Association of cholesteryl ester transfer protein (CETP) gene polymorphism, high density lipoprotein cholesterol and risk of coronary artery disease: a meta-analysis using a Mendelian randomization approach

Several clinical parameters are associated with common diseases and are helpful for predicting and preventing these common diseases. For instance, lipid profiles are well acknowledged to be associated with the risk of coronary artery disease (CAD) and myocardial infarction (MI) [1]. Observational and experimental studies have documented a strong positive association of low density lipoprotein cholesterol (LDL-C) and an inverse association of high density lipoprotein cholesterol (HDL-C) with the risk of CAD [2]. Nevertheless, the causal role of HDL-C in the development of atherosclerosis has not been fully clarified and the evidence available from conventional population studies is scarce and paradoxical [3]-[6]. Many large epidemiological studies, such as the Framingham study [7], have described an increase in HDL-C as being protective to CAD. However, it has been demonstrated that Dalcetrapib and Torcetrapib, two cholesteryl ester transfer protein (CETP) inhibitiors, were shown to elevate circulating HDL-C concentrations substantially, but do not benefit CAD patients in two large randomized controlled trials [8],[9]. Both environmental exposures and genetic factors are thought to contribute to the majority of these inconsistencies. Deciphering the regulation of HDL-C metabolism via the interaction of inherited variations with environmental factors may help explore the underlying pathological mechanism of CAD.

CETP plays a key role in determining the circulating HDL levels and transfers cholesteryl esters from HDL-C to LDL-C, as well as very low density lipoprotein cholesterol (VLDL-C), in exchange for triglyceride rich lipoproteins [10]. An inverse association of CETP activity with HDL-C levels was observed [11]. Some genome-wide association studies have suggested that the correlation of the CETP locus with HDL-C concentrations is more significant than that of any other locus found across the genome [12],[13]. Among these genetic variants, the TaqIB polymorphism (rs708272) in intron 1 [14] has been investigated extensively [15]. Significant associations of the TaqIB polymorphism with variations in CETP promoter activity [16] and circulating HDL-C concentrations [14] were observed, showing lower CETP concentrations and higher HDL-C levels among B2 carriers than among B1B1 homozygotes [14],[17]-[19]. However, the definite relationship between the TaqIB polymorphism and CAD encountered a considerable dispute among the genotype-disease studies [12],[19]-[23], most likely due to relatively small sample sizes, limitations in statistical power and interactions with ethnic descents, population classifications and environment exposures. We hypothesized that a large meta-analysis is a robust method that can reliably appraise the heterogeneity present in genetic association studies and also can expound the divergences. Given that the HDL-C is an intermediate in the causal pathway from the CETP gene to CAD, it would be sensible to conduct a meta-analysis that in some way integrates the triangle relationship: the TaqIB polymorphism-HDL-C (genotype-phenotype), the TaqIB polymorphism-CAD (genotype-disease), and HDL-C-CAD (phenotype-disease). The logic of this approach is greatly strengthened by the appeal to Mendelian randomization [24], which is according to Mendel's second law (the law of independent assortment) [25]. Mendelian randomization means that the inheritance of an individual's genes is independent by a seemingly random process at conception. Theoretically, the genetic polymorphism, which is causally and specifically bound up with the intermediate phenotype and the predicted risk of disease, could be exploited as an instrument to attain an un-confounded estimation of phenotype-disease association. In the present study, we examined whether the increase in circulating HDL-C concentrations due to the TaqIB polymorphism was correlated with a reduced risk of CAD, based on the rationale of Mendelian randomization [26].

Because HDL-C serves an important role in CAD pathogenesis, we expected that the CETP TaqIB polymorphism, which modulates HDL-C, to likewise confer the risk of disease. To our best knowledge, the current meta-analysis is one of the largest systematic reviews of studies investigating the relationship between the CETP polymorphism, HDL-C and potential risk of CAD. The synthesized estimation of the TaqIB polymorphism concerning CAD risk was presented in our meta-analysis containing 50,996 subjects. Subsequently, the possible causal inference of HDL-C on CAD risk was further assessed. The overall OR of the TaqIB polymorphism under a random-effects model was suggestive of a modest protective effect on CAD. Considering that violation of HWE might diminish the total effect of the meta-analysis, we performed a sensitivity analysis by excluding the studies demonstrating a departure from HWE. Trends in the ORs did not change substantially by estimation of the association among only the HWE-confirming studies. We provide robust evidence that the TaqIB polymorphism is associated with CAD and that the HWE-violation only slightly influences the interpretations of the observed results in our meta-analysis. Interestingly, the pre-specified subgroup analyses revealed that the B2 allele carriers had a remarkable higher risk reduction in CAD among Asians compared with Caucasians, in spite of similar frequency of the B2 allele. It remains unclear the real reasons for the divergence across the different ethnic groups, because we did not directly test the TaqIB polymorphism in different ethnic groups. We also did not estimate the association between the TaqIB polymorphism and other phenotypes. Given some GWASs have identified that numerous lipid-associated genetic variations differ with ethnicity [72]-[74], we expect that future investigations of the TaqIB polymorphism will uncover its genetic ancestral backgrounds and pleiotropic effects. In addition, the magnitude of association was greater in H-B studies than in P-B studies; the OR is likely overestimated, since subjects sampled from a single hospital may not reflect the real environmental exposures in the source population. In theory, participants recruited from the community, or from the general population, are more representative and reliable. Prospective studies and studies regarding MI or ACS as endpoints concerning the association between the TaqIB polymorphism and CAD did not provide robust evidence for a statistically significant association [75]. Of note, the variability of environmental exposures may be another source of between-study heterogeneity, as reflected from our meta-regression analysis. The protective effect of the B2 allele on CAD risk was potentiated in a population with low smoking rates and low circulating HDL-C levels. Although meta-regression reflects an ecological correlation, rather than a causal inference, our results imply that the underlying interactions of the TaqIB genotype with lifestyle and phenotype may influence the development of atherosclerosis. Considering a wide range of confidence intervals of the overall evaluation, further data from large-scale and well-designed studies are required to improve the precise of the study effect.

The strength of the instrument (the TaqIB polymorphism in our study) is determined by the absolute magnitude of its association with the intermediate phenotype (HDL-C in our study). A previous individual patient data-based meta-analysis including 7 large population-based studies and 3 randomized placebo-controlled trials (13,677 Caucasian subjects), have provided robust evidence that the TaqIB polymorphism is significantly associated with HDL among Caucasians [18]. Hence, the TaqIB polymorphism was certified as a qualified predictive instrument when using a Mendelian randomization approach among Caucasians because it correlates with a specific phenotype and consequently impacts the disease in the pathway intermediated by the phenotype. Our results were concordant with two previous meta-analyses including 15,704 and 19,035 participants respectively [18],[19]. However, neither of these studies assessed the relationship of HDL-C and CAD after adjustment for TaqIB polymorphism and the triangulation of the TaqIB polymorphism and HDL-C associations with CAD risk remained confounded.

Mendelian randomization, a form of instrumental variable analysis [76], was applied to determine the magnitude of the causal relationship of increased HDL-C with CAD by using the TaqIB polymorphism as the instrument. Interestingly, genetically elevated HDL-C levels did not reduce CAD risk. Our results supplement the evidence challenging conventional views that raising circulating HDL-C levels may translate into protection of CAD risk. Recently, Voight et al. [77] utilized the Asn396Ser polymorphism in the endothelial lipase gene (LIPG) and a genetic score containing 14 common single nucleotide polymorphisms (solely concerning HDL-C) as two instruments for Mendelian randomization. As a result, a specific and substantial increase in HDL-C levels due to LIPG Asn396Ser and the genetic score was verified to be not related to MI. Employing various instruments may contribute to the precision of instrumental variable estimates and may facilitate examination of potential instrumental variable assumptions. Besides LIPG, CETP was acknowledged to have a profound influence on HDL-C metabolism and consequently CAD risk. The TaqIB polymorphism, a silent base change affecting the 277th nucleotide, is almost fully concordant with another promoter variants -629 C-A [16],[78],[79]. The data from in vivo studies have documented that the -629 C-A variant is directly functional in regulating the CETP activity [16], suggesting that the TaqIB polymorphism, as a marker of the -629 C-A polymorphism, may be vital in regulating CETP activity and HDL-C metabolism. In order to maximize statistical power, we exploited the TaqIB polymorphism, rather than the -629 C-A polymorphism, as an instrument because extensive data on the TaqIB polymorphism were available. We suggest a strong possibility that raising HDL-C via specific means of inhibiting CETP will not decrease the risk of CAD. Whether CETP efforts pro- or anti-atherogenic effects, by means of the LDL-C receptor pathway, is still unclear [80],[81]. As a matter of fact, the results from animal studies and cytological studies remain paradoxical. Transgenic mice expressing CETP demonstrate accelerated atherosclerosis compared with non-expressing controls [82]-[84]. However, another in vivo study reported that CETP expression reduced atherosclerosis in lecithin cholesterol acyltransferase (LCAT) transgenic mice [85] and a further study revealed that CETP offset the deleterious effect of LCAT [86].

Furthermore, we restate the underlying restriction of raising circulating HDL-C levels as an effective treatment to reduce residual cardiovascular risk in patients treated by conventional statin therapy. Intense research efforts on therapeutic agents have been conducted to reduce the residual cardiovascular risk by raising circulating HDL-C levels. Statins, fibrates and niacin are regarded as effective pharmacological agents for raising HDL-C. However, the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study documented there was no added clinical benefit in using combination therapy with fenofibrate plus simvastatin compared with simvastatin monotherapy, despite the fact there were significantly increased levels of HDL-C in the combination therapy group [5]. In the Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) trial, the addition of niacin to statin therapy did not reduce the rate of cardiovascular events, despite remarkable improvements in HDL-C [87]. The Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events (ILLUMINATE) trial exhibited an excess of deaths and cardiovascular disease in the group receiving the CETP inhibitor, torcetrapib and atorvastatin compared with atorvastatin alone [8]. Subsequently, another large randomized trial performed in ACS patients showed that another CETP inhibitor, dalcetrapib, increased HDL-C levels, but did not reduce the risk of recurrent cardiovascular events [9]. Recently, a potential explanation was put forward that functional HDL-C properties may play a key role on the development of CAD rather than an indirect one [88]. Cholesterol efflux and reverse cholesterol transport is an important function of HDL-C. In vitro experiments and transgenic animal model studies have revealed that circulating HDL-C concentrations do not necessarily reflect the efficacy and anti-atherogenicity of reverse cholesterol transport [89]. It is likely that the increments of HDL-C generated by the CETP inhibition are either non-functional or pro-inflammatory rather than anti-inflammatory, which impedes their physiologic role in reverse cholesterol transport.

Another possibility is that the composition of HDL-C is altered in CAD patients and they are no longer productive at high levels or after therapeutic intervention [9]. Some studies have indicated that HDL-C and its major structural protein, apolipoprotein A1 are dysfunctional and are extensively oxidized by myeloperoxidase in human atheroma. In vitro oxidation of either apolipoprotein A1 or HDL particles by myeloperoxidase impairs their cholesterol acceptor function [90]. The precise evaluation of functional HDL-C properties and change in HDL-C sub-fractions are recommended for future studies.

Common variants in lipid-associated loci that are also associated with CAD may implicate genes at these loci as possible therapeutic targets. Recently, a large meta-analysis based on GWAS aimed to investigate the genetic markers for blood lipids [91]. A genome-wide association screen for serum lipids (including approximate 2.6 million SNPs) was conducted in 100,000 individuals of European ancestry. Ninety-five loci (of which 59 are novel) were identified to show genome-wide significant association with serum lipid traits. Further, the lead SNPs from the study were tested in 24,607 CAD patients and 66,197 individuals without CAD. They documented that common variants in lipid-associated loci are consistently related to CAD, implying that these loci may be the potential therapeutic targets. They also suggested that some causal genes in lipid-associated loci may have pleiotropic effects on non-lipid parameters that play an important role on the CAD risk reduction. They provided a foundation from which to develop a broader biological understanding of lipoprotein metabolism and to identify potential therapeutic targets for preventing CAD.

There are still limitations using Mendelian randomization analysis. Potential pleiotropic effects of genes may influence the results when a naturally occurring single nuclear polymorphism is used as an instrument to evaluate disease causality. Although single genetic variation is relative to extensive diseases, many of these associations are false positives because the gene has been more generally characterized than most in population investigations of genetic association studies. Canalization or developmental compensation also provides difficulties in interpreting the gene-disease association. These processes, which bring developmental buffering, offset the influence of the genetic variation or environmental forces [92], remain difficult to evaluate. Further experimental and subject-matter studies may provide a more precise estimation. In addition, CAD has numerous clinical features, ranging from stable coronary syndrome to ACS, which is determined by the stability of the atherosclerosis plaque. We could not completely exclude noise in our data when using CAD as the endpoint. Considering more power would be obtained from a more refined phenotype than a more global one, a more precise method that identifies the vulnerable plaque is needed and may be beneficial to estimate the effect of HDL on coronary atherosclerosis.

In conclusion, our results provided robust evidence that the TaqIB polymorphism, which specifically raised HDL-C concentrations, was uniformly associated with a reduction in CAD risk. However, a lack of causal inference in increased HDL-C levels with the pathogenesis of CAD was detected based on the Mendelian randomization approach among Caucasians. We highlight not only the requirements for further validation of the functional role of HDL-C in reverse cholesterol transport and in the etiology of atherosclerosis, but also as a high possibility that lifestyle interventions or pharmacotherapy, which elevates circulating HDL-C levels, cannot be assumed to benefit CAD patients in reality.