Association between KIF6 rs20455 polymorphism and the risk of coronary heart disease (CHD): a pooled analysis of 50 individual studies including 40,059 cases and 64,032 controls

Background The KIF6 rs20455 polymorphism has been verified as an important genetic factor of coronary heart disease (CHD), but with controversial results. The aim of this study was to explore the association between KIF6 rs20455 polymorphism and susceptibility to CHD. Methods All eligible studies were identified by searching Medline (mainly PubMed), EMBASE, the Web of Science, Cochrane Collaboration Database, Chinese National Knowledge Infrastructure, Wanfang Database and China Biological Medicine up to October 5, 2016.Odds ratios (ORs) with 95% confidence interval (CI) were used to explore the association between KIF6 rs20455 polymorphism and CHD risk. Begg’s and Egger’s tests were used to examine the publication bias. Subgroup analysis and sensitivity analysis were performed to test the reliability and stability of the results. All the analyses were carried out by Stata 12.0 software. Results A total of 28 publications including 50 individual studies were analyzed in this present work. There are no significant association found between KIF6 rs20455 polymorphism and CHD risk (Homozygote model: OR = 1.007, 95% CI =0.952–1.066, P = 0.801; Heterozygote model: OR = 1.009, 95% CI = 0.968–1.052, P = 0.636; Dominant model: OR = 1.007, 95% CI = 0.966–1.048, P = 0.753; Recessive model: OR = 0.989, 95% CI = 0.943–1.037, P = 0.655; Allele comparison model: OR = 1.00, 95% CI = 0.971–1.030, P = 0.988). Furthermore, subgroup analyses were performed by ethnicity, source of control. Conclusions Our result suggests that KIF6 rs20455 polymorphism may not be associated with CHD susceptibility. However, additional very well-designed large-scale studies are warranted to confirm our results.


Background
Coronary heart disease (CHD), a multifactorial heart disorder resulting from both environmental and genetic factors [1], is one of the leading causes of disability and death around the world [2]. Epidemiology studies have suggested that hypertension, hyperlipidemia, diabetes mellitus, obesity and smoking are major risk factors for CHD [3]. In recent years, more and more studies reveled that several loci and variants are strongly associated with CHD [4,5]. It has been estimated that approximately 50% of the variability of the major risk factors for CHD is determined by genetics [6].
The KIF6 protein is one of several molecular components that mediate intracellular transport of organelles, protein complexes, and mRNAs. A common Trp719Arg (rs20455) SNP in exon 19 of the KIF6 gene has been identified as a potential risk factor for CHD [7,8]. The KIF6 protein belongs to the kinesin superfamily, which is involved in the intracellular transport in a microtubule and ATP-dependent manner [9]. The rs20455 polymorphism replaces the nonpolar 'Trp' residue in codon 719 with a basic ' Arg' amino acid. This SNP lies near the putative cargo binding taildomain and may alter the cargo activity of KIF6 [10]. Carriers of the 719Arg allele exhibit a 50% increased risk of events compared with non-carriers [8,11]. Up to now, multiple large prospective and case-control studies have reported the association between KIF6 rs20455 polymorphism and the risk of CHD. However, somestudies have not verified inconsistent results. Published studies have generally been restricted in terms of sample size and ethnic diversity, and individual studies may have in-sufficient power to achieve a comprehensive and reliable conclusion. In view of the discrepancies in the findings of previous published studies, we aimed to perform a meta-analysis of the published studies to clarify the association between KIF6 rs20455 polymorphism and CHD to get a better under-standing of this relationship.

Literature search
A comprehensive search for all related studies from both electric databases, such as, Medline (mainly PubMed), Embase, Web of science, China National Knowledge Infrastructure (CNKI) et al., and hand search from references of all eligible literatures. Single or combinations of the following keywords were used: "kinesin like protein 6" or "KIF6" or "rs20455" or "719Arg", "single nucleotide polymorphism, SNP or variation, mutation", "genetic association" and "coronary heart disease" or "CHD". No language and sample size were set. When more than one studies of the same population were included in several publications, only the most recent or complete studies were included in this meta-analysis.

Selection criteria
Articles included should meet following criteria: an appropriate description of KIF6 rs20455 polymorphism in CHD cases and healthy controls; results expressed as odds ratio (OR); and studies with a 95% confidence interval (CI) for OR with sufficient data to calculate these numbers. While for the exclusion criteria provided as follows: studies without raw data; case-only studies, family-based studies, case reports, editorials, and review articles (including meta-analyses). In studies with overlapping cases/controls, the study with the higher quality score or the study with more information on the origin of the cases/controls was included in the meta-analysis.

Data extraction
Two researchers extracted important information independently and carefully from all eligible studies according to the criteria listed above. Any disagreement will be resolved by the two authors through discussion or the third author. The following data were extracted from each included study: first author's surname, year of publication, country, ethnicity, genotyping method, source of control, total number of cases and controls, distributions of KIF6 rs20455 genotypes. Different ethnicity descents were categorized as Caucasian, Asian, and Mixed populations (the original studies didn't clarify the race of the subjects or mixed races).

Statistical analysis
We adopted poled ORs and corresponding 95% confidence interval (CIs) to detect the association between KIF6 rs20455 polymorphism and CHD risk. Heterogeneity was explored by Q statistic [12], and the P value was <0.05 will be considered statistically significant. Heterogeneity was also assessed using the I 2 statistic, which takes values between 0% and 100% with higher values denoting greater degree of heterogeneity (I 2 = 0-25%: noheterogeneity; I 2 = 25-50%: moderate heterogeneity; I 2 = 50-75%: large heterogeneity; I 2 = 75-100%: extreme heterogeneity) [13]. Different statistical models will be selected according to the result of heterogeneity. Random (Der Simonian-Laird method) [14] will be used to calculate the precise results when the P value of heterogeneity was <0.05, or the I 2 > 50%. Otherwise, fixed effects model (Mantel-Haenszel method) will be adopted [15]. Five genetic comparison model were carried out and calculated as follows: homozygote model (GG vs. AA), heterozygote model (AG vs. AA), recessive model (GG vs. AG + AA), and dominant model (GG + AG vs. AA), and allele comparison model (G-allele vs. A-allele). Hardy-Weinberg equilibrium in the control group was tested by the chi-square test for goodness of fit, and a P value of <0.05 was considered significant. Subgroup analyses were performed by ethnicity, source of control, to confirm if our results were stable and robust [16]. Begg's funnel plots [17] and Egger's test [18] were explored to examine if potential publication bias were existed in this study. Sensitivity analysis was carried out by sequentially omitting each study and finding the influence on the overall summary estimate [19]. All the statistical analyses were finished by STATA software (version 12.0; Stata Corporation, College Station, TX). All the P values were two-sided.

Characteristics of all included studies
Totally, 209 potential relevant studies were searched through several databases. Based on the including criteria listed above, only 28 articles including 50 separate studies were included finally [8,. A flow diagram summarizing the process of study selection was present in Fig. 1. The baseline characteristics ofall included studies were listed in Table 1 [41]. Moreover, there were 37 studies from Caucasian decedent, 9 studies from Asian populations and the rest 14 studies from mixed populations. There were 20 population-based (PB) studies, 21 hospital-based (HB) studies and four family based (FB) study, three community based (CB) study, two hospital and community based (H-CB) study. Different ethnicity descents were categorized as Caucasian, Asian and Mix (the original studies didn't clarify the race of the subjects or mixed races).

Sensitivity analysis
The sensitivity analysis was performed to evaluate the influence of each individual study on the pooled OR by omitting every single study. The analysis results reflected that our results were statistically stable and reliable.

Publication bias
There was no significant publication bias found in the meta-analysis, reflected by P values from Begg's correlation (Heterozygote model: P = 0.089; Dominant model: P = 0.061; Allele comparison model: P = 0.052, Fig. 7)    Table 2.

Discussion
Large sample and unbiased epidemiological studies of predisposition genes polymorphisms could provide insight into the in vivo relationship between candidate genes and complex diseases. Many epidemiological studies have investigated the relationship between the KIF6 rs20455 polymorphism and the risk of CHD, but because of small sample size and the low statistical power of individual studies, results have been contradictory. In this present study, we searched all eligible studies to date and got the precise result if KIF6 rs20455 polymorphism could contribute to the risk of CHD. To the best of our knowledge, our present work was the most comprehensive one through enrolling all eligible studies. Herein, we included 50 individual studies, including 40,059 cases and 64,032 controls. Overall, there was no association between KIF6 rs20455 polymorphism and CHD risk. Hamidizadeh et al. found that significant association was found between this gene polymorphism and CHD risk among Caucasian populations [43], and the result was verified in another study through enrolling 143,000 subjects [40]. However, no association was found in a meta-analysis, among South Asians, African-Americans, Hispanics, East Asians, and mixed decedent populations [39]. Furthermore, other recent studies were also found no association existed between this gene polymorphism and CHD risk [25,26,[47][48][49]. When we got the subgroup analyses by ethnicity, there was also no association found among Caucasian and Asian populations. While decreased risk of this gene polymorphism and CHD risk was found among mixed populations. Of note, mixed populations means the original studies didn't clarify the race of the subjects or mixed races. This result may be not provided some useful information for clinical deeds. So, further studies should be performed with clearly race or ethnicity stated in their work.
Publication bias was found in some genetic models. The explanations might arise from some aspects. First, our meta-analysis took into consideration only fully published data, and the abstract and conference papers were excluded. Second, this meta-analysis only focused on papers published in Chinese and English language, and some eligible studies which were reported in other languages might be missed. Third, positive results tend to be accepted by journals while negative results are often rejected or not even submitted. We should point out that the publication bias might partly account for the results, but which were not affected deeply. When we adjusted the results using the trim and fill method, the adjusted risk estimate was attenuated but remained significant, indicating the stability of our results.
Some limitations of this meta-analysis should be addressed. Firstly, heterogeneity is a potential problem when interpreting all the results of meta-analysis. Although we minimized the likelihood by performing a careful search for published studies, using the explicit criteria for study inclusion, the significant between-study heterogeneity still existed in most of comparison. The presence of heterogeneity can result from differences in the age distribution, selection of controls, prevalence lifestyle factors and so on. Secondly, only published studies were included in this meta-analysis. Therefore, potential publication bias was existed in some genetic models. Despite the limitations, our meta-analysis significantly increased the statistical power based on substantial data from different studies. The sensitivity analyses outcomes reflected that our results were statistically stable and reliable.
In conclusion, this present meta-analysis suggests that carriers of KIF6 rs20455 polymorphism may irrelative to     the risk of CHD. We also observed no compelling evidence of an association between the KIF6 rs20455 SNP and CHD in multiple race/ethnic groups. These findings do not support the clinical utility of testing for the KIF6 rs20455 polymorphism in the primary prevention of CHD and indirectly question whether genotype information at this locus is able to identify subjects most likely to benefit from the use of statins.