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Technical Briefs |
1 Institute of Medical and Chemical Laboratory Diagnostics and2 Department of Clinical Neurology, Medical University of Vienna, Vienna, Austria;3 Hospital Barmherzige Brueder, Vienna, Austria;
aaddress correspondence to this author at: Institute of Medical and Chemical Laboratory Diagnostics, Waehringer Guertel 18-20, A-1090 Vienna, Austria; fax 43-1-40400-2097, e-mail christine.mannhalter{at}meduniwien.ac.at
Ischemic stroke is a multifactorial disease and a major cause of death and disability throughout the world. Acquired risk factors (e.g., hypertension, cigarette smoking, and diabetes mellitus) account only for 
69% of the population-attributable risk. Thus, it is likely that other, as yet unidentified, factors contribute to the development of stroke (1). Both epidemiologic and animal-based studies suggest that alterations in a variety of candidate genes, including hemostatic genes, genes controlling homocysteine metabolism, the gene that encodes angiotensin-converting enzyme, and the gene that encodes endothelial nitric oxide synthase, are important in the pathogenesis of ischemic stroke (2)(3). Apparently the genetic influences are polygenic. In addition, ischemic stroke comprises many different phenotypes. According to previous studies, genetic factors seem to have different effects depending on stroke etiology [e.g., lacunar stroke and polymorphisms in the gene encoding interleukin-6 (4)]. In combination with acquired risk factors such as smoking, the presence of one or a combination of several predisposing genes may favor the occurrence of stroke.
Cytochrome P-450 (CYP) 2C9 belongs to a large family of heme-containing enzymes that catalyze the oxidation of various drugs and endogenous substrates. CYP2C9 is produced in the liver, is responsible for 50% of the epoxygenase activity in the human liver, and metabolizes a wide variety of clinically important drugs (5). Interestingly, CYP2C isoforms also seem to play a role in the regulation of vascular tone. CYP2C9 is expressed in the endothelium and, via production of endothelium-derived hyperpolarizing factor (EDHF), may cause vasorelaxation as a result of hyperpolarization of vascular smooth muscle cells by activation of Ca2+-activated K+ channels (6). EDHF production seems to be inhibited by NO and/or prostacyclin and has been described as an important regulator of vascular tone under certain pathologic conditions and in certain vascular beds, such as the coronary microcirculation (7). Decreased concentrations of CYP2C have been shown to attenuate EDHF-mediated vascular response in porcine coronary artery endothelial cells (8); the effect appears to be directly attributable to the regulation of an enzyme homologous to CYP2C9 and generation of the CYP metabolites 11,12-epoxyeicosatrienoic acids (9).
Polymorphisms in the CYP2C9 gene (CYP2C9*2 and CYP2C9*3) influence both the activity and substrate specificity of CYP2C9 (10)(11). We therefore hypothesized that altered CYP2C9 activity may modulate vascular function and influence the risk of vascular disease. In a previous study we observed a protective effect of the CYP2C9 mutant genotype on the development of myocardial infarction (MI) in males (12). This was surprising because one would have expected carriers of CYP2C9 mutant alleles to exhibit a diminished CYP2C9 metabolic capacity leading to decreased endothelial EDHF synthesis and an increased risk for MI. However, CYP enzymes also contribute to oxidative stress via the formation of oxygen radicals in the vasculature (13). Particularly, CYP2C9 has been shown to be a major source of reactive oxygen species within coronary artery endothelial cells (14). Decreased formation of oxygen radicals in carriers of mutant alleles might explain our findings of a protective effect.
In the pathogenesis of stroke, no data are available on the impact of CYP2C9 mutant alleles and decreased CYP2C9 activity. We therefore investigated the influence of the CYP2C9*2 and CYP2C9*3 variants on the development of ischemic stroke in patients from the Vienna Stroke Registry.
A total of 389 patients under the age of 60 years (median age, 52 years; 251 males and 138 females) from the Vienna Stroke Registry (15) and 369 healthy controls (median age, 45 years; 199 males and 170 females), all participants in an official health service program of the city of Vienna, all free of clinically manifest vascular disease in their personal and first-degree family histories, and from the same geographic area, were included in the study. The study was approved by the local ethics committee, and all individuals participating in the study gave written informed consent. Stroke etiology was classified according to the Banff classification as large-vessel disease (ipsilateral carotid stenosis 
70%, presumable local thrombosis of a large intracranial vessel, arterio-arterial embolism from aortic plaques/thrombi), small-vessel disease (clinical lacunar syndrome and no lesion or subcortical lesion <1.5 cm on computed tomography or magnetic resonance imaging), cardioembolic (high-risk source of cardiac embolism), or of undetermined etiology. Arterial hypertension was defined as a history of arterial hypertension, blood pressure values >140/95 mmHg 1 week after the event, or receiving antihypertension medication. The presence of diabetes mellitus was defined by fasting blood glucose concentrations >8.3 mmol/L (150 mg/dL) or a history of diabetes (including patients taking antidiabetes medication). Hyperlipidemia was defined as fasting total serum cholesterol >6.2 mmol/L (240 mg/dL), a history of hyperlipidemia, or use of lipid-lowering medication. Obesity was considered present in individuals with a body mass index 30 kg/m2. Detailed characteristics of the patients and controls are shown in Table 1
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For detection of CYP2C9 genetic polymorphisms, a mutagenic separated PCR assay (MS PCR) was used as described previously (12). The resulting PCR products had lengths of 105 and 114 bp for CYP2C9*2 [wild-type (wt) and mutant allele, respectively] and 159 and 168 bp for CYP2C9*3 (wt and mutant allele, respectively). Because both the CYP2C9*2 and the CYP2C9*3 alleles have been shown to lead to decreased enzyme activity (10)(11) and the frequencies of CYP2C9*2/*2 and *3/*3 were very low, the genotype results were assembled in 2 groups for regression analysis: homozygous carriers of wt alleles (CYP2C9*1/*1 individuals) and individuals carrying 1 or 2 variant alleles (mutant allele carriers).
For statistical analyses, the SPSS 10.0 software package (SPSS Inc.) was used. Continuous data are presented as the median and interquartile range (25th–75th percentiles); discrete data are presented as counts and percentages. Univariate analysis was performed with nonparametric Mann–Whitney U-tests for continuous variables and 
2 tests for dichotomous variables, where applicable. Multivariate logistic regression analysis was used to assess the association of the CYP2C9 genotype and the group classification and to adjust for potentially confounding variables. We used multiplicative interaction terms and log likelihood ratio 2 tests to test for interactions between the CYP2C9 genotype, traditional vascular risk factors, and cerebrovascular events. The significance niveau for possible statistical interaction was a P value <0.1 by the log likelihood ratio test. Results of the logistic regression models are given as the odds ratio (OR) and the 95% confidence interval (95% CI). A 2-sided P value <0.05 was considered statistically significant. Power analysis revealed that our sample size is sufficient to detect a difference with a factor of 1.38 with a power of 80%.
Univariate analysis revealed no differences in the overall CYP2C9 genotype frequencies between patients from the Vienna Stroke Registry and controls (P = 0.31; OR = 0.84; 95% CI, 0.68–1.24; Table 1
); multivariate analysis (adjusted for conventional vascular risk factors) also revealed no significant differences (P = 0.38; OR = 0.84; 95% CI, 0.57–1.24). Genotype frequencies were in Hardy–Weinberg equilibrium in all groups and were in good agreement with data for other European populations (5). We found no association between the CYP2C9 genotype and stroke etiology (P = 0.8; OR = 1.03; 95% CI, 0.79–1.34) or neurologic outcome 1 week after the event (Rankin scale; P = 0.2; OR = 1.09; 95% CI, 0.95–1.26). Because different CYP2C9 genotypes were not associated with stroke severity, we do not assume that the presence of CYP2C9 mutant alleles predisposes to a lethal outcome in stroke patients. We also found no differences between males and females and no significant interactions between CYP2C9 genotype and conventional vascular risk factors (age, hypertension, smoking, diabetes mellitus, hyperlipidemia, and obesity).
On first sight, our results seem contradictory to previously published data from our group, in which we were able to show a significant association of CYP2C9 genotype and MI (12). Although both diseases share the common clinical manifestation of acute thrombotic intraarterial occlusion, the pathogenetic background seems to differ. MI has a rather homogeneous pathogenesis of unstable intermediate vessel arteriosclerosis followed by plaque rupture and subsequent thrombosis (16). In contrast, ischemic stroke is the clinical manifestation of a variety of causes, including thromboembolism, large-vessel arteriosclerosis, or microangiopathy (17). All of these types of stroke differ in their conventional risk profiles. Genetic variations of single risk factors do not significantly influence the overall risk for ischemic stroke. The impact of a single polymorphism on a complex disease such as an ischemic cerebrovascular event may be influenced by patient selection, ethnic background, or sample size, among others. Our collective represents a cross-sectional sample of survivors of cerebrovascular events. Patients who died within 24 h were not included in our study; our findings therefore can be applied only to the survivors of an acute cerebrovascular event. We conclude that, in contrast to MI, the CYP2C9*2 and CYP2C9*3 alleles do not represent risk factors for ischemic stroke.
Acknowledgments
This work was supported by Research Grant 9996 from the Jubilaeumsfonds der Österreichischen Nationalbank.
References
gene polymorphism with cerebral infarction. Brain Res Mol Brain Res 2003;115:50-54.[Medline]
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