This cohort study examines data for participants in the UK Biobank to determine associations of a combined reduction of CETP and PCSK9 concentrations with plasma lipid levels and such clinical outcomes as risk of coronary artery disease and age-related macula degeneration.
Key Points
Question
Is there evidence for nonadditive effects of genetically defined low concentrations of CETP and PCSK9 on plasma lipid concentrations, risk of coronary artery disease, and age-related macular degeneration?
Findings
In this cohort study, a 2 × 2 factorial Mendelian randomization study including 425 354 participants from the UK Biobank, an additive association of a genetically reduced combined concentration of CETP and PCSK9 was found for lipid levels and risk of coronary artery disease, while the association of CETP with age-related macular degeneration was not mitigated.
Meaning
Our findings suggest that joint inhibition of CETP and PCSK9 has additive effects on lipid concentrations and clinical outcomes.
Abstract
Importance
Cholesteryl ester transfer protein inhibition (CETP) has been shown to increase levels of high-density lipoprotein cholesterol (HDL-C) and reduce levels of low-density lipoprotein cholesterol (LDL-C). Current LDL-C target attainment is low, and novel phase 3 trials are underway to investigate whether CETP inhibitors result in reduction of cardiovascular disease risk in high-risk patients who may be treated with PCSK9-inhibiting agents.
Objective
To explore the associations of combined reduction of CETP and PCSK9 concentrations with risk of coronary artery disease (CAD) and other clinical and safety outcomes.
Design, Setting, and Participants
Two-sample 2 × 2 factorial Mendelian randomization study in a general population sample that includes data for UK Biobank participants of European ancestry.
Exposures
Separate genetic scores were constructed for CETP and PCSK9 plasma protein concentrations, which were combined to determine the associations of combined genetically reduced CETP and PCSK9 concentrations with disease.
Main Outcomes and Measures
Blood lipid and lipoprotein concentrations, blood pressure, CAD, age-related macular degeneration, type 2 diabetes, any stroke and ischemic stroke, Alzheimer disease, vascular dementia, heart failure, atrial fibrillation, chronic kidney disease, asthma, and multiple sclerosis.
Results
Data for 425 354 UKB participants were included; the median (IQR) age was 59 years (51-64), and 229 399 (53.9%) were female. The associations of lower CETP and lower PCSK9 concentrations with CAD are similar when scaled per 10-mg/dL reduction in LDL-C concentrations (CETP: odds ratio [OR], 0.74; 95% CI, 0.67 to 0.81; PCSK9: OR, 0.75; 95% CI, 0.71 to 0.79). Combined exposure to lower CETP and PCSK9 concentrations was associated with an additive magnitude with lipids and all outcomes, and we did not observe any nonadditive interactions, most notably for LDL-C (CETP: effect size, −1.11 mg/dL; 95% CI, −1.40 to −0.82; PCSK9: effect size, −2.13 mg/dL; 95% CI, −2.43 to −1.84; combined: effect size, −3.47 mg/dL; 95% CI, −3.76 to −3.18; P = .34 for interaction) and CAD (CETP: OR, 0.96; 95% CI, 0.94 to 1.00; PCSK9: OR, 0.94; 95% CI, 0.91 to 0.97; combined: OR, 0.90; 95% CI, 0.87 to 0.93; P = .83 for interaction). In addition, when corrected for multiple testing, lower CETP concentrations were associated with increased age-related macular degeneration (OR, 1.11; 95% CI, 1.04 to 1.19).
Conclusions and Relevance
Our results suggest that joint inhibition of CETP and PCSK9 has additive effects on lipid traits and disease risk, including a lower risk of CAD. Further research may explore whether a combination of CETP- and PCSK9-related therapeutics can benefit high-risk patients who are unable to reach treatment targets with existing options.
Introduction
Randomized clinical trials (RCTs) and Mendelian randomization (MR) studies have convincingly shown that low-density lipoprotein cholesterol (LDL-C) is causally associated with cardiovascular disease (CVD) risk.1,2 Statin therapy is the cornerstone of CVD prevention, followed by proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors and ezetimibe.3 Recently published clinical guidelines recommend a stringent LDL-C therapeutic target for patients at very high risk (<1.4 mmol/L).4 However, studies have shown that attainment is very low, with only a third of patients who are taking PCSK9 inhibitors meeting their LDL-C targets.5,6,7 Therefore, additional therapies for lowering LDL-C are deemed of importance to address the residual CVD risk.8
Cholesteryl ester transfer protein (CETP) has been widely considered as a therapeutic target, and multiple large RCTs have been conducted to assess whether CETP inhibition results in CVD risk reduction. While CETP inhibitors were initially considered as agents for increasing levels of high-density lipoprotein cholesterol (HDL-C), RCTs and MR studies have shown that CETP inhibitors and varying CETP concentrations affect multiple lipid fractions.9,10 When comparing MR and RCT data, previously investigated CETP inhibitors were found to be associated with heterogeneous effects on blood lipids and (off-target) on blood pressure, which resulted in a compound-related directionally discordant effect on clinical outcomes (ranging from deleterious to beneficial effects).10
Recently, 2 phase 3 RCTs were announced (PREVAIL [NCT05202509] and BROADWAY [NCT05142722]) for investigating the CETP inhibitor obicetrapib. These trials together will include more than 10 000 high-risk patients who are unable to meet their recommended lipid targets despite receiving maximally tolerated lipid-lowering treatment, including PCSK9 monoclonal antibodies.11 Another phase 3 trial studying dalcetrapib is nearly finished.12 As such, exploring the effects of joint inhibition of CETP and PCSK9 will provide information on the putative effects of this combination of therapeutics in high-risk patients who are unable to reach treatment targets with existing therapeutic options.
A recent MR study by Schmidt et al10 provided further support for considering the associations with joint inhibition of CETP and PCSK9 plasma concentrations.10 This article showed that lower concentrations of PCSK9 and CETP had distinct lipidomic fingerprints, with PCSK9 predominantly associated with LDL-C and apolipoprotein B (apo B), while lower CETP concentration was associated with beneficial effect on a wider range of lipids, including intermediate-density and very-low-density lipoprotein cholesterol (ie, remnant-cholesterol). This suggest that while both targets affect coronary artery disease (CAD) risk, they might act through distinct pathways and therefore offer additive benefit.
To formally assess the on-target associations with the combined targeting of PCSK9 and CETP, we conducted the current MR and factorial MR study. Specifically, we evaluated the associations of lower CETP and PCSK9 protein plasma concentrations with lipids, CAD, and a range of clinical outcomes that have been found to be associated with PCSK9 or CETP in previous MR studies.10,13
Methods
Overview
All participants provided informed consent, and the study was approved by the North West Multi-centre Research Ethics Committee. This study was conducted under the UK Biobank (UKB) application number 44972. We used the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) MR checklist to ensure transparent reporting. Further, the studies included in the genome-wide association studies (GWAS) were approved by their respective ethics committees, and all participants provided informed consent.
In this MR study, we used genetic variation to establish the causal associations of lower CETP and PCSK9 protein concentrations with clinically relevant traits such as CAD14,15 (eMethods in the Supplement). In factorial MR, we modeled the expected effects of combined lower PCSK9 and CETP concentrations by combining the 2 separate genetic scores in interaction regression models and by creating multiple subgroups in the investigated population, similar to the factorial design of a trial with 2 or more drug compounds.
This study thus consists of 3 parts (eFigure 1 in the Supplement). First, we investigated the associations of genetically predicted lower CETP or PCSK9 concentrations with lipids and CAD risk using external summary data, to confirm that our genetic scores replicate the expected effects of lower CETP and PCSK9 concentrations as found in previous MR studies. Second, we investigated the associations of individually genetically predicted lower CETP and PCSK9 concentrations with lipids and clinical outcome beyond CAD, using UKB data. Third, we used factorial MR to investigate the associations of the combined exposure to genetically predicted lower CETP and PCSK9 concentrations with lipids and clinical outcome.
Construction of Genetic Scores
We constructed genetic scores using publicly available summary data on genetic associations with PCSK9 concentrations (reported in log[ng/L]) in 3290 participants from the LIFE-HEART study16 and CETP concentrations (reported in ug/L) in 4248 participants from the NEO study,17 by selecting independent (R2<0.1) common variants (minor allele frequency >0.01) within a 100-kb region around the genes. Details on the construction of the genetic scores can be found in the eMethods in the Supplement.
Data Sources
We used summary-level GWAS data to replicate the known associations of genetically predicted lower CETP and PCSK9 with lipids and CAD. We leveraged aggregated genetic association data for the following outcomes: LDL-C, HDL-C, total cholesterol, triglycerides, apo B, apolipoprotein A1 (apo A1),18,19 and CAD20 (eTable 1 in the Supplement).
We used individual participant data from the UKB to further validate our results, to provide data on additional outcomes and for the factorial MR analyses (eFigure 1 in the Supplement).16,17,21 We investigated the associations of lower PCSK9 and CETP concentrations with lipids, blood pressure, CAD, type 2 diabetes, age-related macular degeneration (AMD), ischemic stroke, any stroke, Alzheimer disease, vascular dementia, heart failure, atrial fibrillation, chronic kidney disease, asthma, and multiple sclerosis, based on previously reported associations.10,13 Details about participant selection, genetic scores, allocation to the 2 × 2 factorial groups, and construction of the clinical outcome variables can be found in the eMethods and eTable 2 in the Supplement.
Analysis
Summary-level 2-sample MR was used to validate the known associations of CETP and PCSK9 scores with lipids and CAD, by employing 3 complementary methods to determine the influence of potential horizontal pleiotropy bias (specifically the inverse variance-weighted, Egger, and MR-PRESSO methods). Subsequently, the CETP or PCSK9 genetic scores were used as exposures in linear regression models and quasibinomial logistic regression models in individual participant data from the UKB,22 depending on the type of outcome. The genetic scores were evaluated as continuous exposures and as median-dichotomized exposures.
For factorial MR, the continuous scores were used in interaction regression models, and the median-dichotomized scores were used to generate 4 groups (resulting in groups with high CETP and high PCSK9 concentrations, low CETP and high PCSK9 concentrations, high CETP and low PCSK9 concentrations, and low CETP and low PCSK9 concentrations) (eFigure 1 in the Supplement). Nonadditivity of the associations of combined lower CETP and PCSK9 with any outcome was assessed using interaction P values for the product term of the continuous PCS9 and CETP scores and by comparing the magnitudes of the associations found using the factorial design. To further investigate if the effect of the PCSK9 and CETP genetic scores are mediated through LDL-C or HDL-C (for CETP), we conducted a formal mediation analysis, conditioning the genetic score for plasma protein concentration on a genetic score for LDL-C (for PCSK9) and LDL-C or HDL-C (for CETP) concentration (using the same genetic variants associated with protein concentration but now weighted by their downstream effect on LDL-C and HDL-C from an external GWAS18).
Missing data are reported in eTable 6 in the Supplement; participants with missing data for a certain trait were omitted. We report associations with a P value of 0.05/12 = .0042 as the threshold for multiple testing significance. Age and sex were included as covariates in all models to increase precision. Analyses were performed using R version 4.0.3.23 Summary data were extracted and MR-PRESSO analyses were performed using the TwoSampleMR package. The plots were made using the ggplot2 and meta packages.24,25
Results
Data for 425 354 UKB participants were included; the median (IQR) age was 59 years (51-64), and 229 399 (53.9%) were female. Significant genetic associations with CETP protein concentrations were available for 15 uncorrelated variants, which jointly had an F statistic of 1454 (eTable 3 in the Supplement) and explained 32.4% of the variance. Selecting variants associated with PCSK9 concentrations resulted in 4 variants with a summed F statistic of 202 (eTable 4 in the Supplement), which together explained 6.0% of the variation in PCSK9 concentrations.
The 2 genetic scores for protein concentrations were first validated using external, non-UKB data. Lower plasma concentrations of CETP were associated with decreased concentrations of LDL-C, triglycerides, and apo B and increased HDL-C and apo A1 concentrations (eTable 5 in the Supplement). The associations were found to be robust for any potential pleiotropy using the MR-Egger and MR-PRESSO methods. Lower plasma concentrations of PCSK9 were associated with reduced total cholesterol, LDL-C, apo B, and apo A1 concentrations (eTable 5 in the Supplement); these associations were also robust to pleiotropy in analyses using MR-Egger or MR-PRESSO. Lower concentrations of both CETP and PCSK9 were associated with lower risk of CAD (PCSK9: odds ratio [OR], 0.55; 95% CI, 0.45-0.68; CETP: OR, 0.93; 95% CI, 0.89-0.98), without evidence for pleiotropy.
After validating our genetic scores for CETP and PCSK9, we next leveraged data from 425 534 UKB participants (eTable 6 in the Supplement) to conduct MR and factorial MR analyses. A total of 35 002 participants experienced a first CAD event, and the population contained 21 160 cases of type 2 diabetes, 3674 AMD cases, 5764 ischemic stroke cases, 11 862 cases of any type of stroke, 1052 cases of Alzheimer disease, 582 cases of vascular dementia, 8287 heart failure cases, 20 241 atrial fibrillation cases, 8253 participants with chronic kidney disease, 31 110 participants with asthma, and 1524 participants with multiple sclerosis.
For each genetic score, we divided the entire cohort into 2 groups based on the median genetically predicted protein concentrations by the respective genetic score and measured the differences between the 2 groups (ie, a group with lower genetically predicted protein concentrations and a group with higher genetically predicted protein concentrations). The UKB analyses showed the same lipid-effects profile as found in the summary-data analyses for both CETP and PCSK9 (Figure 1 and eTables 7 and 8 in the Supplement).
Figure 1. Associations of Lower CETP or PCSK9 With Lipids.
Difference in plasma lipid and lipoprotein concentration for the group with lower protein concentrations vs the group with higher protein concentrations, after grouping participants by genetically predicted CETP or PCSK9 concentrations below or above the median. Apo A1 indicates apolipoprotein A1; apo B, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides.
Corrected for multiple testing, genetically predicted lower CETP concentrations were associated with lower odds of CAD (OR, 0.96; 95% CI, 0.94-0.99) and higher odds of AMD (OR, 1.11; 95% CI, 1.04-1.19) (Figure 2). Lower genetically predicted PCSK9 concentrations were also associated with lower odds of CAD (OR, 0.94; 95% CI, 0.92-0.96) (Figure 2). Analyses using the continuous genetic scores showed similar estimates as the dichotomized scores (eTables 9 and 10 in the Supplement). The associations of lower CETP and lower PCSK9 concentrations with CAD are similar when scaled to a 10-mg/dL reduction in LDL-C (for CETP: OR, 0.74; 95% CI, 0.67-0.81; for PCSK9: OR, 0.75; 95% CI, 0.71-0.79; to convert LDL-C to millimoles per liter, multiply by 0.0259). The mediation analysis indicated that the PCSK9 effect on CAD was attributable to the effect on LDL-C (OR of lower LDL-C conditional on PCSK9: 0.57, 95% CI, 0.38-0.86), with inconclusive results for LDL-C or HDL-C mediation by CETP (OR of lower LDL-C conditional on CETP: 1.13, 95% CI, 0.73-1.74; OR of higher HDL-C conditional on CETP: 0.79; 95% CI, 0.62-1.02) (eTable 11 in the Supplement).
Figure 2. Associations of Lower CETP or PCSK9 With Clinical Outcomes.
Odds ratio (OR) for clinical outcomes for the group with lower CETP or PCSK9 concentrations vs the group with higher CETP or PCSK9 concentrations. AMD indicates age-related macular degeneration.
To evaluate the effect of joint inhibition of PCSK9 and CETP, we performed interaction tests and divided UKB participants into 4 groups, based on median genetically predicted CETP and PCSK9 concentrations (Table). We found no evidence for nonadditivity in the association with lipoproteins or lipids, most notably for LDL-C (CETP: effect size, −1.11 mg/dL; 95% CI, −1.40 to −0.82 mg/dL; PCSK9: effect size, −2.13 mg/dL; 95% CI, −2.43 to −1.84 mg/dL; combined: effect size, −3.47 mg/dL; 95% CI, −3.76 to −3.18 mg/dL; P = .34 for interaction). In addition, we found no evidence for nonadditivity in the association with the odds for any clinical outcome (eTables 12 and 13 in the Supplement, Figure 3, and Figure 4).
Table. Characteristics for Participants of the 2 × 2 Factorial Scheme Including Demographic Data at Baseline Visit and Cases at End of Follow-up.
| Referencea | Lower CETP | Lower PCSK9 | Lower CETP and PCSK9 | |
|---|---|---|---|---|
| No. of participants | 109 902 | 108 636 | 103 572 | 103 244 |
| Age, median (IQR), y | 59 (51-64) | 59 (51-64) | 59 (51-64) | 59 (51-64) |
| Male, No. (%) | 50 584 (46.0) | 50 183 (46.2) | 47 630 (46.0) | 47 558 (46.1) |
| Female, No. (%) | 59 318 (54.0) | 58 453 (53.8) | 55 942 (54.0) | 55 686 (53.9) |
| Body mass index, median (IQR)b | 26.73 (24.13-29.86) | 26.68 (24.10-29.82) | 26.75 (24.15-29.89) | 26.72 (24.12-29.88) |
| Blood pressure, median (IQR), mm Hg | ||||
| Systolic | 138 (126-152) | 138 (126-152) | 138 (126-152) | 138 (126-152) |
| Diastolic | 82 (75-89) | 82 (75-89) | 82 (75-89) | 82 (75-89) |
| Alcohol use, No. (%) | ||||
| Prefer not to answer | 69 (0.1) | 80 (0.1) | 80 (0.1) | 74 (0.1) |
| Never | 7388 (6.7) | 7316 (6.7) | 6922 (6.7) | 6823 (6.6) |
| Special occasions only | 11 836 (10.8) | 11 622 (10.7) | 11 261 (10.9) | 11 092 (10.7) |
| 1-3 Times a month | 12 261 (11.2) | 12 050 (11.1) | 11 343 (11.0) | 11 499 (11.1) |
| Once or twice a week | 28 677 (26.1) | 28 325 (26.1) | 27 395 (26.5) | 27 048 (26.2) |
| 3 or 4 Times a week | 26 397 (24.0) | 26 004 (23.9) | 24 672 (23.8) | 24 650 (23.9) |
| Daily or almost daily | 23 274 (21.2) | 23 239 (21.4) | 21 899 (21.1) | 22 058 (21.4) |
| Smoking status, No. (%) | ||||
| Prefer not to answer | 390 (0.4) | 377 (0.3) | 368 (0.4) | 331 (0.3) |
| Never | 58 946 (53.6) | 58 214 (53.6) | 55 670 (53.8) | 55 739 (54.0) |
| Former | 39 122 (35.6) | 38 641 (35.6) | 36 786 (35.5) | 36 410 (35.3) |
| Current | 11 444 (10.4) | 11 404 (10.5) | 10 748 (10.4) | 10 764 (10.4) |
| Medication use at baseline, No. (%) | ||||
| Lipid-lowering therapy | 20 067 (18.3) | 19 061 (17.5) | 17 652 (17.0) | 16 933 (16.4) |
| Blood pressure–lowering therapy | 23 036 (21.0) | 22 280 (20.5) | 21 425 (20.7) | 21 051 (20.4) |
| Insulin therapy | 1166 (1.1) | 1130 (1.0) | 1098 (1.1) | 1046 (1.0) |
| Aspirin | 15 787 (14.4) | 15 183 (14.0) | 14 524 (14.0) | 14 023 (13.6) |
| Laboratory parameters, median (IQR), mg/dL | ||||
| Total cholesterol | 220 (190-250) | 222 (193-252) | 217 (188-246) | 219 (191-249) |
| LDL-C | 138 (116-162) | 137 (115-160) | 136 (114-159) | 135 (113-157) |
| HDL-C | 53 (44-63) | 56 (47-67) | 52 (44-62) | 56 (47-67) |
| Triglycerides | 133 (94-193) | 130 (92-189) | 134 (94-193) | 131 (92-189) |
| Apolipoprotein B | 103 (88-120) | 102 (87-119) | 102 (87-118) | 100 (85-117) |
| Apolipoprotein A1 | 149 (133-167) | 154 (137-173) | 149 (133-167) | 154 (138-173) |
| Genetic scores, median (IQR) | ||||
| PCSK9 | 0.06 (0.05-0.11) | 0.06 (0.05-0.11) | 0.19 (0.16-0.24) | 0.19 (0.16-0.24) |
| CETP | 2.63 (2.30-2.84) | 3.43 (3.23-3.71) | 2.63 (2.30-2.84) | 3.43 (3.23-3.71) |
| Total cases at end of follow-up, No. (%) | ||||
| Coronary artery disease | 9442 (8.6) | 9039 (8.3) | 8419 (8.1) | 8102 (7.8) |
| Type 2 diabetes | 5584 (5.1) | 5305 (4.9) | 5188 (5.0) | 5083 (4.9) |
| Age-related macular degeneration | 900 (0.8) | 1004 (0.9) | 853 (0.8) | 917 (0.9) |
| Ischemic stroke | 1484 (1.4) | 1489 (1.4) | 1418 (1.4) | 1373 (1.3) |
| Any stroke | 3098 (2.8) | 3093 (2.8) | 2914 (2.8) | 2757 (2.7) |
| Alzheimer disease | 278 (0.3) | 274 (0.3) | 244 (0.2) | 256 (0.2) |
| Vascular dementia | 153 (0.1) | 143 (0.1) | 145 (0.1) | 141 (0.1) |
| Heart failure | 2233 (2.0) | 2126 (2.0) | 1929 (1.9) | 1999 (1.9) |
| Atrial fibrillation | 5403 (4.9) | 5167 (4.8) | 4892 (4.7) | 4779 (4.6) |
| Chronic kidney disease | 2199 (2.0) | 2126 (2.0) | 1946 (1.9) | 1982 (1.9) |
| Asthma | 8055 (7.3) | 7860 (7.2) | 7687 (7.4) | 7508 (7.3) |
| Multiple sclerosis | 415 (0.4) | 408 (0.4) | 346 (0.3) | 355 (0.3) |
Abbreviations: CETP, cholesteryl ester transfer protein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9.
SI conversion factors: To convert mg/dL to mmol/L for total cholesterol, LDL-C, and HDL-C, multiply by 0.0259; to convert mg/dL to mmol/L for triglycerides, multiply by 0.0113.
The reference group was higher levels of CETP and PCSK9.
Calculated as weight in kilograms divided by height in meters squared.
Figure 3. Associations of Combined Lower CETP and PCSK9 With Lipids.
Difference in lipids and lipoproteins for the groups with lower CETP, lower PCSK9, or lower CETP and PCSK9 concentrations, vs the group with higher CETP and PCSK9 concentrations (reference). P values are provided for interaction tests evaluating the potential for association with a nonadditive effect of CETP and PCSK9. HDL-C indicates high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.
Figure 4. Associations of Combined Lower CETP and PCSK9 With Clinical Outcome.
Differences in clinical outcome for the groups with lower CETP, lower PCSK9, or lower CETP and PCSK9 concentrations vs the group with higher CETP and PCSK9 concentrations (reference). P values are provided for interaction tests evaluating the potential for association with a nonadditive effect of CETP and PCSK9. OR indicates odds ratio.
Most importantly, we observed an additive pattern for CAD compared with the group with higher CETP and PCSK9 concentrations, with an OR of 0.96 (95% CI, 0.94-1.00); an OR of 0.94 (95% CI, 0.91-0.97) for the group with lower CETP concentrations or PCSK9 concentrations; and an OR of 0.90 (95% CI, 0.87-0.93) for the group with both lower CETP and PCSK9 concentrations (P = .83 for interaction) (Figure 4). Similar additive effects were observed for all other clinical outcomes (Figure 4 and eFigures 2 and 3 in the Supplement). For example, lower CETP concentration was associated with increased risk for AMD, and this association was not modified by lower PCSK9 compared with higher CETP and PCSK9 concentrations (lower CETP: OR, 1.14; 95% CI, 1.04-1.24, lower PCSK9: OR, 1.00; 95% CI, 0.92-1.10, lower CETP and lower PCSK9: OR, 1.09; 95% CI, 0.99-1.19; P = .96 for interaction).
Discussion
In this study, we used MR and factorial MR to anticipate the effects of independent and combined inhibition of CETP and PCSK9. We found that genetically lower CETP and PCSK9 concentrations were associated with lower LDL-C and apo B concentrations and lower odds for CAD. Moreover, when corrected for multiple testing, genetically lower CETP concentrations were associated with increased odds for AMD. Analyses with continuous scores showed a similar pattern. We observed associations with an additive magnitude for combined genetically lower CETP and PCSK9 concentrations on lipids and lipoprotein concentrations and clinical outcomes, underscored by the fact that we did not find any statistical evidence for an interaction between the continuous CETP and PCSK9 scores. To our knowledge, our data provide the first suggestion that the combination of CETP inhibition with PCSK9 inhibition will result in an independent and additive effect on the reduction of atherogenic lipids and a proportional clinically relevant reduction of CAD risk. This is of importance for 2 phase 3 clinical trials currently underway and provides an important baseline against which the effects can be compared of joint prescription of drug compounds inhibiting CETP and PCSK9.
Our data support the findings from multiple previous MR studies, which consistently showed that variants in and around the CETP locus are associated with both a beneficial lipid profile and reduced risk of CVD.10,17,26 Our observation that there are no significant interactions between lowering PCSK9 and CETP concentrations on lipids, lipoproteins, and CAD is different from a previous factorial MR study showing that the combination of genetic variants in the CETP locus with genetic variants in the HMGCR locus (mimicking the effects of on-target statin therapy) resulted in an apo B reduction and cardiovascular event risk reduction that was less than the additive reduction of CETP and HMGCR inhibition alone.26 It was suggested that the failure of evacetrapib was due to an attenuated reduction of apo B concentrations compared with the measured LDL-C reduction, in the setting where nearly all participants were using statins (>96%). CETP has an effect on the composition of atherogenic lipoprotein particles, by transferring cholesteryl esters between HDL-C– and apo B–containing lipoproteins, that substantially differs from the effect of HMGCR or PCSK9 inhibition, which likely complicates the measurement of LDL-C concentrations and precludes the use of LDL-C as solid predictor of CVD risk lowering.27,28 This is exemplified by the results from a recent clinical trial with anacetrapib, where a clinically meaningful difference in LDL-C lowering was observed between a direct LDL-C assay (−41%) and the reference method β-quantification (−17%).29 The 9% relative risk reduction for CVD events observed in patients randomized to receive anacetrapib was not in line with the 41% reduction in LDL-C concentrations, but in light of the apo B and/or non–HDL-C reductions found in this trial, it was comparable with the magnitude of effect seen in statin-therapy trials with similar non–HDL-C reductions.
Scaling the CAD effects to a 10-mg/dL reduction in LDL-C concentrations in this study resulted in comparable estimates for CETP (OR, 0.74; 95% CI, 0.67; 0.81) and PCSK9 (OR, 0.75; 95% CI, 0.71; 0.79), which is consistent with previous MR studies and clinical trials suggesting that the CAD benefit is due to the reduction of LDL-C.30 To explore this more formally, we conducted a mediation analysis, which confirmed that the PCSK9-CAD association was LDL-C mediated but did not show conclusive evidence for a similar LDL-C mediation or HDL-C mediation for the CETP-CAD association. A more detailed mediation analysis conducted by Schmidt et al10 explored effects on additional outcomes.
We observed a significant association between genetically predicted lower CETP levels and higher risk for AMD. However, this effect has not been observed in clinical trials thus far, and hence it requires careful consideration to what extent drug-target protein effects might anticipate the effects of drug compounds targeting the same protein. While there is substantial prior evidence on the association of CETP variants and risk of AMD both from GWAS and MR studies,10,13 this does not necessarily imply that a therapeutic agent targeting CETP will result in increased risk of incident AMD. To the best of our knowledge, it is unknown whether CETP inhibitors given orally gain access to the eye, either directly or indirectly through effects on smaller HDL-C particles known to associate with AMD risk.31 It is therefore essential to acknowledge that the absence of an AMD signal in the trial data (up to 49 months of follow-up)29,32 suggests that either there is no AMD effect of CETP inhibitors or this effect is considerably smaller than the anticipated effects on the CVD end points these trials were powered on.
The observed CETP association with AMD is an important example of how genetic drug target validation can inform clinical trials by highlighting outcomes that warrant special consideration. At the same time, this example emphasizes that associations seen in drug target MR may not be reproduced in trials of compounds targeting the protein of interest because some effects may be contingent on access to particular tissues. Drug target MRs are advantageous in that they can identify potential on-target associations of drug target perturbation, across tissues, and over potentially longer follow-up times than would be available in clinical trials of a drug compound. However, they cannot take the place of drug trials, which provide the essential information on drug dosage and necessary exposure/induction times and assess both on-target as well as off-target compound effects.
Genetic studies and clinical trials have shown that multiple lipid-lowering targets are associated with increased risk for type 2 diabetes.33,34 We did not observe an association between lower PCSK9 concentrations and type 2 diabetes, which is in contrast with previous MR studies.34,35 However, a recent meta-analysis showed only limited increases in plasma glucose and hemoglobin A1c concentrations in patients treated with PCSK9 monoclonal antibodies and failed to show an increased risk for type 2 diabetes (risk ratio, 1.04; 95% CI, 0.96-1.13).36 Secondary analyses of various CETP-inhibitor trials and other MR studies showed beneficial effects on the risk for new-onset type 2 diabetes and related parameters.10,29,37,38 We observed a suggestive association between lower CETP concentrations and type 2 diabetes in concordant direction with the clinical trials, but this association did not reach significance for multiple testing.
This study has a number of strengths in comparison with previous studies: The fact that PCSK9 and CETP are circulatory proteins enabled us to use protein concentrations as exposure variables, which more directly reflects the on-target associations with protein inhibition, compared with previous MR studies often using genetic effect on lipid subfractions as a downstream proxy of protein concentration and activity.26,39,40 Moreover, we only used a 100-kb window on both sides of the gene, as it is estimated that 92% of all lead genetic variants associated with gene expression reside within a 100-kb region around the locus.41 The moderately sized flanking region and selection of scores on protein concentration association decrease the risk of inadvertently inducing horizontal pleiotropy by mixing signals of neighboring genes.42
Limitations
A number of limitations warrant further discussion. First, while we did not observe an interaction between CETP and PCSK9, this does not provide evidence of absence.43 Furthermore, drug compounds may affect off-target pathways, which could result in effects that deviate from the evidence presented here. Additionally, the provided genetic evidence constitutes a small but likely persistent inhibiting association with protein concentration at concentrations where nonadditivity might not be expected, unlike the larger effects seen by pharmaceutically inhibiting a target. Third, to ensure optimal statistical power for interaction analyses, we included participants from White British, White Irish, and other White ancestry in our analyses. The use of a primarily European cohort implies that caution must be taken in translating these results to other populations, and future studies could be conducted in novel biobanks incorporating multiple ancestries, such as the All of Us biobank.44 Fourth, although the provided results were based on the large sample size data from the UKB and replicated previously known associations with CETP and PCSK9, it is likely that future studies will be able to expand on these results by sourcing additional cases, large protein quantitative trait loci data, or whole-genome sequencing data.
Conclusions
Our results suggest that joint inhibition of CETP and PCSK9 has an additive effect on lipid traits and disease risk, including a lower risk of CAD. Further research may explore whether a combination of CETP- and PCSK9-related therapeutics can benefit high-risk patients who are unable to reach treatment targets with existing options.
eMethods
eReferences
eTable 1. Overview of included studies with summary data
eTable 2. Definitions of the clinical outcome parameters
eTable 3. Genetic variants included in the CETP genetic score from the NEO study and their effect on CETP levels
eTable 4. Genetic variants included in the PCSK9 genetic score from the LIFE-HEART study, and their effect on PCSK9 levels
eTable 5. Effect estimates from 2-sample MR analyses using protein-weighted genetic instruments on lipids and CAD
eTable 6. Baseline table and percentage missing values
eTable 7. Associations of dichotomized CETP score with lipids and clinical outcome
eTable 8. Associations of dichotomized PCSK9 score with lipids and clinical outcome
eTable 9. Associations of continuous CETP score with lipids and clinical outcome
eTable 10. Associations of continuous PCSK9 score with lipids and clinical outcome
eTable 11. Mediation analyses
eTable 12. Interaction effect estimates of the combined CETP and PCSK9 genetics scores
eTable 13. 2x2 factorial estimates, all estimates compared to the reference group of higher CETP and PCSK9 levels
eFigure 1. Flowchart of the study
eFigure 2. Associations of lower CETP or PCSK9 with clinical outcome
eFigure 3. Associations of combined lower CETP and PCSK9 with other clinical outcome
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
eMethods
eReferences
eTable 1. Overview of included studies with summary data
eTable 2. Definitions of the clinical outcome parameters
eTable 3. Genetic variants included in the CETP genetic score from the NEO study and their effect on CETP levels
eTable 4. Genetic variants included in the PCSK9 genetic score from the LIFE-HEART study, and their effect on PCSK9 levels
eTable 5. Effect estimates from 2-sample MR analyses using protein-weighted genetic instruments on lipids and CAD
eTable 6. Baseline table and percentage missing values
eTable 7. Associations of dichotomized CETP score with lipids and clinical outcome
eTable 8. Associations of dichotomized PCSK9 score with lipids and clinical outcome
eTable 9. Associations of continuous CETP score with lipids and clinical outcome
eTable 10. Associations of continuous PCSK9 score with lipids and clinical outcome
eTable 11. Mediation analyses
eTable 12. Interaction effect estimates of the combined CETP and PCSK9 genetics scores
eTable 13. 2x2 factorial estimates, all estimates compared to the reference group of higher CETP and PCSK9 levels
eFigure 1. Flowchart of the study
eFigure 2. Associations of lower CETP or PCSK9 with clinical outcome
eFigure 3. Associations of combined lower CETP and PCSK9 with other clinical outcome
