Background Although East Asians carry the cytochrome P450 (CYP) 2C19*2 allele more frequently than do Caucasians, the impact of the CYP2C19*2 allele on clopidogrel pharmacodynamics and clinical outcomes is unknown.
Objective To evaluate the effect of CYP2C19 variants on clopidogrel pharmacodynamics and long-term prognosis in East Asian patients with drug-eluting stents (DES).
Methods DES-treated patients taking dual antiplatelet therapy were enrolled from a Korean multicentre genetic registry. The CYP2C19*2 allele was genotyped using the Taqman method (n=2146), and on-treatment platelet reactivity was measured with the VerifyNow P2Y12 assay (n=1415).
Results 1011 patients (47%) carried at least one CYP2C19*2 allele. The mean on-treatment platelet reactivity was significantly higher in carriers than in non-carriers (250±76 vs 231±83 P2Y12 reaction unit, p<0.001). For up to 12 months' follow-up, the composite of cardiovascular death, non-fatal myocardial infarction and stent thrombosis was significantly higher in carriers of the CYP2C19*2 allele than non-carriers (2.0% vs 0.8%, p=0.02). On landmark analysis, there was no difference in clinical outcome after 12 months between the groups.
Conclusion The CYP2C19*2 genetic variant may be associated with worse outcome in Korean patients treated exclusively with DES and dual-antiplatelet therapy due to a significant increase in cardiac death, myocardial infarction or stent thrombosis.
- CYP2C19 loss of function alleles
- cardiovascular events
- antiplatelet treatment
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Clopidogrel is a prodrug that is converted to an active metabolite by the cytochrome P450 (CYP) system.1 The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function and thus a decreased antiplatelet effect of clopidogrel.2 Specifically, in patients who are carriers of the CYP2C19*2 allele, the conversion of clopidogrel to its active metabolite may be reduced, resulting in decreased response to clopidogrel treatment and worse cardiovascular outcome.3 4 Although a couple of studies have shown that the CYP loss-of-function (LOF) polymorphism may be associated with hard outcome in patients receiving percutaneous coronary intervention (PCI) in general,5–7 the effect of the LOF allele on outcome in a cohort of patients exclusively treated with drug-eluting stents (DES), for whom long-term use of clopidogrel is critical, is unknown.
In addition the frequency of the CYP2C19*2 mutation is known to vary considerably according to ethnicity. Its frequency has been reported to be higher (30%) in Asians compared with healthy Caucasians (∼15%).8 In those with coronary artery disease, its frequency was 28–29% and 45% in Caucasians and Asians, respectively.4 9 10 Previous studies showing an association between CYP2C19*2 LOF and cardiac outcome were all performed in Western populations and it is unknown whether a similar association exists in Asians.
In this study, we evaluated whether the CYP2C19*2 polymorphism was associated with cardiac events up to 1 year post-PCI in a large multicentre Korean cohort of patients exclusively treated with DES.
Between March 2003 and June 2009, 3321 patients who were treated with PCI and agreed to genetic analysis were enrolled in the SKY registry (Seoul National University, G[K] yeongsang National University and Yonsei University). This multicentre registry was designed in 2009; registries from three hospitals were combined to identify classic and unusual risk factors, as well as genetic factors affecting complex phenotypes of coronary thrombosis. For the present analysis, patients treated with anything other than a DES and those that received additional antiplatelet agents in addition to conventional aspirin and clopidogrel (ie, cilostazol) were excluded.
Procedural anticoagulation was achieved with unfractionated heparin as per standard of care, with glycoprotein IIb/IIIa inhibitors used per operator discretion. It is recommended to administer aspirin 300 mg at least 24 h before the index PCI if the patient was not taking aspirin prior to admission. It will be recommended that patients receive an oral 300 mg or 600 mg loading dose of clopidogrel before PCI if the patient was not taking clopidogrel prior to admission. The protocol recommended use of aspirin 100 mg daily indefinitely and clopidogrel 75 mg daily for a minimum of 6 months; a longer duration of clopidogrel use was permitted at the discretion of the treating physicians.
Follow-up was every 3 months, with a detailed report of drug treatment. All clinical events were adjudicated by two independent cardiologists who were blinded to the genetic analysis.
The genotyping of CYP2C19*2 (681G>A; rs4244285) was done using the TaqMan fluorogenic 5′-nuclease assay (ABI, Foster City, California, USA). The final volume of PCR was 5 μl, containing 10 ng of genomic DNA and 2.5 μl TaqMan Universal PCR Master Mix, with 0.13 μl of 40× Assay Mix. Thermal cycle conditions were as follows: 50°C for 2 min to activate the uracil N-glycosylase and to prevent carry-over contamination, 95°C for 10 min to activate the DNA polymerase, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. All PCR were performed using 384-well plates by a Dual 384-Well GeneAmp PCR System 9700 (ABI) and the endpoint fluorescent readings were performed on an ABI PRISM 7900 HT Sequence Detection System (ABI). Duplicate samples and negative controls were included to ensure accuracy of genotyping.
Platelet function test
A blood sample was obtained 12–24 h after clopidogrel loading with whole blood anticoagulated in a sodium citrate bottle exclusively used for the VerifyNow P2Y12 assay. On-treatment platelet reactivity (OPR) after clopidogrel treatment was measured using the VerifyNow P2Y12 assay (Accumetrics, San Diego, California, USA). The instrument measures the changes in light transmission throughout the sample. Technical details and reliability of the test have been reported previously.11 12 The VerifyNow P2Y12 assay reports results as P2Y12 reactivity units (PRU), % inhibition and BASE. Per cent inhibition of the P2Y12 receptor is calculated as [1 – (PRU/BASE)] × 100. High OPR (HOPR) was defined as OPR >240 PRU, as previously reported.13 14
Outcomes and definitions
The primary analysis endpoint was major adverse cardiovascular events (MACE) and the composite hard outcome. MACE was defined as the composite of cardiovascular death, non-fatal myocardial infarction (MI) and repeat revascularisation (including target lesion and target vessel revascularisations), and the composite hard outcome as cardiovascular death, non-fatal MI and stent thrombosis. All deaths were considered as cardiovascular deaths unless a clear non-cardiovascular cause was demonstrated. MI was defined as a recent ischaemic symptom with ECG abnormalities in the ST segment (depression or elevation of at least 0.1 mV) and a positive troponin concentration as defined locally. Repeat revascularisation was defined as any repeat percutaneous intervention or surgical bypass of any vessel. Stent thrombosis was defined as definite or probable ST according to the Academic Research Consortium criteria.15
The baseline characteristics of the patients were compared according to genotype with the t test or one-way ANOVA for continuous variables as appropriate and the χ2 test for categorical variables. The cumulative incidence was analysed from the time of stent implantation to the first event according to the Kaplan–Meier method, and the difference was evaluated by the log-rank test.
Adjusted HRs were calculated with the multivariable Cox model entering eight clinical variables commonly associated with stent thrombosis (age, hypertension, diabetes, dyslipidaemia, chronic kidney disease, sum of length of DES, use of proton-pump inhibitor and smoking) and the CYP2C19*2 carrier status. SPSS V.17.0 was used for all statistical analyses; p<0.05 was considered statistically significant.
The study profile is shown in figure 1 and the baseline clinical, angiographic and procedural characteristics of the patients included in the analysis are shown in table 1 according to the carrier status of CYP2C19*2 allele. We noted no deviation from the expected proportions of genotypes in the population predicted by the Hardy–Weinberg equilibrium for polymorphisms (χ2=1.1, p=0.58). The mean age of the patients was 60.8±9.8 years, and 66% of the patients were male. The clinical characteristics were similar between the two groups. Statins and β-blockers were used by 1836 (86%) and 1568 (73%) patients, respectively, while proton-pump inhibitors and vitamin K antagonists were only rarely used in both groups. Clopidogrel loading was done in 1375 (64%) patients before PCI; the others were mostly those on chronic clopidogrel therapy. There was no difference between CYP2C19 carriers versus non-carriers regarding the timing of clopidogrel loading relative to PCI. The mean duration of clopidogrel treatment was 11.4±5.2 months in the non-carrier group and 11.2±5.1 months in the carrier group (p=0.21). A total of 2061 patients (96.0%) were on maintenance clopidogrel therapy at 3 months post-procedure, and 1887 patients (87.9%) were on maintenance therapy at 6 months follow-up.
CYP2C19*2 genotype status and OPR
Among 2146 patients, OPR was measured in 1415 (66%), using the VerifyNow P2Y12 system. OPR significantly increased as the number of CYP2C19*2 alleles increased (r=0.13, p<0.001). Mean OPR was 231±82, 248±78 and 261±71 PRU in the groups with 0, 1 and 2 CYP2C19*2 alleles, respectively (figure 2). When divided between carriers and non-carriers of CYP2C19*2, mean OPR was significantly higher in carriers compared with non-carriers (250±77 vs 231±82 PRU, p<0.001). Among 1415 patients, 726 (51.3%) patients were classified as high OPR. The frequency of high OPR was significantly higher in carriers than in non-carriers of CYP2C19*2 (56% vs 47%, p<0.001). In the limited number of patients that had OPR measured, there was a mild trend towards a higher risk of clinical events in the HOPR group compared with the not-HOPR group, which did not reach statistical significance (see supplementary online table).
CYP2C19*2 genotype status and clinical outcome
Table 2 shows the association between carrier status of the CYP2C19*2 allele and various clinical outcomes up to 1 year. There was no significant association between carrier status and MACE (MACE rate: 10.7% vs 8.8% for carriers vs non-carriers, RR 1.24, 95% CI 0.93 to 1.65, p=0.14). In contrast, the rate of composite hard outcome was significantly higher in the carrier group compared with the non-carrier group (2.3% vs 0.9%, HR 2.62, 95% CI 1.24 to 5.53, p=0.01). The rate of composite hard outcome was higher in the homozygote carrier group compared with the heterozygote carrier group without statistical significance (2.9 vs 2.2%, p=0.55). All components constituting the composite hard outcome were numerically higher in carriers compared with non-carriers of the CYP2C19*2 allele. Figure 3 shows the Kaplan–Meier curves for the cumulative rates of MACE (figure 3A,B) and composite hard outcome (figure 3C,D) for the entire follow-up period and from 1 year onward. There was no association between the carrier status of the CYP2C19*2 allele and MACE in both the entire analysis and the 1-year landmark analysis. With regard to the composite hard outcome, the CYP2C19*2 allele carrier status was associated with worse outcome only up to 1-year clinical follow-up. In the landmark analysis from 1-year post PCI, the carrier status was not associated with increased composite hard outcome (figure 3D). Most of the curve separation occurred early within the first months (figure 3C).
On multivariate analysis to find independent predictors of the composite hard outcome, carrier status of the CYP2C19*2 allele, dyslipidaemia and chronic kidney disease were independent predictors of the composite hard outcome (table 3).
In this analysis of a large multicentre registry of patients receiving PCI with exclusively DES, we found that CYP2C19*2 polymorphism is significantly associated with both high OPR and hard outcome in Korean patients. On landmark analysis, we found that the association no longer exists after 1 year, during which time most patients discontinued clopidogrel.
The association of CYP2C19 polymorphism with OPR and clinical outcome
CYP2C19 gene polymorphism is known to be associated with decreased clopidogrel active metabolites and thus reduced response to clopidogrel in healthy people and in patients with coronary artery disease.7 16 17 Accordingly, the number of CYP2C19*2 alleles was associated with OPR. Furthermore, we recently reported that CYP2C19 LOF allele was associated with increased OPR in those receiving dual antiplatelet therapy but not in those receiving dual antiplatelet therapy plus additional cilostazol.18 In the present study, the carriers of the CYP2C19*2 allele had significantly higher mean OPR compared with non-carriers. Furthermore, the *2 allele carriers were at significantly increased risk of the composite hard outcomes compared with non-carriers, while the total number of MACE, which included repeat revascularisations, was not statistically different between the two groups. This result in Koreans is again in accord with previous reports that showed a significant association between the CYP2C19*2 variant and clinical outcome.3 6 10 19 Sawada et al reported that a subclinical intra-stent thrombus was detected more frequently in CYP2C19*2 carriers than in non-carriers.20 This result was consistent with our finding that CYP2C19*2 carriers were mostly at increased risk of thrombotic events such as stent thrombosis and MI.
Another interesting finding was the landmark analysis performed from 1 year. The median duration of clopidogrel treatment was 11.6 months (IQR 7.4–13.1) and the mean duration was 11.3 months. What was interesting was that most of the separation in the Kaplan–Meier curves for the composite hard outcome occurred within the first couple of months, where the role of platelets is critical. We therefore performed a landmark analysis from 1 year and found that the association between carrier status of the *2 variant and hard outcomes was valid only within the first year, and that there were no significant differences between carriers and non-carriers after 1 year. These results suggest that the harmful effect of the *2 variant is valid only while clopidogrel is used.
Racial differences in the incidence of CYP2C19*2 genetic variant
Previous studies have demonstrated that carriers of the LOF CYP2C19 allele have significantly lower levels of the active metabolite of clopidogrel, diminished platelet inhibition, and subsequently increased rates of cardiovascular events.3 4 21 22 There seems to be quite a variation in the frequency of the CYP2C19*2 polymorphism according to ethnicity. Its frequency was reported to be higher in East Asians and African-Americans than in Western populations.8 Collins et al showed that although African-Americans were more compliant with clopidogrel, they had a higher rate of DES stent thrombosis,23 which the authors attributed to the higher rate of CYP2C19*2 polymorphism in African-Americans. In the present study, the frequency of CYP2C19*2 carriers in Koreans was 47%, which is higher than that reported in Western populations. What is interesting is that although the rate *2 variant is higher in the Korean population in general, the 1 year clinical event rate post-PCI is more or less similar, if not lower, in Koreans compared with Western populations.24 25 Therefore the association of CYP2C19*2 variant and clinical outcome seems to hold true in Koreans (within the same ethnicity), but the ethnic difference of why the frequency is higher in Koreans but the event rate similar to Western data needs to be evaluated in studies that enrol a robust number of both Asians and Western patients. Inter-ethnically, there may be a slight difference in the degree (strength) of association between the variant and outcome. Another possibility is that there may be other unknown or unexplained protective factors in Koreans that may negate the effects of the *2 variant.
This study has some limitations. First, OPR was not measured at exactly the same time point. Therefore, the time points might affect the value of OPR. However, the difference of OPR according to CYP2C19*2 variation was similar regardless of the time of sampling post-clopidogrel loading. Second, OPR measurement was only available in 1415 (66%) of the 2146 patients available for genotyping. Although the rate of the composite hard outcome was numerically higher in the HOPR group, this did not reach statistical significance. The small number of patients whose OPR was measured could have resulted in diminished statistical power. However, it is important to note that the rates of clinical events were numerically higher in the HOPR group regardless of the event (non-fatal MI, stent thrombosis, composite hard outcome). Third, there are other genetic variants of CYP2C19 that could possibly alter the response to clopidogrel, such as *3, *5 and *17, but only the CYP2C19*2 was addressed in this paper. However, up to now, only the *2 allele has been repeatedly shown to be associated with platelet reactivity and more importantly clinical outcome in Western populations.
In conclusion, the CYP2C19*2 genetic variant may be associated with worse outcome in Korean patients treated exclusively with DES and dual-antiplatelet therapy due to a significant increase in cardiac death, MI or stent thrombosis.
Linked article 227652.
Il-Young Oh and Kyung Woo Park contributed equally to this study.
Funding This study was supported by grants from the Korean Society of Interventional Cardiology (2010), from the Clinical Research Center for Ischemic Heart Disease (A040152), and from the Innovative Research Institute for Cell Therapy, Seoul National University Hospital (A062260), sponsored by the Ministry of Health, Welfare & Family, Korea. Dr. Hyo-Soo Kim is also a professor of Word Class University Program, Molecular Medicine and Biopharmaceutical Sciences, Seoul National University sponsored by the Ministry of Education, Science & Technology, Korea.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Seoul National University Hospital Institutes for Biomedical Research.
Provenance and peer review Not commissioned; externally peer reviewed.
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