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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, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass
Roche Molecular Systems, Alameda, Calif.
Abstract
Background and Purpose— In an Icelandic population, gene variants of the phosphodiesterase 4D, cAMP-specific (PDE4D) gene were reported to be risk predictors for ischemic stroke. Case–control studies in other populations have yielded mixed evidence for association. A recent analysis in a prospective, non-Icelandic study found an association with stroke after stratification by hypertension.
Methods— We evaluated nine PDE4D single nucleotide polymorphisms (SNPs) among 259 incident ischemic stroke cases and 259 controls were matched on age and smoking status and length of follow up since randomization, all drawn from initially healthy white males within the Physicians’ Health Study cohort who were prospectively followed for first-ever stroke events.
Results— Genotype and allele distributions were similar between cases and controls. Results from single-marker conditional logistic regression analysis adjusting for traditional stroke risk factors showed significant association of SNP56 with risk of ischemic stroke (recessive odds ratio [OR], 2.26; 95% confidence interval [CI], 1.11 to 4.61; P=0.03). Among the participants without baseline hypertension, SNP42 (additive OR, 1.68; 95% CI, 0.99 to 2.86, P=0.06), SNP45 (dominant odds ratio, 2.24; 95% CI, 1.00 to 5.00, P=0.05), and SNP56 (additive odds ratio, 1.77; 95% CI, 1.02 to 3.10, P=0.04) showed modest association with increased risk of ischemic stroke.
Conclusions— We found modest associations between several PDE4D gene polymorphisms and risk of incident ischemic stroke in men without baseline hypertension in this prospective, non-Icelandic study. Although of borderline statistical significance, the direction and magnitude of the effect for SNP42 parallels that observed in a recent study evaluating women from an independent, nested case–control study.
Key Words: embolic stroke PDE4D
Introduction
Recent findings from a genomewide linkage investigation and subsequent case–control study in an Icelandic population1,2 suggest that genetic variation of the phosphodiesterase 4D, cAMP-specific (PDE4D)—a key signal transduction molecule in multiple cell types, including vascular cells—may be associated with stroke risk. The direction of effect in these initial findings have generally not been replicated in studies of non-Icelandic populations,3–6 although a possible replication of the association with ischemic stroke was noted.3,5–7 Recently, Brophy et al8a found that individual PDE4D single nucleotide polymorphisms (SNPs) were associated with increased risk of ischemic stroke in nonhypertensive subjects (defined as <160/90 mm Hg) in a prospective study of American women over age 65 (Study of Osteoporotic Fractures ).8a We therefore examined the role of nine PDE4D (gene ID: 5144; chromosome: 5q12) SNPs or haplotypes thereof as risk determinants of incident ischemic stroke in a prospective, nested case–control sample within the Physicians’ Health Study (PHS) cohort. These nine polymorphisms were chosen based on the associations observed in the Icelandic population2 and in SOF8a and map to two regions of the gene: region A (SNPs 9, 26, 32, 34, 42, 45, and 56) at the 5' end of the gene, including the putative promoter and first intron, and region D (SNPs 219 and 222), potentially overlapping regulatory elements for the shorter splice variants.2
Materials and Methods
Study Design
We used a nested case–control design within the PHS,8b a randomized, double-blind, placebo-controlled trial of aspirin and beta-carotene initiated in 1982 among 22 071 male, 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 that was stored for genetic analysis. All participants were free of any prior myocardial infarction (MI), stroke, transient ischemic attacks, and cancer at study entry. 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 (or on antihypertensive medication), diabetes, or hyperlipidemia was defined by self-report of diagnosis collected 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.
Stroke was defined by the presence of a new focal neurologic 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 (CT), or magnetic resonance image (MRI) scanning. For each ischemic stroke case, a control matched by age, smoking history, and length of follow up was chosen among those subjects who remained free of vascular diseases at the time the index event occurred in the case participant. The control participants were selected from those who remained event-free up to the date that the dataset was closed for selection of study participants. The present association study consisted of white men only; 259 incident ischemic stroke case–control pairs were identified for the present investigation.
The study was approved by the Brigham and Women’s Hospital Institutional Review Board for Human Subjects Research.
Genotype Determination
Genotyping was performed using an immobilized probe approach, as previously described (Roche Molecular Systems).9 In brief, each DNA sample was amplified in a multiplex polymerase chain reaction (PCR) using biotinylated primers. Each PCR 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 two independent observers. Discordant results (<1% of all scoring) were resolved by a joint reading and, when necessary, a repeat genotyping. Results were scored blinded as to case–control status.
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 of ischemic stroke associated with each genotype were calculated separately by conditional logistic regression analysis conditioning on the matching by age, smoking status (never, past, current), and length of follow up since randomization and further controlling for randomized treatment assignment (aspirin or beta-carotene) and traditional cardiovascular risk factors such as history of hypertension (140/90 mm Hg), presence or absence of diabetes, and body mass index (BMI, kg/m2), assuming an additive, dominant, or recessive mode of inheritance. Because smoking is a known risk factor for stroke, matching by smoking status improves the efficiency of the analysis to examine the possible association of the gene variants tested and the clinical outcome. Pairwise linkage disequilibrium (LD) was examined as described by Devlin and Risch.10 For comparison with earlier reports,2,8a we also examined haplotypes of five SNPs in region A (SNPs 9, 26, 32, 34, and 42). Haplotype estimation and inference was determined using PHASE v2.1.11,12 Haplotype distributions between cases and controls were examined by likelihood ratio test. The relationship between haplotypes and incident ischemic stroke was examined using a haplotype-based logistic regression analysis with baseline parameterization13 adjusting for the same risk factors. All analyses were carried out using SAS/Genetics 9.1 package (SAS Institute Inc). Based on the notion that potential genetic effects could be more readily discerned in individuals without hypertension,14 the main cardiovascular risk factor for ischemic stroke, and in light of the findings of Brophy et al,8a a stratified analysis by baseline hypertension status (defined as 140/90 mm Hg in PHS) was performed to examine potential effect modification on 302 participants without baseline hypertension. We did not compute risk estimates for participants with baseline hypertension status resulting from small sample size. For each odds ratio (OR), we calculated 95% confidence intervals (CIs). A two-tailed probability value of 0.05 was considered a statistically significant result. Because the present investigation aimed to test positive associations observed in the Icelandic population and in the SOF cohort,8a our data were not corrected for multiple testing.
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 except for SNP219 (P=0.02). Genotyping error is an unlikely explanation because of the stringent genotyping–scoring protocol used. One possible explanation is that the observed Hardy-Weinberg disequilibrium for SNP219 could be the result of chance or underlying association with stroke.15,16
Using a single-marker 2 analysis, genotype and allele distributions were similar between cases and controls (Table 2). Results from the fully adjusted conditional logistic regression analysis, assuming an additive, dominant, or recessive mode of inheritance, showed significant association of SNP56 with increased risk of ischemic stroke (recessive OR, 2.26; 95% CI, 1.11 to 4.61, P=0.03; Table 3). Among those without baseline hypertension, we found an association of SNP45 (dominant OR, 2.24; 95% CI, 1.00 to 5.00, P=0.05) and SNP56 (additive OR, 1.77; 95% CI, 1.02 to 3.10, P=0.04) with increased risk of ischemic stroke (Table 4). Interestingly, the suggestive association for SNP56 under the recessive model shows a higher OR in subjects without baseline hypertension (recessive OR, 3.12; 95% CI, 0.93 to 10.50, P=0.07) than in the unstratified population (recessive odds ratio, 2.26; 95% CI, 1.11 to 4.61, P=0.03). A suggestive association of SNP42 with increased risk was also noted (additive OR, 1.68; 95% CI, 0.99 to 2.86, P=0.06; Table 4). The effect of SNP42 on ischemic stroke risk was in the same direction as that observed in SOF.8a Measures of LD are listed in Table 5.
The overall haplotype distributions were similar between cases and controls. The most frequent haplotypes were A-G-C-C-A and C-A for regions A and D, respectively (Table 6) and hence were used as the referent. The fully adjusted haplotype-based conditional logistic regression analysis showed a suggestive association of haplotype A-A-T-A-G with risk of ischemic stroke (P=0.06), relative to the referent haplotype, in participants without baseline hypertension (data not shown). This haplotype differs from the Icelandic risk haplotype at the last SNP allele2 and from the protective SOF haplotype at two alleles.8a
Discussion
Phosphodiesterase 4D cAMP-specific hydrolyzes cAMP and is a key signal transduction molecule in multiple cell types, including vascular smooth muscle cells. Furthermore, it has been implicated in inflammatory responses, cell migration and proliferation, and in atherosclerosis, the underlying processes leading to stroke.17,18
In this prospective investigation, we examined specific PDE4D SNPs and haplotypes that were associated with either increased or decreased risk of stroke in an Icelandic population or in the SOF (U.S. population). In our data, we observed modest associations between SNPs 42, 45, and 56 and increased risk of incident ischemic stroke. The direction of these effects is opposite to those observed in the Icelandic population,2 which did not stratify by hypertension status. For example, the Icelandic study found that the A-allele of SNP45 was associated with decreased risk, whereas the present investigation found the A-allele was associated with marginally increased risk among those without baseline hypertension. However, the modest effect observed in our data for SNP42 among nonhypertensive participants was in the same direction of effect as that observed in the SOF population.8a By contrast, the SOF data did not find significant associations for SNPs 45 or 56. Thus, stratified analysis of earlier studies according to hypertension status is warranted.
All of the observed associations in the present study are of borderline statistical significance, and thus it is possible that these findings represent the play of chance. However, an alternative explanation for the apparent inconsistency between the present findings and those observed in the Icelandic population and the partial consistency between the PHS and SOF results is that different populations may exhibit heterogeneity of linkage disequilibrium between the putative risk allele and the surrogate markers being tested.19–21 Recently, several independent non-Icelandic association studies examined the associations of specific PDE4D gene variants with risk of ischemic stroke6,22 and/or stroke subtypes (carotid/cardiogenic),3,5–7,23 although these studies did not stratify by hypertension status nor examine a similar set of SNPs. Although the LD pattern among these noncausal SNPs in PHS was more similar to that in SOF than in the Icelandic population (data not shown), we observed only partial congruence between the findings of SOF and findings in the current PHS data. Thus, as shown in Table 7, although we believe it of interest that multiple studies to date have found apparent allelic imbalances, in particular SNP45, within the PDE4D gene between stroke patients and controls, these findings have not been consistent between studies or in direction of effect.
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 the result of bias or unrecognized confounding. Our study cohort consists of entirely white males with distinct socioeconomic status (physicians), so our data cannot be generalized to other ethnic groups or women. Furthermore, because none of the gene variants tested has been found to be functional, we cannot exclude the possibility that the polymorphisms examined are in linkage disequilibrium with an unidentified susceptibility gene(s)/polymorphism(s) that are responsible for the observed significant associations. We have limited information on ischemic stroke-subtype classification; the potential association of these PDE4D polymorphisms/haplotypes with subtypes of ischemic stroke could not be examined in the present investigation. Thus, additional prospective studies are needed to further examine the association of PDE4D gene variation in ischemic stroke.
In conclusion, our prospective investigation found modest evidence for associations of specific PDE4D gene polymorphisms with risk of incident ischemic stroke among men. Like in the analysis of the female SOF cohort,8a the significance of the observed associations for individual SNPs was increased among those without hypertension at baseline, a finding that contrasts in direction of effect reported in the original study from an Icelandic population but that continues to raise hypotheses regarding a potential role for the PDE4D gene in stroke.
Acknowledgments
Sources of Funding
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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