The IL-6 rs1800795 and rs1800796 polymorphisms are associated with coronary artery disease risk

Abstract Studies examining the associations between the interleukin‐6 (IL‐6) rs1800795 and rs1800796 gene polymorphisms and risk of coronary artery disease (CAD) remain controversial. Our aim was to evaluate the accurately determine role of these two polymorphisms in CAD risk. PubMed, Embase, VIP, Wan fang and China National Knowledge Infrastructure databases were searched. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The trial sequential analysis (TSA) was conducted, and bioinformatics tools were employed. A total of thirty‐seven articles were obtained. For the IL‐6 rs1800795 polymorphism, 9411 CAD patients and 3161 controls were included, 4720 patients with CAD, and 5000 controls were included for the IL‐6 rs1800796 polymorphism. In the pooled analysis, significant associations were only observed for the rs1800796 polymorphism (allelic: OR [95%CI] = 1.28 [1.13, 1.44], dominant: OR [95%CI] = 1.35 [1.17, 1.57], recessive: OR [95%CI] = 1.35 [1.18, 1.55], heterozygote: OR [95%CI] = 1.26 [1.15, 1.37], homozygote: OR [95%CI] = 1.62 [1.23, 2.13]). Significant associations were detected in the Asian and Mongoloid populations and ‘more than 500’ subgroup for the rs1800795 polymorphism. TSA confirmed the true‐positive results for the rs1800796 polymorphism. The bioinformatics analysis showed that the two polymorphisms played important roles in the gene transcription. The IL‐6 rs1800796 polymorphism is associated with an increased susceptibility to CAD and is a risk factor for CAD. The IL‐6 rs1800795 polymorphism is associated with an increased risk of CAD in Asians, particularly in Chinese, and a decreased risk of CAD in an African population is remarkably observed.


| INTRODUC TI ON
Coronary artery disease (CAD) is the leading cause of death both in developed and developing countries. 1,2 The aetiology of CAD remains obscure. Environmental and genetic factors, as well as the interactions between them, play a crucial role in the pathophysiology of CAD. 3,4 The heritability of CAD was estimated to range from 40%-60% based on family and twin studies. 5 Furthermore, the greatest genetic influence was observed on early-onset CAD events, 6 which implies a more vital role for genetic factors in determining CAD risk. Genotyping common single nucleotide polymorphisms (SNPs) within a potential CAD-related gene is an essential and efficient method to detect genetic risk markers, and many significant SNPs associated with CAD risk have been reported, such as matrix metalloproteinase-9, 7 interleukin-27 8 and Toll-like receptor 4. 9 Inflammation plays a key role in the pathophysiology of CAD by promoting the development of atherosclerosis. 10 As a pro-inflammatory and immune-regulatory cytokine, IL-6 plays an important role in the genesis and maintenance of the inflammatory response in atherosclerosis. The IL-6 gene is located on chromosome 7p21-24 and comprises 5 introns and 6 exons. 11 Many SNPs in the IL-6 gene related to CAD risk have been reported, including IL-6-174G/C, 12 IL-6-572C/G, 13 IL-6-597G/A, 14 IL-6-634C/G 15 and IL-6+2954G/C 16 ; however, some of them were not associated with CAD risk (IL-6-597G/A and +2954G/C) or only one study reported the increased risk of CAD (IL-6-634C/G). Among them, two common polymorphisms (IL-6 rs1800795 −174G/C and IL-6 rs1800796 −572C/G) have been extensively investigated; however, the results were inconclusive. Several previous studies have been conducted in an attempt to draw significant conclusions, but the limitations in sample size and potential false-positive results caused by systematic errors may bias the results. We therefore performed a study to more accurately determine associations between IL-6 polymorphisms and CAD risk; in addition, the bioinformatics analysis was conducted to explore the potential molecular mechanism.

| Inclusion and exclusion criteria
For inclusion in our analysis, studies must have met the following inclusion criteria: (a) evaluation of the relationship between the IL-6 polymorphisms and coronary artery disease; (b) coronary artery disease was defined as 50% stenosis in the left main coronary artery, or multiple significant (≥70% stenosis) in more than one coronary artery 17 ; (c) a case-control or cohort design; (d) genotype distribution data were able to be acquired to calculate odds ratios (ORs) and 95% confidence intervals (CIs), particularly detailed data from the control group for testing Hardy-Weinberg equilibrium. Exclusion criteria were as follows: (a) duplication of previous studies; (b) comments, reviews and editorials; (c) non-English or non-Chinese articles; and (d) studies lacking controls. Based on the inclusion and exclusion criteria, the first two authors independently reviewed the references and included the relevant studies. Any disagreement was solved by discussion with the third author (Wang).

| Data extraction
For all included studies, the first two authors independently extracted the following data using a standardized form: first author's last name, year of publication, study country, study region, age and body mass index (BMI), source of the control population, genotyping method, sample size and genotype frequency of polymorphisms in the IL6 gene in patients and controls. Disagreement was settled by rechecking the data or discussion with a third author.

| Quality assessment
The quality of the included studies was independently assessed by all the authors according to a set of criteria that were modified based on the Newcastle-Ottawa quality assessment scale (Table S1).

| Statistical analysis
Hardy-Weinberg equilibrium (HWE) was tested in control groups from each study using the chi-squared test, and P < .05 was considered a significant departure from HWE. Odds ratios (ORs) and respectively. Heterogeneity was evaluated using the Q statistic (significance level of P < .1) and I 2 statistic (greater than 50% as evidence of a significant inconsistency). Heterogeneity between studies was evaluated with the I 2 test, and a higher I 2 values indicated higher levels of heterogeneity (I 2 > 90%: extreme heterogeneity; I 2 = 70% to 90%: substantial heterogeneity; I 2 = 50% to 70%: moderate heterogeneity; I 2 < 50%: low heterogeneity). In the heterogeneity evaluation, the fixed-effects model was used when

| Trial sequential analysis
Systematic bias and random errors are inevitable when conducting a meta-analysis because of the sparse data and repeated significance testing; moreover, trials with low methodological quality, publication bias and a small sample size may generate a false-positive result. Trial sequential analysis (TSA) is an approach that provides the required amount of information (number of samples) and further reveals potentially false-positive results in a meta-analysis. 19 Therefore, TSA was employed to calculate the required amount information for obtaining reliable data of our study. 20,21 The TSA was performed by anticipating a 10% relative risk reduction, an overall 5% risk of type I error and a statistical test power of 80%.

| Bioinformatics analysis
Ensembl is a genome browser for vertebrate genomes that supports research in comparative genomics, evolution, sequence variation and transcriptional regulation, and this database provides the genomic context, genes and regulatory elements, flanking sequence, population genetics, phenotype data, sample genotypes, linkage disequilibrium and phylogenetic context of a single nucleotide polymorphism (http://asia.ensem bl.org/index.html). SNPinfo is an important bioinformatics analysis tool that predicts SNP function.
The SNPinfo database can help researches specify genes or linkage regions and select SNPs based on GWAS results, calculate linkage disequilibrium (LD) and predict functional characteristics of both coding and non-coding SNPs (https://snpin fo.niehs.nih.gov/). 22 In addition, the RNAfold web server is one of the core programmes of the Vienna RNA package that has been used to predict the minimum free energy of single sequences that influence the stability of the structure. 23 Therefore, we conducted bioinformatics analyses using the aforementioned databases and methods to identify the potential molecular mechanisms for further research.

| Characteristics of the included studies
The PRISMA flow diagram of our analysis was shown in Table S2.
Two hundred and thirty articles were retrieved by searching the international and Chinese databases. After removing duplicates and screening title and abstracts, 54 articles were subjected to the fulltext assessment and 12 articles were excluded due to the lack of detailed genotype distribution data. Finally, 37 articles [12][13][14]16, were included in the qualitative and quantitative synthesis.
The characteristics of all included studies regarding the associations between IL6 gene polymorphism and coronary artery disease are presented in Table 1. For the IL-6 rs1800795 polymorphism, 33 studies involving 9411 CAD patients and 3161 controls were included; 21 studies of 4720 patients with CAD and 5000 controls were included for the IL-6 rs1800796 polymorphism. Based on the modified Newcastle-Ottawa Quality Assessment Scale, the score of each included study was greater than 7, which implied a sufficient methodological quality for analysis.

| The pooled analysis of IL-6 polymorphisms and CAD risk
The main results of our analysis and the heterogeneity test of the associations between IL-6 gene polymorphisms and coronary artery disease risk are shown in

| Subgroup analyses of the associations between IL-6 polymorphisms and CAD risk
Subgroup analyses were introduced to identify the source of heterogeneity and further reveal additional information about the associations between IL-6 polymorphisms and CAD risk. Table 2 summarizes the results of the subgroup analyses based on HWE, region, ethnicity, the source of controls and sample size.
For the subgroup in accordance with HWE, significant associations were only detected for the IL-6 rs1800796 polymorphism, and all five genetic models indicated strong associations with an increased OR We stratified studies into three subgroups by sample size based on the modified quality scale score (less than 300, between 300 and 500, and greater than 500) to evaluate the effect of sample size on the associations between the two polymorphisms and CAD risk. In the greater than 500 subgroup, significant associations were observed between the rs1800795

| The sensitivity analysis of IL-6 polymorphisms and CAD risk
A sensitivity analysis was conducted by sequentially omitting each individual study to detect the effect of each study on the results of the overall meta-analysis. None of the studies changed the corresponding pooled ORs; thus, the results of our meta-analysis were stable and reliable ( Figure 3A-B).

| Publication bias
The

| Trial sequential analysis
A previous meta-analysis of the associations between IL-6 polymorphisms and CAD risk reported negative results. For our pooled analysis of IL-6 rs1800795 and rs1800796 polymorphisms, significant associations were only observed for the IL-6 rs1800796 polymorphism. Hence, a trial sequential analysis was required to verify that our significant association was not a false-positive result. Similar strength associations were discovered in five different genetic models. The allelic genetic model produced the best value and is a natural model of inheritance with a stronger genotype-phenotype association, which also does not pre-assume any interactions between the numbers of variant alleles. Therefore, we chose the allelic genetic model of the rs1800796 polymorphism to conduct the trial sequential analysis. The results of trial sequential analysis are shown in Figure 4. The x-axis and y-axis represent the number of patients and the cumulative Z score, respectively. Within the designed assumptions of confidence and effect size, the informa-

| Bioinformatics analysis
Based on the genomic context obtained from the Ensembl database, we constructed the summary genetic diagram for the rs1800795 and rs1800796 polymorphisms ( Figure 5A). The two polymorphisms were both located in the promoter region near exon 2, implying that these sequences are potential transcription factor binding sites.
Hence, we analysed the sequences of the two polymorphisms and the results from the SNPinfo database showed both polymorphisms are located in potential transcription factor binding sites ( Figure 5B).
In addition, the secondary structure of DNA at the rs1800795 and

| D ISCUSS I ON
In our study, two polymorphisms (rs1800795 and rs1800796) in the IL-6 gene were analysed for associations with CAD risk. The two common polymorphisms have been extensively studied in depth over the past few decades, providing sufficient enough data for a subgroup analysis designed to discover potential intriguing associations.
Moreover, the two polymorphisms are located in the promoter region of the IL-6 gene, and may influence the expression of the IL-6 gene, and result in susceptibility to CAD. Several meta-analyses have been conducted to explore the associations between the two polymorphisms and CAD risk, but the results were inconsistent. Significant associations between the IL-6 rs1800795 polymorphism and CAD risk were reported in some meta-analyses, 57 these two studies and found that the opposite results may due to the relatively small sample size. Additionally, the adjusted alpha was not used to adjust for multiple tests, and thus, that conclusion that the result was a true positive is questionable. The controversial results from previous meta-analysis and case-control studies prompted us to examine the associations between the IL6 rs1800795 and rs1800796 polymorphisms and CAD risk. Therefore, we chose these two common polymorphisms in the IL6 gene to analyse the potential CAD risk.
No association between the IL-6 rs1800795 polymorphism and CAD risk with high heterogeneity was observed in the pooled results. Hence, we employed a detailed subgroup analysis to determine the potential sources of heterogeneity and associations. For In addition, an analysis of the sequence and secondary structure was performed using the RNAfold web server. The minimum free energy (MFE) and the free energy of the thermodynamic ensemble of the mutant alleles of the rs1800795 and rs1800796 polymorphisms were reduced compared with the wild alleles. The principle of minimum energy states that for a closed system, with constant external parameters and entropy, the internal energy will decrease and approach a minimum value at equilibrium. 66 The mutant allele in a gene sequence may alter the free energy. The minimum free energy and the free energy of the thermodynamic ensemble are two thermodynamics parameters that have been used as a measure the required energy to reach the equilibrium for the stability F I G U R E 5 A, The genetic structure of IL-6 gene. B, The most related Transcription Factor Binding Sites predicted by SNP ratio of a sequence. 23 The reduction implies that less energy is needed to form the secondary structure of the sequence containing the mutant allele, indicating that the sequence of the mutant alleles of the rs1800795 and rs1800796 polymorphisms is easier to disperse from the DNA double helix structure to serve as the template strand during transcription. Hence, these structural changes may affect the expression of the IL-6 gene. However, a bioinformatics prediction is not sufficient, and further fundamental research on the effect of the two polymorphisms on the transcription of the IL6 gene is needed.
Several limitations existed in our study. First, only English and Chinese articles were included as a language restriction, which may bias the results. Second, the number of included studies was relatively small in some subgroups, such as the African population in the subgroup analysis of the IL-6 rs1800795 polymorphism, and thus, the results should be interpreted with caution. Third, only two common SNPs were evaluated in our study and other relevant SNPs in the IL-6 gene that are unknown or understudied may also have potential associations with CAD risk. Forth, the distance between the two common polymorphisms is relatively close, and the potential interactions between the two polymorphisms or other unknown polymorphisms need to be studied. In addition, the potential influence of environmental factors on genotype-CAD associations is worth considering.
In conclusion, the IL-6 rs1800796 polymorphism is associated with an increased susceptibility to CAD and is a risk factor for CAD.
In addition, the IL-6 rs1800795 polymorphism is associated with an increased risk of CAD in Asian, particularly in Chinese volunteers.
Remarkably, a decreased risk of CAD was observed in the African population.

ACK N OWLED G EM ENT
This work is supported by grants from the National Natural Science Foundation of China (Nos. 81170205). The funder, Zhaohui Wang, was responsible for the article.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Yu performed the statistical analysis of evaluating the strengths of associations between these two polymorphisms and CAD risk.

AUTH O R CO NTR I B UTI O N
Zhaohui Wang hosted the meeting addressing the disagreements.
All authors reviewed and revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.

F I G U R E 6
The RNAfold analysis of the IL-6 polymorphisms