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Sinyov et al. mtDNA mutations in buccal epithelium

INTRODUCTION High-resolution B-mode ultrasonography using a linear
vascular transducer at 7.5 MHz with the SonoScape
Investigations of the human genome make the early, SSI-1000 ultrasonic scanner (SonoScape Medical
presymptomatic diagnosis of different pathologies Corp., Shenzhen, China) was used to determine
possible. According to the literature, [1-4] various the degree of arterial stenosis of each participant.
pathologies, including atherosclerosis, may be Intima-medial thickness (IMT) was measured using
associated with mitochondrial mutations. The the program ProSound (R. Selzer, California Institute
phenomenon of heteroplasmy is typical for the of Technology, Pasadena, USA). Ultrasonographic
mitochondrial genome, meaning that a quantitative examination of the carotid arteries included scanning
assessment of mutant alleles in the mitochondrial the left and right carotid arteries with a focus on the
genome is necessary when investigating the back wall of the artery in three fixed projections:
association of mitochondrial mutations with diseases, anterolateral, lateral, and posterolateral. [9]
in particular, the heteroplasmy level of mutations
associated with pathologies should be evaluated. DNA phenol-chloroform extraction

Because of the instability of the mitochondrial genome, Total DNA for this study was isolated from whole blood
both somatic and hereditary mutations often occur. using phenol-chloroform extraction. [10,11] This DNA
These can accumulate, influencing the phenotype of isolation method included the following steps: (1) cell
the carrier. According to the literature, heteroplasmy of lysis (using sodium dodecyl sulfate); (2) enzymatic
the mitochondrial genome is common in normal human degradation of proteins with proteinase K; (3) cell
cells. During human embryogenesis, in the process lysate deproteinization with phenol and chloroform.
[5]
of determining cells and tissues, morphologic and
chemical differences occur between cells. As a result The above-mentioned stages of pure DNA product
of cell division, mitochondria are randomly distributed isolation came amid centrifugation to remove denatured
between cells. This determines the difference between proteins and fragments of cell organelles.
cells in the ratio of normal and mutant molecules
of mitochondrial (mt)DNA. It has previously been Next, DNA was precipitated from the solution using
established that single nucleotide polymorphisms of the ethanol and, following centrifugation, the precipitate
mitochondrial genome are unevenly distributed in the was dissolved in a buffer solution.
tissues and organs of adults. [6-8] This uneven distribution
impedes the use of biomarkers in diagnosing different Polymerase chain reaction
diseases. Therefore, the identification of biomarkers Polymerase chain reaction (PCR) was conducted with
for the non-invasive genetic diagnosis of pathologies the aim of amplifying the short-chain mtDNA fragments
seems an interesting prospect. to achieve a sufficient number of amplicon copies for
further analysis of these fragments. The conditions for
The aim of the present pilot study was to compare PCR were mentioned in previous publications. [4,10-12]
heteroplasmy levels in mtDNA m.12315G>A,
m.3336T>C, m.1555А>G, m.13513G>A, and Pyrosequencing
m.3256C>T between human buccal epithelial cells and Short-chain amplicons obtained during PCR were
whole blood cells in individuals with different degrees subjected to pyrosequencing in order to determine
of predisposition to atherosclerosis. The potential the nucleotide sequences of these fragments and
use of buccal epithelium for the genetic diagnosis assess heteroplasmy levels in the investigated
of atherosclerosis using mutations in mtDNA was mtDNA positions. This work was performed using
assessed.
the PSQ96MA pyrosequencer (Biotage AB, Uppsala,
Sweden), as previously described. [13]
METHODS

This study was carried out according to the Declaration Analysis of heteroplasmy levels in mtDNA
of Helsinki and with permission of an ethics committee mutations
(Russian Cardiology Research and Production Quantitative assessment of the mutant allele involved
Complex, Moscow, Russia). Samples were taken from estimating the heteroplasmy levels of mtDNA
134 donors, all of whom provided a written informed mutations, using the pyrogram peak height of each
consent. sample in the studied region of a single-stranded
PCR fragment of mitochondrial genome. The general
Samples of buccal epithelial and whole blood cells formula for calculating the percentage of heteroplasmy
were obtained from the donors. was as follows: [10,11]
146 Vessel Plus ¦ Volume 1 ¦ September 26, 2017