Effect of polymorphisms in interleukin-18 gene on the susceptibility to coronary artery disease in a Chinese population
Coronary artery disease (CAD) has a high mortality rate in several countries. Interleukin (IL)-18 has been previously correlated with atherosclerotic plaque rupture. In this case-control study, the relationship between -607A/C and -372C/G promoter polymorphisms in IL-18 and risk of CAD development was investigated. A total of 326 CAD patients were consecutively recruited from the First Hospital of Yulin between March 2013 and May 2015. The IL-18 -607A/C and -372C/G polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Patients with CAD had a higher body mass index, a history of hypertension or diabetes (all P < 0.001), cigarette smoking habit (P = 0.002); as well as higher plasma total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels (all P < 0.001) and lower high-density lipoprotein cholesterol (P < 0.001) levels compared to the control subjects. Unconditional logistic regression analysis revealed significant correlation between the CC genotype of IL-18 -607A/C and CAD development, compared to the AA genotype [adjusted odds ratio (OR) = 2.42; 95% confidence interval (CI) = 1.52-3.89; P < 0.001]. The recessive model showed a significant association between the CC genotype of IL-18 -607A/C and an increased risk of CAD, compared to the AA+AC genotype (OR = 2.51, 95%CI = 1.65-3.85). However, IL-18 -372C/G did not contribute to the risk of glioma development in the co-dominant, dominant, and recessive models. Therefore, the IL-18 -607C/A polymorphism was significantly correlated with the risk of CAD development.
Coronary artery disease (CAD) is commonly associated with a high mortality rate in most countries; more than 80% of the CAD cases have been reported in low- to median-income countries (He et al., 2005). CAD develops as a result of vascular tract stenosis or occlusion caused by coronary atherosclerotic lesions and several environmental and lifestyle factors (Campbell et al., 1998; Erbel and Görge, 2014; Bullock-Palmer, 2015). Previous experimental studies at the molecular level have indicated that many genetic factors, including the genes encoding angiotensinogen and angiotensin-converting enzyme, hepatic lipase, insulin receptor substrate-1, kinesin family member 6, cholesteryl ester transfer protein, ATP-binding cassette subfamily A member 1, and apoptotic extrinsic death receptor, have an important role in the risk of CAD development (Bonfim-Silva et al., 2016; Cyrus et al., 2016; Kishore Kumar et al., 2016; Mohammadzadeh et al., 2016; Vatte et al., 2016; Zhang et al., 2016).
The inflammatory response could promote the formation and stability of plaques (Libby et al., 2002). Previous studies have indicated that several inflammatory factors contribute to the development of CAD, such as C-reactive protein, tumor necrosis factor, interleukin-6 (IL-6), IL-17A, and transforming growth factor (Zernecke et al., 2008; Yang et al., 2015; Zheng et al., 2016). An association has also been reported between IL-18 and atherosclerotic plaque rupture (de Nooijer et al., 2004), which, in turn, is believed to influence the occurrence of CAD (Opstad et al., 2011). Polymorphisms in IL-18 are also believed to influence gene transcription, alter IL-18 expression, and promote development of cardiovascular diseases (Liu et al., 2013; Lu et al., 2013; Opstad et al., 2013; Hazzaa et al., 2014). Here, we performed a case-control study to investigate the relationship between the -607A/C and -372C/G polymorphisms in the promoter region of IL-18 and risk of CAD development.
MATERIAL AND METHODS
A total of 326 patients with CAD were consecutively recruited from the First Hospital of Yulin between March 2013 and May 2015. CAD was newly diagnosed and confirmed by coronary angiography in all patients. CAD was characterized by a stenosis diameter of 50% in any of the major coronary arteries, such as the left main, left anterior descending, left circumflex, and right coronary arteries.
Control subjects were randomly selected from among individuals who visited the outpatient clinic of our hospital for a regular health check-up during the same time period. The controls were age- (± 5 years) and gender-matched with the CAD patients. All control subjects were confirmed to be free of CAD or other cardiovascular diseases, chronic or acute infectious diseases, or malignant tumors.
The demographic, environmental, and clinical characteristics of the recruited patients and controls were obtained from medical records. The demographic and environmental factors included the gender, age, body mass index (BMI), history of hypertension and diabetes, habit of cigarette smoking and alcohol consumption levels, and so on. The clinical variables included the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. Signed informed consent forms were obtained from all participants. This study was approved by the Ethics Committee of the First Hospital of Yulin.
Blood samples were collected from the cases and controls in EDTA-coated tubes. Total genomic DNA was extracted from these samples, using the Tiangen® DNA Blood Min kit (Tiangen Biotech Co., Ltd., Beijing, China). The IL-18 -607A/C and -372C/G polymorphic sites were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The forward and reverse primers for IL-18 -607A/C and -372C/G were 5'-GTTGCAGAAAGTGAAAAATTATTAC-3' and 5'-GTTGCAGAAAGTGTAAAAATTATTAA-3' and 5'-CCCCAACTTTTACGGAAGAAAAG-3' and 5'-CCCCAACTTTTACGGAAGAAAAC-3', respectively. The IL-18 -607A/C and -372C/G polymorphic sites were digested with MseI and BfuCI, respectively. The PCR conditions were set as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 45 s, annealing at variable temperatures for 30 s, and extension at 72°C for 45 s. The amplification was verified on a 1.5% agarose gel.
Statistical variations between the demographic and clinical variables of the two study groups were determined using the chi-square (χ2) test for categorical data and the Student t-test for continuous variables. Probable deviations of the IL-18 -607A/C and -372C/G polymorphisms from the Hardy-Weinberg equilibrium (HWE) were determined using the chi-square test, where the observed values were compared with the expected values. The correlation between IL-18 -607A/C and -372C/G polymorphisms and risk of CAD was analyzed by multivariate logistic regression analysis, and the results are reported as odds ratios (ORs) with their corresponding 95% confidence intervals (CI). Interactions between the IL-18 -607A/C and -372C/G polymorphisms and the potential risk factors of CAD were analyzed by the Spearman correlation analysis. Data analysis was performed using SPSS v.17.0 (SPSS Inc., Chicago, IL, USA).
As expected, the CAD patients and controls are comparable in terms of age (χ2 = 0.09, P = 0.75) and gender (χ2 < 0.001, P = 1.00) (Table 1). The chi-square and Student t-tests showed no significant differences in the alcohol consumption levels (χ2 = 1.16, P = 0.28) between the CAD patients and control subjects. The patients with CAD had higher BMI (χ2 = 19.83, P < 0.001), higher probability of a history of hypertension (χ2 = 52.49, P < 0.001) or diabetes (χ2 = 8.65, P = 0.003), cigarette smoking habit (χ2 = 9.29, P = 0.002), as well as higher plasma TC (t = 4.61, P < 0.001), TG (t = 4.70, P < 0.001), and LDL-C (t = 9.01, P < 0.001) levels, and lower HDL-C (t = 12.31, P < 0.001) levels compared to the control subjects.
Demographic, lifestyle, and clinical characteristics of patients with coronary artery disease (CAD) and control subjects included in this study.
||%||χ2 or t-value||P value|
|TC (mM/dL)||195.52 ± 41.67||181.34 ± 36.75||4.61||<0.001|
|TG (mM/dL)||135.34 ± 39.52||121.53 ± 35.40||4.70||<0.001|
|LDL-C (mM/dL)||121.50 ± 34.20||98.64 ± 30.55||9.01||<0.001|
|HDL-C (mM/dL)||36.72 ± 8.63||45.60 ± 9.76||12.31||<0.001|
BMI, body mass index; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.
The AA, AC, and CC genotypes of IL-18 -607A/C were observed in 33.13% (108), 39.26% (128), and 27.61% (90) of all patients, and 38.34% (125), 48.47% (158), and 13.19% (43) of the control subjects, respectively. Alternately, the GG, GC, and CC genotypes of the IL-18 -372G/C polymorphism were observed in 69.02% (225), 27.61% (90), and 3.37% (11) of all patients, and 73.01% (238), 24.54% (80), and 2.45% (8) controls, respectively (Table 2). The chi-square test revealed significant differences between genotype distributions of IL-18 -607A/C (χ2 = 21.00, P < 0.001) and no significant differences between the genotype distributions of IL-18 -372C/G (χ2 = 1.43, P = 0.49). The genotype frequencies of IL-18 -607A/C (χ2 = 0.40, P = 0.53) and -372C/G (χ2 = 0.17, P = 0.68) in the control subjects were in line with the HWE.
Genotype distributions of the interleukin 18 (IL-18) -607A/C and -372C/G polymorphisms in the patients and controls.
||%||χ2||P value||HWE in controls|
HWE, Hardy-Weinberg equilibrium.
Unconditional logistic regression analysis revealed a significant correlation between the CC genotype of IL-18 -607A/C and CAD development, compared to the AA genotype (adjusted OR = 2.42; 95%CI = 1.52-3.89; P < 0.001) (Table 3). Alternately, the recessive model showed a significant correlation between the CC genotype of IL-18 -607A/C and an increased risk of CAD, compared to the AA+AC genotype (OR = 2.51, 95%CI = 1.65-3.85). However, the IL-18 -372C/G did not contribute to the risk of glioma development.
Relationship between IL-18 -607A/C and -372C/G polymorphisms and risk of coronary artery disease (CAD).
||%||Multivariate logistic regression analysis|
|Adjusted OR (95%CI)1||P value|
1Adjusted for body mass index, hypertension, diabetes, cigarette smoking, total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. OR, odds ratio; CI, confidence interval.
Spearman correlation analysis revealed no significant correlation between the IL-18 -607A/C and -372C/G polymorphisms and BMI, hypertension, diabetes, and cigarette smoking habit, and the TC, TG, LDL-C, and HDL-C levels in CAD risk (P values for all correlations >0.05).
The IL-18 -607C/A and -137G/C polymorphisms are located at the junction of the response elements of nuclear factor cAMP binding protein. Previous studies have indicated that the IL-18 -607C/A and -137G/C polymorphisms influence the expression of IL-18 and IFN-γ; moreover, the expression of IL-18 -607CC and -137GG genotypes were significantly higher those of other genotypes (Giedraitis et al., 2001). IL-18 expression is also positively correlated with the expression of IL-18 mRNA (Giedraitis et al., 2001). Additionally, the A and C alleles of IL-18 -607C/A and -137G/C are believed to induce low IL-18 activity (Giedraitis et al., 2001; Liang et al., 2005; Arimitsu et al., 2006). Some studies have also indicated that patients with cardiovascular and cerebrovascular diseases have significantly higher levels of IL-18 in the plasma compared to the healthy controls, which was associated with disease development (Chen et al., 2007; Gao et al., 2010; Liu et al., 2013; Li et al., 2014). Here, we attempted to elucidate the association between the IL-18 -607C/A and -137G/C polymorphisms and risk of CAD; we determined that the CC genotype of IL-18 -607A/C could elevate the risk of CAD, compared to the wild-type genotype.
Previous studies have indicated a close association between functional IL-18 polymorphisms and the risk of developing cardiovascular and cerebrovascular diseases (Thompson et al., 2007; Liu et al., 2009; Pei et al., 2009; Hernesniemi et al., 2010a,b; Zhang et al., 2010; Lu et al., 2013). Liu et al. (2009), in a study conducted in 241 Chinese patients with CAD and 145 control subjects, reported that the IL-18 -137G/C polymorphism influenced IL-18 expression and the occurrence of CAD. Pei et al. (2009) and Lu et al. (2013) reported that the IL-18 -607C/A polymorphism influenced the risk of acute myocardial infarction or ischemic stroke in a northern Chinese Han population. However, other studies did not find any significant association between IL-18 polymorphisms and coronary heart disease and atherosclerosis (Thompson et al., 2007; Hernesniemi et al., 2010a,b). The discrepancies among these studies could be attributed to differences in the diseases investigated, study populations, study design, and sample size.
Three major limitations should be paid attention. Firstly, patients and controls were selected from only one hospital, which could induce selection bias in our study. Secondly, polymorphisms in genes other than IL-18 (-607C/A and -137G/C) may influence the development of this disease, and interact with IL-18.
In summary, we observed a statistically significant relationship between the IL-18 -607C/A polymorphism and risk of developing coronary artery disease; however, the IL-18 -137G/C polymorphism was not correlated with this disease. Our results should be confirmed by further studies.