Prospective Evaluation of the Alcohol Dehydrogenase γ1/γ2 Gene Polymorphism and Risk of Stroke
Background and Purpose— Genetic polymorphism of the alcohol dehydrogenase type 3 gene (ADH1C) has recently been associated with reduced risk of myocardial infarction. However, data on risk of stroke are not available.
Methods— We examined the possible association between the ADH1C γ1/γ2 polymorphism and risk of stroke in a prospective, nested case-control sample from the Physicians’ Health Study of 14 916 apparently healthy men who were followed over a 12-year period. A total of 320 incident stroke cases and 550 age- and smoking-matched controls were genotyped.
Results— All observed genotype frequencies were in Hardy-Weinberg equilibrium. The allele and genotype distributions of the polymorphism tested were similar between cases and controls, such that the relative risk of stroke was 1.04 for ADH1C γ1/γ2 (95% CI=0.85 to 1.28; P=0.65) assuming an additive mode of inheritance. Contrary to prior findings for myocardial infarction, no evidence of association was observed to suggest an effect modification of ADH1C genotypes with the level of alcohol consumption on the risk of stroke. Similar findings were observed in subgroup analysis restricted to ischemic events.
Conclusions— In this large, prospective study, we found little evidence that the ADH1C γ1/γ2 polymorphism is associated with risk of future stroke. These data raise the possibility of important pathologic differences in ischemia between the coronary and cerebral circulations.
Stroke, a major cause of permanent disability, has serious public health complications in developed countries. Several studies have reported an association of light to moderate alcohol consumption with reduced risk of cardiovascular diseases including stroke,1–4 whereas others found an increased risk of stroke with alcohol intake.5,6 A recent meta-analysis of experimental studies has concluded that heavy alcohol consumption increases the relative risk of stroke while light to moderate alcohol consumption may be protective against total and ischemic stroke.7
The class I alcohol dehydrogenase (ADH) enzymes, including ADH type 3, are mainly involved in ethanol oxidation. Genetic variants have been found for the ADH type 3 (ADH1C) locus, resulting in γ1 and γ2 isoenzymes.8 The γ1 subunit differs from γ2 subunit by 2 amino acid substitutions: arginine (R) for glutamine (Q) at position 271, and isoleucine (I) for valine (V) at position 349.8 Pharmacokinetic studies have shown that the γ1 isoenzyme is associated with a fast rate of ethanol oxidation while the γ2 isoenzyme with a slow rate of ethanol oxidation.9 Interestingly, the ADH1C γ2 allele has recently been associated with reduced risk of myocardial infarction (MI), and importantly, to interact with alcohol consumption.10 However, to date, no data are available for the risk of stroke. We therefore investigated the role of the ADH1C γ1/γ2 polymorphism as a risk marker for stroke in the Physicians’ Health Study (PHS).
Subjects and Methods
We employed a nested case-control design within the PHS, a randomized, double-blinded, placebo-controlled trial of aspirin and beta-carotene initiated in 1982 among 22 071 male, predominantly white, US physicians, 40 to 84 years of age at study entry.11 Before randomization, 14 916 participants provided an EDTA-anticoagulated blood sample and stored for genetic analysis. All participants were free of prior MI, stroke, transient ischemic attacks, and cancer at study entry. Yearly follow-up self-report questionnaires provided reliable updated information11 on newly developed diseases and the presence or absence of other cardiovascular risk factors. History of cardiovascular risk factors, such as hypertension, diabetes, or hypercholesterolemia, was defined by self-report of diagnosis at entry into the study.
Stroke was defined by the presence of a new focal neurological deficit, with symptoms and signs persisting for >24 hours, and was ascertained from blinded review of medical records, autopsy results, and the judgment of a board-certified neurologist. Stroke was classified as ischemic, hemorrhagic, or unknown, on the basis of clinical reports, computed tomographic (CT), or magnetic resonance image (MRI) scanning. For the present investigation, 320 incident cases of stroke were identified among those study participants who had stored blood samples.
For each case, 1 or more controls (if available) matched by age, smoking history, and length of follow-up were chosen among those subjects who remained free of vascular diseases (a total of 550 controls). The study was approved by the Brigham and Women’s Hospital Institutional Review Board for Human Subjects Research.
Details of genotype determination have been described previously.12 In brief, the ADH1C γ1/γ2 isoenzyme-polymorphism was determined by polymerase chain reaction–restriction fragment length polymorphism gel electrophoresis. To confirm genotype assignment, scoring was carried out by 2 independent observers. Discordant results (<2% of all scoring) were resolved by a joint reading and, where necessary, a repeat genotyping. Results were scored blinded as to case-control status.
Allele and genotype frequencies among cases and controls were compared with values predicted by Hardy-Weinberg equilibrium using the χ2 test. Relative risks of stroke associated with each genotype, with 95% confidence intervals (CI), were calculated by logistic regression analysis conditioning on the matching by age, smoking status, and length of follow-up since randomization, and further controlling for randomized treatment assignment, hypertension, diabetes, BMI, and level of alcohol consumption. Conditional logistic regression was also used to detect possible effect modification of ADH1C genotypes on the relation between the level of alcohol consumption and the risk of stroke by incorporating interaction terms for each category of alcohol consumption and each genotype. The probability values for trends were determined based on the Wald test. All probability values were 2-tailed.
Baseline characteristics of stroke cases and controls are shown in Table 1. As expected, the case participants had a higher prevalence of hypertension, and diabetes at baseline as compared with controls. As also shown in Table 1, the allele and genotype frequencies were similar between cases and controls. The allele and genotype frequencies for the study population were in Hardy-Weinberg equilibrium.
Although statistically nonsignificant, we observed a trend of risk reduction among men according to the level of alcohol consumption (Table 2). As compared with men who were classified as rare or nondrinkers, men who were classified as monthly drinkers had a relative risk of 0.89, 95% CI 0.54 to 1.50; men classified as weekly drinkers had a relative risk of 0.72, 95% CI 0.49 to 1.06; and men classified as daily drinkers had a relative risk of 0.73, 95% CI 0.48 to 1.11; P for trend=0.10. The level of alcohol consumption did not modify the association between the ADH1C genotype and the risk of stroke (Table 3). Similar null findings were obtained in analyses limited to ischemic events.
In this large, prospective investigation, we found no evidence of association between the ADH1C γ1/γ2 polymorphism and risk of stroke, either in the total cohort or in subgroup analysis limited to ischemic events. Furthermore, we found little evidence of effect modification or interaction of the variant tested with the level of alcohol consumption on the risk of stroke. The current null findings are in contrast to a previous report in this cohort,10 which suggested the ADH1C γ1/γ2 polymorphism as a protective factor against the risk of MI.
We believe chance is an unlikely explanation of this difference for 2 reasons. First, the background allele frequency of the ADH1C polymorphism tested is virtually identical in our nested case-control study and in the previous report by Hines et al,10 thus greatly reducing the possibility of misclassification bias. Second, in our study, we had the ability to detect, with 80% power, at an alpha of 0.05, a risk ratio of >1.35 for γ2 allelic variant with stroke assuming an additive model. By contrast, we believe that the observed difference between studies reflects a relative differential contribution of traditional risk factors between MI and cerebrovascular event. In particular, while LDL cholesterol is an important risk factor for MI, lipid levels are poor predictors of stroke,13 and thus effects of alcohol mediated through lipid changes may be less important for stroke. Conversely, hypertension and diabetes are relatively more powerful predictors of stroke. Several speculative explanations have been suggested including nonclassic atherosclerotic mechanisms and differential effect of plasma lipids on atherogenesis in the intracranial vasculature.13 Thus, if there are as yet unidentified environmental modifiers of the ADH1C gene effect on atherothrombosis, these gene-environment interactions may well differ between MI and stroke.
In conclusion, in this large, prospective, nested case-control study among middle-aged US men, we found no evidence of association between the ADH1C polymorphism tested and the risk of stroke, nor any evidence of an effect modification with the level of alcohol consumption on the risk of stroke. These data contrast with those for MI previously observed in this cohort and thus raise the possibility of important pathophysiologic differences between ischemia in the coronary and cerebral vasculatures.
This study was supported by the National Heart, Lung, and Blood Institute (HL58755, HL63293), the American Heart Association, and the Doris Duke Charitable Foundation (P.M.R.).
- Received September 15, 2003.
- Accepted October 29, 2003.
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