Association of Cytochrome P450 2C19 Genotype With the Antiplatelet Effect and Clinical Efficacy of Clopidogrel Therapy ORIGINAL CONTRIBUTION Association of Cytochrome P450 2C19 Genotype With the Antiplatelet Effect and Clinical Efficacy of Clopidogrel Therapy Alan R. Shuldiner, MD Jeffrey R. O’Connell, DPhil Kevin P. Bliden, BS Amish Gandhi, MD Kathleen Ryan, MPH Richard B. Horenstein, MD Coleen M. Damcott, PhD Ruth Pakyz, BS Udaya S. Tantry, PhD Quince Gibson, MBA Toni I. Pollin, PhD Wendy Post, MD, MS Afshin Parsa, MD Braxton D. Mitchell, PhD Nauder Faraday, MD William Herzog, MD Paul A. Gurbel, MD D UAL ANTIPLATELET THERAPY, including clopidogrel and aspirin, inhibits platelet function, preventing ische- mic events and improving outcomes fol- lowing acute coronary syndromes and percutaneous coronary intervention (PCI).1,2 To exert an antiplatelet effect, clopidogrel requires conversion to an active thiol metabolite (SR 26334) by hepatic cytochrome P450 (CYP) iso- enzymes, which inhibit adenosine di- phosphate (ADP)–stimulated platelet For editorial comment see p 896. Author Affiliations: Division of Endocrinology, Dia- betes and Nutrition, Department of Medicine (Drs Shuldiner, O’Connell, Gandhi, Horenstein, Damcott, Pollin, and Mitchell, Mss Ryan and Pakyz, and Mr Gib- son) and Division of Nephrology, Department of Medi- cine (Dr Parsa), University of Maryland School of Medi- cine, Baltimore; Geriatric Research and Education Clinical Center, Veterans Administration Medical Cen- ter, Baltimore (Dr Shuldiner); Sinai Center for Throm- bosis Research, Sinai Hospital of Baltimore, Baltimore (Mr Bliden and Drs Tantry, Herzog, and Gurbel); Di- vision of Cardiology, Department of Medicine (Drs Post and Herzog); and Department of Anesthesiology and Critical Care Medicine (Dr Faraday), Johns Hopkins Uni- versity School of Medicine, Baltimore. Corresponding Author: Alan R. Shuldiner, MD, Divi- sion of Endocrinology, Diabetes and Nutrition, Uni- versity of Maryland School of Medicine, 660 W Red- wood St, Room 494, Baltimore, MD 21201 (ashuldin @medicine.umaryland.edu). Context Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)–dependent platelet activation. However, nonrespon- siveness is widely recognized and is related to recurrent ischemic events. Objective To identify gene variants that influence clopidogrel response. Design, Setting, and Participants In the Pharmacogenomics of Antiplatelet In- tervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to 429 healthy Amish persons and measured response by ex vivo platelet aggregometry. A genome-wide association study was performed followed by genotyping the loss-of- function cytochrome P450 (CYP) 2C19*2 variant (rs4244285). Findings in the PAPI Study were extended by examining the relation of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an independent sample of 227 patients un- dergoing percutaneous coronary intervention. Main Outcome Measure ADP-stimulated platelet aggregation in response to clo- pidogrel treatment and cardiovascular events. Results Platelet response to clopidogrel was highly heritable (h2= 0.73; P � .001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the CYP2C18–CYP2C19–CYP2C9–CYP2C8 cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (P = 1.5 � 10−13 for rs12777823, additive model). The rs12777823 polymorphism was in strong linkage disequilibrium with the CYP2C19*2 variant, and was associated with diminished clopidogrel response, accounting for 12% of the variation in platelet aggregation to ADP (P = 4.3 � 10−11). The relation between CYP2C19*2 genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (P = .02). Furthermore, patients with the CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95% confidence interval, 1.18-4.99; P = .02). Conclusion CYP2C19*2 genotype was associated with diminished platelet re- sponse to clopidogrel treatment and poorer cardiovascular outcomes. JAMA. 2009;302(8):849-858 www.jama.com ©2009 American Medical Association. All rights reserved. (Reprinted) JAMA, August 26, 2009—Vol 302, No. 8 849 Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 activation by irreversibly binding to platelet P2Y12 receptors. 3-5 Variability in clopidogrel response is well established.6-8 Patients treated with clopidogrel who demonstrate higher ex vivo platelet reactivity are at increased risk of ischemic events.9-12 Variation in platelet function in response to clopi- dogrel has been associated with lipo- philic statins, calcium channel block- ers, proton pump inhibitors, St John’s wort, and smoking.13-16 However, these factors account for only a small frac- tion of the variation in response. Re- cently, the loss-of-function CYP2C19 (GenBank 1557) *2 allele has been shown to be associated with a de- creased activation of clopidogrel17,18 and antiplatelet effect19-23 and with in- creased cardiovascular events in pa- tients receiving clopidogrel.18,24-26 To identify genes associated with variation in clopidogrel response, we performed a genome-wide association study of ADP-stimulated platelet aggregation in response to clopidogrel in the Old Order Amish, a relatively homogeneous founder population in which confounding factors, including medication usage and lifestyle variabil- ity, are minimized. Replication and extension of genetic findings and time-to-event analyses were performed in a population with high risk for car- diovascular disease recruited from a cardiac catheterization laboratory in Baltimore, Maryland. METHODS Study Populations Amish Pharmacogenomics of Anti- platelet Intervention Study. The Amish Pharmacogenomics of Anti- platelet Intervention (PAPI) Study (NCT0079936) recruited 429 gener- ally healthy white participants 20 years or older between August 2006 and October 2008 (for additional details, see eMethods at http://www.jama .org). These individuals comprised a number of relative pairs informative for estimating heritabilities, including 105 parent-offspring pairs, 175 sibling pairs, 1 grandparent-grandchild pair, 48 avun- cular pairs, and 12 first-cousin pairs. Medical and family histories, anthro- pometry, physical examinations, and blood samples after an overnight fast were obtained. Complete blood count with platelet number and levels of se- rum lipids (total cholesterol, high- density lipoprotein cholesterol, and tri- glycerides) were assayed by Quest Diagnostics (Horsham, Pennsylvania); levels of low-density lipoprotein cho- lesterol were calculated using the Friede- wald equation. After baseline platelet aggregation measurements were obtained, partici- pants were given a 300-mg oral load- ing dose of clopidogrel followed by 75 mg per day for 6 days. Follow-up platelet aggregation studies were repeated 1 hour following the last dose of clopidogrel. A second follow-up platelet aggregation measurement was made later the same day, 1 hour after oral ingestion of 324 mg of chewable aspirin. Platelet function was assessed by optical aggregometry with a PAP8E Aggregometer (Bio/Data Corporation, Horsham, Pennsylvania) in platelet- rich plasma, stimulated with ADP (20 µmol/L) or arachidonic acid (1.6 mmol/L) (eMethods). Sinai Hospital of Baltimore Study Patients. The Sinai Hospital of Balti- more Study (NCT00370045) enrolled 227 patients older than 18 years and undergoing nonemergent PCI between January 2004 and May 2007 (eMeth- ods). Of these, 140 (61.7%) were w h i t e , 8 3 ( 3 6 . 6 % ) w e re A f r i c a n American, and 4 (1.8%) were other race/ethnicity. Information on race/ ethnicity was obtained by self-report. On the day of PCI, patients received a 600-mg (n = 112) or 300-mg (n = 25) clopidogrel loading dose; 90 were already receiving maintenance therapy with a 75-mg daily dose at the time of PCI and received no loading dose. There were no differences in baseline characteristics or in the long-term out- comes investigated in stratified analy- ses of acute clopidogrel dosing; thus, these groups were combined for fur- ther analyses. Patients received bivalirudin or hep- arin therapy, either with (n = 107) or without (n = 120) eptifibatide.27,28 An- ticoagulant therapy was discontinued at the completion of the procedure in all patients. All patients received 81 to 325 mg of aspirin daily for at least 1 week prior to PCI and 325 mg on the day of the procedure. To minimize the effects of acute anticoagulant therapy during PCI, platelet function was mea- sured on the day of hospital discharge in patients not treated with eptifi- batide or 5 days or more postdis- c h a rg e i n p a t i e n t s t re a t e d w i t h eptifibatide. In total, results of baseline platelet function studies were available for 143 patients not taking clopidogrel at the time of enrollment, and results of postclopidogrel platelet aggregation s t u d i e s w e r e a v a i l a b l e f o r 1 8 8 patients. Platelet aggregation was assessed in platelet-rich plasma after stimulation with 20 µmol/L of ADP or 2 mmol/L of arachidonic acid using a C h ro n o l o g L u m i - A g g re g o m e t e r (Model 490-4D; Chronolog, Haver- town, Pennsylvania), as described previously (eMethods).7 Aspirin (325 mg/d) and clopidogrel (75 mg/d) were prescribed for all patients at the time of hospital dis- charge, according to American Col- lege of Cardiology/American Heart Association guidelines.1 We assessed medication adherence by self-report and by review of source documents from hospitalizations for ischemic events. The 227 enrolled patients were con- tacted at the end of 1 and 12 months post-PCI to determine the occurrence of postdischarge cardiovascular ische- mic events (eMethods). Of these pa- tients, 95 were still taking clopidogrel after 1 year; 132 were not. A physi- cian, blinded to the study results of the patient, adjudicated all end points through review of source documents obtained from medical records. Post- discharge ischemic events were de- fined as myocardial infarction (the oc- currence of ischemic symptoms and a troponin I value greater than the up- per limits of normal), ischemic stroke, stent thrombosis (definite stent throm- CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY 850 JAMA, August 26, 2009—Vol 302, No. 8 (Reprinted) ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 bosis according to Academic Research Consortium criteria29), unplanned tar- get vessel revascularization (revascu- larization of vessel treated at time of study enrollment), unplanned nontar- get vessel revascularization (revascu- larization of a vessel different from that treated at time of study enrollment), hospitalization for coronary ischemia without revascularization (hospitaliza- tion for chest pain with evidence of is- chemia on electrocardiogram and no evidence of myocardial infarction as measured by troponin I value), and death secondary to any cardiovascular cause. The study protocols were approved by the respective institutional review boards at the University of Maryland and Sinai Hospital of Baltimore. Writ- ten informed consent was obtained from each participant; participants were compensated for their participation. Genotype Analysis Genotyping in the PAPI Study was per- formed with the Affymetrix GeneChip Human Mapping 500K or 1 M (ver- sion 6.0) arrays according to the manu- facturer’s instructions (Affymetrix Inc, Santa Clara, California). Genotype calls were performed using BRLMM (500K array) or Birdseed version 2 (1 M ar- ray). Single-nucleotide polymor- phisms (SNPs) present on both arrays with a minor allele frequency greater than 1% were included in our analy- ses. The mean genotype call rate of these 400 230 SNPs was 98.7%. All SNPs within the region of interest on chromosome 10q24 were in Hardy- Weinberg equilibrium (P � .05), ex- cept rs12572351 (P = 1.89 � 10−5) and rs2860838 (P = 1.03 � 10 − 4 ). Fol- low-up genotyping of the known com- mon loss-of-function CYP2C19*2 vari- ant (rs4244285), as well as other functional variants of CYP2C19 (*3 [rs4986893], *5 [rs56337013], and *17 [rs12248560]) was performed using TaqMan SNP genotyping assays (Ap- plied Biosystems, Foster City, Califor- nia). The genotype concordance rate was greater than 98% in a subset of du- plicate samples. Statistical Analysis Summary statistics (eg, means [SDs]) and frequencies for the Amish PAPI and Sinai Hospital of Baltimore popula- tions were generated using SAS ver- sion 9.1 (SAS Institute Inc, Cary, North Carolina). For both studies, platelet ag- gregation was expressed as the maxi- mum percentage change in light trans- mittance, using platelet-poor plasma as a referent. Amish PAPI Study We assessed the correlates (eg, age, sex, body mass index [BMI], lipid levels, and blood pressure) of clopidogrel re- sponse using a regression-based ap- proach as implemented in the SOLAR version 4.07 (Southwest Foundation for Biomedical Research, San Antonio, Texas),30 in which we accounted for re- latedness among study participants by including a polygenic component as a random effect. Triglyceride levels were logarithm-transformed for analysis and back-transformed for presentation. Dis- tribution analyses were generated in SAS. All statistical tests were 2-sided. Association analyses between SNPs and ADP-stimulated platelet aggrega- tion following clopidogrel administra- tion were performed under a variance component model that assesses the effect of genotype as an additive effect on the quantitative trait, while simultaneously estimating the effects of age, age2, sex, preclopidogrel platelet aggregation, and the aforementioned polygenic component. The polygenic component was mod- eled using the relationship matrix de- rived from the complete Amish pedi- gree structure available through published genealogical records main- tained by the church.31 The heritabil- ity of baseline platelet aggregation and clopidogrel response corresponds to the proportion of the trait variance ac- counted for by the polygenic compo- nent. The genomic control � coeffi- cient was 1.03; thus, the P values reported are unadjusted. A power calculation indicated 80% power to detect SNPs with allele fre- quencies of 0.2 to 0.4 in the initial sample (n = 429), accounting for 8% to 9% of phenotypic variation at � = 10−7. To determine whether the loss-of- function CYP2C19*2 variant could account for the chromosome 10q24 association signal, we estimated the in- dependent effects of both rs12777823— the most highly associated SNP from the genome-wide association analysis— and the CYP2C19*2 variant on plate- let aggregation by including both in the model simultaneously. Pairwise link- age disequilibrium correlation statis- tics (|D’| and r2) were computed using Haploview (http://www.broad.mit .edu). Sinai Hospital of Baltimore Study We estimated the effect of CYP2C19*2 genotype on preclopidogrel and post- clopidogrel ADP-stimulated platelet ag- gregation under an additive genetic model by classifying participants ac- cording to whether they had 0, 1, or 2 risk alleles. The genotype effect was es- timated using analysis of variance with adjustment for age, sex, race, study (Peri-Procedural Myocardial Infarc- tion, Platelet Reactivity, Thrombin Gen- eration, and Clot Strength: Differen- tial Effects of Eptifibatide � Bivalirudin vs Bivalirudin study; Clopidogrel Loading With Eptifibatide to Arrest the Reactivity of Platelets [CLEAR PLATELETS–1] Study), and treat- ment group (clopidogrel dose and use of eptifibatide). We similarly compared platelet ag- gregation values between participants who did and did not experience an event while taking clopidogrel. Lastly, we constructed survival curves to com- pare 1-year cardiovascular event-free survival between participants with ( n = 6 7 ) a n d w i t h o u t ( n = 1 6 0 ) a CYP2C19*2 risk allele. We analyzed the effect of the allele on outcomes in re- lation to ongoing clopidogrel therapy at the time of the event. We used the proportional hazards model to esti- mate the relative hazard associated with having a risk allele on subsequent is- chemic event rates, adjusting for age, sex, and race. This analysis was car- ried out both with and without ADP- CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY ©2009 American Medical Association. All rights reserved. (Reprinted) JAMA, August 26, 2009—Vol 302, No. 8 851 Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 stimulated aggregation included as a mediator variable. S u r v i v a l a n a l y s i s s t r a t i f i e d b y CYP2C19*2 genotype was also per- formed in the subset of 95 patients still taking clopidogrel at the time of event or at 1 year of follow-up and the 132 patients who were not receiving clopi- dogrel at the time of event or at 1 year of follow-up. RESULTS Amish PAPI Study By design, Amish PAPI participants were generally healthy (TABLE 1). There was wide interindividual variability in ADP-stimulated platelet aggregation at baseline and after clopidogrel admin- istration, with no clear cutoff to de- fine resistance (FIGURE 1). Poorer clopidogrel response, as defined by ADP-stimulated aggregation after 7 days of clopidogrel administration, was as- sociated with increasing age (3.8% of variance; 95% confidence interval [CI], 3.6%-4.1%; P � .001), greater BMI (2.3% of variance; 95% CI, 2.2%- 2.4%; P = .005), higher triglyceride lev- els (1.3% of variance; 95% CI, 1.28%- 1.35%; P = .01), and nominally lower levels of high-density lipoprotein cho- lesterol (1.0% of variance; 95% CI, 0.98%-1.03%; P = .04) (TABLE 2). The variation explained by these variables combined was less than 10%. The heri- tability of ADP-stimulated platelet ag- gregation at baseline and in response to clopidogrel was 0.33 (SE, 0.13; 95% CI, 0.08-0.58; P = .005) and 0.73 (SE, 0.12; 95% CI, 0.49-0.97; P � .001), re- spectively, suggesting a substantial ge- netic component. A genome-wide association analysis revealed a cluster of 13 SNPs spanning 1.5 megabases on chromosome 10q24, showing strong evidence for associa- tion with clopidogrel response, with P values �10−7 (FIGURE 2 and eTable 1; Q-Q plot shown in eFigure). These SNPs were in strong linkage disequi- librium with each other, eg, pairwise r2= 0.35 to 0.99 with rs12777823, the most significantly associated SNP ( P = 1 . 5 � 1 0 − 1 3 f o r t h e a d d i t i v e model). The 9 participants homo- z y g o u s f o r t h e m i n o r a l l e l e o f rs12777823 had the poorest response to clopidogrel, while the 300 homo- zygous for the major allele had the best response; the 117 heterozygous p a r t i c i p a n t s w e r e i n t e r m e d i a t e between the 2 homozygous groups. None of the chromosome 10q24 SNPs associated with platelet aggre- gation after clopidogrel administra- tion was associated with baseline platelet aggregation measures, con- sistent with this locus as a true de- terminant of clopidogrel response. No other genomic region revealed a s s o c i a t i o n s i g n a l s t h a t m e t o r exceeded genome-wide significance ( P � 1 . 0 � 1 0 − 7 ) ( F i g u r e 2 a n d eTable 1). The full results of the genome-wide association study are a v a i l a b l e a t h t t p : / / m e d s c h o o l . u m a r y l a n d . e d u / e n d o c r i n o l o g y /supplementalInfo.asp. The cluster of SNPs on chromo- some 10q24 most significantly associ- ated with clopidogrel response is lo- cated within and immediately flanking the CYP2C18–CYP2C19–CYP2C9– CYP2C8 gene cluster, which encodes a group of cytochrome P450 enzymes t h a t p l a y a n i m p o r t a n t r o l e i n drug metabolism, including conver- Table 1. Characteristics of Amish PAPI Study and Sinai Hospital of Baltimore Study Participants Characteristic, Units Amish PAPI a Sinai Hospital of Baltimore Men Women Men Women No. (%) 214 (49.9) 215 (50.1) 136 (59.9) 91 (40.1) Age, mean (SD), y 43.9 (13.2) 47.3 (14.4) 62.5 (11.4) 67.0 (11.0) White, No. (%) 214 (100) 215 (100) 93 (68.4) 47 (51.6) Body mass index, mean (SD) b 26.0 (3.7) 28.3 (5.6) 30.1 (6.3) 30.9 (7.1) Blood pressure, mean (SD), mm Hg Systolic 116.6 (12.0) 117.4 (14.0) 138.0 (19.9) 144.6 (19.9) Diastolic 70.8 (7.4) 69.7 (7.5) 73.6 (13.8) 70.3 (14.2) Hypertension, No. (%) c 11 (5.1) 13 (6.0) 102 (75.0) 72 (79.1) Lipids, mean (SD), mg/dL Total cholesterol 206.0 (44.8) 217.9 (52.2) NA NA LDL-C 137.7 (41.5) 141.0 (48.9) NA NA HDL-C 54.7 (15.0) 61.6 (15.2) NA NA Triglycerides d 68.1 (40.4) 76.0 (39.4) NA NA Hypercholesterolemia, No. (%) e 52 (24.3) 59 (27.4) 111 (81.6) 72 (79.1) Taking aspirin, No. (%) 6 (2.8) 3 (1.4) 136 (100) 91 (100) Self-reported diabetes, No. (%) 1 (0.5) 2 (0.9) 39 (28.7) 44 (48.4) Hematocrit, mean (SD), % 41.3 (2.4) 37.7 (2.3) 41.8 (5.0) 37.9 (4.5) White blood cell count, median (IQR), �1000/µL 5.8 (5.1-6.7) 5.8 (5.1-6.8) 6.8 (5.7-8.2) 7.5 (6.0-9.2) Platelet count, mean (SD), �100 000/µL 240.9 (43.7) 248.1 (50.3) 223.6 (68.5) 252.2 (71.4) Current smoker, No. (%) f 38 (17.8) 0 40 (29.4) 18 (19.8) Abbreviations: HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; LDL-C, low-density lipoprotein cho- lesterol; NA, not available; PAPI, Pharmacogenomics of Anti-Platelet Intervention. SI conversion factors: To convert HDL-C, LDL-C, and total cholesterol values to mmol/L, multiply by 0.0259; triglyc- eride values to mmol/L, multiply by 0.0113. a For PAPI Study, all participants were withdrawn from prescription and nonprescription medications, vitamins, and supplements 7 days prior to and for the duration of the study. Participants taking prescription antihypertensive, lipid- lowering, and diabetes medications accounted for 0%, 1.6%, and 0% of participants, respectively. b Calculated as weight in kilograms divided by height in meters squared. c Defined as systolic blood pressure greater than 140 mm Hg or diastolic blood pressure greater than 90 mm Hg or taking prescription medication for previously diagnosed hypertension. d Logarithm-transformed for analysis and back-transformed for presentation. e Defined as LDL-C level greater than 160 mg/dL or taking prescription medication for previously diagnosed hyper- cholesterolemia. f Self-reported history of smoking cigarette, pipe, or cigar. CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY 852 JAMA, August 26, 2009—Vol 302, No. 8 (Reprinted) ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 sion of clopidogrel to its active metabo- lite.4,5,17,18,32-35 A guanine�adenine mutation in exon 5 of CYP2C19 ( r s 4 2 4 4 2 8 5 , a l s o k n o w n a s CYP2C19*2) creates an aberrant splice site that leads to an altered reading frame at amino acid 215 and a prema- ture stop codon 20 amino acids down- stream, producing a nonfunctional truncated protein, lack of translation re- sulting from nonsense-mediated mes- senger RNA decay, or both. The frequency of CYP2C19*2 was 0.17 in the Amish PAPI population, similar to that found in other white populations. CYP2C19*2 was in high linkage disequilibrium with the clus- ter of SNPs on chromosome 10q24 identified in the genome-wide asso- c i a t i o n s t u d y ( e g , r 2 = 0 . 8 7 w i t h rs12777823). CYP2C19*2 was associ- ated with ADP-stimulated platelet aggregation after clopidogrel adminis- tration, with a high degree of statisti- cal significance (P = 4.3 � 10−11 for the additive model) (FIGURE 3). ADP- stimulated platelet aggregation was reduced to 40.7%, 47.1%, and 65.4% of baseline in response to clopidogrel i n p a r t i c i p a n t s w i t h 0 , 1 , a n d 2 CYP2C19*2 alleles, respectively. The CYP2C19*2 genotype, present in its heterozygous and homozygous state in 30.8% and 2.1% of the PAPI study population, accounted for 12% of the variation in clopidogrel response. When we included CYP2C19*2 as a covariate, the association between chromosome 10q24 SNP rs12777823 and clopidogrel response was mark- edly attenuated (P = 1.5 � 10−13 with- out adjustment for CYP2C19*2 geno- type to P = 2.5 � 10−3 with adjustment for CYP2C19*2 genotype). These f i n d i n g s s h o w t h a t t h e l o s s - o f - f u n c t i o n C Y P 2 C 1 9 * 2 m u t a t i o n accounts for most or all of the origi- nal 10q24 association signal. Other previously described CYP2C19 loss-of-function variants, CYP2C19*3 and *5, were not polymorphic in the Amish population, but there may be ad- ditional variation in CYP2C19 or a nearby gene, further contributing to relatively small effects on response and accounting for the remainder of the as- sociation signal. The gain-of-function variant CYP2C19*17 had an allele fre- quency of 0.25 and was not associated with ADP-stimulated platelet aggrega- tion, either at baseline or in response to clopidogrel (eTable 2). Predictors (age, BMI, and levels of high-density lipoprotein cholesterol and triglycerides) and heritability (h2= 0.83 [SE, 0.12], P � .001) of ADP- stimulated platelet aggregation dur- ing clopidogrel administration were very similar after a single 324-mg dose of aspirin was added. Addition of aspirin to clopidogrel resulted in potent inhibition of platelet aggrega- tion in response to arachidonic acid, indicating effective inhibition of the cyclooxygenase pathway. CYP2C19*2 genotype had no effect on aspirin- induced inhibition of platelet aggrega- tion of this pathway (eTable 3). How- ever, the association observed between C Y P 2 C 1 9 * 2 g e n o t y p e a n d A D P - stimulated platelet aggregation and clopidogrel persisted after administra- tion of aspirin (P = 7.9 � 10−13), sug- gesting that this drug combination, which is standard of care in patients with acute coronary syndromes and following PCI, does not overcome the Figure 1. Distribution of Adenosine Diphosphate (ADP)–Stimulated (20 µmol/L) Platelet Aggregation Before and After 7 Days of Clopidogrel Administration in 429 Members of the Amish Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study 40 15 10 5 20 25 35 30 0 ADP-Stimulated Platelet Aggregation, % % o f S tu d y G ro u p Postclopidogrel 0 10 20 30 40 50 60 70 80 90 100 40 15 10 5 20 25 35 30 0 ADP-Stimulated Platelet Aggregation, % % o f S tu d y G ro u p Preclopidogrel 0 10 20 30 40 50 60 70 80 90 100 Class intervals include data greater than the lower limit and equal to the upper limit of each interval. Table 2. Multivariate Analysis of Clopidogrel Response as Measured by Adenosine Diphosphate–Stimulated Platelet Aggregation in PAPI Study Participants (n = 429) Characteristic � (SE) P Value a Variance of Significant Predictors, % Age 0.19 (0.04) �.001 3.8 Sex 1.73 (1.15) b .13 Body mass index 0.35 (0.12) .005 2.3 Lipids Total cholesterol 0.01 (0.01) .27 HDL-C −0.08 (0.04) .04 1.0 LDL-C 0.02 (0.01) .17 Log triglycerides 0.03 (0.01) .01 1.3 Blood pressure Systolic 0.0 (0.05) .21 Diastolic 0.07 (0.08) .42 Abbreviations: HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PAPI, Pharma- cogenomics of Anti-Platelet Intervention. a Adjusted for age, sex, and preclopidogrel adenosine diphosphate (20 µmol/L)–stimulated platelet aggregation; age adjusted for sex and preclopidogrel platelet aggregation; sex adjusted for age and preclopidogrel platelet aggregation. b Indicates that women tend to respond less well than men. CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY ©2009 American Medical Association. All rights reserved. (Reprinted) JAMA, August 26, 2009—Vol 302, No. 8 853 Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 effect of the CYP2C19*2 genotype on platelet function. In addition to the CYP2C19*2 vari- ant, Simon et al25 identified a variant in ABCB1 (GeneID 403879), which en- codes a transporter that modulates clo- pidogrel absorption, to be associated with poorer clinical outcomes in pa- tients receiving clopidogrel. In our study, this variant (rs1045642) was not associated with ADP-stimulated plate- let aggregation at baseline or after clo- pidogrel treatment (P = .60). Sinai Hospital of Baltimore Study We next sought to replicate and ex- tend our findings in a group of indi- viduals from the general US popula- tion with a clinical indication for clopidogrel who underwent PCI and were recruited from a cardiac catheter- ization laboratory in Baltimore. Char- acteristics of these participants are shown in Table 1. Similar to the Amish PAPI Study group, those with the CYP2C19*2 genotype had no differ- ence in baseline platelet aggregation (P = .58) but demonstrated greater re- sidual platelet aggregation after clopi- dogrel therapy (P = .02) compared with those without the loss-of-function al- lele (Figure 3). After 1 year of follow- up, carriers of the CYP2C19*2 geno- type had higher cardiovascular event rates compared with noncarriers (20.9% vs 10.0%; hazard ratio [HR], 2.42; 95% CI, 1.18-4.99; P = .02) (FIGURE 4). The increased event rate conferred by the CYP2C19*2 genotype was limited to the 95 participants still taking clopidogrel when the event occurred (HR, 3.40; 95% CI, 1.36-8.46; P = .004), with no in- crease in event rate in those not taking clopidogrel when the event occurred or at 1 year of follow-up (HR, 1.39; 95% CI, 0.39-4.88; P = .60). Inclusion of ADP-stimulated platelet aggregation as a covariate in the regression model markedly reduced the relation be- tween the CYP2C19*2 genotype and cardiovascular outcome (HR, 1.58; 95% CI, 0.68-3.66; P = .29), suggesting that the genotype effect on clinical out- comes is mediated through decreased inhibition of platelet function. Figure 2. Genome-Wide Association Study of Adenosine Diphosphate–Stimulated Platelet Aggregation in Response to Clopidogrel −l o g 1 0 (P V al u e) 6 8 10 12 4 2 0 14 22212019181716151413121110987654321 −l o g 1 0 (P V al u e) 6 8 10 12 14 4 2 0 96 600 000 96 900 00095 700 00095 400 000 96 000 000 96 300 000 A Chromosome CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster Chromosome 10 location, bp PDE6C LGI1 PIPSL NOC3L HELLS CYP2C19 CYP2C8 CYP2C9CYP2C18TBC1D12PLCE1TMEM20C10orf4 C B Linkage disequilibrium (r2) 0 10.5 1 rs 1 0 1 0 9 2 0 4 rs 2 0 2 5 4 4 5 rs 1 3 2 6 8 3 7 rs 1 2 7 7 7 8 2 3 rs 7 1 7 2 3 8 rs 2 8 6 0 8 3 8 rs 2 8 6 0 9 0 3 rs 9 3 3 2 1 0 5 rs 9 3 3 2 1 1 3 rs 1 2 5 7 2 3 5 1 rs 1 0 5 0 9 6 7 9 rs 1 9 3 4 9 5 1 rs 1 9 3 4 6 8 0 2 3 4 5 6 7 8 9 10 11 12 13 A, Association (plotted as −log P value) of individual single-nucleotide polymorphisms distributed across the 22 autosomes. Horizontal dotted line indicates P = 1.0 � 10−7. B, Enlargement of 1.5-megabase region on chro- mosome 10q24. Genes encoded in the region are shown below the plot. C, Linkage disequilibrium (r2) among the 13 single-nucleotide polymorphisms showing genome-wide significance with clopidogrel response. In- creasing shades of gray represent increasing linkage disequilibrium, from white (r2= 0) to black (r2= 1). CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY 854 JAMA, August 26, 2009—Vol 302, No. 8 (Reprinted) ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 COMMENT Our study in a relatively large healthy drug-naive population indicates that clopidogrel response as defined by ADP- stimulated platelet aggregation is a nor- mally distributed trait, with no clear cutoff to define resistance. Others have reported similar findings in patient populations with wide variation in ath- erosclerotic burden, medication us- age, concurrent illnesses, and compli- ance—all potential confounding influences affecting clopidogrel re- sponse. Thus, clopidogrel response is likely caused by multiple factors, in- cluding potentially genetic factors. We found that increased age, BMI, and tri- glyceride levels and decreased levels of high-density lipoprotein cholesterol are predictors of poorer clopidogrel re- sponse; however, these factors com- bined account for less than 10% of the variation, suggesting other, undiscov- ered factors that contribute to varia- tion in clopidogrel response. Since all Amish persons are related, we were uniquely able to estimate heri- tability of clopidogrel response by de- termining the extent of variation in ADP-stimulated platelet aggregation that could be attributed to familial re- latedness. Clopidogrel response was highly heritable. Indeed, in an agnos- tic genome-wide association analysis, we found compelling evidence for a ma- jor locus on chromosome 10q24 that influences clopidogrel response. This locus extends across the CYP2C18– CYP2C19–CYP2C9–CYP2C8 gene clus- ter. CYP2C19 was a strong biological candidate because this enzyme is a key activator of the antiplatelet function of clopidogrel. Indeed, follow-up geno- typing indicated that the common loss- of-function CYP2C19*2 variant was as- sociated with clopidogrel response and could account for most of the associa- tion signal detected in the initial ge- nome-wide association study. The CYP2C19*2 genotype accounts for ap- proximately 12% of the variation in clo- pidogrel response. With age, BMI, and lipid levels, approximately 22% of the variation in clopidogrel response can be explained. Although substantial and highly sig- nificant, the majority of the variation in platelet response to clopidogrel re- mains unexplained. Presumably, other unmeasured factors that influence clo- pidogrel response (including poten- tially additional genetic variants, given the high heritability estimate) remain to be identified. CYP2C19*2 genotype was not associated with preclopido- grel platelet aggregation measures, and the postclopidogrel measures were as- sociated with genotype even after ad- justing for preclopidogrel measures, indicating a true association with clo- pidogrel response. Due to linkage dis- equilibrium across this region, we can- not rule out that a variant other than CYP2C19*2 may be causative, but this is unlikely in light of the unequivocal effect of this mutation on enzyme pro- duction. Furthermore, it is possible that other variants in CYP2C19 or other CYP2C genes at this locus may also con- tribute to clopidogrel response. Our study is unique in that we used an agnostic genome-wide approach. In our genome-wide association study, we detected no other region associated with clopidogrel response at or exceeding a genome-wide level of statistical signifi- cance, suggesting that common vari- ants in other parts of the genome with similar or greater effect size are un- Figure 3. Association of CYP2C19*2 (rs4244285) Loss-of-Function Variant With Adenosine Diphosphate–Stimulated Platelet Aggregation Before and After Clopidogrel Administration in Participants in the Amish Pharmacogenomics of Antiplatelet Intervention (PAPI) Study and Sinai Hospital of Baltimore Study Preclopidogrel 60 40 80 100 20 0 No. of participants P la te le t A g g re g at io n , % No. of CYP2C19∗2 Alleles 0 102 1 37 2 4 60 40 80 100 20 0 No. of participants P la te le t A g g re g at io n , % Postclopidogrel No. of CYP2C19∗2 Alleles 0 288 1 132 2 9 Preclopidogrel 60 40 80 100 20 P = .58 P = .92 0 No. of participants P la te le t A g g re g at io n , % No. of CYP2C19∗2 Alleles 0 288 1 132 2 9 60 40 80 100 20 0 No. of participants P la te le t A g g re g at io n , % Postclopidogrel No. of CYP2C19∗2 Alleles 0 131 1 54 2 3 Amish PAPI Study Sinai Hospital of Baltimore Study P = .02 P = 4.3 × 10–11 The horizontal line in the middle of each box indicates the median; the top and bottom borders of each box indicate the interquartile range (IQR). The whiskers above and below the box indicate plus/minus 1.5 IQRs, respectively; the points beyond the whiskers indicate outliers beyond 1.5 IQRs, except for those carrying 2 alleles in which all data points are plotted. CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY ©2009 American Medical Association. All rights reserved. (Reprinted) JAMA, August 26, 2009—Vol 302, No. 8 855 Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 likely. However, we cannot rule out the possibility that common SNPs or other types of variants (eg, copy number vari- ants, insertions/deletions), or rare vari- ants with large effect size not tagged by those SNPs genotyped, may have been missed. Genome-wide association studies are prone to false-positive results, owing to the large number of statistical tests per- formed. It is unlikely that our finding represents a false-positive result, be- cause we replicated the association be- tween CYP2C19*2 genotype and ADP- stimulated platelet aggregation in an independent sample. In addition to the replication of the initial association be- tween CYP2C19*2 genotype and clo- pidogrel response, the Baltimore Sinai Hospital cohort demonstrates general- izability to an outbred population with significant atherosclerotic disease. Im- portantly, the association between CYP2C19*2 genotype and cardiovas- cular event–free survival in this high- risk population provides evidence for clinical relevance. A limitation is that the Sinai Hospital cohort was a mixed population that received differing regi- mens of antiplatelet agents in the acute setting (1-3 days). However, our strati- fied analyses did not detect any signifi- cant effect of acute management on the longer-term outcomes investigated. Furthermore, we cannot rule out the possibility that a subset of patients were nonadherent to clopidogrel. Our data show that there is no relationship be- tween CYP2C19 genotype and platelet aggregation in the absence of clopido- grel. Thus, the differences in platelet ag- gregation we observed between geno- types once clopidogrel treatment was initiated strongly suggest that a large proportion of the population did in- deed adhere to the clopidogrel regi- men. Lastly, owing to limitations in sample size, we could not determine if CYP2C19 genotype was related to ad- verse bleeding events. Using a candidate gene approach, Hu- lot et al22 were the first to report an as- sociation between CYP2C19*2 geno- type and ADP-stimulated platelet aggregation in response to clopidogrel. More recently, CYP2C19*2 genotype was associated with poorer clinical out- comes in patients with coronary artery disease treated with clopidogrel and as- pirin.18,24-26 In patients taking clopido- grel and aspirin following myocardial in- farction, those carrying the CYP2C19*2 genotype were more likely to experi- ence a second cardiovascular event, with an HR (3.69) similar to that found in our study.24 In the Therapeutic Outcomes by Optimizing Platelet Inhibition With Pra- sugrel–Thrombolysis in Myocardial In- farction (TRITON-TIMI) 38 trial, pa- tients carrying the CYP2C19*2 genotype had lower plasma levels of the active me- tabolite of clopidogrel, reduced maxi- mal platelet aggregation in response to clopidogrel, and a 53% higher compos- ite primary efficacy outcome of the risk of cardiovascular events and death.18 In a nationwide French registry of pa- tients with acute myocardial infarction treated with clopidogrel, poorer out- comes were observed in patients who carried 2 CYP2C19 loss-of-function alleles (HR, 1.98).25 Unlike our study and the other studies, heterozygous per- sons from this French registry did not appear to have a significantly increased event rate, suggesting a more modest effect of the genotype on clopidogrel re- sponse in these patients. The loss-of-function CYP2C19*2 genotype is common in diverse popu- lations. In white populations, approxi- mately 24% have at least 1 CYP2C19*2 allele. The frequency of this allele is somewhat lower in Mexican Ameri- cans (� 18% with at least 1 CYP2C19*2 allele), higher in African Americans (� 33% with at least 1 copy), and mark- edly higher in Asian populations (� 51% with at least 1 copy).36-38 Thus, clopidogrel resistance due to this vari- ant may be particularly important in Asian and African American popula- tions. However, the strength of effect of CYP2C19*2 genotype on clopido- grel response may depend on other fac- tors such as genetic background or en- vironmental exposures, which may differ among ethnic groups. Unfortu- nately, our sample size was not suffi- cient to examine ethnicity-specific dif- Figure 4. Event-Free Survival Over 1 Year of Follow-up in Sinai Hospital of Baltimore Patients Treated With Clopidogrel Following Percutaneous Coronary Intervention, Stratified by CYP2C19*2 Genotype 50 40 10 20 30 0 0 0 No. at risk No. of CYP2C19∗2 alleles 1 0 158 67 90 154 Days All patients % E xp er ie n ci n g E ve n t 270 144 180 150 360 143 61 5356 50 No. of CYP2C19∗2 alleles 0 1 92 40 90 90 Days Patients not taking clopidogrel at time of event 270 87 180 88 360 86 38 3638 33 66 27 90 Days Patients taking clopidogrel at time of event 270180 360 64 5862 57 23 1718 16 Postdischarge ischemic events were defined as myocardial infarction (the occurrence of ischemic symptoms and a troponin I value greater than upper limits of normal), ischemic stroke, stent thrombosis (definite stent thrombosis according to the Academic Research Consortium criteria29), unplanned target vessel revasculariza- tion (revascularization of vessel treated at time of study enrollment), unplanned nontarget vessel revascular- ization (revascularization of a vessel different from that treated at time of study enrollment), hospitalization for coronary ischemia without revascularization (hospitalization for chest pain with evidence of ischemia on electrocardiogram and no evidence of myocardial infarction as measured by troponin I value), and death sec- ondary to any cardiovascular cause. Patients were further stratified into those who were taking clopidogrel when the event occurred or at 1 year of follow-up and those who were not. All analyses adjusted for age, sex, and race. For all patients, hazard ratio (HR) = 2.42 (95% confidence interval [CI], 1.18-4.99; P = .02); for pa- tients taking clopidogrel at time of event, HR = 3.40 (95% CI, 1.36-8.46; P = .004); for patients not taking clo- pidogrel at time of event, HR = 1.39 (95% CI, 0.39-4.88; P = .60). CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY 856 JAMA, August 26, 2009—Vol 302, No. 8 (Reprinted) ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 ferences in CYP2C19 genotype effects on clopidogrel response. Additional studies in diverse populations will be necessary. The commonly prescribed proton pump inhibitors omeprazole and esomeprazole, and some other medica- tions commonly coprescribed with clo- pidogrel, such as cimetidine and fluoxi- tine, are potent inhibitors of CYP2C19, introducing the possibility that they may attenuate the antiplatelet activity of clopidogrel, especially in individuals heterozygous or homozygous for the CYP2C19*2 genotype.14,39-41 We could not test this hypothesis directly, be- cause all participants in the Amish PAPI Study were drug-naive, and the num- ber of participants taking proton pump inhibitors in the Sinai Hospital of Bal- timore sample was too small. CYP2C19 genotype may prove use- ful in helping clinicians choose the most effective antiplatelet therapy and dose for a given individual. Those with the CYP2C19*2 genotype may benefit more from an antiplatelet regi- men that does not include clopido- grel, such as the third-generation thienopyridine prasugrel, or ticagrelor and cangrelor. Like clopidogrel, these agents inhibit ADP-stimulated platelet aggregation but are not as dependent on CYP2C19 for activation. Genotype- directed decisions regarding which antiplatelet agent to use in a specific patient may also have an important economic impact if costs of equally efficacious medications differ greatly. Whether CYP2C19*2 carriers may benefit from increased dosing of clo- pidogrel is not yet known. CONCLUSIONS We report the first genome-wide asso- ciation study of clopidogrel response and show that the common loss-of- function CYP2C19*2 variant is a ma- jor determinant of ADP-stimulated platelet aggregation. Individuals with this genotype have reduced protec- tion from clopidogrel in preventing car- diovascular disease–related events fol- lowing PCI. Prospective randomized clinical trials will be necessary to de- termine the efficacy of CYP2C19 geno- type–directed therapy in evidence- based clinical decision making. Author Contributions: Drs Shuldiner and Gurbel had full access to all of the data in the study and take re- sponsibility for the integrity of the data and the ac- curacy of the data analysis. Study concept and design: Shuldiner, Post, Faraday, Herzog, Gurbel. Acquisition of data: Shuldiner, Bliden, Gandhi, Horenstein, Damcott, Pakyz, Tantry, Herzog, Gurbel. Analysis and interpretation of data: Shuldiner, O’Connell, Bliden, Ryan, Damcott, Tantry, Gibson, Pollin, Post, Parsa, Mitchell, Faraday, Herzog, Gurbel. Drafting of the manuscript: Shuldiner, Bliden, Tantry, Herzog, Gurbel. Critical revision of the manuscript for important in- tellectual content: Shuldiner, O’Connell, Gandhi, Ryan, Horenstein, Damcott, Pakyz, Tantry, Gibson, Pollin, Post, Parsa, Mitchell, Faraday, Herzog, Gurbel. Statistical analysis: O’Connell, Bliden, Mitchell, Gurbel. Obtained funding: Shuldiner, Gurbel. Administrative, technical, or material support: Shuldiner, Bliden, Ryan, Damcott, Pakyz, Tantry, Gibson, Herzog, Gurbel. Study supervision: Shuldiner, Bliden, Faraday, Gurbel. Financial Disclosures: Dr Faraday reported that he is a coinventor of a patent application on novel anti- thrombotic agents and their methods of use. Dr Gurbel reported receiving grant support from Schering- Plough, AstraZeneca, Bayer Healthcare, Sanofi- Aventis, Portola Pharmaceuticals, Daiichi-Sankyo, and Lilly; and receiving honoraria/consulting income from Schering-Plough, AstraZeneca, Bayer Healthcare, Sanofi-Aventis, Portola Pharmaceuticals, Daiichi- Sankyo, Lilly, and Pozen. No other authors reported disclosures. Funding/Support: This study was supported by Na- tional Institutes of Health grants NIH U01 GM074518 and U01 HL084756, the Clinical Nutrition Research Unit of Maryland (P30 DK072488), the University of Maryland General Clinical Research Center (M01 RR 16500), the Baltimore Veterans Administration Geri- atric Research and Education Clinical Center, and Si- nai Hospital of Baltimore. Role of the Sponsors: The funding organizations had no role in the design and conduct of the study; the collection, analysis, and interpretation of the data; or the preparation, review, or approval of the manu- script. Additional Information: eMethods, eTables 1 through 3, and eFigures 1 and 2 are available at http://www .jama.com. Additional Contributions: We gratefully acknowl- edge our Amish liaisons and field workers and the ex- traordinary cooperation and support of the Amish com- munity, without which these studies would not have been possible. REFERENCES 1. Antman EM, Hand M, Armstrong PW, et al; Ca- nadian Cardiovascular Society; American Academy of Family Physicians; American College of Cardiology; American Heart Association. 2007 focused update of the ACC/AHA 2004 guidelines for the management of patients with ST-elevation myocardial infarction: a report of the American College of Cardiology/ American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2008;51(2):210- 247. 2. King SB III, Smith SC Jr, Hirshfeld JW Jr, et al; 2005 WRITING COMMITTEE MEMBERS. 2007 Focused Up- date of the ACC/AHA/SCAI 2005 Guideline Update for Percutaneous Coronary Intervention: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines: 2007 Writing Group to Review New Evidence and Update the ACC/AHA/SCAI 2005 Guideline Update for Per- cutaneous Coronary Intervention, Writing on Behalf of the 2005 Writing Committee. Circulation. 2008; 117(2):261-295. 3. Gurbel PA, Tantry US. Clopidogrel resistance? Thromb Res. 2007;120(3):311-321. 4. Hollopeter G, Jantzen HM, Vincent D, et al. Iden- tification of the platelet ADP receptor targeted by an- tithrombotic drugs. Nature. 2001;409(6817):202- 207. 5. Savi P, Pereillo JM, Uzabiaga MF, et al. Identifica- tion and biological activity of the active metabolite of clopidogrel. Thromb Haemost. 2000;84(5):891- 896. 6. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, et al. Variability in individual responsiveness to clopido- grel: clinical implications, management, and future perspectives. J Am Coll Cardiol. 2007;49(14):1505- 1516. 7. Gurbel PA, Bliden KP, Hiatt BL, O’Connor CM. Clo- pidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation. 2003;107(23):2908-2913. 8. Gurbel PA, Tantry US. Drug insight: clopidogrel nonresponsiveness. Nat Clin Pract Cardiovasc Med. 2006;3(7):387-395. 9. Angiolillo DJ, Alfonso F. Platelet function testing and cardiovascular outcomes: steps forward in iden- tifying the best predictive measure. Thromb Haemost. 2007;98(4):707-709. 10. Bliden KP, Dichiara J, Tantry US, Bassi AK, Chaganti SK, Gurbel PA. Increased risk in patients with high plate- let aggregation receiving chronic clopidogrel therapy undergoing percutaneous coronary intervention: is the current antiplatelet therapy adequate? J Am Coll Cardiol. 2007;49(6):657-666. 11. Buonamici P, Marcucci R, Migliorini A, et al. Im- pact of platelet reactivity after clopidogrel adminis- tration on drug-eluting stent thrombosis. J Am Coll Cardiol. 2007;49(24):2312-2317. 12. Gurbel PA, Bliden KP, Guyer K, et al. Platelet re- activity in patients and recurrent events post- stenting: results of the PREPARE POST-STENTING Study. J Am Coll Cardiol. 2005;46(10):1820- 1826. 13. Farid NA, Small DS, Payne CD, et al. Effect of a torvastatin on the pharmacokinetics and pharmaco- dynamics of prasugrel and clopidogrel in healthy subjects. Pharmacotherapy. 2008;28(12):1483- 1494. 14. Gilard M, Arnaud B, Le Gal G, Abgrall JF, Boschat J. Influence of omeprazol on the antiplatelet action of clopidogrel associated to aspirin. J Thromb Haemost. 2006;4(11):2508-2509. 15. Lau WC, Gurbel PA, Watkins PB, et al. Contri- bution of hepatic cytochrome P450 3A4 metabolic ac- tivity to the phenomenon of clopidogrel resistance. Circulation. 2004;109(2):166-171. 16. Siller-Matula JM, Lang I, Christ G, Jilma B. Calcium- channel blockers reduce the antiplatelet effect of clopidogrel. J Am Coll Cardiol. 2008;52(19):1557- 1563. 17. Kim KA, Park PW, Hong SJ, Park JY. The effect of CYP2C19 polymorphism on the pharmacokinetics and pharmacodynamics of clopidogrel: a possible mechanism for clopidogrel resistance. Clin Pharma- col Ther. 2008;84(2):236-242. 18. Mega JL, Close SL, Wiviott SD, et al. Cyto- chrome P-450 polymorphisms and response to clopidogrel. N Engl J Med. 2009;360(4):354-362. 19. Brandt JT, Close SL, Iturria SJ, et al. Common poly- morphisms of CYP2C19 and CYP2C9 affect the phar- macokinetic and pharmacodynamic response to clo- pidogrel but not prasugrel. J Thromb Haemost. 2007; 5(12):2429-2436. 20. Fontana P, Hulot JS, De Moerloose P, Gaussem CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY ©2009 American Medical Association. All rights reserved. (Reprinted) JAMA, August 26, 2009—Vol 302, No. 8 857 Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021 P. Influence of CYP2C19 and CYP3A4 gene polymor- phisms on clopidogrel responsiveness in healthy subjects. J Thromb Haemost. 2007;5(10):2153- 2155. 21. Frere C, Cuisset T, Morange PE, et al. Effect of cytochrome p450 polymorphisms on platelet re- activity after treatment with clopidogrel in acute coro- nary syndrome. Am J Cardiol. 2008;101(8):1088- 1093. 22. Hulot JS, Bura A, Villard E, et al. Cytochrome P450 2C19 loss-of-function polymorphism is a major de- terminant of clopidogrel responsiveness in healthy subjects. Blood. 2006;108(7):2244-2247. 23. Malek LA, Kisiel B, Spiewak M, et al. Coexisting polymorphisms of P2Y12 and CYP2C19 genes as a risk factor for persistent platelet activation with clopidogrel. Circ J. 2008;72(7):1165-1169. 24. Collet JP, Hulot JS, Pena A, et al. Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study. Lancet. 2009;373(9660):309-317. 25. Simon T, Verstuyft C, Mary-Krause M, et al; French Registry of Acute ST-Elevation and Non-ST- Elevation Myocardial Infarction (FAST-MI) Invest- igators. Genetic determinants of response to clopido- grel and cardiovascular events. N Engl J Med. 2009; 360(4):363-375. 26. Trenk D, Hochholzer W, Fromm MF, et al. Cy- tochrome P450 2C19 681G�A polymorphism and high on-clopidogrel platelet reactivity associated with adverse 1-year clinical outcome of elective percuta- neous coronary intervention with drug-eluting or bare- metal stents. J Am Coll Cardiol. 2008;51(20):1925- 1934. 27. ESPRIT Investigators. Novel dosing regimen of ep- tifibatide in planned coronary stent implantation (ESPRIT): a randomised, placebo-controlled trial. Lancet. 2000;356(9247):2037-2044. 28. Lincoff AM, Bittl JA, Harrington RA, et al; REPLACE-2 Investigators. Bivalirudin and provisional glycoprotein IIb/IIIa blockade compared with hepa- rin and planned glycoprotein IIb/IIIa blockade d u r i n g p e r c u t a n e o u s c o r o n a r y i n t e r v e n t i o n : REPLACE-2 randomized trial. JAMA. 2003;289 (7):853-863. 29. Applegate RJ, Sacrinty MT, Little WC, Santos RM, Gandhi SK, Kutcher MA. Incidence of coronary stent thrombosis based on academic research consortium definitions. Am J Cardiol. 2008;102(6):683-688. 30. Almasy L, Blangero J. Multipoint quantitative- trait linkage analysis in general pedigrees. Am J Hum Genet. 1998;62(5):1198-1211. 31. Agarwala R, Biesecker LG, Hopkins KA, Francomano CA, Schäffer AA. Software for constructing and verify- ing pedigrees within large genealogies and an applica- tion to the Old Order Amish of Lancaster County. Ge- nome Res. 1998;8(3):211-221. 32. Brandt JT, Payne CD, Wiviott SD, et al. A com- parison of prasugrel and clopidogrel loading doses on platelet function: magnitude of platelet inhibition is related to active metabolite formation. Am Heart J. 2007;153(1):66.e9-66.e16. 33. Takahashi M, Pang H, Kawabata K, Farid NA, Kurihara A. Quantitative determination of clop- idogrel active metabolite in human plasma by LC-MS/MS. J Pharm Biomed Anal. 2008;48(4): 1219-1224. 34. Umemura K, Furuta T, Kondo K. The common gene variants of CYP2C19 affect pharmacokinetics and phar- macodynamics in an active metabolite of clopidogrel in healthy subjects. J Thromb Haemost. 2008;6 (8):1439-1441. 35. Wallentin L, Varenhorst C, James S, et al. Prasu- grel achieves greater and faster P2Y12 receptor- mediated platelet inhibition than clopidogrel due to more efficient generation of its active metabolite in aspirin-treated patients with coronary artery disease. Eur Heart J. 2008;29(1):21-30. 36. Luo HR, Poland RE, Lin KM, Wan YJ. Genetic poly- morphism of cytochrome P450 2C19 in Mexican Americans: a cross-ethnic comparative study. Clin Phar- macol Ther. 2006;80(1):33-40. 37. Xiao ZS, Goldstein JA, Xie HG, et al. Differences in the incidence of the CYP2C19 polymorphism af- fecting the S-mephenytoin phenotype in Chinese Han and Bai populations and identification of a new rare CYP2C19 mutant allele. J Pharmacol Exp Ther. 1997; 281(1):604-609. 38. Takakubo F, Kuwano A, Kondo I. Evidence that poor metabolizers of (S)-mephenytoin could be iden- tified by haplotypes of CYP2C19 in Japanese. Pharmacogenetics. 1996;6(3):265-267. 39. Gurbel PA, Lau WC, Tantry US. Omeprazole: a possible new candidate influencing the antiplatelet effect of clopidogrel. J Am Coll Cardiol. 2008; 51(3):261-263. 40. Ho PM, Maddox TM, Wang L, et al. Risk of ad- verse outcomes associated with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome. JAMA. 2009;301(9):937- 944. 41. Small DS, Farid NA, Li YG, et al. Effect of raniti- dine on the pharmacokinetics and pharmacodynam- ics of prasugrel and clopidogrel. Curr Med Res Opin. 2008;24(8):2251-2257. In order to be a realist, you must believe in miracles. —David Ben-Gurion (1886-1973) CYTOCHROME P450 2C19 GENOTYPE AND CLOPIDOGREL THERAPY 858 JAMA, August 26, 2009—Vol 302, No. 8 (Reprinted) ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ by a Carnegie Mellon University User on 04/05/2021