The risk of coronary heart disease (CHD) varies considerably in different ethnic groups, which may be due to genetic factors 1. Thus, one of the most important are the new genetic risk factors of CHD, associated with immune inflammation and intravascular thrombosis 2.

Especially important to study gene-to-gene interactions that increase the risk of early debut of CHD and myocardial infarction 3. The integration of immune response, haemorheology disorders and endothelial dysfunction occur through pleiotropy effects of peroxisome proliferator-activated receptor-α (PPAR- α) 4.

Activation of PPAR-α contributes to the suppression of various mechanisms of immune inflammation through a system of nuclear factor κ-β: production of proinflammatory cytokines (interleukin 6, 8, 1-β, interferon - γ, tumor necrosis factor-α, VCAM-1), the adhesion and the migration of mononuclear cells into the subendothelium, suppression of pro-inflammatory activity of the endothelium and the production of acute phase proteins 5. PPAR-α have a layered effect on lipid metabolism: increase the synthesis of high-density lipoprotein cholesterol, stimulate reverse cholesterol transport, lower triglyceride levels 6.

PPAR-α gene localized in the long arm of 22nd chromosome. L162V polymorphism PPAR-α gene is characterized by replacement of leucine to valine at 162 position and is due for replacement of G to C 484 5-position of gene's exon. L162V genotype PPAR-α gene was associated with components of the metabolic syndrome 7, with the early development of CHD (Northwick Park Heart Survey), occurs four times more frequently in patients with angiographically verified coronary atherosclerosis than in the control group 8. This genotype was associated with the early development of diabetes 9. Carriers of L162V genotypes with diabetes melitus type 2 had higher levels of total cholesterol and apolipoprotein AI 9.

One of the important pathophysiological mechanisms of activation of PPAR-α receptor is suppressing of tissue factor expression. Tissue factor is a primary element of coagulation cascade and is directly relevant to thrombogenesis. In case of cell's damage part of tissue factor molecules are tightly associated with the coagulation factor VIIa, supporting its function in an external way the accelerator of blood coagulation. A603G polymorphism tissue factor gene has prognostic value in acute coronary syndrome 10, therefore, its study with a clinical position is extremely important.

Tissue factor serum levels depend on external factors as well as genetic factors. T. Ott et al have found that carriage of G allele gene polymorphism A603G tissue factor is associated with higher concentrations of tissue factor in the blood plasma and increased risk of myocardial infarction 11.

A603G gene polymorphism of tissue factor was associated with death and reinfarction in patients with myocardial infarction 12. In addition, this polymorphism is associated with an increased risk of venous thrombosis and CHD 13.

It is found that PPAR-α decrease tissue factor expression and activity in human mononuclear cells 14. Therefore, the study of gene-gene interactions of PPAR-α and tissue factor genes is extremely important.

The goal of this study is to determine the association of L162V polymorphism of PPAR-alpha gene, A603G polymorphism of tissue factor gene and the risk of coronary heart disease development in Russian population.  

The current study included 414 patients with CHD (men and women) aged 33 to 80 years (mean age – 61,6 ± 0,48 years). The control group consisted of 220 people without CHD of comparable age (mean age – 60,09 ± 0,72 years, p = 0.081).

The majority of the surveyed patients had a history of myocardial infarction – in 295 (71,3%) of all patients with CHD

Traditional CHD risk factors in different age groups of CHD onset are shown in Table 1. Such traditional CHD risk factors as hypertension, smoking, obesity or overweight, dyslipidemia, were detected more frequently than in half of observed cases.

There were no significant differences in the frequency of occurrence of such traditional factors of CHD as hypertension, family history, fasting hyperglycemia and dyslipidemia in patients with the debut of the disease in different age (p>0,05, Table 1). However, a history of smoking were detected significantly more often in the group of studied patients with CHD debut at the age of 46 – 59 years compared with the group of patients with debut of the disease at the age of 60 years and older (p = 0,0071, Table 1). In addition, diabetes mellitus type 2, obesity or overweight were more common in patients with CHD debut at the age of 59 years and less compared with the group with debut of disease at the age of 60 years and older (p<0,017, Table 1).

Molecular-genetic examination of patients with CHD and a control group of comparable age without CHD was performed. Deoxyribonucleic acid (DNA) extraction from venous blood leukocytes was carried out on the column "K-SORB-100" ("Syntol", Russian Federation).

To determine the polymorphic variants of L162V PPAR-α gene polymorphism PCR was performed on an automated thermocycler Tertsik on a thermal cycler (DNA Technology, Moscow).

Amplification of the fragment was produced using the following sequence of synthetic oligonucleotides (Beagle, Russian Federation):

• forward primer: 5'GACTCAAGCTGGTGTATGACAAGT-3;

• reverse primer: 5'CGTTGTGTGACATCCCGACAGAAT-3.

The reaction mix for restriction analysis included 18 mkl of the PCR product, 1mkl of restriction enzyme Hinf I (SibEnzyme, US LLC), buffer O. Incubation of mix produces at 37 °C for 12 hours.

Restriction analysis was performed using a vertical electrophoresis in 8% polyacrylamide gel followed by staining with ethidium bromide and visualization in the ultraviolet.

Polymorphic variants of A603G polymorphism of tissue factor is determined by PCR. Amplification of the studied fragment of tissue factor gene was carried out using synthetic oligonucleotides shown below (Beagle, Russian Federation):

• forward primer: 5'AGTCACTATCTCTGGTCGTA-3;

• reverse primer: 5'CTTCCCTTCCATTTGGTGAT-3.

Restriction analysis was performed with the use for the preparation of the reaction mix 1 mkl restriction enzyme Bst2UI (SibEnzyme, US LLC), 2 mkl tenfold buffer G, 0,2 mkl of the BSA. Incubating the mix was performed at 60^оC for five hours, followed by visualizing a 2% agarose gel stained with ethidium bromide, using UV light.

Statistical analysis was performed using the statistical software package Statistica 10 (Stat Soft Inc., version 10.0.228.8, Oklahoma, USA). Analysis of qualitative binary signs held by means of Fisher's exact test.

A comparative analysis of the distribution of L162L, L162V genotypes and frequency of alleles L and V of L162V polymorphism of PPAR-α gene in patients with CHD and in the control group of comparable age without clinical and angiographic evidence of CHD. Analytical data presented in Table 2.

Analysis of the distribution L162L, L162V L162V polymorphism genotypes PPAR-α gene in patients with coronary heart disease and in the control group without CHD revealed the following statistically significant differences. Distribution in CHD patients was as follows: L162L genotype – 358 of 414 (86%), L162V genotype – 56 of 414 (14%). In the control group without CHD distribution was as follows: L162L genotype – 206 of 220 (93,6%), L162V genotype – 14 of 220 (6,4%) (p=0,004, Table 2). Thus, L162V genotype of PPAR-α gene was significantly more common in patients with CHD than in the control group without CHD. Thus, the carrier L162V genotype was associated with an increased risk of CHD in 2,13 times in comparison with the control group without CHD (OR=2,13; CI:1,16÷3,9; p=0,008, Table 2). The incidence V allele was higher in CHD patients than in the control group and the carriage V allele associated with an increased CHD risk by 2,21 times compared with the control group without CHD (OR = 2,21; CI : 1,21 ÷ 4,01, Table 2).

(Table 2) – Distribution of L162L and L162V genotypes and frequency of alleles L and V of L162V polymorphism of PPAR-α gene in CHD patients and in the control group without CHD

The patients were divided into three groups: those with coronary heart disease debut at the age of 45 years or less, aged 46 – 59 years, 60 years of age and older. Results of the analysis of genotypes and alleles distribution of L162V polymorphism (PPAR-α gene) in patients with CHD with the debut of the disease at different ages are presented in Table 3.

L162V heterozygous genotype carried 20 of 58 (34,4%) patients with CHD with the debut of the disease at the age of not more than 45 years, 22 of 203 (10,8%) – at the age of 46 – 59 years. Thus, L162V genotype PPAR-α gene was detected significantly more often in patients with coronary heart disease debut at age of 45 or younger (p = 0,00002) and was associated with an increased risk of CHD at the age of 45 years or less in 4,68 times (OR = 4,68; CI: 2,3 ÷ 9,52, Table 3). V allele was detected significantly more often in patients with CHD incidence aged 45 years or less (p = 0,00005, OR = 3,88; CI: 2,02 ÷ 7,46).

An assessment tissue factor level in the serum of 110 patients with CHD and 60 people in the control group without clinical and angiographic evidence of CHD.

In the group of CHD patients tissue factor level was 197,3 ± 9,2 pg/ml, while in the group without CHD – 132,4 ± 7,6 pg/ml. Thus, the level of tissue factor was significantly higher in CHD patients than in the control group (p = 0,000004, Figure 1).

The distribution A603A, A603G and G603G genotypes of tissue factor gene in CHD patients differed from distribution of above mentioned genotypes of control group without clinical and angiographic evidence of CHD. Thus, G603G genotype was detected significantly more often in CHD patients than in the control group without CHD, and his carriage increased the risk of CHD (OR = 2,68, CI: 1,78 ÷ 4,01, p<0,0001, Table 4). G allele was significantly more often detected in CHD patients compared with the control group and was associated with an increased risk of CHD in 4 times.

An assessment of tissue factor level and distribution A603A, A603G, G603G genotypes of A603G polymorphism of tissue factor in patients with CHD and in the control group without CHD. The results are shown in Table 5.

The level of tissue factor was significantly higher in patients with CHD – carriers G603G genotype compared with carriers A603A genotype (217,9±15,2 pg/ml and 152,6±30,4 pg/ml, respectively, p=0,04), but not significantly different when comparing the groups of patients with CHD – carriers A603G A603A and genotypes as well as genotypes of A603G and G603G A603G gene polymorphism tissue factor (p> 0,05). In the control group the levels of tissue factor were not significantly different between carriers of different genotypes under study tissue factor gene polymorphism (p> 0,05, Table 4, Table 2).

Evaluation of distribution of A603A, A603G and G603G genotypes of tissue factor gene was performed between patients with CHD debut at the age of 45 years and younger and at the age of 46 – 59 years who were homogeneous with respect to the traditional cardiovascular risk factors. As the group surveyed patients with CHD debut at the age of 60 years and older was significantly different from the above groups on traditional cardiovascular risk factors such as diabetes, obesity or overweight, and smoking , it was excluded from the analysis.

Analysis of the distribution of genotypes of A603G polymorphism of tissue factor gene in patients with different age of CHD debut showed the following: G603G genotype was detected in 28 of 58 (48%) patients with age of onset of the disease of 45 years or younger, while only in 74 of 203 (36 %) persons with a debut at the age of 46 – 59 years. Thus, G603G genotype was significantly more frequent in patients with the debut of the disease at the age of 45 years and less than in the group with the older age, with no statistically significant effect on the risk (p = 0,049; OR=1,61, CI:0,83÷3,1, Table 6).

One of the most important pathophysiological mechanisms of activation of PPAR-α is the suppression of the expression of tissue factor, resulting in an extremely important study of gene-to-gene interactions of L162V polymorphism of PPAR-α gene and A603G polymorphism of tissue factor gene.

The combination of L162V genotype of PPAR-α gene and G603G genotype of tissue factor gene was detected significantly more often in CHD patients compared to the control group without CHD than other combinations of genotypes of observed gene polymorphisms (L162V / G603G genotypes – 17 of 314 (5,4%) of CHD patients and in 4 of 220 (2,5%) individuals in the control group, and was associated with an increased risk of CHD development in 3,04 times (OR = 3,04; CI: 1,01 ÷ 9,16).

A carriage of V allele of L162V polymorphism of PPAR-α gene and G allele of A603G polymorphism of tissue factor gene, as well as their combination are associated with an increased CHD risk, especially at age 45 years or less.