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the Center for Cardiovascular Disease Prevention, the Donald W. Reynolds Center for Cardiovascular Research, the Leducq Center for Molecular and Genetic Epidemiology (R.Y.L.Z., H.H.H., P.M.R.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass
the Roche Molecular Systems (S.C., H.A.E.), Alameda, Calif.
Abstract
Background and Purpose— Recent findings have implicated specific gene polymorphisms of arachidonate 5-lipoxygenase–activating protein (ALOX5AP), and 2 at-risk haplotypes (HapA, HapB) in myocardial infarction and stroke. To date, no prospective data are available.
Methods— We evaluated 10 specific Icelandic ALOX5AP gene variants among 600 male participants with incident atherothrombotic events (myocardial infarction [MI] or ischemic stroke) and among 600 age- and smoking-matched male participants, all white, who remained free of reported cardiovascular disease during follow-up within the Physicians’ Health Study cohort.
Results— Overall allele, genotype, and haplotype distributions were similar between cases and controls. Single-marker conditional logistic regression analysis adjusted for potential risk factors found no association with risk of atherothrombotic events. Further investigation using a haplotype-based approach showed similar null findings with MI (HapA: odds ratio [OR]=1.18, 95% CI, 0.76 to 1.85; P=0.46; HapB: odds ratio=0.62, 95% CI, 0.36 to 1.07; P=0.08), and with ischemic stroke (HapA: odds ratio=1.11, 95% CI, 0.65 to 1.89; P=0.71; HapB: odds ratio=0.82, 95% CI, 0.47 to 1.42; P=0.47).
Conclusions— We found no evidence for an association of the specific Icelandic ALOX5P gene variants/at-risk haplotypes tested with risk of incident MI nor ischemic stroke in this prospective, non-Icelandic study.
Key Words: ALOX5AP haplotypes MI risk factors stroke
Introduction
Cardiovascular diseases, including myocardial infarction (MI) and ischemic stroke, are the leading causes of mortality and morbidity in western countries. The underlying pathogenesis is likely to be mediated by both genetic and environmental risk factors. The initial report,1 in an Icelandic population, of a significant association of genetic variants of arachidonate 5-lipoxygenase-activating protein (ALOX5AP) with increased risk of MI and stroke has attracted great interest. In their study, Helgadottir and coauthors reported a linkage and association of a 4–single-nucleotide polymorphism (SNP) haplotype, HapA, of ALOX5AP gene with risk of MI and stroke.1 In addition, they reported an association of a different 4-SNP haplotype, HapB, with risk of MI in a British population.1 Helgadottir and coauthors further assessed the contribution of ALOX5AP variants, in particular the HapA, and HapB haplotypes, to stroke, in a Scottish population, and found that the HapA haplotype confers a relative risk of 1.36 assuming a multiplicative model (P=0.007) for stroke.2 However, they found no association for HapB. Subsequent studies by others in several non-Icelandic populations have since yielded conflicting results.3,4
To date, no prospective genetic-epidemiological data are available on risk of MI, and ischemic stroke. We therefore simultaneously evaluated the role of 10 ALOX5AP (GeneID: 241; Chromosome: 13q12) SNPs (SG13S25, SG13S377, SG13S106, SG13S114, SG13S89, SG13S30, SG13S32, SG13S41, SG13S42, and SG13S35), and specific haplotypes thereof, in particular HapA, and HapB at-risk haplotypes, as risk determinants of incident MI, and ischemic stroke in a prospective, nested case-control sample within the Physicians’ Health Study (PHS) cohort. These polymorphisms (except SG13S106, SG13S30, and SG13S42: unpublished data from deCODE Genetics) were chosen based on the associations observed in the Icelandic study.1
Materials and Methods
Study Design
We used a nested case-control design within the PHS,5 a randomized, double-blinded, placebo-controlled trial of aspirin and beta carotene initiated in 1982 among 22 071 males, predominantly white (>94%), US physicians, 40 to 84 years of age at study entry. 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. As the study participants were all US male physicians, yearly follow-up self-report questionnaires provide reliable updated information on newly developed diseases and the presence or absence of other cardiovascular risk factors. History of cardiovascular risk factors, such as hypertension (>140/90 mm Hg or on antihypertensive medication), diabetes or hyperlipidemia (>240 mg/dL), was defined by self-report of diagnosis at entry into the study. For all reported incident vascular events occurring after study enrollment, hospital records, death certificates, and autopsy reports were requested and reviewed by an end-points committee using standardized diagnostic criteria.
The diagnosis of MI was confirmed by evidence of symptoms in the presence of either diagnostic elevations of cardiac enzymes or diagnostic changes on electrocardiograms. In the case of fatal events, the diagnosis of MI was also accepted based on autopsy findings. 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, on the basis of clinical reports, computed tomographic, or MRI scanning.
For each case (MI or ischemic stroke), a control matched by age, smoking history (never, past, or current) and length of follow-up were chosen among those subjects who remained free of vascular diseases. The present association study consisted of 341 MI case-control pairs, and 259 ischemic stroke case-control pairs, all white males.
The study was approved by the Brigham and Women’s Hospital Institutional Review Board for Human Subjects Research.
Genotyping Determination
Genotyping was performed using an immobilized probe approach, as previously described (Roche Molecular Systems).6 In brief, each DNA sample was amplified in a multiplex polymerase chain reaction using biotinylated primers. Each polymerase chain reaction product pool was then hybridized to a panel of sequence-specific oligonucleotide probes immobilized in a linear array. The colorimetric detection method was based on the use of streptavidin-horseradish peroxidase conjugate with hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine as substrates.
To confirm genotype assignment, scoring was carried out by 2 independent observers. Discordant results (<1% of all scoring) were resolved by a joint reading, and where necessary, a repeat genotyping. Results were scored blinded as to case-control status. Overall completion rate of genotyping determination was 95%.
Statistical Analysis
Allele and genotype frequencies among cases and controls were compared with values predicted by Hardy-Weinberg equilibrium using the 2 test. Relative risks associated with each genotype were calculated separately by conditional 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, history of hypertension, presence or absence of diabetes, and body mass index, assuming an additive, dominant, or recessive mode of inheritance. Pairwise linkage disequilibrium (LD) was examined as described by Devlin and Risch.7 For comparison with published reports by others, we examined 2 previously described at-risk haplotypes: HapA (SG13S25G-SG13S114T-SG13S89G-SG13S32A), and HapB (SG13S377A-SG13S114A-SG13S41A-SG13S35G). Haplotype estimation and inference was determined using PHASE v2.1.8,9 Haplotype distributions between cases and controls were examined by likelihood ratio test. The relationship between haplotypes and clinical outcomes was examined using a haplotype-based logistic regression analysis with baseline-parameterization,10 adjusting for the same risk factors. All analyses were carried out using SAS/Genetics 9.1 package (SAS Institute, Inc). For each odds ratio (OR), we calculated 95% CIs. A 2-tailed P value of 0.05 was considered a statistically significant result.
Results
Baseline characteristics of cases and controls are shown in Table 1. As expected, the case participants had a higher prevalence of traditional cardiovascular risk factors at baseline as compared with controls. The genotype frequencies for the polymorphisms tested were in Hardy-Weinberg equilibrium in the control group and in the case group.
Using a single-marker 2 analysis, allele and genotype distributions were similar between cases and controls (Table 2). Results from the adjusted conditional logistic regression analysis, assuming additive, dominant, or recessive mode of inheritance, showed no significant association of the variants tested with the clinical outcomes (P0.07; data not shown). In general, the polymorphisms tested were in LD (supplemental Table I, available online at http://stroke.ahajournals.org). The overall haplotype distributions between cases and controls were similar (MI: HapA region, P=0.79, HapB region, P=0.94; ischemic stroke: HapA region, P=0.77, HapB region, P=0.26; supplemental Table II, available online at http://stroke.ahajournals.org). The most frequent haplotypes were G-T-G-C, and G-T-A-G for HapA region, and HapB region, respectively (supplemental Table II), and thus were used as the referents. Results from the adjusted haplotype-based conditional logistic regression analysis again showed similar null findings (supplemental Table III, available online at http://stroke.ahajournals.org).
Discussion
The present prospective investigation provides no evidence for an association of the specific gene variants, nor at-risk haplotypes of the ALOX5AP gene, previously suggested as genetic risk determinants, with MI or stroke in a non-Icelandic white population.
In the initial Icelandic report,1 a 4-SNP haplotype (HapA) was found to be associated with a 2x greater risk of MI, and an almost 2x greater risk of stroke. The same group also reported an association of a different 4-SNP ALOX5AP haplotype (HapB) with risk of MI in a British sample population1 (Table 3). A subsequent report by Helgadottir and coauthors found an association between HapA and an increased risk of ischemic stroke (relative risk=1.35; P=0.02), and an over-representation of HapB (relative risk=1.65; P=0.02) with ischemic stroke in a Scottish male sample population2 (Table 3). Recently, Lohmussar and coauthors3 reported that sequence variants in the ALOX5AP gene are significantly associated with stroke, particularly in males, in a Central European sample population. A nominally significant association with stroke was observed for SG13S114 (OR=1.24; P=0.017), and SG13S100 (OR=1.26; P=0.024). However, they found no association of HapA with stroke risk.3 More recently, Meschia and coauthors conducted the first replication study using a North American sample population, and found no association between ALOX5AP gene variants and stroke, although MI was not investigated in their study.
Given this situation, a possible explanation for the apparent discrepancies is that the observed allele, genotype, and at-risk haplotype frequencies for the SNPs examined may differ between studies, which could be the result of population/ethnic differences. As previously suggested,3,4 the ALOX5AP gene variation may play a substantial role in risk of MI, and stroke in Iceland (an isolate population), but a lesser role in non-Icelandic populations because of different population LD structures. These recent results are consistent with the initial report that different at-risk haplotypes were found between the Icelandic and British study populations.1
As shown in Table 3, not all of the published reports examined the same set of SNPs, nor did all of the reported studies examine the association of ALOX5AP variants with MI and stroke simultaneously. Further, not all published studies presented information on allele, genotype and at-risk haplotype frequencies, LD structure, and risk estimates, thus making a direct comparison and informative interpretation across studies difficult.
It has been noted in the initial report1 that variants of ALOX5AP gene are involved in the pathophysiology of MI and stroke by increasing the production of leukotriene B4, a critical regulator in the 5-lipoxygenase pathway, and a proinflammatory agent. Leukotrienes are arachidonic acid metabolites, which have been implicated in various inflammatory conditions, including asthma, arthritis, psoriasis, and atherosclerosis.11,12 Notably, a recent article by the same Icelandic group found a haplotype (HapK) of the gene encoding leukotriene A4 hydrolase, a protein in the same biochemical pathway of ALOX5AP, confers ethnicity-specific (particularly in blacks) risk of MI.13
The prospective nature of the PHS study and the use of a closed population sampling scheme in which subsequent case status was determined solely by the development of disease strongly reduce the possibility that our findings are attributable to bias or confounding. Our study cohort consists of entirely white males with distinct socioeconomic status (physicians), so our data cannot be generalized to other ethnic groups and women. In our study, we had the ability to detect, based on the present sample sizes, assuming 80% power, at an of 0.05, a risk ratio of >1.54 (MI), and 1.64 (ischemic stroke) if the minor allele frequency is 0.50, and of >2.26 (MI), and 2.49 (ischemic stroke) if the minor allele frequency is 0.05 assuming a univariable-additive mode. Thus, we cannot rule out a modest risk of cardiovascular disease associated with the polymorphisms/haplotypes tested. It is important to recognize that association studies like this one can only examine the possible association between phenotype and the tested polymorphisms. Our study therefore cannot exclude the possibility that examination of different polymorphisms/loci, which would by definition have to be in linkage disequilibrium with the ones tested, might obtain different results.
In conclusion, our prospective study found no evidence for an association of specific Icelandic ALOX5AP gene polymorphisms/at-risk haplotypes examined with risk of atherothrombotic events. If corroborated in other non-Icelandic prospective studies, our data suggest that ALOX5AP gene variation is not informative for risk assessment of atherothrombosis in non-Icelandic populations.
Acknowledgments
We thank Michael Grow and Houman Khakpour (both at Roche Molecular Systems) for their expertise and efforts in developing the genotyping reagents used for this study. We thank Anna Helgadottir at deCODE Genetics for sharing unpublished data.
Sources of Funding
This work was supported by grants from the National Heart Lung and Blood Institute (HL-58755 and HL-63293), the Doris Duke Charitable Foundation, the American Heart Association, and the Donald W. Reynolds Foundation, Las Vegas, Nevada.
Disclosures
None.
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