ASSOCIATION OF PSYCHOSOCIAL FACTORS WITH DOPAMINE RECEPTOR D 4 ( DRD 4 ) , DAT GENE POLYMORPHISM AND CARDIOVASCULAR INCIDENCE RISK

Solely adverse environmental factors can hardly be fully responsible for the development of elevated levels of trait anxiety [1-6]. The study of 8-16-year-old twins from Great Britain showed genetic correlation between anxiety and depression [7]. The genetic correlation coefficient was as high as 80%, whereas factors of general environment accounted for the rest 20% [7]. Anxiety can be caused by the abnormal dopamine synthesis, [8–10] although the study regarding the relationships between anxiety traits and variable number of tandem repeat (VNTR) polymorphisms in the dopamine D4 receptor (DRD4) and the dopamine transporter (DAT) genes gained controversial results [11-14]. Great interest in studying anxiety is driven also by the fact that anxiety is considered as an independASSOCIATION OF PSYCHOSOCIAL FACTORS WITH DOPAMINE RECEPTOR D4 (DRD4), DAT GENE POLYMORPHISM AND CARDIOVASCULAR INCIDENCE RISK

Solely adverse environmental factors can hardly be fully responsible for the development of elevated levels of trait anxiety [1][2][3][4][5][6].The study of 8-16-year-old twins from Great Britain showed genetic correlation between anxiety and depression [7].The genetic correlation coefficient was as high as 80%, whereas factors of general environment accounted for the rest 20% [7].Anxiety can be caused by the abnormal dopamine synthesis, [8][9][10] although the study regarding the relationships between anxiety traits and variable number of tandem repeat (VNTR) polymorphisms in the dopamine D4 receptor (DRD4) and the dopamine transporter (DAT) genes gained controversial results [11][12][13][14].Great interest in studying anxiety is driven also by the fact that anxiety is considered as an independ-

ORIGINAL ARTICLES
ent risk factor for cardiovascular morbidity and mortality [15][16][17][18].To our knowledge, no available prospective population-based study in the literature describes similar data obtained by using the World Health Organization (WHO) programs.
The objectives of our study were to determine trait anxiety levels in an open population of 25-64-year-old males; to carry out an association analysis of trait anxiety and VNTR polymorphisms in the DRD4 and DAT genes; and to calculate Hazard ratio (HR) for developing arterial hypertension (AH), myocardial infarction (MI), and stroke, depending on the anxiety levels over a 24-year period of the study.

Material and methods
Three screening studies were conducted in a framework of the World Health Organization Multinational Monitoring of Trends and Determinants in Cardiovascular Disease program (MONICA) [8][9][10] and MONICA-psychosocial subprogram [21] in 1984, 1988, and 1994, respectively.A total of 2149 males aged 25-64 years, residents of one district of the city of Novosibirsk, were examined.The response rate was 82,1%.Anxiety levels were evaluated by using the Spielberger's test [22].Spielberger's inventories were filled out by each participant individually.Genotyping of the gene polymorphism was performed in the Molecular Genetics Laboratory of the Research Institute of Internal Medicine by using the methods described in detail elsewhere [23][24][25][26].Frequency distribution of the variable number of tandem repeat (VNTR) polymorphisms in the DRD4 and DAT genes in a population of 25-64-year-old males is shown in Table 1 and Table 2.The cohort for prospective study (n=1423) screened out all male participants diagnosed with ischemic heart disease, cerebrovascular pathology, arterial hypertension (AH), myocardial infarction (MI), and diabetes.During the entire 24 years of the study from 1984 to 2008, all first time MI events (n=104) were registered by using the WHO Acute Myocardial Infarction Register program, whereas the arterial hypertension (n=162) and stroke (n=76) events were documented in the process of the yearly observations on the cohort.
Statistical analysis of data was performed by using the SPSS (Statistical Package for Social Sciences) software package version 11.5.Chi square (χ 2 ) statistic was used to investigate whether distributions of categorical variables differed from one another in between the groups.The stratified Cox proportional regression model was used for determination of the HR adjusted for different data collection dates.A value of p<0,05 was considered statistically significant [27][28].

Discussion
More than half of 25-64-year-old males in the study population had HLA.Carriers of the DRD4 genotypes 4/4 and 2/4 were found more often in MLA group, whereas males with the genotype 4/6 were found more often in HLA group.We observed similar frequency distribution pattern for the DRD4 alleles.
Carriers of the DAT genotype 10/10 were found more often in MLA group than in HLA group.A pattern of frequency distribution among the carriers of the genotypes 9/10 and 9/9 was the opposite, namely: these genotypes were found more often in HLA group than in MLA group.The other genotypes in males with various levels of trait anxiety were found significantly rarer with the prevalence rates ranging from 2% to 5%.The ratios of the alleles 9 and 10 in males with trait anxiety were similar to the ratios of the corresponding genotypes.
Our data provided evidence that trait anxiety significantly increased the risk of developing CVD.Maximal risks for developing AH (HR AH =6,8) and stroke (HR S =6,4) were observed in males with HLA as early as within the first five years of the study compared to MLA group.Relative risks for developing AH and stroke in HLA group decreased with the course of time (for ten-year period: HR AH =5 and HR S =3,8; for 20-year period: HR AH =5 and HR S =3,8).At the same time, HR for developing MI showed a different pattern, namely: maximal risk for developing MI was found within 10 years of the study (HR=3,1); 20-year period revealed some downward trend in MI HR (HR=2,7); both 10-year and 20-year period indices exceeded the HR rates for developing MI within the first five years of the study (HR=2,5).
The differently directed HR trends in developing AH and stroke versus MI can be explained by the fact that HLA, as a cause of AH and stroke, was found more often in the older groups.Further decrease in HR for 10-year and 20-year periods was caused by reduction in a cohort size due to adverse outcomes in these groups.At the same time, HLA, as a cause of MI development, was found more often in the younger age groups, obviously resulting in a different HR trend pattern, namely: maximal HR was registered for 10-year period, whereas minimal HR was found for the first five years [29][30][31].The results of our study are consistent with data obtained by other authors [15].Meta analysis of 20 studies, conducted from 1980 to 2009, showed that anxiety in originally healthy individuals increased risk for developing coronary artery disease (HR=1,26, 95% CI=1,15-1,38, p<0,0001) independently of demographic factors, biological risk factors, and lifestyle.

Conclusion
1. Prevalence of high level of anxiety in 25-64-yearold male population of Western Siberia metropolis (the city of Novosibirsk) was as high as 50,9%.
2. High level of anxiety in 25-64-year-old male population was associated with the DRD4 genotype 4/6 and the DAT genotype 9/9.
3. High level of anxiety in 25-64-year-old male population caused maximal risk of developing arterial hypertension and stroke within the first five years of observation.
4. High level of anxiety in 25-64-year-old male population resulted in maximal risk of developing myocardial infarction within 10-year period, whereas risk of myocardial infarction events during the first five years of observation was minimal.

Table 3 Distribution of genotype and allele frequencies of the dopamine D4 receptor gene and prevalence of trait anxiety
Abbreviations: LLA -low level of anxiety, MLA -moderate level of anxiety, HLAhigh level of anxiety.

Table 4 Distribution of genotype and allele frequencies of the DAT gene and prevalence of trait anxiety
Abbreviations: LLA -low level of anxiety, MLA -moderate level of anxiety, HLAhigh level of anxiety.