It is not known whether genetic variants in the cholesteryl ester transfer protein (CETP) gene are associated with recurrent coronary heart disease events or mortality in secondary prevention patients. Among 3,717 patients with acute coronary syndrome or coronary artery bypass grafting (CABG) enrolled in a prospective genetic registry, we evaluated whether CETP gene variants previously shown to be associated with reduced CETP activity and high-density lipoprotein cholesterol increase (“A” allele for both Taq IB [rs708272] and rs12149545) are associated with a reduction in recurrent myocardial infarction (MI), recurrent revascularization, or death. At 4.5 years of follow-up, 439 recurrent MI, 698 recurrent revascularizations, and 756 deaths occurred. Using an additive model of inheritance, the “A” allele for rs708272 was not associated with recurrent MI (hazard ratio [HR] 0.95, 95% confidence interval [CI] 0.78 to 1.17 for AG; HR 0.89, 95% CI 0.67 to 1.19 for AA; compared with GG genotype), recurrent revascularization (HR 1.13, 95% CI 0.95 to 1.33 for AG; HR 1.05, 95% CI 0.84 to 1.32 for AA), or mortality (HR 1.02, 95% CI 0.86 to 1.19 for AG; HR 1.11, 95% CI 0.91 to 1.37 for AA) in the overall cohort. Similar results were seen for the “A” allele for rs12149545. In the CABG subgroup, AG genotype for rs708272 was associated with an increased mortality (HR 1.38, 95% CI 1.06 to 1.79) compared with GG genotype. Results remained consistent using dominant model of inheritance. In conclusion, genetic CETP variants were not associated with recurrent MI or recurrent revascularization in overall cohort with a possible mortality increase in patients who underwent CABG.
Cholesteryl ester transfer protein (CETP) inhibition has been suggested to be an important therapeutic target with several drugs in development. Most of these trials have targeted patients with established atherosclerosis. Two of these CETP inhibitors (torcetrapib and dalcetrapib) failed to show an improvement in cardiovascular outcomes with an increase in adverse outcomes with torcetrapib. Several reasons have been postulated to explain these findings, for example, increase in blood pressure and possibly, differences in the high-density lipoprotein cholesterol (HDL-C)–raising or low-density lipoprotein cholesterol–lowering efficacy of these drugs. Some have also questioned whether CETP inhibition itself is a viable therapeutic option. Mendelian randomization studies have been shown to explain whether an increased risk of coronary heart disease (CHD) events seen with a risk marker is indeed causal. Because the inheritance of a genetic trait is independent of environmental and behavioral factors, the associations seen in these studies are more robust compared with classic epidemiological studies in which confounding and reverse causation remain a concern. Genotypic polymorphisms leading to an alteration in the level of a risk marker and cardiovascular outcomes are more likely to indicate a causal association. Therefore, using principles of Mendelian randomization, we evaluated whether single nucleotide polymorphisms (SNPs) shown to be associated with lower levels of CETP and increased HDL-C levels are associated with a decreased incidence of recurrent myocardial infarction (MI), recurrent revascularization, or mortality in patients with established atherosclerosis (those presenting with acute coronary syndrome [ACS] or those undergoing coronary artery bypass grafting [CABG]).
Methods
TexGen is a prospective, collaborative genetic registry enrolling patients with atherosclerotic CHD who seek care at multiple institutions within the Texas Medical Center. For the present analyses, we restricted our patient population to those patients presenting with ACS or those undergoing CABG (with or without concomitant valve surgery) enrolled in the TexGen registry from September 2001 through September 2008. Preoperative, intraoperative, and postoperative characteristics of these patients were obtained through a clinical research database maintained at the St. Luke’s Episcopal Hospital. The variables used for our analyses included age, gender, history of hypertension, diabetes mellitus, smoking, presence of renal insufficiency, New York Heart Association functional class, and the use of aspirin, β blockers, or statins.
We evaluated 1 SNP in the TaqIB polymorphism from intron 1 (rs708272) and 1 SNP in the promoter region G-2708A (rs12149545) of the CETP gene. These SNPs in the CETP gene have been known to be associated with HDL-C levels in previous studies. Genotyping (details provided as Supplemental text ) was performed in a multiplex reaction using the MassARRAY system and iPLEX Gold Complete Genotyping Reagent Set (Sequenom Inc., San Diego, California). Both SNPs had a missing rate of 2.7%. Quality control concordance for 37 replicated samples that were randomly distributed multiple times throughout the plate set was 100%.
Prospective follow-up of outcomes in the TexGen registry includes annual follow-up telephone calls by the research nurses and annual surveys mailed to each patient. In addition, any hospitalization for patients enrolled in the database is also verified using hospitalization records. Our primary outcome of interest was the incidence of recurrent MI in the entire cohort (those presenting with ACS or those undergoing CABG). Our secondary outcomes included the need for recurrent revascularization procedures (recurrent percutaneous coronary intervention, recurrent CABG after the index ACS event, or CABG surgery) and all-cause mortality. We included all-cause death as one of the secondary outcomes because some of the patients experiencing MI may suffer a sudden arrhythmic death and could possibly be misclassified if only recurrent MI was used as the outcome measure.
Recurrent MI was defined as new ST-segment elevation in 2 contiguous leads in the setting of ischemic symptoms or new horizontal or down-sloping ST depression of ≥0.05 mV in 2 contiguous leads; and/or T-wave inversions of ≥0.1 mV in 2 contiguous and serum troponin I greater than ninety-ninth percentile of the upper normal limit. In-hospital MI after CABG was defined as the increased levels of creatine kinase-MB isoenzyme with appearance of new Q waves on electrocardiogram, a new regional wall motion abnormality (other than paradoxical septal motion), or the presence of MI at autopsy. Recurrent revascularization was defined as the receipt of another PCI or CABG after the index ACS event or CABG surgery. Vital status was ascertained on patients using data from the Texas State Bureau of Vital Statistics.
Each of the 2 SNPs were studied separately. Our hypothesis was that the presence of genetic polymorphisms associated with reduced CETP activity (and higher HDL-C levels) will be associated with a lower risk of recurrent CHD events and mortality. We initially performed a log-rank test to evaluate the univariate association between each of the 2 SNPs (rs708272 and rs12149545) and the risk of recurrent MI. Cox proportional hazards regression models (adjusted for age, gender, hypertension, diabetes mellitus, current smoking, renal insufficiency, New York Heart Association functional class, and aspirin, β-blocker, and statin use) were subsequently performed to determine whether “A” allele for each of the 2 SNPs (known to be associated with a reduced CETP activity and therefore, increased HDL-C levels) was independently associated with a reduction in the incidence of recurrent MI. Cox regression analyses were also performed to study whether the “A” for each of the SNPs was also associated with a reduction in the recurrent revascularization or mortality. We initially used an additive model of inheritance as the risk ratios in previous studies followed an additive genetic pattern, although results are also presented using a dominant genetic model as Supplemental material . Analyses were also performed separately for TexGen patients presenting with ACS or those who underwent CABG. Finally, although SNPs in the CETP gene have shown to be associated with HDL-C levels in ethnicities other than whites (e.g., blacks or Asians ), we also performed analyses in those TexGen participants who self-identified their race as white.
Statistical analyses were performed using SAS statistical software, version 9.1 (SAS Institute, Inc., Cary, North Carolina). All analyses were performed using 2-tailed tests for significance. Because we evaluated SNPs affecting a CETP locus with future risk of MI, revascularization, or death on an a priori basis and did not perform a genome-wide association, adjustments were not made for multiple testing. The study protocol was approved by the Institutional Review Board at Baylor College of Medicine. The investigators are solely responsible for the design and conduct of the study, all analyses, the drafting and editing of the report, and its final contents.
Results
We included 3,717 patients who either presented with ACS or underwent CABG. Of this cohort, 2,926 patients presented with ACS and 1,569 patients underwent CABG during the index hospitalization. Table 1 describes the baseline characteristics of the study cohort. Mean age was 62.5 years with a predominance of men (73%). Diabetes and hypertension were prevalent in 34.3% and 78.7% of patients, respectively. Sixteen percentage of the patients had a history of renal insufficiency and 50.6% had New York Heart Association functional class III/IV symptoms.
| Characteristic | n (%) |
|---|---|
| Age, mean ± SD (yrs) | 62.51 ± 10.92 |
| Men | 2,712 (73) |
| Family history of premature coronary artery disease | 1,248 (33.6) |
| Diabetes mellitus | 1,274 (34.3) |
| Hypertension (by history) | 2,925 (78.7) |
| Transient ischemic attacks or stroke | 333 (9) |
| Renal insufficiency | 599 (16.1) |
| Smoker | 812 (21.9) |
| New York Heart Association functional class III/IV symptoms | 1,882 (50.6) |
| β-Blocker use | 2,779 (74.8) |
| Statin use | 2,708 (73) |
| Aspirin use | 3,144 (84.6) |
| History of ACS | 2,926 ∗ (78.7) |
| History of CABG | 1,569 ∗ (42.2) |
∗ Numbers add up to >3,717 as some patients with ACS underwent CABG.
In the overall cohort, the frequencies of GG, AG, and AA genotypes for rs708272 were 33%, 50%, and 17%, respectively. For rs12149545, the prevalences of GG, AG, and AA genotypes were 52%, 41%, and 7%, respectively. Overall, 67% of the patients carried at least 1 copy of the “A” for rs708272 and 48% of the patients carried at least 1 copy of the “A” allele for rs12149545. The association between rs708272 and the primary and secondary outcomes for the entire cohort at a mean follow-up of 4.5 years from the index ACS event or CABG surgery is listed in Table 2 . A total of 1,893 patients (51%) suffered an adverse CHD event (439 [11.8%] had recurrent MIs, 698 [18.8%] underwent recurrent revascularization, and 756 [20.3%] died). Using an additive model of inheritance, the presence of AG or AA genotype for rs708272 was not associated with a significantly lower incidence of recurrent MI compared with GG genotype in adjusted models. In addition, AG and AA genotypes were not associated with a lower incidence of recurrent revascularization or mortality compared with the GG genotype. Similar nonsignificant results were seen for the AG or AA genotypes (compared with GG genotype) for rs12149545 for either primary or secondary outcomes.
| rs708272 ( Taq IB) ∗ | |||
|---|---|---|---|
| Genotype (n) | Recurrent MI (%) | Unadjusted HR (95% CI) | HR (95% CI) † |
| GG (1,259) | 153 (12.2) | Referent | Referent |
| AG (1,840) | 218 (11.9) | 0.95 (0.77–1.16) | 0.95 (0.78–1.17) |
| AA (618) | 68 (11) | 0.87 (0.66–1.16) | 0.89 (0.67–1.19) |
| Recurrent revascularization (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,259) | 219 (17.4) | Referent | Referent |
| AG (1,840) | 363 (19.8) | 1.13 (0.96–1.34) | 1.13 (0.95–1.33) |
| AA (618) | 116 (18.8) | 1.05 (0.84–1.31) | 1.05 (0.84–1.32) |
| Death (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,259) | 247 (19.6) | Referent | Referent |
| AG (1,840) | 367 (20) | 0.99 (0.84–1.16) | 1.02 (0.86–1.19) |
| AA (618) | 142 (23) | 1.12 (0.91–1.37) | 1.11 (0.91–1.37) |
| rs12149545 ∗ | |||
|---|---|---|---|
| Genotype (n) | Recurrent MI (%) | Unadjusted HR (95% CI) | HR (95% CI) † |
| GG (1,921) | 240 (12.5) | Referent | Referent |
| AG (1,514) | 171 (11.3) | 0.87 (0.72–1.06) | 0.88 (0.72–1.07) |
| AA (282) | 28 (9.9) | 0.77 (0.52–1.14) | 0.81 (0.55–1.20) |
| Recurrent revascularization (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,921) | 341 (17.8) | Referent | Referent |
| AG (1,514) | 301 (19.9) | 1.11 (0.95–1.29) | 1.10 (0.94–1.29) |
| AA (282) | 56 (19.9) | 1.07 (0.81–1.42) | 1.10 (0.83–1.46) |
| Death (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,921) | 380 (19.8) | Referent | Referent |
| AG (1,514) | 310 (20.5) | 1.02 (0.87–1.18) | 1.02 (0.88–1.18) |
| AA (282) | 66 (23.4) | 1.14 (0.88–1.48) | 1.13 (0.87–1.47) |
∗ “A” allele shown to be associated with reduced CETP activity and increased levels of HDL-C.
† Adjusted for age, gender, hypertension, diabetes, current smoking, renal insufficiency, presence of New York Heart Association functional class III/IV symptoms, aspirin use, β-blocker use, and statin use.
We also evaluated the association between rs708272 or rs12149545 and the primary or secondary outcomes in the subgroup of patients admitted with ACS or those undergoing CABG. AG or AA genotypes for rs708272 or rs12149545 were not associated with recurrent MI or the need for recurrent revascularization in patients who presented with ACS ( Table 3 ) or those who underwent CABG ( Table 4 ) in fully adjusted models. Contrary to our hypothesis, AG phenotype for rs708272 was associated with an increased risk of mortality (hazard ratio 1.38, 95% confidence interval [CI] 1.06 to 1.79) compared with GG genotype in the CABG subgroup ( Table 4 ). No significant association between CETP genotypes and increased mortality was seen for patients in the ACS subgroup.
| rs708272( Taq IB) ∗ | |||
|---|---|---|---|
| Genotype (n) | Recurrent MI (%) | Unadjusted HR (95% CI) | HR (95% CI) † |
| GG (1,011) | 133 (13.2) | Referent | Referent |
| AG (1,446) | 191 (13.2) | 0.96 (0.77–1.23) | 0.98 (0.79–1.22) |
| AA (469) | 59 (12.6) | 0.91 (0.67–1.23) | 0.92 (0.68–1.26) |
| Recurrent revascularization (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,011) | 199 (19.7) | Referent | Referent |
| AG (1,446) | 328 (22.7) | 1.14 (0.96–1.36) | 1.12 (0.94–1.34) |
| AA (469) | 103 (22) | 1.06 (0.84–1.35) | 1.06 (0.83–1.34) |
| Death (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,011) | 201 (19.9) | Referent | Referent |
| AG (1,446) | 269 (18.6) | 0.90 (0.74–1.07) | 0.96 (0.80–1.15) |
| AA (469) | 111 (23.7) | 1.09 (0.86–1.37) | 1.10 (0.87–1.39) |
| rs12149545 ∗ | |||
|---|---|---|---|
| Genotype (n) | Recurrent MI (%) | Unadjusted HR (95% CI) | HR (95% CI) † |
| GG (1,522) | 210 (13.8) | Referent | Referent |
| AG (1,179) | 150 (12.7) | 0.88 (0.72–1.09) | 0.90 (0.73–1.11) |
| AA (225) | 23 (10.2) | 0.71 (0.46–1.09) | 0.73 (0.47–1.12) |
| Recurrent revascularization (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,522) | 309 (20.3) | Referent | Referent |
| AG (1,179) | 271 (23) | 1.11 (0.94–1.31) | 1.10 (0.94–1.29) |
| AA (225) | 50 (22.2) | 1.03 (0.77–1.39) | 1.04 (0.78–1.41) |
| Death (%) | Unadjusted HR (95% CI) | HR (95% CI) † | |
|---|---|---|---|
| GG (1,522) | 296 (19.5) | Referent | Referent |
| AG (1,179) | 234 (19.9) | 0.99 (0.84–1.18) | 1.04 (0.88–1.24) |
| AA (225) | 51 (22.7) | 1.09 (0.81–1.47) | 1.15 (0.85–1.55) |
∗ “A” allele shown to be associated with reduced CETP activity and increased levels of HDL-C.
† Adjusted for age, gender, hypertension, diabetes, current smoking, renal insufficiency, presence of New York Heart Association functional class III/IV symptoms, aspirin use, β-blocker use, and statin use.
| rs708272( Taq IB) ∗ | |||
|---|---|---|---|
| Genotype (n) | Recurrent MI (%) | Unadjusted HR (95% CI) | HR (95% CI) † |
| GG (526) | 52 (9.9) | Referent | Referent |
| AG (770) | 69 (8.9) | 0.91 (0.63–1.30) | 0.90 (0.62–1.28) |
| AA (273) | 20 (7.3) | 0.74 (0.44–1.24) | 0.75 (0.45–1.25) |
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