Abstract
We conducted a case-control study to estimate the association between IL-17A rs2275913, rs3819025 and rs3748067 polymorphisms and development of coronary artery disease. A total of 415 patients with coronary artery disease and 448 health controls were recruited during the period of March 2013 and October 2014. Genotyping of IL-17A rs2275913, rs3819025 and rs3748067 were analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism. By logistic regression analysis, we found that individuals with the AA genotype (OR, 2.18; 95% CI, 1.35-3.56) and the GA+AA genotype (OR, 1.39, 95% CI, 1.06-1.84) of rs2275913 were associated with an increased risk of coronary artery disease when compared with the GG genotype. Individuals carrying the GA+AA genotype of rs2275913 were more likely to have a higher risk of coronary artery disease in those with hypertension and smoking habit, and the adjusted ORs (95% CI) were 3.92 (2.13-6.82) and 2.74 (1.71-4.40). In conclusion, we suggest that individuals with the AA genotype and the GA+AA genotype of rs2275913 are associated with an increased risk of coronary artery disease, especially in those with hypertension and smoking habit.
Keywords: Interleukin-17, polymorphism, coronary artery disease
Introduction
Coronary artery disease is one of the main causes of morbidity and mortality worldwide, and more than 80% of the coronary artery diseases occur in low-to-middle income countries [1]. The etiology of coronary artery disease is not well understood, and the process of coronary artery disease is caused by many environmental factors, such as male gender, hypertension, smoking, drinking and diabetes mellitus as well as high serum cholesterol [1,2]. However, not all of the individuals who exposed to similar environmental factors of coronary artery disease would suffer from coronary artery disease, which suggests that genetic factors have an important role in the susceptibility to coronary artery disease.
It is well known that inflammation plays an important role in the development of atherosclerosis [3]. Interleukin-17 (IL-17) is a novel family cytokine that consists of six protein members (from IL-17A to IL-17F), which plays an important role in many chronic inflammatory diseases [4,5]. IL-17 is always produced by activated CD4+ T cells (Th17 cells) and other leukocytes, such as T cells, natural killer cells (NK cells), lymphoid tissue inducer-like cells (LTi-like cells), and neutrophils [6]. IL-17A is the most important member of IL-17, and it locates in 6q12 and constitute by three exons and two introns. IL-17A rs2275913, rs3819025 and rs3748067 are three common SNPs, and two previous studies have reported the association between IL-17 gene polymorphisms and risk of cardiovascular disease [7,8], but the results are inconsistent. Therefore, we conducted a case-control study to estimate the association between IL-17A rs2275913, rs3819025 and rs3748067 polymorphisms and development of coronary artery disease.
Materials and methods
Subjects
A total of 462 patients with coronary artery disease were recruited during the period of March 2013 and October 2014 in the Affiliated Hospital of Inner Mongolia Medical University. All the cases were newly diagnosed by angiography, and the coronary artery disease was defined as a diameter stenosis of 50% in any of the main coronary arteries, including left main, left anterior descending, left circumflex artery or right coronary artery. The exclusion criteria were subjects who had myocardial spasms or a myocardial bridge, congenital heart disease, peripheral artery disease, autoimmune-related disease and renal as well as liver deceases or cancers. Finally, 415 patients were included into our study, with a participation rate of 89.83%.
Control subjects were randomly recruited from individuals who received a regular health-check up in our hospital during the same period. All the control subjects were diagnosed to have no history of coronary artery disease, atherosclerosis, had myocardial spasms or a myocardial bridge, congenital heart disease, peripheral artery disease, autoimmune-related disease and renal diseases. A total of 448 control subjects were collected into the Affiliated Hospital of Inner Mongolia Medical University and the Second Affiliated Hospital of Inner Mongolia Medical University.
The social-demographic characteristics were collected from patients with coronary artery disease and control subjects using a standardized questionnaire, such as age, gender, body mass index, hypertension, diabetes mellitus, alcohol drinking and tobacco smoking. Smoking status was based on self-reported smoking, and the subjects who had never smoked less than 100 cigarettes in their lives were classified as non-smokers. Drinking status was defined as non-drinker and drinker. The clinical data were collected from medical records, including total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c) and triglyceride (TG).
A written informed consent was gained from each subject before entering the study group. The collection of blood samples for this study was previously approved by ethics committee of our hospital.
Genetic analysis
Each participant was asked to provide a 5-ml peripheral venous blood sample after enrolling into our study. The DNA was extracted from peripheral blood by salt extraction. Genotyping of IL-17A rs2275913, rs3819025 and rs3748067 was analyzed by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). The primers for IL-17A rs2275913, rs3819025 and rs3748067 were as follows: rs2275913, 5’-GCAGCTCTGCTCAGCTTCTAA-3’ (forward) and 5’-TTCAGGGGTGACACCATTTT-3’ (reverse); rs3819025, 5’-GGTGTCACCCCTGAACCCACT-3’ (forward) and 5’-CATGCCCACGGTCCAGAAATA-3’ (reverse); rs3748067, 5’-AAGCAGGGAGCCTGCAGAGTG-3’ (forward) and 5’-GGCACCACACAACCCAGAAAG-3’ (reverse). The PCR fragments of the investigated polymorphisms were subsequently digested with their specific restriction enzyme, and BstENI for rs3748067C>T, MaeII for rs3819025 and AvaII for rs2275913. The amplification conditions were 95°C for 5 min, then 30 cycles of 94°C for 0.5 min, 60°C for 0.5 min and 72°C for 1 min, at last 72°C for 10 min. Digestion products were separated by electrophoresis on ethidium bromide stained agarose gel and visualized under UV light.
Statistical analysis
All the statistical analyses were conducted with the SPSS 16.0 statistical software (SPSS, Chicago, IL). The distributions of demographic and clinical characteristics of patients with coronary artery disease and control subjects were compared by Pearson χ2 test or student t test. The distribution of genotypes in controls was tested for deviation from Hardy-Weinberg equilibrium. Multiple logistic regression models were established to estimate relative risks of IL-17A rs2275913, rs3819025 and rs3748067 for coronary artery disease after adjustment for confounding factors. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated. All tests were two-sided with a significant level of P-value <0.05.
Results
The characteristics of patients with coronary artery disease and control subjects were shown in Table 1. No significant difference was found in age between patients with coronary artery disease and control subjects, and the mean ages of patients with coronary artery disease and control subjects were 59.20±10.85 and 60.10±10.35 years, respectively (P>0.05). By χ2 test, we found that patients with coronary artery disease were more likely to be males, suffer from hypertension and diabetes, and be smokers and drinkers (P<0.05). Moreover, patients with coronary artery disease had higher TC, LDL-c and TG and have lower HDL-c when compared with control subjects (P<0.05).
Table 1.
Characteristics of patients with coronary artery disease and control subjects
| CAD cases | % | Controls | % | χ2 test or t test | P value | |
|---|---|---|---|---|---|---|
|
|
|
|||||
| N = 415 | N = 448 | |||||
| Mean age, years | 59.20±10.85 | 60.10±10.35 | 1.25 | 0.11 | ||
| <60 | 225 | 54.22 | 215 | 47.99 | 11.9 | |
| ≥60 | 190 | 45.78 | 233 | 52.01 | 3.34 | 0.07 |
| Sex | ||||||
| Male | 321 | 77.35 | 265 | 59.15 | ||
| Female | 94 | 22.65 | 183 | 40.85 | 11.65 | 0.001 |
| BMI, kg/m2 | 23.74±3.20 | 23.47±3.12 | 1.25 | 0.11 | ||
| <24 | 285 | 68.67 | 246 | 54.91 | ||
| ≥24 | 130 | 31.33 | 102 | 22.77 | 0.36 | 0.55 |
| Hypertension | ||||||
| No | 283 | 68.19 | 352 | 78.57 | ||
| Yes | 132 | 31.81 | 96 | 21.43 | 11.93 | 0.001 |
| Diabetes mellitus | ||||||
| No | 305 | 73.49 | 384 | 85.71 | ||
| Yes | 110 | 26.51 | 64 | 14.29 | 19.98 | <0.001 |
| Alcohol drinking | ||||||
| Non-drinkers | 181 | 43.61 | 247 | 55.13 | ||
| Drinkers | 234 | 56.39 | 201 | 44.87 | 11.44 | 0.001 |
| Tobacco smoking | ||||||
| Non-smokers | 233 | 56.14 | 295 | 65.85 | ||
| Smokers | 182 | 43.86 | 153 | 34.15 | 8.54 | 0.003 |
| TC, mmol/dL | 198.40±45.75 | 174.65±32.50 | 8.84 | <0.001 | ||
| LDL-c, mmol/dL | 117.52±26.55 | 97.42±23.64 | 11.76 | <0.001 | ||
| HDL-c, mmol/dL | 37.35±9.10 | 44.25±8.25 | 11.68 | <0.001 | ||
| TG, mmol/dL | 137.41±38.50 | 117.66±29.75 | 8.47 | <0.001 |
The genotype distribution of rs2275913 and rs3819025 confirmed with Hardy-Weinberg equilibrium in the control group (P values were 0.40 and 0.07, respectively), but rs3748067 were not (P value for HWE was 0.03). By χ2 test, the genotype distributions of rs2275913 was significant different between patients with coronary artery disease and control subjects (χ2 = 11.62, P value = 0.003). By logistic regression analysis, we found that individuals with the AA genotype and the GA+AA genotype of rs2275913 were associated with an increased risk of coronary artery disease when compared with the GG genotype, and the adjusted ORs (95% CI) were 2.18 (1.35-3.56) and 1.39 (1.06-1.84) for the AA genotype and the GA+AA genotype, respectively (Table 2). However, no significant association was found between rs3819025 and rs3748067 polymorphisms and risk of coronary artery disease.
Table 2.
Genotype distributions of IL-17A rs2275913, rs3819025 and rs3748067 and their association with risk of coronary artery disease
| IL-17 gene | Base change | Patients | % | Controls | % | P value for HWE | OR (95% CI)1 | P value |
|---|---|---|---|---|---|---|---|---|
| rs2275913 | ||||||||
| GG | 168 | 40.48 | 220 | 49.11 | 1.0 (Ref.) | - | ||
| GA | 187 | 45.06 | 196 | 43.75 | 1.25 (0.93-1.68) | 0.12 | ||
| AA | G>A | 60 | 14.46 | 36 | 8.04 | 0.4 | 2.18 (1.35-3.56) | 0.001 |
| GA+AA | 247 | 59.52 | 232 | 51.79 | 1.39 (1.06-1.84) | 0.02 | ||
| rs3819025 | ||||||||
| AA | 179 | 43.13 | 212 | 47.32 | 1.0 (Ref.) | - | ||
| AG | 177 | 42.65 | 180 | 40.18 | 1.16 (0.86-1.57) | 0.3 | ||
| GG | A>G | 59 | 14.22 | 56 | 12.50 | 0.07 | 1.25 (0.81-1.93) | 0.29 |
| AG+GG | 236 | 56.87 | 236 | 52.68 | 1.18 (0.90-1.56) | 0.22 | ||
| rs3748067 | ||||||||
| TT | 301 | 72.53 | 339 | 75.67 | 1.0 (Ref.) | - | ||
| TC | 91 | 21.93 | 95 | 21.21 | 1.08 (0.77-1.52) | 0.65 | ||
| CC | C>T | 23 | 5.54 | 14 | 3.13 | 0.03 | 1.85 (0.89-3.96) | 0.07 |
| TC+CC | 114 | 27.47 | 109 | 24.33 | 1.18 (0.86-1.62) | 0.29 |
Adjusted for sex, age, hypertension, diabetes mellitus, alcohol drinking, tobacco smoking, TC, LDL-c, HDL-c and TG.
We also conducted gene-environmental analysis to investigate the association between rs2275913 polymorphism and demographic characteristics of subjects in the risk of coronary artery disease. We found individuals carrying the GA+AA genotype of rs2275913 were more likely to have a higher risk of coronary artery disease in those with hypertension and smoking habit, and the adjusted ORs (95% CI) were 3.92 (2.13-6.82) and 2.74 (1.71-4.40), respectively (Table 3). However, we did not find significant association of rs2275913 polymorphism with diabetes mellitus and alcohol drinking in the development of coronary artery disease (P value >0.05).
Table 3.
Interaction between rs2275913 polymorphism and demographic characteristics of subjects in the risk of coronary artery disease
| Variables | rs2275913 | OR (95% CI)1 | P value | |||
|---|---|---|---|---|---|---|
|
| ||||||
| TT | TC+CC | |||||
|
| ||||||
| Case | Control | Case | Control | |||
| Hypertension | ||||||
| No | 125 | 184 | 158 | 168 | 1.34 (0.94-1.85) | 0.07 |
| Yes | 43 | 63 | 89 | 33 | 3.92 (2.13-6.82) | <0.001 |
| Diabetes mellitus | ||||||
| No | 123 | 210 | 182 | 174 | 1.75 (0.96-2.13) | 0.06 |
| Yes | 45 | 37 | 65 | 27 | 1.85 (0.94-4.10) | 0.05 |
| Alcohol drinking | ||||||
| Non-drinkers | 75 | 135 | 106 | 112 | 1.71 (0.92-2.05) | 0.08 |
| Drinkers | 93 | 112 | 141 | 89 | 1.69 (0.81-2.48) | 0.1 |
| Tobacco smoking | ||||||
| Non-smokers | 112 | 163 | 121 | 132 | 1.33 (0.93-1.91) | 0.1 |
| Smokers | 56 | 84 | 126 | 69 | 2.74 (1.71-4.40) | <0.001 |
Adjusted for sex and age.
Discussion
It is well known that genetic susceptibility to cardiovascular disease has gain an increasing attention to investigate the polymorphisms of genes involved in the development of coronary artery disease. Inflammation and related cytokines are involved in both innate and acquired immune responses, and play an important role in the inflammatory response that contributes to the development of cardiovascular disease.
IL-17A is a relatively novel cytokine family contains six homologous members, and IL-17A is an important inflammatory cytokine to connect the innate and adaptive immunity [9]. Molecular studies have shown that IL-17 is an essential proinflammatory cytokine to evoke lots of cytokines and chemokines secretion through different cell types, such as mesenchymal cells and myeloid cells to promote monocytes and neutrophils into the microenvironment of inflammation [10].
Currently, many epidemiological studies have investigated the association between IL-17 gene and development of cardiovascular and cerebrovascular diseases [11-14]. Lv et al. conducted an experimental study, and reported that IL-17 could cause inflammatory immune responses and neuronal damage, such as cerebral ischemia/reperfusion injury [11]. Li et al. reported that Th17/Treg cells were imbalanced in atherosclerotic cerebral infarction patients, and contributed to the occurrence of atherosclerotic cerebral infarction, suggesting their pathogenetic role in atherosclerotic cerebral infarction [13]. Márquez et al. conducted a meta-analysis with 1266 biopsy-proven giant cell arteritis patients and 3779 healthy controls, and they reported that IL-17 rs2275913 and rs4711998 polymorphisms were associated with increased risk of giant cell arteritis [14].
For the association between IL-17 gene polymorphisms and development of cardiovascular diseases, only two previous studies reported their association [15,16]. Pei et al. conducted a case-control association in a Chinese population, and they have indicated that IL-17F rs763780 polymorphism is unlikely to contribute to the development of myocardial infarction [15]. Another study also conducted a case-control study in a Chinese population, and has reported that IL-17A rs8193037 is associated with an increased risk of coronary artery disease and contributes to increased expression of IL-17A in acute myocardial infarction [16]. In our study, we found that individuals with the AA genotype and the GA+AA genotype of rs2275913 were associated with an increased risk of coronary artery disease when compared with TT genotype, which is inconsistent with the results of previous studies. The discrepancies of these results may be caused by differences in ethnicities, study design, and sample size as well as by chance.
Moreover, our study found that individuals carrying the GA+AA genotype of rs2275913 were more likely to have a higher risk of coronary artery disease in smokers. Previous studies reported that IL-17 gene polymorphism was associated with smoking related diseases [17-19]. Further studies are greatly needed to confirm our study.
In conclusion, we suggest that individuals with the AA genotype and the GA+AA genotype of rs2275913 were associated with an increased risk of coronary artery disease, especially in those with hypertension and smoking habit. Further genetic studies with large sample size are greatly needed to confirm the association between IL-17 gene polymorphisms and risk of coronary artery disease.
Disclosure of conflict of interest
None.
References
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