Mutations of mtDNA, due to their higher frequency of occurrence compared to nuclear DNA mutations, are the most promising biomarkers for assessing predisposition of the occurrence and development of atherogenesis. The aim of the present article was an analysis of correlation of several mitochondrial genome mutations with carotid atherosclerosis. Leukocytes from blood of study participants from Moscow polyclinics were used as research material. The sample size was 700 people. The sample members were diagnosed with “atherosclerosis” on the basis of ultrasonographic examination and biochemical and molecular cell tests. DNA was isolated from blood leukocyte samples of the study participants. PCR fragments of DNA, containing the region of 11 investigated mutations, were pyrosequenced. The heteroplasmy level of these mutations was detected. Statistical analysis of the obtained results was performed using the software package SPSS 22.0. According to the obtained results, an association of mutations m.652delG, m.3336C>T, m.12315G>A, m.14459G>A m.15059G>A with carotid atherosclerosis was found. These mutations can be biomarkers for assessing predisposition to this disease. Additionally, two single nucleotide substitutions (m.13513G>A and m.14846G>A), negatively correlating with atherosclerotic lesions, were detected. These mutations may be potential candidates for gene therapy of atherosclerosis and its risk factors.
In recent years, more and more attention is paid to molecular genetic diagnostics of polygenic multifactorial diseases, including cardiovascular pathologies and atherosclerosis. Atherosclerosis occurs in many men and women of middle age [
MtDNA mutations, due to their higher frequency of occurrence compared to nuclear DNA mutations, are the most promising biomarkers for assessing predisposition of the occurrence and development of atherogenesis. Each cell of a human organism, depending on the tissue, which it belongs to, has form one to several hundred mitochondria. In each of mitochondria, there are some copies of mitochondrial genome. Mitochondrial genome is characterized by the maternal type of inheritance. Somatic mutations often occur in mtDNA, the expressivity of which notably depends on the heteroplasmy level.
Even though the linkage of autosome mutations with atherosclerosis has been reported [
Leukocytes from blood of patients from Moscow polyclinics were used as research material. The sample size was 700 people. Men were older than 40 years, and women were older than 50. The sample members were diagnosed with “atherosclerosis” on the basis of ultrasonographic examination and biochemical and molecular cell tests [
There were 700 participants selected for the study, which made up two approximately equal groups:
Conventionally healthy patients;
Study participants which were at high risk of occurrence and development of atherosclerotic lesions.
The following methods of investigation were used as the following:
Isolation of DNA using the method of phenolchloroform extraction [
Polymerase chain reaction (PCR) in order to obtain DNA fragments containing the region of the investigated mutations [
Pyrosequencing of PCR fragments [
Analysis of the heteroplasmy level in the investigated mutations using the original quantitative method previously developed by the authors of this article on the basis of pyrosequencing technology [
Statistical analysis of the obtained results by using the software package SPSS 22.0 [
During the statistical analysis of results, Mann–Whitney
Eleven mitochondrial genome mutations, detected during the analysis of affected by atherosclerosis segments of aortas, were decided to be analyzed in blood cells of 700 study participants from the Moscow region, in which during clinical and ultrasonographic examinations atherosclerotic lesions of carotid arteries were found. The average age of the participants was 64.7 years.
A statistically significant positive correlation of atherosclerotic plaques with the heteroplasmy level mutations m.652delG, m.3336C>T, m.12315G>A, and m.14459G>A and a negative correlation with mutation m.14846G>A (
Bootstrap analysis of correlation of individuals’ atherosclerotic plaques with the heteroplasmy level of mitochondrial mutations.
Mutation  Correlation coefficient value  Asymptotical significance (2tailed) 

m.652delG  0,464  0,010 
m.652insG  −0,319  0,060 
m.1555A>G  −0,163  0,247 
m.3256C>T  0,274  0,101 
m.3336T>C  0,593  0,002 
m.5178C>A  0,305  0,064 
m.12315G>A  0,612  0,001 
m.13513G>A  −0,201  0,187 
m.14459G>A  0,605  0,001 
m.14846G>A  −0,452  0,010 
m.15059G>A  0,116  0,212 
Antiatherogenic effect of allele A at position 13,513 was shown at the heteroplasmy level above 65% in atherosclerotic plaques (
Association of 65% heteroplasmy level of allele 13513A with the absence of atherosclerotic plaques in carotid arteries (APCA).
Spearman correlation  65% heteroplasmy level of m.13513G>A  APCA  

65% heteroplasmy level of m.13513G>A  Correlation coefficient  1000  −0,204 
Significance (2tailed)  —  0,054  
Number of valid cases  90  90  


APCA  Correlation coefficient  −0,204  1000 
Significance (2tailed)  0,054  —  
Number of valid cases  90  502 
As the effect of different mutations is multidirectional, it is necessary to consider the cumulative impact of 11 studied mutations or total mutational burden. This characteristic was assessed in two stages:
the construction of logistic regression model (Tables
the construction of ROC curves (Table
Summary of a linear regression model of interconnection of mutational burden with atherosclerotic plaque in carotid arteries.
Model  Minus twice the log likelihood  Cox and Snell 
Nagelkerke 

1  147,273  0,358  0,481 
Classification of cases of atherosclerotic plaque association with a total burden of 11 mutations.
Model  Detected cases  Predicted cases  

Association of atherosclerotic plaques with a total burden of 11 mutations  Percentage of correct predictions  
0,00  1,00  
1  Association of atherosclerotic plaques with a total burden of 11 mutations  0,00  44  24  64,7 
1,00  24  68  73,9  
Total percentage value  70,0 
The analysis of included variables and the coefficient of link force and direction.
Analyzed variables  

Mutations  B  S.E.  Wald  df  Sig.  Exp (B)  
Model 1  m.1555A>G  −0,163  0,042  14,952  1  0,000 
0,850 
m.3256C>T  0,033  0,051  0,417  1  0,519  1033  
m.14846G>A  −0,026  0,029  0,845  1  0,358  0,974  
m.5178C>A  0,034  0,045  0,560  1  0,454  1034  
m.652delG  0,052  0,022  5761  1  0,016 
1054  
m.12315G>A  0,122  0,027  20,958  1  0,000 
1130  
m.13513G>A  −0,046  0,017  6951  1  0,008 
1047  
m.14459G>A  0,030  0,015  3971  1  0,046 
0,970  
m.15059G>A  0,052  0,020  6836  1  0,009 
1054  
m.652insG  0,077  0,081  0,901  1  0,343  1080  
m.3336T>C  0,052  0,028  3420  1  0,064 
1054  
Constant  −2384  1308  3321  1  0,068 
0,092 
Coefficient B indicates the link direction;
ROC analysis of interconnection of mutational burden with atherosclerotic plaques in carotid arteries.
Probability of faultless prognosis  

Area under the curve  Standard error  Asymptomatic significance  Asymptomatic confidence interval 95%  
Lower than 95%  Higher than 95%  
0,842  0,030  0,001  0,784  0,900 
A predictor in the analysis of ROC curves was a probability belonging to one category or another (0 (no atherosclerotic plaques) or 1 (the presence of atherosclerotic plaques of any size)).
On the basis of values of the included variables, with the use of the model, the estimation of probability belonging to a category “0” or “1” for each study participant was performed. The data obtained on the probability, which can be considered as a measure of the relative risk, were used for ROC analysis (Figure
ROC curve for the analysis of total mutation burden of 11 mitochondrial genome mutations as genetic markers of the presence of atherosclerotic plaques in carotid arteries.
According to the data of ROC analysis, the model turned out to be significant. The threshold value of 0.54 was chosen; it corresponded to the sensitivity value of 0.739 and specificity value of 0.735 (
Therefore, the predictive and explanatory ability of the model for total mutation burden in atherosclerotic plaques of any size was significantly higher than that of the models made separately for each mutation. The total mutational burden of 11 investigated mitochondrial genome mutations was associated with 84.2% of atherosclerotic plaques in the carotid arteries in human.
According to the statistical data (Table
Bootstrap analysis of сorrelation of mitochondrial genome mutations with the IMT CA.
Mutation  Correlation coefficient  Asymptotical significance (2tailed) 

m.652delG  0,161  0,206 
m.652insG  0,017  0,722 
m.1555A>G  0,009  0,865 
m.3256C>T  0,329  0,091 
m.3336T>C  0,081  0,383 
m.5178C>A  0,318  0,103 
m.12315G>A  0,619  0,001 
m.13513G>A  −0,478  0,050 
m.14459G>A  0,157  0,236 
m.14846G>A  −0,493  0,045 
m.15059G>A  0,529  0,028 
A significant negative correlation with the present parameter was found for mutations m.13513G>A and m.14846G>A (
Due to the fact that the effect of different mutations on a change of the IMT CA was multidirectional, it was necessary to consider the cumulative impact of 11 studied mutations or total mutational burden. This characteristic was assessed in two stages, as it was in section
Summary of linear regression model of interconnection of mutational burden with IMT CA.
Model  Minus twice the log likelihood  Cox and Snell 
Nagelkerke 

1  65,002  0,260  0,382 
Classification of cases of IMT CA association with a total burden of 11 mtDNA mutations.
Model  Detected cases  Predicted cases  

Association of IMT CA with a total burden of 11 mutations  Percentage of correct predictions  
0,00  1,00  
1  Association of IMT CA with a total burden of 11 mutations  0,00  55  2  96,5 
1,00  11  9  45,0  
Total percentage value  83,1 
The analysis of included variables and the coefficient of link force and direction.
Analyzed variables  

Mutations  B  S.E.  Wald  df  Sig.  Exp(B)  
Model 1  m.1555A>G  −0,177  0,079  5017  1  0,025 
0,838 
m.3256C>T  0,098  0,071  1916  1  0,166  1103  
m.14846G>A  −0,108  0,051  4483  1  0,034 
0,898  
m.5178C>A  0,079  0,078  1024  1  0,312  0,924  
m.652delG  0,006  0,036  0,032  1  0,857  0,994  
m.12315G>A  0,027  0,030  0,806  1  0,369  1028  
m.13513G>A  −0,023  0,023  1001  1  0,317  1023  
m.14459G>A  0,018  0,026  0,463  1  0,496  0,982  
m.15059G>A  0,026  0,023  1273  1  0,259  1027  
m.652insG  −0,187  0,156  1439  1  0,230  1205  
m.3336T>C  0,028  0,028  1036  1  0,309  1028  
Constant  1350  1913  0,498  1  0,480  3858 
Coefficient B indicated the link direction;
Analysis of ROC curves is presented in Figure
The ROC curve for the analysis of total mutation burden of 11 mitochondrial genome mutations as genetic markers of the presence of thickening of the intimamedial layer of carotid arteries.
ROC analysis of interconnection of mutational burden with IMT CA.
Probability of faultless prognosis  

Area under the curve  Standard error  Asymptomatic significance  Asymptomatic confidence interval 95%  
Lower than 95%  Higher than 95%  
0,849  0,055  0,001  0,742  0,956 
On the basis of values of the included variables, the model estimated the probability belonging to a category 0 or 1 for each study participant. The data on the probability, which can be considered as a measure of relative task, were used for the ROC analysis (Figure
The area under the curve was 0.849, consequently the model turned out to be significant. Sensitivity was 0.700; specificity was 0.900 (
The evaluation of the predictive and explanatory power of the model for total mutational burden in IMT CA allowed us to consider that the predictive and explanatory power in the used model was significantly higher than that in the models constructed individually for each mutation. Total mutational burden of 11 studied mitochondrial genome mutations was associated with 84.9% of thickening of the intimamedial layer of carotid arteries in humans.
Therefore, an analysis of mtDNA mutations in a large representative sample of 700 study participants, which included patients with atherosclerosis and conventionally healthy individuals, was carried out. The sample members were diagnosed with “atherosclerosis” on the basis of ultrasonographic examination and biochemical and molecular cell tests. Four mutations of the mitochondrial genome, highly significantly linked with the presence of atherosclerotic plaques in patients (m.652delG, m.3336C>T, m.12315G>A, and m.14459G>A) were identified. Two single nucleotide substitutions of mtDNA were significantly linked with the absence of atherosclerotic plaques in individuals (m.13513G>A and m.14846G>A). Additionally, two mitochondrial mutations were detected (m.12315G>A and m.15059G>A), which significantly positively correlated with the thickening of the intimamedial layer of carotid arteries. Two other mutations significantly negatively correlated with the thickening of the intimamedial layer of carotid arteries (m.13513G>A and m.14846G>A). These mutations are localized in MTRNR1, MTTL2, MTND1, MTND6, and MTCYTB. It suggests an idea of an important role of genes of subunits of respiratory chain enzymes of mitochondria and ribosomal RNA and also an important role of transfer RNAleucine in atherogenesis processes. A negative correlation of mutations m.13513G>A (MTND6) and m.14846G>A (MTCYTB) with atherosclerosis may indicate that these mutations can lead to enzyme stabilization and can make their work more efficient. The total mutational burden of eleven studied mitochondrial mutations was associated with more than 84% cases of occurrence of atherosclerotic plaques and pathological thickening of the intimamedial layer of carotid arteries, which indicates the high diagnostic value of complex analysis of these mutations in atherosclerosis. Therefore, the most optimal for gene diagnostics of atherosclerosis would be the use of all these eleven investigated mtDNA mutations, as it enables the evaluation of predisposition to atherosclerosis and its early diagnosis in the maximum number of patients.
However, we should acknowledge certain limitations of this study. Although the study was performed in rather large sample of atherosclerotic patients and apparently healthy nonatherosclerotic individuals, it should be noted that using the same samples in whom the described association was originally identified may artificially augment the strength of prediction. With such level of prediction, it may be suggested that mitochondrial mutations are extremely powerful risk factors, but the effect size of these mutations may be probably a lot lower and artificially inflated using the same data. Therefore, replication studies in independent naïve cohorts are necessary to confirm the findings.
It is necessary to mention that although the investigated sample consisted of 700 study participants, the statistical processing of the results included a bootstrap analysis during which the manyfoldincreased sample of patients with atherosclerosis was compared with manyfoldincreased sample of conditionally healthy donors.
We suppose that in our further studies, in which a much larger sample of study participants will be analyzed, it will not be necessary to resort to bootstrap analysis.
The importance of detecting mtDNA mutations linked with occurrence and development of atherosclerotic lesions in humans does not admit of doubt due to the fact that the mortality from atherosclerosis is at a very high level.
In the present study, an association of five mutations with carotid atherosclerosis was found (m.652delG, m.3336C>T, m.12315G>A, m.14459G>A m.15059G>A). These mutations can be biomarkers for assessing predisposition to this disease. Additionally, two single nucleotide substitutions (m.13513G>A and m.14846G>A), negatively correlating with atherosclerotic lesions, were detected. These mutations may be potential candidates for gene therapy of atherosclerosis and its risk factors.
The results obtained in this study may be useful to medical geneticists, specializing in the evaluation of the predisposition to atherosclerosis and other vascular diseases and also in the early diagnosis of these pathologies.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the Russian Science Foundation (Grant no. 151510022).