Association between genetic polymorphisms of cytochrome P450 2C19 and the risk of cerebral ischemic stroke in Chinese
- Shuzhen Gu†1, 2,
- Yan Sun†3,
- Ruifa Han4,
- Lin Wang4,
- Dongliang Wang1,
- Jizuo Wang1 and
- Xin Li1Email author
© Gu et al.; licensee BioMed Central Ltd. 2014
Received: 27 October 2013
Accepted: 9 July 2014
Published: 17 July 2014
Cytochrome P450 (CYP) 2C19 is a very important drug metabolizing enzyme. Although the single nucleotide polymorphisms (SNPs) of CYP2C19 G681A and G636A have been suggested that they may increase the incidence of cardiovascular events, the relationship between SNPs in CYP2C19 and cerebral ischemic stroke (CIS) are unclear. The aim of this study was to investigate the correlation between the distribution of G681A and G636A polymorphisms in CYP2C19 gene and the risk of CIS in Chinese.
The peripheral blood DNA was extracted from 299 patients with CIS and 295 healthy controls. The genotyping was conducted using the polymerase chain reaction-restriction fragment length polymorphism. The sampled sequencing was applied to verify the correctness of genotyping results. Both the genotype and allele distributions were compared in patients with CIS and healthy controls.
The frequencies of CYP2C19 681AA (11.7% vs. 2.7%; P = 0.000), 636AA (4.0% vs. 0.7%; P = 0.007), 636AG (7.0% vs. 2.2%; P = 0.038) genotype, CYP2C19 681A (30.9% vs. 20.8%; P = 0.000) and 636A (13.0% vs. 5.8%; P = 0.000) allele in the CIS group are significantly higher than those in the controls. The frequencies of CYP2C19 681AA (16.7% vs. 8.6%; P = 0.036), CYP2C19 636AA (7.0% vs. 2.2%; P = 0.038) genotype, CYP2C19 681A (36.4% vs. 27.6%; P = 0.023) and CYP2C19 636A (17.5% vs.10.3%; P = 0.010) allele in the recurrent stroke group are significantly higher than those in the first onset group. Multivariate logistic regression analysis of risk factors for cerebral ischemic stroke and recurrent stroke respectively suggests that the CYP2C19 681AA genotype may be an independent risk factor for CIS (OR = 6.179, 95% CI: 2.285 ~ 16.708; P = 0.000) and recurrent stroke (OR = 2.305, 95% CI: 1.121 ~ 4.743; P = 0.023).
The AA genotype and A allele of CYP2C19 G681A may be related to the occurrence and recurrence of cerebral ischemic stroke.
KeywordsCerebral ischemic stroke CYP2C19 Genetic polymorphism
The occurrence of cerebral ischemic stroke is influenced by eating habits, environment, genetic factors and so on. Fundamentally, genetic factors involve in a series of key enzymes and receptors on many metabolic pathways. Therefore, the study of the genetic risk factor for cerebral ischemic stroke has become a hot spot currently.
Cytochrome P450(CYP) 2C19 is a very important drug metabolizing enzyme, which involves in approximately 2% of the clinical drug metabolism. The activity of CYP2C19 enzyme not only exhibits a significant ethnic heterogeneity, but also has an obvious differences between individuals. It is believed that the differences are mainly caused by genetic variations. Many researchers have investigated the molecular mechanism of CYP2C19 enzyme polymorphisms, which include the wild-type CYP2C19*1, CYP2C19*2, CYP2C19*3, CYP2C19*4, CYP2C19*5, CYP2C19*6, CYP2C19*7, CYP2C19*8, etc. CYP2C19*2 and CYP2C19*3 are the main variants in CYP2C19, while others are relatively rare in humans.
Studies have shown that the single nucleotide polymorphisms (SNPs) of CYP2C19*2(CYP2C19 G681A, rs4244285) and CYP2C19*3(CYP2C19 G636A, rs4986893) may increase the incidence of cardiovascular events [1–3]. Whereas there are less research about the relationship between SNPs in CYP2C19 and cerebral ischemic stroke. So we carried out a case-control study to investigate the relationship between the distribution of G681A and G636A polymorphisms in CYP2C19 gene and cerebral ischemic stroke in Chinese Han population.
From May 2011 to March 2013, we recruited 299 patients with acute cerebral ischemic stroke who were admitted to the Neurology Department of the second hospital, Tianjin Medical University, Tianjin, China. All patients were diagnosed as acute cerebral ischemic stroke, which was diagnosed by a neurologist according to the diagnostic criteria determined by the guidelines for the primary prevention of stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association  and confirmed by computed tomographic (CT) scan and/or conventional magnetic resonance imaging (MRI) of the brain; the time from incidence to hospitalization was less than seven days. We excluded patients who had cerebral hemorrhage, transient ischemic attack (TIA), cerebral venous thrombosis, nervous system infection, neurodegenerative disease, hepatic and renal dysfunction, thrombocytopenia and tumor. In addition, patients were excluded when they took PPI, tricyclic antidepressants, antiepileptics and antipsychotics.
Controls enrolled in this study may have high blood pressure, diabetes, smoking and other vascular risk factors, excluding history of cerebral ischemic stroke, TIA, atrial fibrillation, myocardial infarction and venous thrombosis.
All subjects were unrelated Chinese Han. There were no significant differences in the age and sex distributions. Informed consent was obtained from all subjects, and the study was approved by the Ethical Committee of Tianjin Medical University.
All subjects underwent a comprehensive medical history, physical examination and clinical chemistry analysis before enrollment. Diagnosis of hyperlipidemia was based on China’s adult dyslipidemia prevention guide . Hypertension was defined as a blood pressure greater than 140/90 mmHg on at least two independent readings, exclude secondary hypertension. Diagnosis of ischemic heart disease according to the WHO diagnostic criteria . Diagnosis of diabetes mellitus according to the WHO diagnostic criteria , exclude type 1 diabetes mellitus and secondary diabetes.
Blood samples were collected in tubes containing EDTA. The genomic DNA was extracted using a commercially available DNA isolation kit (Cwbiotech, Beijing, China) according to the manufacturer’s instruction.
The primers of CYP2C19*2 and CYP2C19*3 alleles were designed and synthesized by BGI Beijing Corporation. The forward primer of 5′- ACC AGA GCT TGG CAT ATT GTA TCT -3′ and the reverse primer of 5′-GAT TCT TGG TGT TCT TTT ACT TTC T-3′ were used for the amplification of the CYP2C19*2 allele. For the CYP2C19*3 variant, the forward primer was 5′- TTT CAT CCT GGG CTG TGC TC -3′ and the reverse primer was 5′- TGT ACT TCA GGG CTT GGT CAA T -3′. The PCR was performed with an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, elongation at 72°C for 30 s, and a final extension at 72°C for 10 min. The amplified products of CYP2C19*2 (192bp) and CYP2C19*3 (234bp) were respectively digested with SmaI and BamHI fast-digest restriction enzyme (Takara biotechnology, Dalian).
Samples from each genotype of CYP2C19 were selected randomly and submitted for direct DNA sequencing which confirmed the results.
Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables were reported as counts and percentages. Analyses of t-tests and chi-square tests were used to test for differences between groups for continuous and categorical variables, respectively. Multivariate logistic regression analysis was used to identify independent predictors of cerebral ischemic stroke. Analyses were performed using SPSS version 19.0 statistical software. A value of P < 0.05 (two-sided) was considered statistically significant. The p value is accurate to three decimal places when we calculated using SPSS.
A total of 299 patients were enrolled in this study, including 177 cases of male, 122 cases of female, the first onset group were 185 cases, the recurrent stroke group were 114 cases, the mean ages were (67.86 ± 11.472) years. And a total of 295 controls were enrolled, including 159 males, 136 females, the mean ages were (66.45 ± 12.972) years.
Demographic and clinical information of the cerebral ischemic stroke and the control group
The cerebral ischemic stroke (n = 299)
The control group (n = 295)
67.86 ± 11.472
66.45 ± 12.972
Male (n, %)
Hypertension (n, %)
Diabetes (n, %)
Hyperlipidemia (n, %)
Ischemic heart disease (n, %)
Smoking (n, %)
Drinking (n, %)
Systolic blood pressure (mmHg)
153.01 ± 22.593
127.82 ± 18.146
Diastolic blood pressure (mmHg)
86.21 ± 12.644
76.51 ± 9.684
Total cholesterol (mmol/L)
5.033 ± 1.366
5.211 ± 1.030
1.654 ± 1.116
1.537 ± 1.038
High-density lipoprotein cholesterol (mmol/L)
1.113 ± 0.562
1.316 ± 0.452
Low--density lipoprotein cholesterol (mmol/L)
3.157 ± 1.061
3.078 ± 0.815
Fasting blood-glucose (mmol/L)
7.504 ± 3.461
5.538 ± 0.980
Demographic and clinical information of the recurrent stroke group and the first onset group
The recurrent stroke group (n = 114)
The first onset group (n = 185)
Age (years, mean ± SD)
69.37 ± 11.132
68.28 ± 11.149
Male (n, %)
Hyperlipidemia (n, %)
Diabetes (n, %)
Hyperlipidemia (n, %)
Ischemic heart disease (n, %)
myocardial infarction (n, %)
Smoking (n, %)
Drinking (n, %)
Systolic blood pressure (mmHg, mean ± SD)
150.99 ± 23.368
154.26 ± 22.074
Diastolic blood pressure (mmHg, mean ± SD)
85.82 ± 11.937
86.46 ± 13.086
Total cholesterol (mmol/L, mean ± SD)
4.990 ± 1.619
5.059 ± 1.188
Triglyceride (mmol/L, mean ± SD)
1.567 ± 0.938
1.708 ± 1.212
High-density lipoprotein cholesterol (mmol/L, mean ± SD)
1.056 ± 0.303
1.148 ± 0.672
Low--density lipoprotein cholesterol (mmol/L, mean ± SD)
3.148 ± 1.325
3.163 ± 0. 862
Fasting blood-glucose (mmol/L, mean ± SD)
7.733 ± 3.798
7.363 ± 3.239
Frequency of CYP2C19 genotypes and alleles
Frequencies of the CYP2C19 polymorphism in cerebral ischemic stroke patients and healthy controls (n, %)
(n = 299)
(n = 295)
Frequencies of the CYP2C19 polymorphism in recurrent stroke group and the first onset group (n, %)
The recurrent stroke group
(n = 114)
The first onset group
(n = 185)
Frequencies of the CYP2C19 polymorphism in different NIHSS groups in patients
(n = 161)
(n = 84)
(n = 54)
Correlation between CYP2C19 genetic polymorphisms and cerebral ischemic stroke
Multivariate logistic regression analysis of independent risk factor for cerebral ischemic stroke
Independent risk factor
Ischemic heart disease
Systolic blood pressure
CYP2C19 is located within a cluster of cytochrome P450 genes on chromosome 10q24, which contains nine exons and eight introns. The gene encodes a 490-aa long protein of approximately 56kDa, which is a member of the cytochrome P450 superfamily of enzymes. This protein localizes to the endoplasmic reticulum and is known to metabolize many xenobiotics, including the anticonvulsive drug mephenytoin, omeprazole, diazepam and some barbiturates. Polymorphism within this gene is associated with variable ability to metabolize mephenytoin, known as the poor metabolizer and extensive metabolizer phenotypes. CYP2C19*2 has been shown to be a G → A transition at 681bp in exon 5 of wild-type CYP2C19*1. This variant results in an aberrant splice site and shifts the reading frame, thereby producing an early-stop codon and a truncated protein . The CYP2C19*3 involves a G → A variant at 636bp in exon 4, that also creates a premature stop codon and a truncated protein . The CYP2C19*2 is the main genetic defect allele of CYP2C19, which accounts for 75% ~ 85% of poor metabolizers (PM) in both white and Oriental populations . The second variant accounts for the remaining defective alleles in Oriental populations PM, but appears to be extremely rare in white persons .
Yin et al.  found that the allele frequencies of CYP2C19*2 were respectively 29.7%, 32.4% and 18.2% in Han, Hui and Mongolian, the allele frequencies of CYP2C19*3 were respectively 7.9%, 10.2% and 11.2% in Han, Hui and Mongolian. Other literatures [14, 15] reported that the allele frequencies of CYP2C19*2 were 11.1% for Western Africa, 14.0% for Western Europe and 16.3% for Northern Europe respectively, and the allele frequencies of CYP2C19*3 were 0% for Western Africa, 0.2% for Western Europe and 0.2% for Northern Europe respectively. In our study, the allele frequencies of CYP2C19*2 and CYP2C19*3 were 25.9% and 9.4% separately. Our result is close with that in Yin’s report.
Studies have shown that the poor metabolizer gene of CYP2C19 was related to the occurrence of coronary heart disease and stroke. Through the research of 654 patients with coronary heart disease, Mao Chen et al.  found that the homozygous CYP2C19*2/*2 genotype was an independent determinant of adverse vascular events in Chinese patients with coronary artery disease (CAD). Mega et al.  reported that carriers of a reduced-function CYP2C19 allele had a higher rate of major adverse cardiovascular events than did noncarriers. Actually our research also found that CYP2C19 681AA genotype, CYP2C19 636AA and CYP2C19 636AG genotype were related with cerebral ischemic stroke. Multivariate logistic regression analysis showed that the CYP2C19 681AA genotype may be an independent risk factor for cerebral ischemic stroke. We postulate that the weak metabolic gene of CYP2C19 may be related to the formation of cerebral artery atherosclerosis, thereby causing the incidence of cerebral ischemic stroke.
Clopidogrel is a prodrug requiring metabolism by CYP2C19 enzyme, in order to be active, which can play the role of anti-platelet aggregation. Clopidogrel is widely used in the secondary prevention of cerebral ischemic stroke, but its clinical efficacy has individual differences. Many studies have shown that CYP2C19 polymorphism is associated with reduced clopidogrel response [18–23]. Our study suggested that the CYP2C19 681AA genotype was an independent risk factor for recurrent stroke, since carriers of a reduced-function CYP2C19 allele had a two-fold risk with recurrent stroke than did noncarriers.
In summary, the AA genotype and A allele of CYP2C19 G681A may be associated with the occurrence and recurrence of cerebral ischemic stroke. In addition, we find that the genotypes of CYP2C19 G681A and G636A have nothing to do with the severity of cerebral ischemic stroke. Since our study has limited sample size, further studies in a large population are needed to confirm these findings.
Cerebral ischemic stroke
Single nucleotide polymorphisms
Magnetic resonance imaging
Transient ischemic attack
High-density lipoprotein cholesterol
This work was supported by the applied basic research programs and cutting-edge technology of Tianjin, China (09JCYBJC11400), the science and technology program of Tianjin, China (12ZCZDSY03100), the education social science key program Tianjin, China (2011ZD016).
- Jang JS, Cho KI, Jin HY, Seo JS, Yang TH, Kim DK, Kim DS, Seol SH, Kim DI, Kim BH, Park YH, Je HG, Jeong YH, Lee SW: Meta-analysis of cytochrome P450 2C19 polymorphism and risk of adverse clinical outcomes among coronary artery disease patients of different ethnic groups treated with clopidogrel. Am J Cardiol. 2012, 110 (4): 502-508.View ArticlePubMedGoogle Scholar
- Yamamoto K, Hokimoto S, Chitose T, Morita K, Ono T, Kaikita K, Tsujita K, Abe T, Deguchi M, Miyagawa H, Saruwatari J, Sumida H, Sugiyama S, Nakagawa K: Impact of CYP2C19 polymorphism on residual platelet reactivity in patients with coronary heart disease during antiplatelet therapy. J Cardiol. 2011, 57 (2): 194-201.View ArticlePubMedGoogle Scholar
- Simon T, Verstuyft C, Mary-Krause M, Quteineh L, Drouet E, Meneveau N, Steg G, Ferrieres J, Danchin N, Becquemont L: Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med. 2009, 360: 363-375.View ArticlePubMedGoogle Scholar
- Goldstein LB, Bushnell CD, Adams RJ, Appel LJ, Braun LT, Chaturvedi S, Creager MA, Culebras A, Eckel RH, Hart RG, Hinchey JA, Howard VJ, Jauch EC, Levine SR, Meschia JF, Moore WS, Nixon JV, Pearson TA: Guidelines for the primary prevention of stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2011, 42 (2): 517-584.View ArticlePubMedGoogle Scholar
- Chinese adult dyslipidemia prevention guidelines establish joint committee: Chinese adult dyslipidemia prevention guidelines. Chin J Cardiol. 2007, 35: 390-419. in ChineseGoogle Scholar
- Joint report of the International Society of Cardiology Association/World Health Organization clinical named standardized thematic group: The naming and standards of the diagnosis of ischemic heart disease. Intern J Cardiol. 1979, 6: 365-366. in ChineseGoogle Scholar
- World Health Organization: Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia: report of a WHO/IDF consultation. 2006, Geneva: World Health OrganizationGoogle Scholar
- Marler JR, Tilley BC, Lu M, Brott TG, Lyden PC, Grotta JC, Broderick JP, Levine SR, Frankel MP, Horowitz SH, Haley EC, Lewandowski CA, Kwiatkowski TP: Early stroke treatment associated with better outcome: the NINDS rt-PA stroke study. Neurology. 2000, 55: 1649-1655.View ArticlePubMedGoogle Scholar
- De Morais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA: The major genetic detect responsible for the polymorphism of S-mephenytoin metabolism in humans. J Biol Chem. 1994, 269 (22): 15419-15422.PubMedGoogle Scholar
- De Morais SM, Wilkinson CR, Blaisdell J, Meyer UA, Nakamura K, Goldstein JA: Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese. Mol Pharmacol. 1994, 46 (4): 594-598.PubMedGoogle Scholar
- De Morais SM, Goldstein JA, Xie HG, Huang SL, Lu YQ, Xia ZS, Ile N, Zhou HH: Genetic analysis of the S-mephenytoin polymorphism in a Chinese population. Clin Pharmacol Ther. 1995, 58 (4): 404-411.View ArticlePubMedGoogle Scholar
- Halling J, Petersen MS, Damkier P, Nielsen F, Grandjean P, Weihe P, Lundblad MS, Brosen K: Polymorphism of CYP2D6, CYP2C19, CYP2C9 and CYP2C8 in the Faroese population. Eur J Clin Pharmacol. 2005, 61 (7): 491-497.View ArticlePubMedGoogle Scholar
- Yin SJ, Ni YB, Wang SM, Wang X, Lou YQ, Zhang GL: Differences in genotype and allele frequency distributions of polymorphic drug metabolizing enzymes CYP2C19 and CYP2D6 in mainland Chinese Mongolian, Hui and Han populations. J Clin Pharm Ther. 2012, 37 (3): 364-369.View ArticlePubMedGoogle Scholar
- Ghodke Y, Joshi K, Arya Y, Radkar A, Chiplunkar A, Shintre P, Patwardhan B: Genetic polymorphism of CYP2C19 in Maharashtrian population. Eur J Epidemiol. 2007, 22: 907-915.View ArticlePubMedGoogle Scholar
- Sistonen J, Fuselli S, Palo JU, Chauhan N, Padh H, Sajantila A: Pharmacogenetic variation at CYP2C9, CYP2C19 and CYP2D6 at global and microgeographic scales. Pharmacogenet Genomics. 2009, 19: 170-179.View ArticlePubMedGoogle Scholar
- Chen M, Liu XJ, Yan SD, Peng Y, Chai H, Li Q, Wei JF, Xu YN, Huang DJ: Association between cytochrome P450 2C19 polymorphism and clinical outcomes in Chinese patients with coronary artery disease. Atherosclerosis. 2012, 220 (1): 168-171.View ArticlePubMedGoogle Scholar
- Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS: Cytochrome P-450 Polymorphisms and Response to Clopidogrel. N Engl J Med. 2009, 360 (4): 354-362.View ArticlePubMedGoogle Scholar
- Harmsze AM, van Werkum JW, Bouman HJ, Ruven HJ, Breet NJ, Ten Berg JM, Hackeng CM, Tjoeng MM, Klungel OH, de Boer A, Deneer VH: Besides CYP2C19*2, the variant allele CYP2C9*3 is associated with higher on-clopidogrel platelet reactivity in patients on dual antiplatelet therapy undergoing elective coronary stent implantation. Pharmacogenet Genomics. 2010, 20 (1): 18-25.View ArticlePubMedGoogle Scholar
- Lee JB, Lee KA, Lee KY: Cytochrome P450 2C19 polymorphism is associated with reduced clopidogrel response in cerebrovascular disease. Yonsei Med J. 2011, 52 (5): 734-738.View ArticlePubMedPubMed CentralGoogle Scholar
- Subraja K, Dkhar SA, Priyadharsini R, Ravindra BK, Shewade DG, Satheesh S, Sridhar MG, Narayan SK, Adithan C: Genetic polymorphisms of CYP2C19 influences the response to clopidogrel in ischemic heart disease patients in the South Indian Tamilian population. Eur J Clin Pharmacol. 2013, 69 (3): 415-422.View ArticlePubMedGoogle Scholar
- Hwang SJ, Jeong YH, Kim IS, Koh JS, Kang MK, Park Y, Kwak CH, Hwang JY: The cytochrome 2C19*2 and *3 alleles attenuate response to clopidogrel similarly in East Asian patients undergoing elective percutaneous coronary intervention. Thromb Res. 2011, 127 (1): 23-28.View ArticlePubMedGoogle Scholar
- Kim IS, Choi BR, Jeong YH, Kwak CH, Kim S: The CYP2C19*2 and CYP2C19*3 polymorphisms are associated with high post-treatment platelet reactivity in Asian patients with acute coronary syndrome. J Thromb Haemost. 2009, 7 (5): 897-899.View ArticlePubMedGoogle Scholar
- Lee JM, Park S, Shin DJ, Choi D, Shim CY, Ko YG, Kim JS, Shin ES, Chang CW, Lee JE, Jang Y: Relation of genetic polymorphisms in the cytochrome P450 gene with clopidogrel resistance after drug-eluting stent implantation in Koreans. Am J Cardiol. 2009, 104 (1): 46-51.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/83/prepub
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