Association between interleukin 6 and interleukin 16 gene polymorphisms and coronary heart disease risk in a Chinese population

Abstract

Objective

To investigate the role of interleukin 6 (IL6) and IL16 single nucleotide polymorphisms (SNPs) in coronary artery disease (CAD) risk in a Chinese population.

Methods

Patients with CAD and healthy control subjects were recruited. IL6 (rs1800795 and rs1800796) and IL16 (rs8034928, rs3848180, rs4577037, rs1131445, rs4778889 and rs11556218) genotyping was performed on the MassARRAY® platform (Sequenom®, San Diego, CA, USA).

Results

Frequencies of rs8034928 variant C allele and rs11556218 variant T allele were higher in patients with CAD (n = 326) than controls (n = 341). The rs8034928 C/C genotype (odds ratio [OR] 2.03; 95% confidence intervals [CI] 1.16, 3.62) and C allele (OR 1.97; 95%CI 1.15, 3.45) were associated with increased risk of CAD compared with wild type. Similarly, the rs11556218 T/T genotype (OR 2.44; 95%CI 1.15, 5.44) and T allele (OR 2.37; 95%CI 1.13, 5.24) were associated with increased CAD risk compared with wild type.

Conclusion

The SNPs rs8034928 and rs11556218 are associated with CAD risk in the Chinese population, and may be useful predictive markers for CAD susceptibility.

Introduction

Coronary artery disease (CAD) is a major health problem worldwide, and has a complex aetiology determined by factors including inflammation, sex, age, smoking, hypertension, diabetes and genetic susceptibility.^1,2 The underlying pathological process of CAD is atherosclerosis, which is characterized by chronic inflammation, due primarily to the deposit of oxidized lipids on the inner layer of the arterial wall. ² Studies have indicated that inflammation-related genes may be associated with CAD risk.^3–7

Elevated concentrations of interleukin 6 (IL6) and its final product, C reactive protein, are associated with increased CAD risk. ² Interleukin (IL) 16 is a proinflammatory and immunoregulatory cytokine with multiple functions in the genesis and maintenance of the inflammatory response. It is produced by diverse cell types including fibroblasts, monocytes, adipocytes and endothelial cells. ⁸ Genetic variants of IL16 are associated with the risk of several cancers and autoimmune diseases.^9–11 Studies investigating the relationship between CAD risk and IL6 or IL16 single nucleotide polymorphisms (SNPs) are inconclusive, however. The IL6 SNPs 174 G > C (rs1800795) and 572 G > C (rs1800796), in addition to IL16 270 T > C (rs8034928), have been shown to be associated with CAD risk,^12,13 but a meta-analysis revealed no association between IL6 174 G > C (rs1800795) and CAD risk, ¹⁴ and no relationship was found between CAD risk and IL6 or IL16 polymorphisms in three large sample studies.^10,12,13 The aim of the present study was to investigate the relationships between SNPs of IL6 (rs1800795 and rs1800796) and IL16 (rs8034928, rs3848180, rs4577037, rs1131445, rs4778889 and rs11556218) and CAD risk, in a Chinese population.

Patients and methods

Study population

This case–control study recruited patients first diagnosed with CAD at the Department of Cardiology, Beijing Chaoyang Hospital, Beijing, China, between February 2008 and December 2011. Inclusion criteria were angiographic evidence of ≥70% stenosis of one major coronary artery, or ≥50% stenosis of the left main coronary artery. Exclusion criteria were: current heparin treatment; autoimmune disease; congenital heart disease; severe kidney or liver disease; malignancy.

Age-matched control subjects were recruited from individuals attending Beijing Chaoyang Hospital, Beijing, China, for routine health examination. Control subjects with known CAD or any other heart disease were excluded from the study.

All study participants completed a structured questionnaire during a face-to-face interview with medical staff, and 5 ml venous blood was collected in ethylenediamine tetra-acetic acid (EDTA) coated tubes and stored at −20℃ before use. Body mass index (BMI) was calculated as weight (kg)/height (m²). Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were determined in serum. Hypertension was defined as systolic blood pressure ≥140 mmHg and/or diastolic pressure ≥90 mmHg. Diabetes was defined as a fasting plasma glucose level of ≥7 mmol/l and/or a 2-h plasma glucose level of ≥11.1 mmol/l during oral glucose tolerance testing.

The study was approved by the Ethics Committee of Beijing Chaoyang Hospital of Capital Medical University, and all participants provided written informed consent.

Genotyping

Blood samples, collected and stored as described above, were collected from all study participants in EDTA-coated tubes and centrifuged at 13 400 g for 3 min at room temperature. The buffy coat was collected and total DNA was extracted using a TIANamp blood DNA kit (Tiangen Biotech, Beijing, China). Genotyping of IL6 (rs1800795 and rs1800796) and IL16 (rs8034928, rs3848180, rs4577037, rs1131445, rs4778889 and rs11556218) was performed in a 384-well plate format on the MassARRAY® platform (Sequenom®, San Diego, CA, USA), which combines polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry technologies. PCR single base extension (SBE) primers (Table 1) were designed using Sequenom® Assay Design, Version 3.1 software (Sequenom®), according to the manufacturer’s instructions. Each PCR reaction mix comprised 50 ng genomic DNA, 200 µM dNTP, 2.5 U Taq DNA polymerase (Promega, Madison, WI, USA) and 200 µM primers, in a total volume of 20 µl. The cycling programme involved preliminary denaturation at 94℃ for 2 min, followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 64℃ for 30 s, and extension at 72℃ for 1 min. PCR products were verified by 1.0% agarose gel electrophoresis and visualized via ethidium bromide staining and ultraviolet light. Genotyping was performed without knowledge of the case/control status of the subjects, and reproducibility was confirmed by repeat analysis of a randomly chosen subgroup of 5% of study participants.

Table 1.

Sequences of primers used for polymerase chain reaction single base extension amplification of interleukin 6 (IL6) and IL16 single nucleotide polymorphisms (SNPs).

SNP	Sequence
	Primer 1	Primer 2
rs1800795	5′-GGAGTCACACACTCCACCT-3′	5′-CTGATTGGAAACCTTATTAAG-3′
rs1800796	5′-GGAGACGCCTTGAAGTAACTGC-3′	5′-GAGTTTCCTCTGACTCCATCGCAG-3′
rs8034928	5′-TTCCATTTGAAGAGAGCCTTTTAGC-3′	5′-GGAGACAGAAAACCCAGGTTCGG-3′
rs3848180	5′-CCTCCAGTTCACAGCATCA-3′	5´-GCTCTACGTTAGTTCCCTTCTA-3´
rs4577037	5′-CAAATCCTCCAGTTCACAG-3′	5′-TGCTCTACGTTAGTTCCCT-3′
rs1131445	5′-TTGATGTTGGCTGGGAACT-3′	5′-CACGCTTTGAGCTTGGTG-3′
rs4778889	5′-CTCCACACTCAAAGCCTTTTGTTCC-3′	5′-CCATGTCAAAACGGTAGCCTCAAGC-3′
rs11556218	5′-GCTCAGGTTCACAGAGTGTTTCCATA-3′	5′-TGTGACAATCACAGCTTGCCTG-3′

Statistical analyses

Continuous variables were presented as mean ± SD and analysed using the independent-samples t-test. Categorical variables were presented as n (%) of subjects and analysed using the χ²-test. The Hardy–Weinberg equilibrium and between-group comparison of genotype distribution were analysed using the χ²-test. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were used to assess the effect of each SNP on CAD risk. Multivariate logistic regression analysis was performed to calculate the OR (95% CI) after adjusting for sex, smoking status, BMI, hypertension, diabetes, TC, TG, LDL-C and HDL-C. A P-value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS® software, version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows®.

Results

The study included 326 patients with CAD (243 males/83 females; mean age 61.4 ± 8.7 years; age range 46–79 years) and 341 healthy control subjects (210 males/131 females; mean age 60.6 ± 9.6 years; age range 43–76 years). Baseline clinical characteristics of the study population are shown in Table 2. Patients with CAD were significantly more likely to be male and to have a higher BMI and TG levels compared with those without CAD. In addition, a higher prevalence of smoking, diabetes and hypertension was observed in patients with CAD than in control subjects (P < 0.05 for all comparisons; Table 2). Control subjects had significantly higher TC, HDL-C and LDL-C levels than patients with CAD (P < 0.05 for all comparisons; Table 2), due to the high prevalence of lipid metabolism disorders within the patient group.

Table 2.

Demographic and clinical characteristics of patients with coronary artery disease (CAD) and healthy control subjects, included in a study investigating the role of interleukin 6 (IL6) and IL16 single nucleotide polymorphisms in CAD risk.

Characteristic	CAD patients n = 326	Controls n = 341	Statistical significance a
Age, years	61.4 ± 8.7	60.6 ± 9.6	NS
Sex
Male	243 (74.6)	210 (61.6)	P < 0.05
Female	83 (25.5)	131 (38.4)
BMI, kg/m2	23.2 ± 3.1	22.7 ± 2.8	P < 0.05
Smoking
Ever	135 (41.4)	93 (27.3)	P < 0.05
Never	191 (58.6)	248 (72.7)
Type 2 diabetes
Yes	136 (41.7)	67 (19.6)	P < 0.05
No	190 (58.3)	274 (80.4)
Hypertension
Yes	159 (48.8)	128 (37.5)	P < 0.05
No	167 (51.2)	213 (62.5)
TC, mmol/l	4.20 ± 0.98	4.65 ± 1.01	P < 0.05
HDL-C, mmol/l	1.19 ± 0.35	1.52 ± 0.42	P < 0.05
LDL-C, mmol/l	2.51 ± 0.91	2.90 ± 0.94	P < 0.05
TG, mmol/l	1.97 ± 1.05	1.75 ± 0.91	P < 0.05

Data presented as mean ± SD or n (%) of subjects.

a

Continuous variables analysed using independent-samples t-test; categorical variables analysed using χ²-test.

NS, not statistically significant (P ≥ 0.05); BMI, body mass index; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride.

Genotype distributions of the eight SNPs are shown in Table 3. In control subjects, the minor allele frequencies (MAFs) were consistent with published MAFs (available at http://www.ncbi.nlm.nih.gov/snp/), and were in Hardy–Weinberg equilibrium.. The rs8034928 T > C and rs11556218 G > T genotype frequencies were significantly different between the CAD and control groups, with the rs8034928 C allele and rs11556218 T allele frequencies being significantly higher in the CAD group than controls (P < 0.05 for each comparison; Table 3). There were no significant between-group differences in the frequencies of rs1800795 G > C, rs1800796 G > C, rs3848180 T > G, rs4577037 T > G, rs1131445 T > C or rs4778889 C/T (Table 3).

Table 3.

Distribution of interleukin 6 (IL6) and IL16 single nucleotide polymorphisms in patients with coronary artery disease (CAD) and healthy control subjects.

SNP	Gene	Published MAF a	MAF		CAD patients n = 326			Controls n = 341			Statistical significance b
			Cases	Controls	A/A c	A/a d	a/a e	A/A c	A/a d	a/a e
rs1800795 G > C	IL6	0.251	0.251	0.212	201 (61.7)	87 (26.7)	38 (11.7)	220 (64.5)	98 (28.7)	23 (6.7)	NS
rs1800796 G > C	IL6	0.284	0.284	0.296	179 (54.9)	110 (33.7)	37 (11.3)	180 (52.8)	120 (35.2)	41 (12.0)	NS
rs8034928 T > C	IL16	0.327	0.312	0.254	167 (51.2)	106 (32.5)	53 (16.3)	193 (56.6)	124 (36.4)	25 (7.3)	P < 0.05
rs3848180 T > G	IL16	0.477	0.477	0.471	105 (32.2)	132 (40.5)	90 (27.6)	110 (32.3)	140 (41.1)	90 (26.4)	NS
rs4577037 T > G	IL16	0.177	0.177	0.152	234 (71.8)	69 (21.2)	23 (7.1)	253 (74.2)	73 (21.4)	15 (4.4)	NS
rs1131445 T > C	IL16	0.346	0.346	0.328	157 (48.2)	112 (34.6)	56 (17.2)	171 (50.1)	116 (34.0)	54 (15.8)	NS
rs4778889 C > T	IL16	0.315	0.307	0.255	170 (52.1)	106 (32.5)	50 (15.3)	201 (58.9)	107 (31.4)	33 (9.7)	NS
rs11556218 G > T	IL16	0.211	0.204	0.154	223 (68.4)	69 (21.2)	34 (10.4)	248 (72.7)	80 (23.5)	12 (3.5)	P < 0.05

Data presented as frequency or n (%) of subjects.

a

MAF (minor allele frequency), published at http://www.ncbi.nlm.nih.gov/snp/

b

χ²-test.

c

Wild type.

d

Heterozygous variant.

e

Homozygous variant.

NS, not statistically significant (P ≥ 0.05).

Table 4 shows the results of multivariate logistic regression analysis of the effects of rs8034928 and rs11556218 on CAD risk, adjusted for sex, BMI, smoking status, diabetes, hypertension, and levels of TC, HDL-C, LDL-C and TG. The rs8034928 variant C/C genotype was associated with a significantly increased risk of CAD than the wild-type T/T genotype. In addition, C allele carriers showed a significantly increased CAD risk in a recessive model. The rs11556218 variant T/T genotype was associated with increased CAD risk, with T allele carriers having a significantly increased CAD risk, in a recessive model.

Table 4.

Multivariate logistic regression analysis of the effects of the interleukin 16 (IL16) single nucleotide polymorphisms rs8034928 and rs11556218 on coronary artery disease risk.

SNP	Genotype	Minor allele		Odds ratio (95% confidence intervals) a
		Cases, n	Controls, n	Codominant	Dominant	Recessive
rs8034928	T/T	167	193	–	–	–
T/C	116	124	1.08 (0.77, 1.52)	1.24 (0.90, 1.70)	1.97 (1.15, 3.45)
C/C	44	25	2.03 (1.16, 3.62)
rs11556218	G/G	220	248	–
G/T	80	80	1.13 (0.77, 1.64)	1.30 (0.92, 1.84)	2.37 (1.13, 5.24)
T/T	26	12	2.44 (1.15, 5.44)

a

Adjusted for sex, body mass index, smoking status, diabetes, hypertension, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglyceride.

The results of multivariate logistic regression analysis of the combined effect of rs8034928 and rs11556218 on CAD risk, adjusted for sex, BMI, smoking status, diabetes, hypertension, TC, HDL-C, LDL-C and TG are given in Table 5. Individuals with both rs8034928 variant C allele and rs11556218 wild-type homozygotes (G/G) had a significantly increased risk of CAD compared with those with homozygous wild-type rs8034928 (T/T) and rs11556218 (G/G). In addition, individuals carrying the rs8034928 variant C allele and rs11556218 variant T allele had a significantly increased risk of CAD than double wild-type homozygotes.

Table 5.

Multivariate logistic regression analysis of the combined effect of the interleukin 16 (IL16) single nucleotide polymorphisms rs8034928 and rs11556218 on coronary artery disease risk.

SNP		Cases, n	Controls, n	OR (95% CI) a
rs8034928	rs11556218
T/T	G/G	75	114	–
T/T	T allele	92	107	1.11 (0.72, 1.71)
C allele	G/G	96	79	1.84 (1.19, 2.86)
C allele	T allele	64	42	2.32 (1.38, 3.88)

a

Adjusted for sex, body mass index, smoking status, diabetes, hypertension, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglyceride.

OR, odds ratio; 95% CI, 95% confidence intervals.

Discussion

The IL16 SNPs rs8034928 and rs11556218 were associated with CAD risk in a Chinese Han population in the present study, but there was no relationship between IL6 rs1800795 and rs1800796, and IL16 rs3848180, rs4577037, rs1131445 or rs4778889 and CAD. These findings suggest that rs8034928 and rs11556218 may be useful genetic susceptibility markers for CAD, allowing for identification of high-risk individuals and the development of targeted therapies.

The IL16 gene contains 7 exons and 6 introns, covering approximately 12.8 kb of genomic DNA. ¹⁵ The SNPs rs11556218, rs8034928, rs3848180 and rs4577037 lie within the intron region, whereas rs1131445 and rs4778889 are located in the 3′ untranslated region. The rs11556218 SNP has been shown to be associated with allergic contact dermatitis, colorectal cancer, endometriosis, gastric cancer, systemic lupus erythematosus, nasopharyngeal carcinoma and renal cell carcinoma,^{10,11,16–20} in addition to CAD risk.^12,21 In accordance with the present study findings, the rs11556218 TG/GG genotypes were previously associated with 1.77-fold increased risk of CAD compared with the TT genotype. ²¹

The rs4778889 polymorphism has been associated with Graves’ disease and asthma, ⁹ systemic lupus erythematosus, ¹¹ and colorectal and gastric cancer. ¹⁰ There was no association between rs4778889 and CAD in the present study, however. This may be explained by differences in population background, the source of control subjects and sample size. Further studies are required to confirm the present findings.

A study including a large number of patients with CAD revealed significant between-group differences in rs8034928 allele frequencies, with the T/C and C/C genotypes associated with increased CAD risk. ⁸ These data are in accordance with the findings of the present study, where the T/C genotype was associated with a 2.14-fold increased risk of CAD, indicating that the IL16 rs8034928 T > C SNP has a role in the development of CAD.

The current study has several limitations. First, the study was conducted in a single hospital in China and the cohort may not be representative of China as a whole. Secondly, CAD is induced by multiple genes and environmental factors, none of which were considered in the present study.

In conclusion, the present study shows that rs8034928 and rs11556218 are associated with CAD risk in a Chinese population. The IL6 SNPs rs1800795 and rs1800796, and the IL16 SNPs rs3848180, rs4577037, rs1131445 and rs4778889, were not associated with CAD risk in this population. Further large-scale studies are required, to elucidate whether these IL6 and IL16 SNPs interact with environmental factors in the development of CAD.