Using the large UK Biobank resource, supported by cholesterol efflux measurements in the MHI Biobank, we report the effect of genetically lower CETP on lipid biomarkers, cholesterol efflux, CRP, and cardiovascular outcomes. We assessed how sex and BMI changed the effect of a genetically predicted decrease of CETP on those measurements and outcomes. We report significant modulatory effect of both sex and BMI on the association of CETP with biomarkers, but we were unable to show that these differences resulted in an effect on cardiovascular outcomes.
In our analyses, we observed that a genetically predicted lower CETP concentration was strongly associated with higher HDL-c and apoA levels and, to a lesser extent, to lower LDL-c and apoB levels. These results are concordant with previous reports of the effect of CETP on lipids and lipoproteins 23,24. We also observed lp(a) levels to be slightly, but significantly, lower in individuals with a genetically predicted reduction in CETP. This observation is interesting as lp(a) is an important risk factor for CAD that is largely independent of other lipoproteins. Previous studies have reported that both torcetrapib and anacetrapib could reduce lp(a) levels 17,19,25. The added genetic support could be indicative of a class effect of CETP inhibitors. In a Mendelian randomization study of lp(a) levels, a 10 mg/dl genetic reduction in lp(a) was associated with an OR of 0.942 for coronary heart disease supporting the causal role of lp(a) in coronary heart disease 26. In our study, we show that a 1 s.d. decrease in the genetic CETP score was associated with a reduction in lp(a) of about 2 mg/dl which corresponds to an OR for coronary artery disease of 0.988 based on this previous MR study.
Genetically lower CETP was not associated with C-reactive protein. In the dal-OUTCOMES trial of dalcetrapib and the ACCELERATE trial of evacetrapib, CETP inhibition was associated with an increase in C-reactive protein, but there was no significant difference in the DEFINE trial of anacetrapib 15,16,27. Given our high power to detect an association with biomarker measurements in the UK Biobank, it is unlikely that a lifelong, genetically lower CETP level has an effect on C-reactive protein levels.
We have also found that genetically lower CETP was associated with higher levels of cholesterol efflux. This observation is concordant with previous reports of increased cholesterol efflux in patients treated with dalcetrapib and anacetrapib 28,29. Genetically lower CETP was also associated with lower rates of cardiovascular outcomes, with 2.5% fewer CAD events per s.d. decrease in the CETP genetic score. The observed protective effect was robust to adjustment for observed apoB levels at baseline in the UK Biobank, supporting that the protective effect of CETP may not be exclusively mediated by apoB levels. However, adjusting for observed apoB levels at a single time point may not completely control for a lifetime reduction in apoB levels by CETP genetic variants. Nonetheless, we presented a unified portrait of the effect of genetically lower CETP which may help better understand on-target effects of CETP inhibitors and the diversity of pathways through which genetic variants in CETP may exert a protective effect on CAD.
The value of using human genetic variants to predict the effect of pharmacological modulation of drug targets is gaining recognition as more examples of drug target discoveries and predictions for ongoing randomized trials are reported 2,30. Human genetics can also inform on subgroup effects in the context of precision medicine, for clinical trial design, or to estimate external validity of drug effects to other patient populations. Genetic studies of CETP variants have highlighted possible effect modification by sex on HDL-c levels, carotid intima-media thickness, the HDL-c / apoAI ratio and on the dynamics of postprandial triglyceride levels 31–35. Sex differences were also observed in many traits thought to be involved in the atheroprotective effect of CETP such as the apoAI and apoA-II composition of HDL and CETP mediated cholesterol efflux 36,37. Whether this translates to cardiovascular outcomes remains unknown 38. Considering that female sex is underrepresented in the majority of cardiovascular clinical trials, sex differences may have important clinical implications as inferences drawn from the unbalanced trial populations are used to inform treatment.
In our analyses, we found sex to be a strong modulator of the effect of genetically lower CETP on lipid biomarkers and cholesterol efflux. Women with CETP lowering genetic variants had lower LDL-c and apoB levels and higher HDL-c, apoA and cholesterol efflux. In a substudy of the DEFINE trial, anacetrapib increased cholesterol efflux in men, but not women 29. In that study, cholesterol efflux capacity was measured using fluorescently labeled cholesterol which mostly captures efflux through the ABCA1 pathway. Here, we used radiolabeled cholesterol and our measurements in cAMP-stimulated conditions also include the contributions of ABCG1 and SR-BI. In healthy subjects, serum from women had more SR-BI mediated efflux capacity whereas serum from men had more ABCA1-mediated efflux capacity 39. We suggest that, on average, with genetically lower CETP, women have more overall cholesterol efflux capacity than men, but that specific pathways may show the inverse relationship. The modulatory effect of sex did not translate to strong differences in cardiovascular outcomes in our analysis even though the additive interaction model suggests that men had a larger relative risk of “hard” CAD than women for a same increase in the CETP score. Because the UK Biobank contains relatively few cardiovascular events, especially in women, and the genetic variants in CETP have a limited effect size, a replication from a large, well-powered study is warranted to confirm these findings. A previous study also reported sex differences in the association between CETP genetic variants and cardiovascular disease40, but sex interaction was not formally assessed and the sample size and power may have been limited in that study (n = 866).
We conducted a meta-analysis of the sex-stratified results of three RCTs of CETP inhibitors and although we did observe a nominally greater protective effect in women (RR = 0.92) than in men (RR = 0.96), the difference in effect was not statistically significant (p = 0.50). A total of 10,769 women pooled across all three studies were included in the meta-analysis for a total of 58,412 participants, which is still lower than the number of individuals included in the smallest study (n = 12,092 for ACCELERATE), suggesting limited power to identify a subgroup effect in women. We conclude that there is some indication of a stronger beneficial effect of CETP inhibition in women, but that confirmation using genetic datasets enriched for cardiovascular events or clinical trials with a greater representation of women would be needed.
Plasma CETP is mainly secreted by macrophages in the liver (Kupffer cells) and adipose tissue does not appear to be a clinically relevant contributor to circulating CETP levels 41,42. However, higher BMI may affect hepatic and systemic inflammation and result in changes in lipid homeostasis that could alter the function of CETP without affecting plasma CETP concentration. There is also previous evidence of an interaction between obesity status and a CETP variant on acute coronary syndrome from a smaller study (n = 474) 43.
In our analyses, the atheroprotective profile of lipoproteins attributable to genetically lower CETP levels was stronger in individuals with lower BMI, when compared to individuals with higher BMI, showing lower levels of LDL-c, apoB and lp(a) and higher levels of HDL-c and apoA with genetically lower CETP. Similar results were observed in models allowing for nonlinear effects on the biomarkers. The modulatory effect of BMI on the relationship between CETP and biomarkers did not translate to cardiovascular outcomes, however, a larger dataset may be needed to assess the possible impact on outcomes with sufficient power.
We also found evidence of three-way interactions of sex, BMI and genetically predicted CETP on LDL-c, apoB levels and cholesterol efflux. The attenuated effect of lower CETP on LDL-c reduction with increasing BMI was specific to men. In women, the increase in cholesterol efflux by genetic reduction in CETP was attenuated with increasing BMI, but this effect was sex-specific.
Our study had some limitations. We relied on common genetic variants to model pharmacological CETP inhibition, but these variants do not include rare mutations which can have much stronger effects on CETP function. CETP activity can also be modulated in more subtle ways than complete inhibition, with molecules such as dalcetrapib preserving pre-β-HDL formation, a function that is inhibited by anacetrapib 44. This effect is due to differences in the CETP-mediated HDL remodeling through homotypic transfer of cholesteryl esters and may play an important role in atherosclerosis as pre-β-HDL are important acceptors for ABCA1-mediated cholesterol efflux. Our genetic study could not distinguish between CETP modulation or inhibition, as measurements of ABCA1-mediated efflux or HDL subtypes were not available. CETP may also have an important intracellular role in storing triglycerides and cholesteryl esters in lipid droplets. 45,46. Whether this activity was altered in our genetic models or played a role in the effect modification by BMI remains to be determined. Also, the estimated effects derived from the genetic variants relate to lifelong exposure to lower CETP concentrations, which may differ from the effects of short-term exposure to pharmacological inhibition of CETP. In addition, although the UK Biobank offers large numbers of study participants, the cohort has a limited number of cardiovascular events. A case-control cohort with larger numbers of CAD events may help increase power to assess the translation of the detected modulatory effects of sex and BMI on lipid biomarkers and cholesterol efflux to cardiovascular outcomes. We did not adjust for multiple testing of phenotypes and effect modifiers in this study which evaluated several correlated phenotypes. We reported confidence intervals and provided power analyses to support the interpretation of results. The genetic variants at the CETP gene have concurrent effects on multiple biomarkers, making it difficult to disentangle the effect of the individual biomarkers on cardiovascular outcomes. The polygenic modeling of multivariable exposures may be an interesting approach to consider for this purpose 47.
In this study, we have evaluated the effect of a genetically predicted reduction in CETP concentration on lipoproteins, lipid fractions, cholesterol efflux and C-reactive protein. We have found results to be largely concordant with those obtained from clinical trials. Using statistical interaction models, we found that sex and BMI are modifiers of the effect of CETP on lipid biomarkers and cholesterol efflux.