesis according to which, each mutation was detected, after which the occurrence
if a mutation occurs in the mitochondrial genome, and development of atherosclerotic lesions begins in
the ATP synthesis is disturbed. The cells containing an individual, and for anti-atherogenic mutations, an
mitochondria with mutant mtDNA begin to suffer a antipathological effect begins to manifest. It should be
shortage of energy and proliferate unlimitedly. The emphasized that the choice of the optimal threshold
number of defective mitochondrial genome copies value was based on simultaneous maximization of
becomes greatly increased. Finally a mutation sensitivity and specificity. The revealed good agreement
reaches the threshold heteroplasmy level and the of the threshold heteroplasmy percentage value in the
pathological process starts. This results in the 11 mitochondrial genome mutations for atherosclerotic
occurrence and development of atherosclerotic plaques and the thickening of the carotid intimal-medial
lesions. When the threshold heteroplasmy level is layer can be indicative of general pathophysiological
reached by antiatherogenic mutations, they begin to mechanisms of the formation of lesions in intimal
show a protective effect. That is why, in people with a carotid arteries.
high heteroplasmy level for antiatherogenic mutations,
atherosclerosis is absent. The investigated mutations are localized in the coding
region of mitochondrial genome. Let’s consider each
For heteroplasmy level evaluation of investigated of them.
samples, a new original method of quantitative
assessment of mutant allele in mitochondrial genome, Mutation m.652delG was localized in the MT-
based on pyrosequencing technology, was developed RNR1 gene. It causes a structural defect of the
by the author and her colleagues [16] . With the use of 12S rRNA subunit, which can lead to a partial or
this method, it is possible to measure the heteroplasmy complete dysfunction of the mitochondrial ribosome.
level of both hereditary and somatic mitochondrial A consequence of this mutation may be a decrease
genome mutations, occurring during the lifetime of in synthesis of protein subunits of the enzymes of
an individual or in pathologic processes. The method the mitochondrial respiratory chain. As a result, the
of quantitative assessment of the mutant allele of synthesis of ATP may decrease, leading to an energy
mitochondrial genome has a number of significant failure in the mitochondria and intimal cells of arteries. It
advantages compared to other quantitative methods can, as a compensatory mechanism, lead to unlimited
such as invasive cleavage of the oligonucleotide probe proliferation of mitochondria and cells. The result of
(Invader), high-performance liquid chromatography, this process can be the occurrence of atherosclerotic
heteroduplex analysis, the analysis of heteroplasmy plaques and thickening of the intima-medial layer of
using Surveyor nuclease, ARMS, SNaPshot, HRM, human arteries.
TGGE, Sanger sequencing, NGS using 454/Roche
equipment, Applied Biosystems SOLiD, Illumina Probably, mutation m.652insG (MT-RNR1 gene)
equipment series used for the analysis of mutations [35-39] . associated with the absence of atherosclerosis stabilizes
subunit 12S of rRNA and improves the functions of
Pyrosequencing has the smallest number of defects ribosomes. Perhaps, in case of occurrence of this
and the greatest number of advantages, compared insertion, the synthesis of protein chains in enzymes
to other methods of measuring the percentage of mitochondrial respiratory chains increases. It leads
of heteroplasmy of mitochondrial genome [40-44] . It to an increase in the synthesis of ATP and protects
provides a unique opportunity to analyze a very short mitochondria and cells from oxidative stress. Therefore,
DNA fragment containing the region of the investigated this mutation can perform protective functions.
mutation. The size of such a DNA fragment is, on
average, 5-10 bp, it significantly reduces the probability Mitochondrial genome mutation m.1555A>G is also
of mistakes made during the analysis. As a result, encoded by MT-RNR1 gene.The mutation has an anti-
the method developed by the author with colleagues, atherogenic effect. It also leads by far the most to the
based on pyrosequencing technology, can serve as stabilization of the mitochondrial ribosome.
the “gold standard” for all other methods of determining
the percentage of mitochondrial genome heteroplasmy In case of a single-nucleotide substitution of
and it should be used to verify the heteroplasmy level m.3256C>T (MT-TL1 gene), transport RNA-Leucine
of mutations detected by other methods. dysfunction (recognition codon UUR) can occur. If
leucine is the last in the protein subunit of the enzyme,
With the aim of subsequent molecular genetic diagnosis then without this tRNA, the protein cannot be separated
of atherosclerosis in blood cells of the study participants, from the ribosome. It can lead to a dysfunction of

188 Vessel Plus ¦ Volume 1 ¦ December 28, 2017