1 Institute of Human Genetics, University of Newcastle-upon-Tyne, UK
2 Clinical Trial Service Unit and Epidemiological Studies Unit, Nuffield Department of Clinical Medicine, University of Oxford, UK
3 Department of Public Health, University of Cambridge, UK
4 Tropical Metabolism Research Unit, University of the West Indies, Kingston, Jamaica
5 Centre National de Genotypage, Paris
6 For information on collaborators and participating centres see end of paper
Correspondence: Bernard Keavney, Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle-upon-Tyne, NE1 3BZ, UK. E-mail: b.d.keavney{at}ncl.ac.uk
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Abstract |
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Methods The present study involves 4685 cases of myocardial infarction (MI) and 3460 unrelated controls without diagnosed cardiovascular disease. Six polymorphisms of four lipid-related genes were genotyped.
Results For the apolipoprotein E 2/
3/
4 polymorphism, the average increase in the plasma ratio of apolipoprotein B to apolipoprotein A1 (apoB/apoA1 ratio) among controls was 0.082 (s.e. 0.007) per stepwise change from
3/
2 to
3/
3 to
3/
4 genotype (trend P < 0.0001). The case-control comparison yielded a risk ratio for MI of 1.16 (95% CI: 1.06, 1.27; P = 0.001) per stepwise change in these genotypes. But, this risk ratio was not as extreme as would have been expected from the corresponding differences in plasma apoB/apoA1 ratio between genotypes. Hence, following adjustment for the measured level of the plasma apoB/apoA1 ratio, the direction of the risk ratio per stepwise change reversed to 0.83 (95% CI: 0.74, 0.92; P < 0.001). Similarly, for the apolipoprotein B Asn4311Ser and Thr71Ile polymorphisms, genotypes associated with more adverse plasma apolipoprotein concentrations were associated with significantly lower risk of MI after adjustment for the apoB/apoA1 ratio. The B2 allele of the cholesteryl ester transfer protein TaqIb polymorphism was associated with a significantly lower plasma apoB/apoA1 ratio, but with no significant difference in the risk of MI. Finally, the lipoprotein lipase Asn291Ser and T4509C (PvuII) polymorphisms did not produce clear effects on either the plasma apoB/apoA1 ratio or the risk of MI.
Conclusions It remains unresolved why some of these genetic factors that produce lifelong effects on plasma lipid concentrations have significantly less than the correspondingly expected effects on CHD rates in adult life.
Accepted 2 June 2004
Genetic factors that affect blood lipids may have either a greater or a lesser than expected effect on vascular disease risk if lifelong differences in blood lipids have a greater or lesser effect on disease than is seen in epidemiological studies of adults, or if those genetic factors also affect other disease mechanisms. If the effects of a particular genetic polymorphism on disease risk are not large, then although studies of only moderate size may well suffice to assess reliably the effects of genotype on blood lipids (the intermediate phenotype), they will not suffice to assess reliably the effects of genotype on disease risk. For genetic polymorphisms that have only moderate effects on blood lipids (so that the means of the values for people with different genotypes all lie well within the normal range of values), reliable direct evidence of whether the effects on risk are appreciably greater or lesser than expected requires studies that involve much larger numbers of cases of vascular disease than is currently customary.1
The two main plasma lipid fractions have opposite effects on the incidence of coronary heart disease (CHD): low-density lipoprotein (LDL) particles, which carry an apolipoprotein B (ApoB) molecule on their surface, are atherogenic; whereas high-density lipoprotein (HDL) particles, which carry an apolipoprotein A1 (ApoA1) molecule on their surface, are cardioprotective. Consequently, the ratio (ApoB/ApoA1) of these plasma apolipoprotein levels is a very strong predictor of CHD rates.2 Some genetic polymorphisms have been identified that have a moderate but definite effect on plasma lipid levels, and hence on the apoB/apoA1 ratio. But, although many studies have investigated the relationship between polymorphisms of some such lipid-related candidate genes and plasma concentrations of lipids,317 few have involved sufficient cases of CHD to assess reliably the relationships between polymorphisms of these genes and disease risk. Moreover, no previous study has been large enough to determine whether any differences in the risk of CHD associated with these lipid-related genotypes are commensurate with their effects on plasma lipid concentrations.
In the present study, comparisons among 3460 control individuals free of cardiovascular disease yielded the associations between different genotypes for each of six lipid-related polymorphisms and the measured plasma apoB/apoA1 ratio. Case-control comparisons involving 4685 patients with confirmed myocardial infarction (MI) and these 3460 controls provided reliable estimates of the associations between genotype and MI risk. Finally, adjustment for the plasma apoB/apoA1 ratio yielded estimates of the associations of genotype with risk among individuals at a given level of plasma lipoproteins.
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Methods |
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Laboratory methods
DNA was extracted from frozen buffy-coat samples, as previously described.18 Six polymorphisms of four candidate genes coding for proteins involved in lipid metabolism were genotyped: apolipoprotein E (APOE) 2/
3/
4; apolipoprotein B (APOB) Asn4311Ser and Thr71Ile; cholesteryl ester transfer protein (CETP) TaqIb; and lipoprotein lipase (LPL) Asn291Ser and T4509C (PvuII). Published primer pairs and conditions were used for PCR amplification of genomic DNA, followed by either allele-specific oligonucleotide hybridisation or restriction enzyme digestion and agarose gel electrophoresis.12,2225 DNA extraction and genotyping were carried out without knowledge of disease status, with samples both from cases and from controls distributed within each of the 96-well genotyping plates. Internal controls were used to check consistency of genotyping between plates. Beckman (Brea, CA, USA) autoanalysers measured plasma concentrations (in g/l) of apolipoproteins A1 and B (using Immuno [Vienna, Austria] reagents), with an initial blank reading subtracted from the final reading to correct for any discoloration from haemolysis.26 Samples from a large plasma pool were included in each analytical run, yielding coefficients of variation of 4% for each apolipoprotein. Repeat samples were obtained from 1038 controls at 23 years after the first sample, and the self-correlations between the measured plasma apolipoprotein values in the original and the repeat samples were used to help take appropriate account of regression dilution when calculating the effects on risk of long-term differences in the usual plasma apoB/apoA1 ratio (see below).27,28
Statistical analysis
MI risk is positively associated with plasma levels of apolipoprotein B and negatively associated with apolipoprotein A1 levels, and the apoB/apoA1 ratio embodies both of these effects simply in a single statistic. In the paired samples from 1038 controls, this ratio was found to have a higher self-correlation (RBA = 0.820) than plasma apolipoprotein B or A1 (0.747 and 0.679 respectively) considered on their own. As a consequence, it should tend to produce less underestimation of associations of MI risk with long-term usual plasma levels of apolipoproteins. The associations between genotypes and the plasma apoB/apoA1 ratio in the unrelated controls were calculated using linear regression. The age- and sex-adjusted MI risk ratios comparing different genotypes were calculated from the case-control comparisons using logistic regression, fitted by unconditional maximum likelihood. The impact of adjusting the associations between genotype and MI risk for the measured level of the apoB/apoA1 ratio was assessed using straightforward regression without correction for regression dilution bias. Subsidiary analyses were conducted (1) with adjustment for the estimated usual levels of the apoB/apoA1 ratio by augmenting the effect of adjustment by the factor 1/RBA,27,28 and (2) with adjustment for separate values of measured apolipoprotein B and apolipoprotein A1. Floating absolute risks and CI were used in order to share the variances of the log risk ratios appropriately between the different genotypes.29 The analyses included all 1923 female controls but only those 1537 male controls who were unrelated to an MI case except by marriage (with adjustment for age as a continuous variable), and are given with 95% CI. (Subsidiary analyses involving both unrelated and related controls found similar associations, albeit with slightly smaller standard errors due to the larger numbers: available on request.) Analyses of genotype associations with MI risk within various subgroups (e.g. with respect to sex, age, smoking, and alcohol consumption) are given with 99% CI to make some allowance for their exploratory nature. Among the cases of confirmed MI, the log-rank test was used to test for differences between genotypes in 6-month survival after admission to hospital (chiefly to exclude the possibility that case fatality rates might differ sufficiently to bias case-control comparisons in this retrospective study).
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Results |
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Figure 2 does not indicate strong evidence of heterogeneity between the MI risk ratios associated with each of these polymorphisms for men versus women, for those at younger versus older ages, for smokers versus non-smokers, or for drinkers of alcohol versus non-drinkers, with the possible exception of alcohol consumption and the LPL Asn291Ser polymorphism. Only small numbers of individuals had the rare Ser allele of this polymorphism, so this apparent heterogeneity of effect on risk may reflect the play of chance (especially when allowance is made for multiple comparisons). Differences between genotypes in case-fatality rates following MI could potentially bias the case-control comparisons in this retrospective study. Six-month follow-up of the cases did not, however, indicate any significant differences in mortality following admission to hospital (data not shown). Nor was there any suggestion that the genotype frequencies differed between patients who were admitted soon after the onset of MI and those who were admitted later after symptom onset, which might have been expected if the early case fatality rates differed substantially (data not shown).
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These adjustments involved only a single measurement of plasma apolipoprotein A1 and B in each individual, which will tend to underestimate the relevance of long-term usual differences in apolipoprotein levels because of random errors in their measurement.27,28 Analyses of the impact of adjustment for estimated usual levels of the plasma apoB/apoA1 ratio on the risk ratio for MI per allele change were performed. But, as a consequence of the high self-correlation coefficient for the apoB/apoA1 ratio (Methods), risk ratio adjusted for the estimated usual levels of the apoB/apoA1 ratio were only slightly more extreme than those adjusted just for the measured levels of the apoB/apoA1 ratio (Table 5).
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Discussion |
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Previous studies
Other studies have reported associations of these genotypes with plasma lipid concentrations that are of similar strength to those found in the present study.8,9,12,13,25,3049 The observed association of the APOE 2/
3/
4 genotype with MI risk is also compatible with the risk ratio of 1.28 (95% CI: 1.07, 1.53) for the
3/4 genotype relative to the
3/3 genotype found in a meta-analysis of 10 studies involving a total of 2494 coronary disease cases.48 But, previous studies have typically involved too few cases of CHD to assess the associations between genotype, plasma lipid concentrations, and disease risk sufficiently reliably. For example, in the PDAY study of atheromatous lesions in 720 young males who died from non-cardiac causes38 (which found larger atherosclerotic lesions among individuals having the
3/4 genotype), adjustment for total cholesterol concentrations did not appreciably change the associations of APOE genotype with atheroma. This contrast with the present findings may reflect the limited sample size, as well as the use of total cholesterol (which is more weakly related to risk than are apolipoproteins A1 and B).
Previous studies have also found the two APOB polymorphisms that we investigated to be associated with differences in plasma apolipoprotein B levels, although none had reliably assessed the association between genotypes at these polymorphisms and MI risk. The present study confirms, with much narrower confidence limits, the genotype-associated differences in apolipoprotein B levels. The two APOB polymorphisms studied are in tight linkage disequilibrium (with the Ser allele of the Asn4311Ser polymorphism occurring mainly on chromosomes also bearing the Ile allele of the Thr71Ile polymorphism), and each was associated with significant differences in the plasma apoB/apoA1 ratio among controls in the present study. But, among individuals with the same plasma apolipoprotein concentrations, these genotypes were found to be associated with MI risk in a direction that is opposite to what might have been expected from their effects on plasma lipid concentrations.
The potentially misleading effects of inadequate sample sizes in previous studies may have been further aggravated by unduly selective emphasis on the apparent results in particular subgroups. For example, one study tested the Ile405Val polymorphism of CETP in 1446 CHD cases, and found no apparent effect of the polymorphism on either plasma HDL cholesterol or CHD among men.50 By contrast, among premenopausal women and postmenopausal women in that study who were not taking hormone replacement therapy, it was reported that carriers of the valine (Val) allele had higher HDL cholesterol levels and, in the postmenopausal subgroup, a higher incidence of CHD (odds ratio = 1.65; 95% CI: 1.06, 2.58). But, there were only 424 female cases in that study and an even smaller number in the postmenopausal subgroup alone. Similarly, in another study only 576 individuals, the Val allele of the Ile405Val polymorphism was associated with higher HDL levels, overall but with a higher CHD risk only in a retrospectively selected subgroup of 47 hypertriglyceridaemic cases.51 Such retrospective emphasis on apparently extreme results in relatively small subgroups may well not be reliable.18,52
Selective survival
The present study involved cases who had survived sufficiently long from the onset of MI to be admitted to hospital. As MI carries a significant early mortality, there is the potential for survivor bias in the case ascertainment for such a retrospective study. But, whereas none of these genotypes appeared to have large effects on the risk of non-fatal MI, there would need to be a substantial effect of genotype on the risk of fatal MI to produce any material bias in these case-control comparisons. There were, however, no significant differences between the genotype frequencies of patients who were admitted within just a few hours of symptom onset and those who were admitted later. Nor did mortality rates during the 6 months following admission to hospital differ significantly between genotypes. Consequently, it does not seem likely that the present findings have been materially influenced by genotype-associated differences in case-fatality rates. A prospective study would avoid the potential for such survivor bias, but it would need to involve hundreds of thousands of healthy individuals followed for several years in order to yield similar number of cases of premature MI (and, hence, similar statistical power).
Potential confounding factors
Differences in plasma lipid concentrations are important determinants of MI risk, so it is perhaps surprising that the relationships between genotypes and risk in the present study were, in general, weaker than would have been expected given the relationships between genotypes and plasma lipoprotein levels, and between plasma lipoprotein levels and disease risk. Various explanations can be considered, though none is clearly correct. First, the strength of the relationship between the apoB/apoA1 ratio and MI risk may have been overestimated in our data due to some effect of confounders, and the genedisease associations may accurately reflect the contribution to risk of the corresponding genetically determined differences in plasma apolipoprotein concentrations. Although this is possible, adjustment for the available potential confounding non-lipid risk factors only accounted for a part of these discrepancies. For example, whereas adjustment for recorded smoking and BMI reduced the estimate of risk per 0.1 g/L higher plasma apoB/apoA1 ratio from 1.63 to 1.48, differences between APOE genotypes in the apoB/apoA1 ratio corresponded to a risk of 1.20 (95% CI: 1.07, 1.34) per 0.1 g/l change (Figure 1). Second, genetic factors act during development as well as in adult life, and if the genetically determined differences in the apoB/apoA1 ratio during adult life are also present during development then there might be some adaptation to them that limits their later effects on the incidence of MI. Third, there may beas yet unclearbiological reasons for these quantitative discrepancies, such as pleiotropic effects of these polymorphisms (or polymorphisms in tight linkage disequilibrium with them) on factors other than plasma lipid concentrations. For example, the apolipoprotein E molecule has many biological activities that might be relevant to the development and rupture of atherosclerotic plaques, including antioxidant properties, inhibition of platelet aggregation, and modulation of inflammatory cytokine release.5357 The 2 allele of the APOE polymorphism is also associated with delayed clearance of atherogenic remnant lipoproteins, which may offset the benefits of lower apolipoprotein B and higher apolipoprotein A1 concentrations.54
Mendelian randomization
It has previously been suggested that comparisons of the relationships between genotype, intermediate phenotype, and disease risk might allow the approach of Mendelian randomization to identify causal associations between hypothesized novel risk factors and particular diseases.58,59 Among the necessary conditions for such an approach to be unconfounded is that differences between genotypes at polymorphisms of interest produce differences only in the levels, rather than in the biological function, of the risk factor. In the present study, however, the polymorphisms were either coding polymorphisms (as in the case of APOB) that might affect the biological function of the protein, or they were related to plasma apolipoprotein concentrations via complex intermediary pathways. Thus, genotypes at these polymorphisms may well influence not only the plasma concentrations of the apolipoproteins, but also the atherogenicity of the plasma at given apolipoprotein concentrations. The apparent discrepancies in the present study between the genotype-associated disease risks and those anticipated from the observed differences in apolipoprotein concentrations should not, therefore, be interpreted as a failure of the Mendelian randomization approach. Rather, these findings suggest that further investigation of the effects of these polymorphisms on more detailed lipid phenotypes (such as LDL particle size and density, triglycerides, remnant lipoprotein metabolism, and HDL subfraction concentrations) may provide important information about the ways in which these genes influence atherogenesis.
Genetic risk prediction
Whatever the explanation(s) may be, however, these discrepancies do have apparently paradoxical consequences for genetic risk prediction. For, at given plasma lipoprotein concentrations, genotypes that adversely affected lipoprotein levels actually involved a lowerrather than a higherrisk of MI. So, for example, among individuals with similar plasma lipoprotein concentrations, the presence of the CETP TaqIb B1/B1 genotype did notby contrast with a previous suggestion60identify individuals at higher risk who might require particularly aggressive management. Instead, these individuals were at slightly lower risk than would have been expected from their lipid profile alone. More generally, the present findings emphasize the need to assess the relevance of genes to disease risk directly in studies involving very much larger numbers of cases than has hitherto been customary (especially if genegene and geneenvironment interactions are to be assessed appropriately reliably), rather than just extrapolating from the observed effects of genotypes on plasma lipoprotein levels, or on other single traits that lie on (or close to) a causal pathway to disease.
Key Messages
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Acknowledgments |
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References |
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