Associations of Cholesteryl Ester Transfer Protein TaqIB Polymorphism with the Composite Ischemic Cardiovascular Disease Risk and HDL-C Concentrations: A Meta-Analysis

Background: Previous studies have evaluated the associations between the cholesteryl ester transfer protein (CETP) TaqIB polymorphism (rs708272), the risk of developing composite ischemic cardiovascular disease (CVD) and the concentration of high-density lipoprotein cholesterol (HDL-C), but results remain controversial. The objective of this study was to investigate whether a relationship exists between these factors. Methods: We conducted a meta-analysis of available studies to clarify the associations of the CETP TaqIB polymorphism with HDL-C concentration and the composite ischemic CVD risk in both Asians and Caucasians. All statistical analyses were done with Stata 12.0. Results: Through utilization of the Cochrane Library, Embase, PubMed, Web of Science, Springer, China Science and Technology Journal Database, China National Knowledge Infrastructure, Google Scholar, and Baidu Library, a total of 45 studies from 44 papers with 20,866 cases and 21,298 controls were combined showing a significant association between the CETP TaqIB variant and composite ischemic CVD risk. Carriers of allele TaqIB-B1 were found to have a higher risk of composite ischemic CVD than non-carriers: OR = 1.15, 95% CI = 1.09–1.21, p < 0.001. Meanwhile, 28 studies with 23,959 subjects were included in the association between the CETP TaqIB polymorphism and the concentration of HDL-C. Results suggested that carriers of the B1B1 genotype had lower concentrations of HDL-C than those of the B2B2 genotype: SMD = 0.50, 95% CI = 0.36–0.65, p < 0.001. Conclusions: The synthesis of available evidence demonstrates that the CETP TaqIB polymorphism protects against composite ischemic CVD risk and is associated with a higher HDL-C concentration in both Asians and Caucasians.


Criteria Brief Description of How the Criteria Were Handled in the Meta-Analysis Reporting of background should include
 Problem definition CETP TaqIB polymorphism is closely associated with HDL-C level and various diseases including CAD, IS and MI. However, the associations between CETP TaqIB polymorphism and serum HDL-C level and susceptibility to AS were inconsistent in previous studies.

 Hypothesis statement
It is likely that CETP TaqIB polymorphism may influence the serum HDL-C level and susceptibility of AS.  Description of study outcomes Atherosclerosis We selected possibly relevant articles in the Cochrane Library, Embase, PubMed, Web of Science, Springer, China Science and Technology Journal Database (CSTJ), China National Knowledge Infrastructure (CNKI), Google Scholar and Baidu Library (last search conducted in January 2016) with search strategy: ("Cholesterol ester transfer protein" OR "CETP") and ("variation" OR "variant" OR "mutation" OR "polymorphism" OR "genotype") and ("CAD" OR "coronary artery disease" OR "coronary heart disease" OR "CHD" OR "myocardial infarction" OR "MI" OR "ischemic cardiovascular disease" OR "IS") and ("high-density lipoprotein cholesterol" OR "HDL-C" OR "blood lipid" OR "serum lipid").


Databases and registries searched

Description of any contact with authors
If necessary data were not reported in the primary manuscripts, we contacted the corresponding authors by email to request the missing data. Reporting of methods should include  Description of relevance or appropriateness of studies assembled for assessing the hypothesis to be tested Eligibility criteria: The eligibility criteria for including articles in the present meta-analysis were as following major criteria: (1) the publication evaluating the associations of the CETP TaqIB polymorphism with AS or HDL-C level; (2) all atherosclerosis cases were diagnosed were made according to the internationally recognized diagnostic criterion as follows: criteria of World Health Organization (WHO), criteria of American College of Cardiology/American Heart Association (ACC/AHA), criteria of European Society of Cardiology (ESC), or angiographic coronary stenosis (generally defined as at least 50% stenosis of one major coronary artery); (3) published in either Chinese or English; (4) for CAD association, sufficient published data for calculating odds ratios (ORs) with their 95% confidence intervals (CIs); for HDL-C level association, the number of population, the mean of HDL-C level and the standard deviations (SD) by genotypes should be available. Exclusion criteria: The exclusion criteria were as follows: (1) Duplicate publications; (2) incomplete information; (3) insufficient or insignificant statistical data; (4) review articles.


Rationale for the selection and coding of data For the association between CETP TaqIB polymorphism and AS, We used the crude ORs and 95% CIs for meta-analysis. If the studies did not provide crude ORs and 95% CIs, we calculated the ORs and 95% CIs by the total numbers of cases and controls, and frequencies of CETP TaqIB polymorphism in cases and controls. For the association between CETP TaqIB polymorphism and HDL-C, a pooled standardized mean difference (SMD) and its 95% CIs were used for the meta-analysis.
 Assessment of confounding NOS rating system was used to assess the confounder. Subgroup analyses were performed and sensitivity analyses were also performed.


Assessment of study quality, including blinding of quality assessors; stratification or regression on possible predictors of study results We assessed the methodological qualities of included studies by the description of study population, the set of controls and cases and related statistical methods. We carried out sensitivity analysis.
 Assessment of heterogeneity Heterogeneity was assessed by the Q-test and I 2 statistic, p < 0.10 and I 2 > 50% indicated evidence of heterogeneity.

Description of statistical methods in sufficient detail to be replicated
Methods of heterogeneity test, quantitative synthesis, assessments of publication bias, sensitivity analyses were reported in detail in the methods section.

Provision of appropriate tables and graphics
We provided flow chart to explain literature searching and selection ( Figure 1); forest plots for the total analysis, (Figures 2 and 3, Figures S1-S5); study characteristics and allele/genotype frequencies (Tables 1 and 2).

Reporting of results should include
Graph summarizing individual study estimates and overall estimate Graph summarizing individual study estimates and overall estimate are presenting in Figures 2 and 3, Figures S1-S5.  Results of sensitivity testing The results of sensitivity analysis were described in results section. Table 3 provided detailed results for the sensitivity analyses.

Indication of statistical uncertainty of findings
The results of heterogeneity test, pooled ORs, 95% confidence intervals and p value for Z test were presented with all pooled analyses. Reporting of discussion should include  Quantitative assessment of bias We evaluated the publication bias by funnel plots, egger's test.

 Justification for exclusion
Based on our preliminary search criteria, a total of 478 publications were eligible. Among these studies, 279 records were excluded (reviews, no out of interest, meta-analysis, duplicate publication and records not published in Chinese and English) and 134 articles without original data were excluded. 

Assessment of quality of included studies
We discussed the results of sensitivity analyses and described the limitations of included studies.

Consideration of alternative explanations for observed results
We discussed that potential unmeasured confounders and explained the limitations of this meta-analysis. We reminded readers that caution should be made when interpreting this metaanalysis.

 Generalization of the conclusions
Our meta-analysis suggested that the CETP TaqIB polymorphism were associated with serum HDL-C level and the susceptibility to AS.
 Guidelines for future research Larger sample-size studies with homogeneous AS patients and well-matched controls are required.

Rationale 3
Coronary artery disease (CAD) and Myocardial infarction (MI) have become the serious public health problems in the world because of its high morbidity and mortality [1,2]. However, its exact mechanisms are still unclear. For a long time, atherosclerosis has attracted more attention because of it's the pathological foundation of CAD and MI. Abnormal cholesterol metabolism was considered to be main factor for atherosclerosis, and many epidemiological evidence has shown that low concentration of serum high-density lipoprotein cholesterol (HDL-C) was considered as an independent risk factors for atherosclerosis [3,4]. High-density lipoprotein (HDL) was demonstrated to play a pivotal role in mediating the transfer of cholesterol from extra hepatic tissues to the liver, and reducing the deposition of cholesterol in the artery wall [5]. Cholesterol ester transfer protein (CETP) gene located on chromosome 16q21, and encodes the key plasma protein that mediate the transfer of esterified cholesterol from HDL to apolipoprotein B-containing particles in exchange for triglycerides [6,7]. CETP gene mutation may affect the transcription and expression of the protein, thereby affecting serum HDL-C level [8]. CETP TaqIB (rs708272) polymorphism as the most common of CETP gene polymorphism loci has received a great deal of attention [9]. In recent years, numerous studies have showed relationship the CETP gene TaqIB polymorphism in the synthesis of HDL-C and AS risk, however, still remain inconsistent, possibly due to small sample sizes in the individual studies.  [11]. However, they were not observed the relationship between CETP TaqIB polymorphism and CAD. Cao et al. and Wang et al. performed meta-analysis to evaluate the association the CETP TaqIB variant and MI, their results shown that the CETP TaqIB-B2 allele is a protective factor to against the development of MI [12,13]. Considering the above four meta-analyses only focused on the association of CETP TaqIB polymorphism with the single atherosclerotic disease, we therefore performed this meta-analysis to clarify the role of the CETP gene TaqIB polymorphism in the synthesis of HDL-C and AS risk.

METHODS Protocol and registration
5 No protocol and registration.

Eligibility criteria 6
The eligibility criteria for including articles in the present meta-analysis were as following major criteria: (1) the publication evaluating the associations of the CETP TaqIB polymorphism with AS or HDL-C level; (2) all atherosclerosis cases were diagnosed were made according to the internationally recognized diagnostic criterion as follows: criteria of World Health Organization (WHO), criteria of American College of Cardiology/American Heart Association (ACC/AHA), criteria of European Society of Cardiology (ESC), or angiographic coronary stenosis (generally defined as at least 50% stenosis of one major coronary artery); (3) published in either Chinese or English; (4) for CAD association, sufficient published data for calculating odds ratios (ORs) with their 95% confidence intervals (CIs); for HDL-C level association, the number of population, the mean of HDL-C level and the standard deviations (SD) by genotypes should be available. The exclusion criteria were as follows: (1)  Following Medical Subject Heading (MeSH) terms and/or text words were used for searching: ("Cholesterol ester transfer protein" OR "CETP") and ("variation" OR "variant" OR "mutation" OR "polymorphism" OR "genotype") and ("CAD" OR "coronary artery disease" OR "coronary heart disease" OR "CHD" OR "myocardial infarction" OR "MI" OR "ischemic cardiovascular disease" OR "IS") and ("high-density lipoprotein cholesterol" OR "HDL-C" OR "blood lipid" OR "serum lipid"). 3

Data collection process 10
Data were independently extracted from original publications by two reviewers (Minghong Yao and Yusong Ding) according to the inclusion criteria listed above. Discrepancy between the reviewers was resolved by consensus or a third reviewer (ShuXia Guo). 4

Data items 11
Data, including name of the first author, year of publication, study population (country, ethnicity), source of controls, case/control sample size, minor allele frequency (MAF), genotype counts in the cases/controls, and evidence of Hardy-Weinberg equilibrium (HWE), the population number, the mean of HDL-C level and its SD by genotypes, were extracted from each study. 4

Risk of bias in individual 12
The Newcastle-Ottawa Scale (NOS) was used to assessed the methodologic quality of the individual studies by two reviewers studies (Minghong Yao and Yizhong Yan) [16]. Each study was evaluated and scored based on three criteria: selection (4 stars), comparability (2 stars), and exposure (3 stars). The NOS point ranges between zero up to nine stars, and the studies with a score of equal to or higher than seven stars was considered to be of high quality. Any disagreement was resolved by discussion with a third reviewer (Jiaming Liu).

Summary measures 13
The strength of associations between the CETP TaqIB polymorphism and atherosclerosis were assessed by summary odds ratios (ORs) with their 95% confidence intervals (CIs). A pooled standardized mean difference (SMD) and its 95% CIs were used for the meta-analysis of HDL-C level and the CETP TaqIB

Additional analyses 16
For the AS, Subgroup analyses were performed based on ethnicity (Caucasians and Asians), atherosclerotic disease (CAD, MI and IS), source of controls (population-based studies and hospital-based studies) and study type (case control study and cohort study); for the HDL-C, Subgroup analyses were performed based on ethnicity (Caucasians and Asians). Sensitivity analyses were performed by limiting the metaanalysis to studies conforming to HWE and sample size. the Galbraith plot and meta-regression were performed subsequently to explore the heterogeneity sources [22].

16-19 Risk of bias within studies 19
The NOS results were shown in Table 1 and Table2. The NOS results showed that the average scores were 6.8 and 6.4, respectively.

16-19
Results of individual studies 20 The main results of individual studies were shown in Figures 2 and 3 (Figures 2 and 3: Forest plots for the relationship between CETP TaqIB variant and AS risk and HDL-C).

Risk of bias across studies 22
Begg's funnel plot and Egger's regression test were performed to assess potential publication bias. For the CETP polymorphism and atherosclerosis risk (B1 vs. B2), the shape of the funnel plot ( Figure 3) did not reveal obvious asymmetry which means no publication bias. Then, it was confirmed by Egger's test (p = 0.074); For the CETP polymorphism and HDL-C (B1B1 vs. B2B2), both of the shape of the funnel plot ( Figure 4) and Egger's test (p = 0.058) did not reveal obvious asymmetry which means no publication bias.

7, 24
Additional analysis 23 Subgroup analyses: For the association between the CETP TaqIB polymorphism and atherosclerosis risk, Subgroup analyzes by ethnicity showed the significant associations in Asians ( Table 3.  Figures S4 and S5). Sensitivity analyses: Sensitivity analysis was performed to determine whether the robustness of the study results. The included studies were limited to those conforming to HWE and sample size. Overall, the corresponding pooled ORs and SMD were not materially altered, either for the CETP TaqIB polymorphism and atherosclerosis risk or CETP TaqIB polymorphism and HDL-C. Results of the sensitivity analysis suggested that the overall results of this meta-analysis were relatively robust and credible. The main results of sensitivity analyses were shown in Table 3 and Figure S6-S11) Heterogeneity analysis: For the relationship between the CETP TaqIB polymorphism and atherosclerosis, significant heterogeneity among the available studies were observed in the overall comparisons (for allelic model:  Figures S12 and S13). Figures S14-S16 shows that association between the CETP TaqIB polymorphism and HDL-C level after exclusion of these outlier studies. However, the significant association between the CETP polymorphism and HDL-C level was unchanged both in Asian subgroup (B1B2 vs.

Summary of evidence 24
In the present meta-analysis, a total of 45 studies from 44 papers with 20,866 cases and 21,298 controls, we found that the TaqIB-B2 allele was significantly associated with reduced of atherosclerosis both in Caucasians and Asians. Additionally, 28 studies with 23,959 subjects were included in the analysis of association between the CETP TaqIB polymorphism and HDL-C level. According to the results, the TaqIB-B2 allele was significantly associated with a higher level of HDL-C both in Caucasians and Asians. Therefore, it is reasonable to assume that the CETP TaqIB polymorphism is probably by influencing the HDL-C metabolism to play protective factors for the development of atherosclerosis. 7

Limitations 25
Limitations: There are several potential limitations in our present meta-analysis should be acknowledged. Firstly, there was significant heterogeneity in our study. Although we have used appropriate meta-analytic techniques, we cannot completely exclude the influence of the heterogeneity. Secondly, it might miss the eligible articles that reported in other languages because our study only focused on articles published in English and Chinese languages. Thirdly, the sample sizes of some studies were rather small. In summary, it is well known that AS was affected by multiple environmental and genetic factors, we are only discussed a single gene polymorphism and not analyzed environmental factors, there are still many unclearly environmental and genetic factors and their interactions. Thus, it remains to be detected. 8

Conclusions 26
Conclusion：In conclusion, the present meta-analysis shows that the CETP TaqIB-B2 allele is associated with a higher serum HDL-C level and a protective role for AS risk both in Asians and Caucasians. Further investigations with the consideration of gene-gene and gene-environment interactions are needed.

Funding 27
This work was supported by National Science and Technology Support Projects for the "Eleventh Five-Years Plan" of China (No. 2009BAI82B04), National Natural Science Foundation of China (No. 81560551) and Special Fund for Investigation of chronic heart and lung disease in Tibet and Xinjiang of China (No. 201402002).
Online submission system