Interleukin-6 polymorphisms and risk of coronary artery diseases in a Chinese population: A case-control study

Objective: We aimed to evaluate the relationship between IL-6-174G>C, -572G>C and -597G>A polymorphisms and development of coronary artery diseases in a Chinese population. Methods: A total of 275 patients with coronary artery disease and 296 healthy control subjects were collected between January 2013 and November 2014. The IL-6 genotyping for -174G>C, -592G>C and -597G>A polymorphic sites was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) according to the manufacturer instructions. Results: Using unconditional regression analysis, we observed that the AC and CC genotypes of IL-6 -592A>C were associated with the increased risk of developing coronary artery disease when compared with the AA genotype, and the adjusted ORs (95%CI) were 1.63(1.12-2.38) and 2.70(1.57-4.67), respectively. Additionally, the C allele of IL-6 -592A>C (OR=1.65, 95%CI=1.29-2.11) was correlated with a higher risk of developing coronary artery disease in comparison to the A allele. However, no relationship was found between IL-6-174G>C and -597G>A polymorphisms and coronary artery disease susceptibility. Conclusion: This study suggests that IL-6 -592G>C polymorphism is correlated with the risk of coronary artery disease. More well-designed prospective studies based on large sample size, multiple SNPs or haplotypes are required to confirm the current findings.

ester transfer protein, interleukin-10 (IL-10) and scavenger receptor class B type 1. [4][5][6][7][8][9] Molecular mechanisms underlying inflammation-correlated diseases included DNA damage, disruption of the immune response and alternation of normal cell microenvironment, which were all closely related with disequilibrium of inflammatory cytokines.Interleukin-6 (IL-6) is such a multifunctional pro-inflammatory cytokine produced by activated T cells, B cells, monocytes as well as cancerous cells.6][17][18][19] We carried out a study to evaluate the relationship between IL-6-174G>C, -572G>C and -597G>A polymorphisms and development of coronary artery disease in a Chinese population.

METHODS
This is a case-control study conducted between January 2013 and November 2014.Two hundred seventy five patients with coronary artery disease were enrolled from the First Hospital of Shanxi Medical University.All the new patients with coronary artery diseases were diagnosed using coronary angiography.The diagnosis of coronary artery disease was as follows: luminal stenosis above 50% in one of the main coronary arteries (left main coronary stenosis, anterior descending branch, circumflex coronary artery and right coronary artery) or their branch retinal arteries, and exhibiting stable angina, unstable angina pectoris and acute myocardial infarction.Patients who had a history of percutancous coronary intervention or coronary artery bypass graft intervention, congenital heart disease, rheumatic valvular heart disease, cardiomyopathy, severe heart failure, endstage renal and liver disease, serious infections, malignant tumor, thyroid disease, connective tissue disease or immune system diseases were excluded A total of 296 healthy control subjects were also enrolled from individuals who visited outpatient clinics at the First Hospital of Shanxi Medical University during the same time period.The control subjects were confirmed to be free of coronary artery diseases and other cardiovascular diseases, end-stage renal and liver disease, serious infections, malignant tumor, thyroid disease and immune system diseases.
The baseline variables of coronary artery disease patients and control subjects, including age, gender, tobacco smoking, alcohol consumption and body mass index (BMI) as well as family history of coronary artery diseases, were collected with a self-designed questionnaire.The clinical data of coronary artery disease patients and control subjects, including hypertension, type 2 diabetes mellitus, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c) as well as high-density lipoprotein cholesterol (HDL-c), were collected from medical records.The study was carried out with the permission of the ethics committee of the First Hospital of Shanxi Medical University.A written informed consent was obtained from all the subjects before enrollment into this study.The ethical standards adopted for this study were in agreement with the standards of the Declaration of Helsinki.DNA extraction and genotyping: Five blood samples were collected from each investigated subject.The peripheral blood was kept in tube with EDTA and stored at -20°C until using.The extraction of the genomic DNA was done from the peripheral blood using QIAamp DNA Blood Mini PCR was performed in a 25 μl reaction mixture containing 10.5μl ddH 2 O, 12.5μl PCR MIX, 0.375μl of each primer (10pmol/μl), 0.25μl Taq enzyme (5U/μl) and 1μl DNA template (50ng/μl).The PCR condition was set at initial denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 60 seconds, and a final elongation of 5 mins at 72°C.Qualitative and quantitative estimation of DNA was carried out with agarose gel electrophoresis and spectrophotometric estimation.Statistical analysis: The differences within demographic, lifestyle and genotype frequencies between the two groups were analyzed using chisquare tests (χ 2 test) or student t test.The correlation between IL-6-174G>C, -572G>C and -597G>A genotype polymorphisms and susceptibility to coronary artery disease was evaluated using unconditional logistic regression analysis.Whether the IL-6-174G>C, -572G>C and -597G>A genotype frequencies were in agreement with the Hardy-Weinberg equilibrium was evaluated using chisquare tests.The results were estimated using Odds ratio (ORs) along with 95% Confidence Intervals (CI), and the results were adjusted for possible confounder.All the statistical analysis was done using IBM SPSS Statistics for Windows, Version 20.0.(Armonk, NY: IBM Corp).P-value <0.05 was regarded as statistically significant.
The relationship between IL-6-174G>C, -592G>C and -597G>A polymorphisms and development of coronary artery diseases is described in Table -IV.Using unconditional regression analysis, we observed that the AC and CC genotypes of IL-6 -592A>C were associated with the increased risk of developing coronary artery disease when compared with the AA genotype, and the adjusted ORs (95%CI) were 1.63(1.12-2.38)and 2.70(1.57-4.67),respectively.Additionally, the C allele of IL-6 -592A>C (OR=1.65,95%CI=1.29-2.11)was correlated with an elevated risk of developing coronary artery disease in comparison to the A allele.However, no relationship was found between IL-6-174G>C and -597G>A polymorphisms and coronary artery disease susceptibility.

DISCUSSION
Coronary artery disease is the one of the leading causes of death, and is a serious public health problem worldwide.So far, the identified factors correlated with coronary artery disease were environment, ethnicity, family history and genetic mutation.2][23][24] In our study, we found that the IL-6 gene polymorphisms is the risk factor of coronary artery disease susceptibility, and we observed that IL-6 -592A>C polymorphism was correlated with higher risk of coronary artery disease in the Chinese population.
The IL-6 gene, located at chromosome 7p21-24, is composed of 4 introns and 5 exons.IL-6 could be considered as a major regulator of cardiovascular disease initiation and progression through circulating levels alternation.Al Shahi et al. carried out a study with 20 acute coronary syndrome patients and 50 stable coronary artery disease patients, and discovered that the higher plasma levels of IL-6 was associated with the pathogenesis of atherosclerosis. 25Eiras et al. reported that epicardial adipose tissue of coronary artery diseases was associated with higher levels of IL-6 mRNA than that of non-coronary artery disease patients. 26Another study was carried out in The Netherlands, and reported that plasma levels of IL-6 was significant higher in coronary artery disease compared to healthy controls. 27enetic polymorphisms can change the structure and quantity of the gene product, ultimately affecting the function of the product. 28e IL-6 genetic polymorphisms may influence the expression and function of IL-6 protein, and thus affect the susceptibility to cardiovascular diseases.Previous studies have investigated the association between IL-6 genetic polymorphisms and development of cardiovascular diseases, but the results are conflicting. 14,16,18,22,29Li et al. carried out a study in Chinese population, and discovered that the CC genotype of IL-6-174G/C and the GG genotype ofIL-6-572C/G are correlated with elevated risk of coronary artery disease. 16Wang et al. carried out a study with 402 patients with coronary artery disease and 402 control individuals, and reported a significant association between IL-6 -174G>C polymorphism and coronary artery disease susceptibility, whereas no association was observed between IL-6-572C/G and this disease. 18 Chinese study found a significant correlation between IL-6 -174G>C genomic variation and onset of cardiovascular events in patients receiving hemodialysis, but no correlation was observed in IL-6-572C/G genomic variation and risk of this disease. 22Gigante et al. carried out a study in two large populations, and reported that IL-6 haplotypes could regulate the circulating levels of inflammatory biomarkers and affect the susceptibility to coronary artery disease. 29Buraczynska et al. reported that C allele of the IL-6 -174G>C was correlated with a significantly higher risk of cardiovascular disease compared to the G allele. 14Our study indicated that a significant relationship between IL-6-572C/G genetic polymorphism and coronary artery disease susceptibility.The discrepancies of the above mentioned studies may be attributed to differences in populations, disease status of coronary artery disease or sample size.
Limitations of the study.First, study subjects were selected from only one hospital, which may cause selection bias.However, the genotype frequencies of IL-6-174G>C, -592G>C and -597G>A confirmed with the HWE, which suggests the genotype frequencies has representative of the general population.Second, our study may overlook the possibility of gene-gene interactions.Third, the sample size of our study is relatively small, which may limit the statistical power to find differences between groups.Further investigations with more sample size are needed to confirm our findings.

Table -
II: The demographic, lifestyle and clinical characteristics of study subjects.

Table -
III: Genotype frequencies of IL-6-174G>C, -592G>C and -597G>A in the two study groups 1Ajusted for age, gender, BMI, family history of coronary artery disease, hypertension, type 2 diabetes mellitus, TC, LDL-c and HDL-c.OR: odds ratio; CI: confidence interval.