a Department of Biological Sciences, University of Durham, Durham, UK
Present address: Department of Obstetrics and Gynaecology, University of Glasgow, Royal Infirmary, Glasgow, UK
b Division of Cardiology, University of Leicester, Glenfield Hospital, Leicester, UK
c Robertson Centre for Biostatistics, University of Glasgow, Glasgow, UK
d Department of Human Genetics, Roche Molecular Systems, Inc, Alameda, California, USA
e Department of Pathological Biochemistry, Glasgow Royal Infirmary University NHS Trust, Glasgow, UK
* Correspondence to: Dr D. J. Freeman, University of Glasgow, Obstetrics and Gynaecology, 3rd Floor Queen Elizabeth Building Royal Infirmary, Glasgow G31 2ER, UK. Tel: +44 141 211 4706; Fax: +44 141 552 0873
E-mail address: d.freeman{at}clinmed.gla.ac.uk
Received 14 April 2003; revised 22 July 2003; accepted 31 July 2003
Abstract
Aim The association of cholesteryl ester transfer protein (CETP) gene polymorphisms with risk of a cardiovascular event and whether any association was explained by an influence on high-density lipoprotein (HDL) levels or low-density lipoprotein (LDL) size was tested in the West of Scotland Coronary Prevention Study (WOSCOPS). Gene-smoking and gene-treatment interactions were investigated.
Methods and results Cases (n=498) and controls (n=1108) were typed for TaqIB, C(631)A, C(629)A, I405V and D442G CETP polymorphisms. Homozygotes for the TaqIB2 allele (B2B2) had a 30% reduced risk of a cardiovascular event (odds ratio [OR] 0.70, CI950.510.96, P=0.03) compared to B1B1 homozygotes. Inclusion of HDL or LDL diameter in multivariate analysis only marginally attenuated the relationships. Non-smokers, but not smokers, showed a dose-dependent association of risk with TaqIB genotype. Treatment benefit was not significantly different in B1B1 (OR 0.71, pravastatin vs placebo), B1B2 (OR 0.68) and B2B2 (OR 0.61) individuals. The other CETP polymorphisms studied had no significant association with cardiovascular risk. Haplotype analysis did not add to the information given by the individual polymorphisms.
Conclusion The association between CETP TaqIB genotype and cardiovascular risk is primarily in non-smokers, is not fully explained by effects on HDL levels or LDL size, and the benefit of pravastatin treatment was not influenced by this polymorphism.
Key Words: Atherosclerosis Enzymes Genetics Lipoproteins CETP WOSCOPS
1. Introduction
There is interest in the role of genetic polymorphisms in predicting susceptibility to disease and responsiveness to dietary and drug interventions. Small-scale studies have looked at the effects of polymorphisms on physiological or biochemical factors and have provided useful information on possible mechanistic links between variation at the gene level and risk factors for cardiovascular disease (CVD). Unfortunately these studies often lack power, have insufficient information on confounding lifestyle variables or are from ethnically non-homogenous populations. This has lead to confusion as to which polymorphisms have significant and clinically relevant influences on risk for disease that are independent of environmental variables.
The TaqIB polymorphism of the CETP gene is consistently associated with high-density lipoprotein (HDL)levels and was associated with coronary heart disease risk in the Framingham Study1but not with risk of a myocardial infarction in the Physicians Health Study.2The TaqIB polymorphism3is found in intron 1 and the rarer B2 allele has been found to be associated with raised plasma HDL cholesterol levels and reduced plasma CETP mass.46Lifestyle factors such as cigarette smoking, alcohol consumption and body mass index (BMI) interact with this association.7,8The relationship between cholesteryl ester transfer protein (CETP) genotype and HDL is strongest in non-smokers5,7and in those with high alcohol consumption.8The TaqIB polymorphism has not been shown to have direct effects on CETP levels or function.
Several other polymorphisms in the CETP gene have been identified. Some are rare, such as the D442G mutation close to the active site which leads to reduced plasma CETP mass and specific activity.9The more common I405V polymorphism, which affects CETP primary structure but not plasma CETP mass,9is associated with vascular disease.10Promoter polymorphisms at position 631 and 629 have been identified.11The C(629)A polymorphism affects promoter activity and hence CETP mass and is in linkage disequilibrium with the TaqIB polymorphism.12,13A report of linkage between the CETP gene and low-density lipoprotein (LDL) particle size raises another possibility for the association between polymorphisms in the CETP gene and cardiovascular risk as both low HDL and increased small dense LDL particles are associated with cardiovascular risk.14
Clinical trials testing the efficacy of treatments for CVD provide a rich resource for studying genetic factors. These studies are large, collect detailed information on lifestyle factors and have clinically relevant end-points. In the Regression Growth Evaluation Statin Study (REGRESS) the CETP TaqIB B1 allele was associated with higher CETP concentration, lower HDL cholesterol and greater progression of disease as assessed by a decrease in mean luminal diameter.15This association was abolished by pravastatin treatment; pravastatin appeared to slow progression in B1B1 but not B2B2 patients. Similarly, in the Veterans Affairs HDL Cholesterol Intervention Trial (VA-HIT) the CETP B1B1 genotype was associated with lower HDL, higher cardiovascular risk and greatest triglyceride-lowering response to gemfibrozil.16
The West of Scotland Coronary Prevention Study (WOSCOPS) was a primary prevention study in 4564-year-old men that demonstrated a significant reduction of cardiovascular morbidity and mortality by pravastatin treatment.17,18During the 5-year course of the study, 580 cardiovascular events (fatal and non-fatal) occurred. We used a nested case control study design, matching two controls to every case by age and smoking habit, to assess the ability of the CETP TaqIB genotype, in addition to the C(629)A, C(630)A, I405V and D442G polymorphisms, to predict cardiovascular events and to examine interactions with smoking and pravastatin treatment. We also examined whether any effect of genotype on risk could be explained by its effect on HDL level or LDL diameter.
2. Methods
2.1. Study subjects
Baseline characteristics of the WOSCOPS participants have been detailed previously.17The study complies with the Declaration of Helsinki, was approved by the local Ethics Committee and participants gave informed consent. The WOSCOPS cohort comprised 6595 males age 4565 (mean 55.2 years) with a mean baseline total cholesterol of 7.0mmol/l, HDL cholesterol of 1.2mmol/l and mean triglycerides of 1.8mmol/l. Recruits had no history of myocardial infarction and had normal renal and hepatic function. Individuals who, during the course of the 5-year study, experienced a definite or suspect fatal or non-fatal myocardial infarction, sudden coronary death or required coronary artery bypass graft or angioplasty were defined as cases (n=580). Each patient was matched with two controls (also drawn from the original cohort of 6595 men), for a total of 1160 controls, on the basis of age (using 2-year age categories) and smoking status, with subject categorized as either non-smokers (those who had never smoked or who had quit smoking) or current smokers who were event free during the 5-year study as described previously.19A proportion of samples (6.7%, n=116) were missing from the Biobank and not available to prepare DNA.
2.2. Plasma lipids
Plasma lipids and lipoproteins were measured in fasting plasma samples according to the protocols of the Lipid ResearchClinics.20LDL peak particle diameter, reported as the particle diameter of the major LDL fraction, was used as an index of LDL size distribution and was determined as described previously21with the exception that 210% polyacrylamide gel electrophoresis was used (Alamo Gels, San Antonio, Texas).
2.3. Polymorphism detection
DNA was extracted from packed blood cells stored at 70°C .22The TaqIB polymorphism was detected as described.8The C(631)A, C(-629)A, I405V, and D442G CETP gene polymorphisms were simultaneously genotyped using a novel multilocus assay, essentially as described by Cheng et al.23DNA was amplified using a multiplex PCR reaction with biotinylated primers. The amplified PCR products were detected colorimetrically by temperature sensitive hybridization to immobilized sequence specific oligonucleotide probes arrayed in a line format on nylon membrane strips and correct allele identification was confirmed by direct sequencing. For the rarest alleles [(631)A,(629)A] synthetic oligonucleotide templates were used to validate the probes. Due to the proximity of the C(631)A and C(629)A polymorphisms, the two-site haplotypes were detected directly. Genotypes were not obtained in 1.1% of the samples due to failed polymerase chain reaction.
2.4. Statistical methods
Differences in baseline characteristics were assessed using conditional logistic regression. Linkage disequilibrium coefficients were estimated using the ASSOCIATE vs 2.35 programme (J. Ott, http://linkage.rockefeller.edu/ott/linkutil.htm#ASSOCIATE). Coefficients were reported as D' where D'=DAB/Dmax[Dmax=min{pAqB, qApB} if D>0 and Dmax=min{pApB,qAqB} if D<0]. Haplotypes were reconstructed using the PHASE program (version 1.0).24Differences in haplotype frequency were tested using a chi-squared test. Differences between cases and controls were compared using a t-test or Wilcoxon test. The effects of the covariates, including CETP genotype, were examined both univariately and simultaneously by conditional logistic regression. Statistical significance was calculated using Wald tests and Wald's confidence intervals are quoted. Tests for interactions between CETP genotype and each of smoking and pravastatin treatment were carried out using likelihood-ratio tests. Additionally, the mean levels of HDL were compared amongst the three genotypes by analysis of variance. All analyses were carried out using the SAS statistical package (version 8.02).
3. Results
3.1. Study subjects
The study comprised 498 case and 1108 control males, matched for age and smoking habit, and baseline characteristics are shown in Table 1. Cases had higher body mass index, triglyceride and LDL cholesterol and lower HDL cholesterol than controls. There was no difference in alcohol consumption, total cholesterol and LDL diameter between the groups.
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4. Discussion
In this case control study derived from the WOSCOP Study the CETP TaqIB genotype was predictive of cardiovascular events in a univariate analysis. Individuals with B2B2 genotype had a significantly reduced risk of 30% (CI95449%) of suffering a cardiovascular event that was independent of alcohol consumption and BMI. The previously recognised interaction of the TaqIB genotype with smoking was observed.5,7The association between TaqIB genotype and cardiovascular risk was limited to non-smokers and, in this subgroup, a dose-dependent relationship between CETP TaqI B genotype and cardiovascular risk emerged. The analysis of the C(631)A, C(629)A and I405V polymorphisms did not add any further information. The small differences in haplotype frequency distribution between cases and controls did not identify a particular haplotype that was markedly over- or under-represented in the cases.
Our data are in agreement with the Framingham, REGRESS and VA-HIT studies.1,15,16The B1B1 genotype was associated with greater progression of coronary atherosclerosis in REGRESS and more cardiovascular end points in VA-HIT than the B2B2 genotype. This is consistent with our observations on clinical endpoints. In each study B1B1 individuals had lower HDL cholesterol levels than B2B2 individuals. These data are inconsistent with that derived from the Physicians Health Study2where no association between the TaqIB polymorphisms and myocardial infarction was found despite a relationship with HDL levels. The fact that the Physicians Health Study comprised a low risk group with a small number of events might explain these discordant results.
The TaqIB polymorphism was in strong linkage disequilibrium with the promoter polymorphisms in agreement with previous observations12,13and may explain why a non-functional TaqIB polymorphism is associated with CETP mass.6However there is evidence that the C(-629)A polymorphism has independent associations with HDL and CETP concentration.12The C(629)A rare A allele is associated with reduced promoter activity and lower CETP mass11and in the present study is associated with a trend towards a reduced risk of a cardiovascular event. The TaqIB and C(629)A polymorphisms have a similar magnitude of association with CVD risk reflected in similar odds ratios and confidence intervals. The C(631)A rare homozygote is associated with a greater CVD risk but the relationship does not reach significance perhaps due to the lack of power associated with a five-fold reduced rare allele frequency compared to the former two polymorphisms. It is possible that other functional polymorphisms in the CETP gene may explain the association between genetic variation at the CETP gene and plasma HDL, CETP levels or both. Two studies published after completion of our analysis have identified a number of new polymorphisms in the promoter of the CETP gene.12,25Klerkx et al.12suggested that the linkage disequilibrium of the C(629)A polymorphism with the TaqIB polymorphism explains its association with HDL cholesterol and CETP concentrations in the REGRESS study. However, Lu et al25found that neither the C(629)A nor the TaqIB polymorphisms were strongly associated with HDL levels and CETP concentrations in a population of 357 elderly Japanese men. Instead, a novel C(2505)A polymorphism seemed to best explain CETP concentrations while a [gaaa](n) repeat and/or the C(2505)A polymorphism independently determined HDL cholesterol levels in this population.
The exact mechanism whereby HDL protects against CVD is not known but its role in reverse cholesterol transport is of paramount importance. Thus a likely mechanism for the association of CETP genotype with cardiovascular risk is the influence on plasma HDL. Variation in the CETP gene is associated with plasma CETP mass and HDL cholesterol as evidenced in the REGRESS study. In the presence of triglyceride-richlipoproteins, CETP removes cholesteryl ester from HDL and replaces it with triglyceride. In combination with hepatic lipase (HL) activity this results in smaller HDL particles26that are more rapidly cleared from the circulation.27Hence high CETP levels are associated with low HDL levels. Because plasma samples that had not been freeze-thawed were unavailable for the present study, CETP levels were not measured. The observation that the relationship between CETP genotype and cardiovascular risk was independent of lifestyle factors but not of HDL might be consistent with a mechanism involving HDL. However, it should be noted that the association between TaqIB genotype and risk of an event is only partially confounded by HDL (OR changed from 0.70 to 0.79). In addition, there was no difference in risk for a cardiovascular event between B1B2 heterozygotes (OR 1.01) and B1B1 homozygotes (Table 3) despite heterozygotes having intermediate HDL levels (Table 4). This suggests that other as yet unknown mechanisms may also be involved in the association of CETP genotype with cardiovascular risk.
Increased small, dense LDL concentrations are associated with increased risk of cardiovascular disease. A linkage of the CETP gene to LDL particle size has been reported and this may also be a potential mechanistic link between variation at the CETP gene and cardiovascular risk.14When LDL diameter was included in a multivariate model there was no significant attenuation of risk. It is noteworthy that LDL diameter did not differ between cases and controls and therefore did not represent a significant risk factor in WOSCOPS. Similarly in VA-HIT, LDL size did not differ between TaqIB genotype groups.16It was reported that the TaqIB polymorphism is not associated with LDL size while the I405V polymorphism is,28suggesting independent relationships between specific CETP gene variants and HDL cholesterol and LDL size.
The mechanism for the interaction of the CETP genotype with smoking is unclear. Smoking interferes with the activity of plasma enzymes involved in HDL metabolism, namely CETP, lecithin cholesterol acyl transferase (LCAT), lipoprotein lipase (LPL), HL and phospholipid transfer protein (PLTP). The circulating levels of HDL may be dependent in part on the relative activity of these enzymes. Smoking appears to impede the HDL-raising effect of the TaqIB2 allele. This could potentially be due to smoking directly counteracting the low CETP levels normally associated with the TaqIB2 allele. However there is only one report of increased CETP concentrations in smokers29and several reports of either neutral or CETP reducing effects of smoking.3032Alternatively smoking may interfere with other enzymes in such a manner as to neutralize the potential benefits of a reduced CETP activity e.g. inhibition of LCAT30,33,34or post-prandial PLTP activity in smokers.32The complex interaction between HDL, smoking and plasma enzyme activities has been discussed elsewhere.31,32
In WOSCOPS 50% of the subjects received pravastatin therapy, a treatment associated with reduced CHD events and mortality.18In the subset of patients comprising the present study pravastatin was associated with a 34% reduction in cardiovascular events (P<0.001). There was no evidence for a differential benefit with therapy in B2B2 compared to B1B1 homozygotes. Similarly there was no difference in benefit between the genotype groups of the other polymorphisms studied. These data are at odds with the REGRESS study where investigators found that pravastatin therapy slowed progression of coronary atherosclerosis in B1B1 but not B2B2 individuals. On the basis of this observation the authors argued that B2B2 individuals did not benefit from treatment which prompted some debate in the literature.35This contradiction might be explained by the different end-points used: cardiovascular events in WOSCOPS and mean luminal diameter in REGRESS which may not correlate strongly to clinical outcome.36In our population the benefit of pravastatin did not differ between genotype groups despite there being differences in HDL levels suggesting that the beneficial effects of pravastatin may be independent of its effects on HDL.
We conclude that variation at the CETP gene, in this male West of Scotland population, has significant impact on cardiovascular risk, which may be independent of its influence on HDL levels and LDL size and is restricted to non-smokers. We found no evidence that any of the CETP genotypes failed to benefit from pravastatin therapy.
Appendix A: West of Scotland Coronary Prevention Study Group
Executive Committee
James Shepherd (chairman), Stuart M. Cobbe, A. Ross Lorimer, James H. McKillop, Ian Ford, Christopher J. Packard, Peter W. Macfarlane, Christopher Isles.
Data and Safety Monitoring Committee
Michael F. Oliver (chairman), Anthony F. Lever, Byron W. Brown, John G. G. Ledingham, Stuart J. Pocock, Basil M. Rifkind.
Cardiovascular End-points Committee
Stuart M. Cobbe (chairman), Barry D. Vallance, Peter W. Macfarlane.
Adverse Events Review Board
A. Ross Lorimer, James H. McKillop, David Ballantyne.
Data centre staff
Liz Anderson, David Duncan, Sharon Kean, Audrey Lawrence, June McGrath, Vivette Montgomery, John Norrie.
Population screening
Melvyn Percy.
Clinical coordination, monitoring, and administration
Elspeth Pomphrey, Andrew Whitehouse, Patricia Cameron, Pamela Parker, Fiona Porteous, Leslie Fletcher, Christine Kilday.
Computerized ECG analysis
David Shoat (deceased), Shahid Latif, Julie Kennedy
Laboratory operations
M. Anne Bell, Robert Birrell
Company liaison and general support
Margot Mellies, Joseph Meyer, Wendy Campbell.
Acknowledgments
We acknowledge the generous support of a British Hyperlipidaemia Association Research Award and the British Heart Foundation (97/160). The West of Scotland Coronary Prevention Study and its follow up were supported by an educational grant from Bristol-Myers Squibb Co. Research.
References