Association between interleukin-8 gene -251 A/T polymorphism and the risk of coronary artery disease: A meta-analysis.

BACKGROUND: The association between interleukin-8 (IL-8) gene polymorphism -251 A>T and susceptibility to coronary artery disease (CAD) has been investigated previously; however, results remain controversial. Thus, a meta-analysis was conducted to reassess the effects of this polymorphism on CAD risks.
METHODS: The PubMed, Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched for relevant studies published up to December, 2018. The pooled odds ratios (OR) were calculated using STATA 13.0 software for allelic (A vs T) as well as homozygote (AA vs TT), heterozygote (AT vs TT), recessive (AA vs AT + TT), and dominant (AA + AT vs TT) genotype models, respectively.
RESULTS: Ten case-control studies (3744 cases and 3660 controls) were included. Overall, a significant association of IL-8 gene -251 A > T polymorphism with an increased risk of CAD was only observed in the dominant genotype model (OR = 1.48), but not others. In the subgroup analysis, significantly increased risks were also found for Chinese (OR = 1.64), polymerase chain reaction-restriction fragment length polymorphism genotyping (OR = 1.61), acute coronary syndrome (ACS) type (OR = 1.92 for 3 datasets; OR = 1.88 for 4 datasets), high quality (OR = 1.64), and age/gender matching status (OR = 1.55) under the dominant model. Furthermore, significantly increased risks were also found for ACS type under allelic (OR = 1.32 for 3 datasets; OR = 127 for 4 datasets), homozygote (OR = 1.64 for 3 datasets; OR = 1.50 for 4 datasets), heterozygote (OR = 1.32 for 3 datasets; OR = 1.30 for 4 datasets), and recessive (OR = 1.40 for 3 datasets; OR = 1.28 for 4 datasets) models.
CONCLUSION: This meta-analysis suggests that Chinese patients carrying -251A allele of IL-8 may have an increased risk for the development of CAD, especially ACS.

Introduction

Cardiovascular disease (CVD) is ranked as the first leading cause of death worldwide, with an estimated annual mortality of 23.3 million people by the year 2030.[ Coronary artery disease (CAD) is the most common form of CVD, accounting for approximately one-third of all deaths.[ Although diabetes mellitus, hypertension, dyslipidemia, smoking, alcohol consumption, and obesity have been demonstrated as main risk factors, several studies suggested that individuals may be genetically predisposed to developing CAD.[ Thus, investigation of key genetic variants underlying CAD may be of significance to develop efficient strategies for predicting, preventing and treating CAD.

There is an increasing amount of evidence to indicate that inflammation plays important roles in the pathogenesis of CAD, specifically in the process of atherosclerosis.[ Progressively increased inflammation may contribute to endothelial dysfunction and then facilitate the deposition of local lipid within the arterial wall, ultimately resulting in plaque formation and vascular stenosis followed by the development of CAD and even sudden death.[ Interleukin-8 (IL-8) is an important pro-inflammatory mediator produced by macrophages and its level has been reported to be increased in patients with CAD.[ Elevated IL-8 in plasma was also proved to be an independent predictor for long-term all-cause mortality in patients with acute coronary syndrome (ACS) that include unstable angina and acute myocardial infarction (MI).[ These findings imply genetic variants that cause the differences in the production of IL-8 levels may be associated with CAD susceptibility. This hypothesis has been implicated in recent studies: Zhang et al found the promoter region of IL-8 had a remarkable single nucleotide polymorphism (SNP) structure (IL-8 −251A/T, rs4073). IL-8 level was the highest among patients carrying the AA genotype, followed by AT and then TT genotype. IL-8 −251 A/T polymorphism was associated with increased susceptibility to ACS (odds ratio [OR] = 1.30, 95% confidence interval [CI]: 1.12–1.53; P = .004).[ The study of Zhang et al also showed patients with AT (OR = 1.59, 95% CI = 1.01–2.57; P = .04) and AA (OR = 2.06, 95% CI = 1.21–3.52; P = .005) genotypes were at an increased risk for developing CAD compared to those with the TT genotype.[ However, subsequent research by Kaur et al[ and Chang et al[ suggested that the T allele may be a risk factor for CAD, while Yang et al,[ Ren et al,[ and Wang et al[ found no association of IL-8 −251 A/T polymorphism with CAD risks. These controversial conclusions may be partially attributed to small sample size of individual studies. Therefore, we aimed to re-evaluate the effects of IL-8 −251 A/T polymorphism on CAD risks by performing a meta-analysis that can synthesize data from all eligible case-control studies and may achieve a more convincing conclusion. To our knowledge, this related meta-analysis has not been reported previously.

Materials and methods

Search strategy

Articles were identified by an electronic search on PubMed, the Cochrane Library, the China National Knowledge Infrastructure (Chinese), and Wanfang (Chinese) databases using the following keywords interleukin-8 (OR IL-8) AND coronary artery disease (OR CAD OR coronary heart disease OR CHD OR myocardial infarction OR MI OR ischemic heart disease OR IHD OR acute coronary syndrome OR ACS OR angina OR atherosclerosis OR cardiovascular) AND single nucleotide polymorphism (OR SNP OR variation OR variant OR mutation). The searching time was up to December, 2018. Furthermore, additional relevant studies on this topic were identified by a hand search of references cited by retrieved articles. Searches were limited to papers published in the English and Chinese language. This search followed the Guidelines of the preferred reporting items for systematic review and meta-analysis statement.

Selection criteria

All the articles were eligible if they met the following inclusion criteria:

assessing the association between IL-8 −251A/T polymorphism and CAD;

being human case-control studies;

providing sufficient information on the genotypes or alleles for calculating the OR and its corresponding 95% CI; and

providing the genotype distribution of control groups conformed to the assumptions of the Hardy–Weinberg equilibrium (HWE).

Studies were excluded when they were

duplicated data;

meeting abstracts, case reports/series, review articles, and editorial comment; and

not meeting all of the inclusion criteria.

Data extraction

The following characteristics were extracted from the eligible studies: author's name, year of publication, country of origin, type of CAD, genotyping method, the source of controls, sex and age matching, HWE test, sample size, genotype and allele frequencies of cases and controls. The quality of included studies was evaluated based on the Newcastle–Ottawa scale (NOS)[ that assessed 3 aspects:

subject selection (0–4 points),

comparability of subject (0–2 points), and

clinical outcome (0–3 points).

A high-quality study was defined as a score of ≥7. Studies with NOS scores ≥6 were considered to be of high quality. Two authors independently reviewed, extracted, and assessed the quality of the data. All disagreements were discussed and resolved with consensus.

Statistical analysis

The analyses were conducted using the STATA software (version 13.0; STATA Corporation, College Station, TX). The overall strength of an association between −251 A/T polymorphism and CAD risk was determined by calculating the crude ORs with 95% CIs for allelic (A vs T) model as well as homozygote (AA vs TT), heterozygote (AT vs TT), recessive (AA vs AT + TT), and dominant (AA + AT vs TT) genotype models, respectively. The z-test was used to measure the statistical significance of the pooled OR and P < .05 was considered to be statistically significant. Furthermore, the subgroup analysis was also performed according to ethnicity (Chinese or Indian), genotyping method (polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] or others), type of CAD (CAD, ACS, or MI), study quality (low quality: quality score <7; high quality: quality score ≥7), and the matching status of age and gender (yes or no).

Heterogeneity among studies was assessed with the Cochran Q (Chi-squared) statistic and the I2 statistic (P < .10 and I2 > 50% indicated evidence of heterogeneity). A random-effects (heterogeneous) or fixed-effects (homogeneous) model was used to calculate pooled effect estimates. The estimate of potential publication bias was evaluated by the Egger regression test (P < .05 was set as the significance level) and funnel plots. Sensitivity analysis was performed by omitting each study at a time to find potential outliers.

Results

Characteristics of eligible studies

The flow diagram of study selection is shown in Figure 1. According to the inclusion and exclusion criteria, 10 case–control studies (including 3744 cases and 3660 controls) were eligible for this meta-analysis[ (Table 1). The selected papers were published between 2010 and 2018. Most of the included studies (90%, 9/10) were performed in the Chinese population[ and only 1 (10%, 1/10) was in Indian.[ In addition, there were 8 studies conducted by PCR-RFLP,[ 1 performed by amplification refractory mutation system-PCR[ and only 1 used MassARRAY[ to detect the SNPs. Of these included studies, 6 studies reported about overall CAD (in which 1 was also divided into ACS and non-ACS cases[), 1 about ACS (which included 2 datasets)[ and 1 about MI (which was not reported to be acute or old).[ Coronary angiography was the mainly used method to diagnose CAD, which was defined as more than 50% or 70% stenosis in at least 1 coronary artery. All of studies provided the distributions of genotype and conformed to HWE. The genotype distribution and allele frequencies in cases and controls are listed in Table 2. According to the NOS criteria, most of the included studies were considered to be of high methodological quality other than one that was performed in Indian[ (Table 3).

Figure 1

Flow diagram of study identification.

Table 1

Characteristics of included studies in the meta-analysis.

Table 2

IL-8 −251A/T polymorphisms genotype and allele distribution in cases and controls.

Table 3

Quality of included studies was evaluated based on the Newcastle–Ottawa Scale (NOS).

Meta-analysis

The results of the association between IL-8 −251A/T polymorphism and CAD and the heterogeneity test are displayed in Table 4. Overall, a significant association of IL-8 gene −251 A > T polymorphism with an increased risk of CAD was only observed in the dominant model (AA + AT vs TT: OR = 1.48, 95% CI = 1.08–2.02; P = .015) (Fig. 2), but not in the allelic (A vs T: OR = 1.08, 95% CI = 0.87–1.33; P = .498), homozygote (AA vs TT: OR = 1.14, 95% CI = 0.77–1.69; P = .510), heterozygote (AT vs TT: OR = 1.04, 95% CI = 0.79–1.37; P = .802), and recessive (AA vs AT + TT: OR = 1.14, 95% CI = 0.91–1.42; P = .262) models.

Table 4

Meta-analysis results.

Figure 2

Forest plots of the association of IL-8 gene −251 A > T polymorphism with an increased risk of CAD under the dominant genotype model (AA + AT vs TT). Squares indicate OR; horizontal lines indicate 95% CI; hollow diamond indicates the pooled OR and its 95% CI. CAD = coronary artery disease, CI = confidence intervals, OR = odds ratio.

In the subgroup analysis, significantly increased risks were also found for Chinese (OR = 1.64, 95% CI, = 1.29–2.08; P < .001), PCR-RFLP genotyping (OR = 1.61, 95% CI, = 1.24–2.10; P < .001), ACS type (OR = 1.92, 95% CI, = 1.63–2.57 for 3 datasets; OR = 1.88, 95% CI, = 1.58–2.24.88 for 4 datasets; both P < .001), high quality (OR = 1.64, 95% CI, = 1.29–2.08; P < .001), and age/gender matching status (OR = 1.55, 95% CI, = 1.16–2.06; P = .003) under the dominant model. Furthermore, significant associations were similarly identified for ACS type under allelic (A vs T: OR = 1.32, 95% CI = 1.17–1.48 for 3 datasets; OR = 1.27, 95% CI = 1.13–1.42 for 4 datasets; both P < .001), homozygote (AA vs TT: OR = 1.64, 95% CI = 1.28–2.10, P < .001 for 3 datasets; OR = 1.50, 95% CI = 1.28–2.10, P = .001 for 4 datasets), heterozygote (AT vs TT: OR = 1.32, 95% CI = 1.10–1.58 for 3 datasets; OR = 1.30, 95% CI = 1.10–1.53 for 4 datasets; both P = .002) and recessive (AA vs AT + TT: OR = 1.40, 95% CI = 1.12–1.75, P = .047 for 3 datasets; OR = 1.28, 95% CI = 1.01–1.63, P = .040 for 4 datasets) models.

Publication bias

The evaluation of publication bias for AA + AT vs TT model using the Egger test indicated that the publication bias was nonsignificant (P = .370). Also, no obvious asymmetry was observed in the funnel plot (Fig. 3). These results revealed no evidence of publication bias.

Figure 3

Egger funnel plot of the overall analysis for the assessment of potential publication bias (dominant genotype model). Each circle represents a separate study. CI = confidence intervals, SND = standard normal deviation.

Sensitivity analyses

As presented in Figure 4, although each study was successively removed, the overall results did not alter obviously, which indicated the high stability of the meta-analysis results.

Figure 4

Sensitivity analysis of the overall analysis for the assessment of influence of each study (dominant model). Every hollow round indicates the pooled OR when the left study is omitted in this meta-analysis. The 2 ends of every broken line represent the 95% CI. The horizontal axis is ln (OR). CI = confidence intervals, OR = odds ratio.

Discussion

On the basis of 10 case–control studies, our meta-analysis revealed IL-8 −251A/T polymorphism was significantly associated with the susceptibility of ACS. Patients with A allele or AA, AT and AA + AT genotypes had a significantly increased risk of developing ACS. Furthermore, the AA + AT genotype was also associated with an increased risk of CAD in overall analysis and this association was consistently significant in subgroups of Chinese, PCR-RFLP genotyping, high quality, and age/gender matching status.

The IL-8 gene, located on chromosome 4q12–21, is composed of 4 exons, 3 introns, and a proximal promoter region.[ Although several polymorphisms had been reported in these genomic structures of IL-8 gene, including +781C/T, −353A/T, +678T/C, +1633C/T, −251A/T, and +394 T/G,[ only −251A/T[ and +394 T/G[ polymorphisms were studied to investigate their associations with CAD. Also, −251A/T polymorphism in the promoter region was shown to be related to the expression alteration of IL-8, with AA genotype contributing to a significantly increased level of IL-8 compared with that of the AT or TT genotype.[ Moreover, higher IL-8 mRNA levels were also reported in who presented the TA genotype compared with the TT genotype.[ Thus, −251A/T polymorphism may be a biomarker associated with a serial of inflammatory diseases, which had been demonstrated in cancer,[ Alzheimer's disease,[ and CAD.[ In line with these studies, we also found AA + AT genotype was associated with an increased risk of overall CAD and all genetic models including mutant A-251 conferred susceptibility to ACS risk. This finding was also consistent with the fact that the cytokine related immune activity (exhibiting elevated level of IL-8, IL-18, IL-1β, IL-16, etc) was higher in the ACS patients compared with stable angina and normal controls.[

Although the mechanism remains unclear, it is supposed the higher IL-8 may contribute to the development and progression of CAD via the following potential mechanisms:

cholesterol efflux: Chen et al used the human IL-8-neutralizing antibody to demonstrate that IL-8 may inhibit cholesterol efflux and promote lipid accumulation, leading to the development of atherosclerosis[;

angiogenesis: Kyriakakis et al reported that antigen-activated invariant natural killer T cells could release IL-8 and then up-regulate the expression of surface IL-8 receptors (C-X-C motif chemokine receptor 2 and vascular endothelial growth factor receptor 2) to promote endothelial cell migration and sprouting, influencing the stability of atherosclerotic plaques[; and

apoptosis: IL-8 is involved in the initiation and amplification of acute inflammatory reactions and then induces injured apoptosis of endothelial cells.[

There are several limitations in this meta-analysis. First, CAD is a multifactorial disease, which may be influenced by interactions between gene-gene as well as gene-environment. However, unadjusted ORs estimates were only used in this study due to the lack of the link between compounding factors (ie, diabetes mellitus, hypertension, dyslipidemia, smoking, alcohol consumption, and obesity) and genotype distribution in the original data of the eligible studies. Second, the number of cases and controls in some specific subgroups was relatively small, which may not provide sufficient statistical power to estimate the correlation between the IL-8 gene polymorphisms and the susceptibility to CAD. Third, only 2 Asian countries were included in the analysis and most of the data (90%) were from China. Fourth, only English or Chinese published studies were included in this meta-analysis. Non-significant or negative findings in other language may be missed.

In conclusion, this meta-analysis provides robust evidence that Chinese patients carrying -251A allele of IL-8 may have an increased risk for the development of CAD, especially ACS.

Author contributions

Conceptualization: Quanfang Zhang.

Data curation: Quanfang Zhang, Zhexun Lian.

Formal analysis: Quanfang Zhang, Zhexun Lian.

Investigation: Wugang Wang, Wei Wang.

Methodology: Wugang Wang, Zuoyuan Chen.

Software: Yan Cui.

Validation: Wenzhong Zhang.

Visualization: Jun Wu.

Writing – original draft: Quanfang Zhang.

Writing – review and editing: Quanfang Zhang.