Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome

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1 c Indian Academy of Sciences RESEARCH ARTICLE Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome XU LIU 1,2,YULUO 3,YANLAI 3,YIANYAO 3, JIMIN LI 3, YUNKAI WANG 3,S.LILLYZHENG 2,6, JIANFENG XU 1,2,4,5,6 and XUEBO LIU 3, 1 State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, and 2 Fudan Center for Genetic Epidemiology, School of Life Sciences, Fudan University, Shanghai , People s Republic of China 3 Department of Cardiovascular Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai , People s Republic of China 4 Fudan Institute of Urology, Huashan Hospital, Fudan University, Shanghai , People s Republic of China 5 Center for Genomic Translational Medicine and Prevention, School of Public Health, Fudan University, Shanghai , People s Republic of China 6 NorthShore Research Institute, NorthShore University Health System, Evanston 60201, Illinois, USA Abstract Polymorphisms of CYP2C19 are associated with platelet response to clopidogrel. This study was conducted to evaluate the contribution of the previously identified polymorphisms to the response of clopidogrel in a cohort of Chinese Han patients. A total of 222 acute coronary syndrome patients undergoing percutaneous coronary intervention treated with clopidogrel were enrolled from September 2012 to June Residual platelet aggregations for all patients were measured by the VerifyNow P2Y12 system. Sixteen single-nucleotide polymorphisms among nine genes were genotyped including CYP2C19, ABCB1 and PON1. In this study, CYP2C19*2 and CYP2C19*17 were strongly associated with higher platelet aggregation and lower platelet aggregation to clopidogrel treatment, respectively (P <0.001). Patients with CYP2C19*2 allele had a higher risk of high on-treatment platelet reactivity than non carriers (adjusted OR, 5.434; 95% CI, , P =0.01). The coexistence of CYP2B6*9 (rs ) and P2Y12 (rs ) and the coexistence of CYP2B6*1B (rs ) and P2Y12 (rs ) were also associated with poor response to clopidogrel. No significant relation of CYP2C19*3 and other polymorphisms to the platelet aggregation was found. In conclusion, CYP2C19*2, CYP2C19*17 coexistence of CYP2B6*9 (rs ) and P2Y12 (rs ) and coexistence of CYP2B6*1B (rs ) and P2Y12 (rs ) were identified to be associated with response to clopidogrel treatment in Chinese Han patients. [Liu X., Luo Y., Lai Y., Yao Y., Li J., Wang Y., Zheng S. L., Xu J. and Liu X Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome. J. Genet. 95, ] Introduction Clopidogrel is a thienopyridine antiplatelet drug used to inhibit platelet function through a P2Y 12 adenosine diphosphate (ADP) receptor. Genetic polymorphisms in different genes have been reported to be associated with the variability of reactivity and clinical outcomes to clopidogrel (Shuldiner et al. 2009; Mega et al. 2009; Price et al. 2012; Perry and Shuldiner 2013). Clinical Pharmacogenetics Implementation Consortium (CPIC) has provided evidence For correspondence. Xuebo Liu, lxb70@hotmail.com; Jianfeng Xu, jxu8088@gmail.com. Xu Liu and Yu Luo contributed equally to this work. and guidelines for CYP2C19 genotype and clopidogrel therapy (Scott et al. 2013). Most common loss-of-function (LOF) alleles are CYP2C19*2 and CYP2C19*3, which have been reported to be associated with higher levels of ADPinduced platelet aggregation and a higher risk of major adverse cardiovascular events in clopidogrel treated patients (Simon et al. 2009; Hwang et al. 2011; Cuisset et al. 2011). The gain-of-function (GOF) allele CYP2C19*17 is associated with an increased function to clopidogrel treatment (Sibbing et al. 2010). The carriage prevalence of the CYP2C19*2 and CYP2C19*3 among Asians are significantly higher than in Caucasians and Africans, while CYP2C19*17 is relatively rare in Asians (Scott et al. 2013; Man et al. 2010). The conversion of clopidogrel to its active Keywords. clopidogrel; CYP2C19 gene; acute coronary syndrome; VerifyNow P2Y12 system; coexisting polymorphisms. Journal of Genetics, Vol. 95, No. 2, June

2 Xu Liu et al. metabolite requires two sequential oxidative steps involving several CYP450 enzymes to modulate clopidogrel s metabolic activation, including CYP2B6, CYP2C9 and CYP2C19 (Gladding et al. 2009). Other risk polymorphisms in ABCB1, which is involved in the intestinal absorption of clopidogrel (Wang et al. 2015), and PON1, which is also involved in the transformation of clopidogrel into its active state (Bouman et al. 2011) may also play roles in the antiplatelet effect of clopidogrel. However, whether these polymorphisms are related to high on-treatment platelet reactivity (HPR) in clopidogrel treated Chinese patients is less known (Wu et al. 2012; Zhang et al. 2013). The purpose of this study was to evaluate the effect of previously identified polymorphisms and coexisting polymorphisms of different genes to the response of clopidogrel in a cohort of Chinese Han acute coronary syndrome (ACS) patients treated with clopidogrel. Materials and methods Study population From September 2012 to June 2013, 222 ACS patients undergoing percutaneous coronary intervention (PCI) with drug-eluting stent (DES) were enrolled in this prospective observational study. The patients had been diagnosed with ACS and all procedures were conducted at the Department of Cardiology, EAST Hospital, Shanghai, China. ACS included unstable angina with ST segment changes, nonst-segment elevation acute myocardial infarction (NSTEMI), and STsegment elevation acute myocardial infarction (STEMI). Patients received a loading dose of 300-mg clopidogrel at 6 24 h before undergoing coronary angiography, followed by a daily regimen of 75-mg clopidogrel and 100-mg aspirin. The ADP-induced P2Y12 receptor-mediated platelet aggregation was tested after days maintenance dose of clopidogrel administration. The exclusion criteria were as follows: concomitant administration of cilostazol, concomitant administration of calcium-channel blockers, use of anticoagulation drugs, stable coronary artery disease, treatment with ticlopidine, ticagrelor or prasugrel, allergies or contraindications to either aspirin or clopidogrel, in-stent restenosis, malignancies, left ventricular ejection fraction less than 30%, pregnancy, severe renal or hepatic insufficiency (serum creatinine of more than 2 mg per deciliter or alanine aminotransferase level >3 times upper normal), total platelet count less than 100*10 9 /L, and haematologic disorder, stroke or transient ischaemic attack within three months. Patients with periprocedural administration of GP IIb/IIIa inhibitors (tirofiban) were enrolled due to its short effective half-life. The study protocol was approved by the hospital s medical ethics committee and informed consent was obtained from each patient. Platelet function All patients received platelet function testing using the VerifyNow P2Y12 system (Accumetrics, San Diego, USA) which is a whole-blood, point-of-care system that assesses responsiveness to P2Y12 antagonists (Kim et al. 2010). The assay contains 20 μmol ADP and 22 nmol prostaglandin E1 to reduce the activation contribution from ADP binding to P2Y1 receptors. Results are reported as P2Y12 reaction units (PRU), baseline and per cent inhibition. PRU indicates the amount of ADP-mediated aggregation specific to the platelet P2Y12 receptor. The per cent inhibition is calculated as %Inhibition = (baseline PRU)/baseline 100, which indicates the difference between pretreatment and posttreatment values. Genotyping for SNPs By collecting data from previous literatures about clopidogrel, 16 SNPs within nine genes were selected: CYP2C19*2 (rs ), CYP2C19*3 (rs ), CYP2C19*4 (rs ), CYP2C19*5 (rs ), CYP2C19*7 (rs ), CYP2C19*8 (rs ), CYP2C19*17 (rs ), CY P2C9*3 (rs ), ABCB1 (rs ), ITGB3 (rs5918), PON1 (rs662), P2RY12 (rs ), CYP2B6*1B (rs ), CYP2B6*9 (rs ), P2Y12 (rs ) and CES1 (rs ). Genomic DNA was extracted from the peripheral whole blood (2 ml) of each patient. SNPs were genotyped using the Sequenom MassARRAY (Sequenom, San Diego, USA) platform. Statistical analysis Statistical tests were performed using SPSS Statistics 16.0 (SPSS, Chicago, USA). Categorical variables were expressed as frequencies and percentages, and compared with chisquare test. Continuous variables were expressed as mean ±SD and compared between groups with one-way ANOVA and Kruskal Wallis test. After demonstrating significant differences among variables by the ANOVA test, post hoc comparisons between the groups were performed with the Student Newman Keuls test for multiple comparisons. To evaluate an independent association of CYP2C19 allele carriage with P2Y12 reaction, multivariate linear regression analysis was performed adjusting for confounding factors: age, gender, body mass index (BMI), hypertension, dyslipidemia, diabete, heart disease history, stroke history and chronic renal failure (CRF). To determine the impact of polymorphisms on HPR, a multivariate logistic regression analysis was performed adjusting for confounding factors. Hardy Weinberg equilibrium (HWE) was tested using a chi-square test. A two-sided P value was used to test for significance (P < 0. 05). Results Patient characteristics and SNP genotyping The mean age of patients was 64.6±9.5 years of which 69.8% of them were male (table 1). We did not find carriers of the CYP2C19*4 (rs ), CYP2C19*5 232 Journal of Genetics, Vol. 95, No. 2, June 2016

3 Effect of genetic polymorphisms and clopidogrel in Chinese Han Table 1. Baseline characteristics according to CYP2C19 genotype. Variable Total (n =222) UM (n =5) EM (n =91) IM (n =95) PM (n =28) P value Age (years) 64.6± ± ± ± ± BMI (kg/m 2 ) 25.0± ± ± ± ± Male 155 (69.8) 3 (60.0) 65 (71.4) 63 (66.3) 21 (75.0) Hypertension 142 (64.0) 2 (40.0) 65 (71.4) 55 (57.9) 17 (60.7) Diabetes 69 (31.1) 1 (20.0) 32 (35.2) 26 (27.4) 10 (35.7) Dyslipidemia 21 (9.5) 1 (20.0) 11 (12.1) 8 (8.4) 1 (3.6) Heart desease history 30 (13.5) 0 (0) 14 (15.4) 9 (9.5) 7 (25.0) Stroke history 23 (10.4) 0 (0) 14 (15.4) 6 (6.3) 3 (10.7) CRF 60 (27.0) 0 (0) 21 (23.1) 30 (31.6) 8 (28.6) Base 251.0± ± ± ± ± PRU 166.9± ± ± ± ±43.1 <0.001 Inhibition (%) 32.8± ± ± ± ±13.5 <0.001 Values are mean ±SD or n (%). BMI, body mass index; CRF, chronic renal failure; PRU, P2Y12 reaction units, UM, ultrarapid s; EM, extensive s; IM, intermediate ; PM, poor. Table 2. Distribution of variant alleles among the study population. Frequency HWE, Gene SNP Allele (%) P value CYP2C19*2 rs A CYP2C19*3 rs A CYP2C19*17 rs T ABCB1 rs T ITGB3 rs5918 C PON1 rs662 A P2RY12 rs A CYP2C9*3 rs C CYP2B6*1B rs C CYP2B6*9 rs T P2RY12 rs C Table 3. Genotypic and phenotypic distributions of CYP2C19 variant. Distribution, Predicted Group by Genotype n (%) function CPIC *1/*1 91 (41.6) Normal Extensive *1/*17 5 (2.3) Increased Ultrarapid *1/*2 84 (38.4) Decreased Intermediate *1/*3 10 (4.6) Decreased Intermediate *2/*2 24 (11.0) Decreased or absent Poor *2/*3 3 (1.4) Decreased or absent Poor *3/*17 1 (0.5) Decreased Intermediate *3/*3 1 (0.5) Decreased or absent Poor CPIC: Clinical Pharmacogenetics Implementation Consortium (rs ), CYP2C19*7 (rs ), CYP2C19*8 (rs ), CES1 (rs ) and excluded these SNPs from our analysis. The other SNPs were in HWE in the study population (table 2). No significant deviations from HWE were observed for other SNPs (P > 0.05). The prevalence of CYP2C19*2, *3 and *17 were 31.1%, 3.9% and 1.3%, respectively in the present cohort. The genotypic and phenotypic distributions of CYP2C19 alleles inthis study population are shown in table 3. According to Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2C19 (Scott et al. 2013) and carriage of the CYP2C19 variant allele in this cohort, enrolled patients were classified into four groups: ultrarapid s (UMs) (*1/*17: 2.3%), extensive s (EMs) (*1/*1: 41.6%), intermediate s (IMs) (*1/*2, *1/*3 and *3/*17: 43.4%) and poor s (PMs) (*2/*2, *2/*3 and *3/*3: 12.8%). Baseline characteristics of the study population according to CYP2C19 genotype are shown in table 1. Genotypes and postclopidogrel platelet reactivity The platelet function measurements were determined in all enrolled patients. The PRU ranged from 7 to 322 among all patients. PRU increased according to the number of CYP2C19 LOF/GOF alleles (96.8 ± 50.7 in UMs versus ± 57.2 in EMs versus ± 56.3 in IMs versus ± 43.1 in PMs, P < 0.001) (table 1). There were significant differences between UMs and the other three groups (UMs versus EMs, P = 0.034; UMs versus IMs, P = 0.002; UMs versus PMs, P < 0.001); EMs and IMs, PMs (P = and P < 0.001, respectively) (figure 1a). No difference was observed between IMs and PMs (P = 0.130). A strong difference between different CYP2C19 LOF allele numbers is shown in figure 1b. Patients without LOF alleles had lower PRU than with one or two LOF alleles (148.1±58.0 for zero LOF carriers versus 177.1±56.3 for one LOF carriers versus 195.1± 43.1 for two LOF carriers, P <0.001). Strong gene-dose relationship could be found between the CYP2C19*2/*17 and PRU. Patients with one or two CYP2C19*2 alleles compared with zero CYP2C19*2 allele exhibited higher PRU (194.3± 45.9 in two alleles carriers versus 178.1±55.8 in one allele carriers versus 151.4±58.1 in no variant carriers, P <0.001, figure 1c). Patients with CYP2C19*17 variant had decreased PRU compared with noncarriers (97.2±45.4 in CYP2C19*17 carriers versus 169.0±57.3 in no variant carriers, P = 0.004, figure 1d). Another LOF allele CYP2C19*3 had no significant relationship with PRU (P = 0.377, figure 1e). No Journal of Genetics, Vol. 95, No. 2, June

4 Xu Liu et al. Figure 1. Distribution of PRU according to degree of CYP2C19 function (a), number of CYP2C19 LOF alleles (b) and different CYP2C19 genotypes(c e). The central box represents the values between the lower and upper quartiles, and the band inside the box shows the second quartile (the median). The lines extending vertically indicate the minimum to maximum value, and outside values are displayed as separate points. association was found between other variants and platelet aggregation (P > 0.01). Relationship between genotypes and HPR HPR cut-off point has been described as 230 PRU in previous studies (Price et al. 2009). Due to the low average PRU of all patients (166.9), the cut-off point for PRU was defined as 208 according to the previous reported literature (Price et al. 2011) and the upper quartile (approximately equal to 208 PRU) of the present cohort. A total of 55 (24.8%) patients met the definition of HPR. A multivariate logistic regression analysis was used to determine the genetic factors that affect HPR with the adjustment of age, 234 Journal of Genetics, Vol. 95, No. 2, June 2016

5 Effect of genetic polymorphisms and clopidogrel in Chinese Han Table 4. Predictors for HPR by univariate and multivariate logistic regression models according to genotypes. Variant SNP No. HPR events HPR events OR P Adjusted OR Adjusted (rs ID) (n =167) n (%) (n =55) n (%) (95% CI) value (95% CI) P value CYP2C19*2 (rs ) GA 61 (36.5) 26 (47.3) ( ) ( ) AA 12 (7.2) 12 (21.8) ( ) ( ) CYP2C19*3 (rs ) GA 12 (7.2) 2 (3.6) ( ) ( ) AA 0 1 (1.8) CYP2C19*17 (rs ) CT 6 (3.6) CYP2C19 genotype UMs 5 (3.0) IMs 67 (40.1) 28 (50.9) ( ) ( ) PMs 15 (9.0) 13 (23.6) ( ) ( ) CYP2C19 LOF allele number 1 67 (40.1) 28 (50.9) ( ) ( ) (9.0) 13 (23.6) ( ) ( ) ABCB1 (rs ) CT 83 (49.7%) 24 (43.6%) ( ) ( ) TT 27 (16.2%) 7 (12.7%) ( ) ( ) ITGB3 (rs5918) TC 1 (0.6) PON1 (rs662) AG 72 (43.1) 23 (41.8) ( ) ( ) GG 65 (38.9) 19 (34.5) ( ) ( ) P2RY12 (rs ) GA 62 (37.1) 26 (47.3) ( ) ( ) AA 10 (6.0) 3 (5.5) ( ) ( ) CYP2C9*3 (rs ) AC 16 (9.6%) 3 (5.5%) ( ) ( ) CYP2B6*1B (rs ) TC 83 (49.7) 26 (47.3) ( ) ( ) CC 34 (20.4) 9 (16.4) ( ) ( ) CYP2B6*9 (rs ) CT 36 (21.6) 16 (29.1) ( ) ( ) TT 6 (3.6) 4 (7.3) ( ) ( ) P2RY12 (rs ) TC 36 (21.6) 18 (32.7) ( ) ( ) CC 3 (1.8) 2 (3.6) ( ) ( ) gender, BMI, hypertension, dyslipidemia, diabete, heart disease history, stroke history and CRF (table 4). Frequency of the CYP2C19*2 allele was remarkably higher in patients with HPR events. Patients with *2/*2 alleles had higher risk of HPR than noncarriers (adjusted OR, 5.434; 95% CI, , P =0.01). The carriers of two CYP2C19 LOF alleles could also predict higher risk of HPR (adjusted OR, 5.373; 95% CI, , P =0.01), with the impact mainly attributing to CYP2C19*2. However, the carriage of CYP2C19*3 or CYP2C19*17 could not predict HPR (P>0.05) due to the low frequency of risk alleles. No significant correlation was found for other genetic variants and HPR. Coexsisting polymorphisms on platelet reactivity on clopidogrel Effects of coexisting polymorphisms on platelet reactivity on clopidogrel were tested by dividing each pair of two SNPs into four groups: group 1: SNP1 wild type / SNP2 wild type; group 2: SNP1 risk allele / SNP2 wild type; group 3: SNP1 wild type / SNP2 risk allele; group 4: SNP1 risk allele / SNP2 risk allele (Tang et al. 2013). Coexistences of CYP2C19*2 (A allele) or CYP2C19*17 (T allele) with polymorphism of other SNPs were associated with poor response to clopidogrel in Chinese patients with ACS (table 1 in electronic supplementary material at in/jgenet/). For other coexisting polymorphisms, carriers of CYP2B6*9 (rs ) (T allele) and P2Y12 (rs ) (C allele) (group 4) had the highest level of ADP-induced platelet aggregation to clopidogrel (157.3±55.7 in group 1 versus 173.4±57.4 in group 2 versus 177.1±60.9 in group 3 versus 193.9±58.2 in group 4, P =0.04) (figure 1a in electronic supplementary material). Coexistence of CYP2B6*1B (rs ) and P2Y12 (rs ) was also associated with poor response to clopidogrel although CYP2B6*1B (rs ) (C allele) alone had lower PRU (179.2±56.2 in Journal of Genetics, Vol. 95, No. 2, June

6 Xu Liu et al. group 1 versus 153.9±55.1 in group 2 versus 166.6±56.8 in group 3 versus 187.3±61.2 in group 4, P =0.004) (figure 1b in electronic supplementary material). Discussion The genetic impacts on clopidogrel response and clinical outcomes have been studied in previous studies. But less evidence has been reported for Chinese Han patients under clopidogrel treatment. The present study identified the impacts of CYP2C19*2 on higher ADP-induced platelet aggregation and increased risk for HPR in the Chinese Han patients treated with clopidogrel which was consistent with previous studies (Chan et al. 2012). According to previous studies and CPIC guidelines, CYP2C19*2 has a higher frequency in most populations especially among Asians (29 35% in Asians, 15% in Caucasians and in Africans) (Scott et al. 2013). In this study, the frequency of CYP2C19*2 was 31.1% and 50.8% of the patients had at least one risk allele. Platelet aggregation was significantly different among four CYP2C19 genotype groups. As compared with wild-type carriers (EMs), UMs with GOF allele (*17) had lower PRU while IM and EM with LOF alleles (*2/*3) had higher PRU. Only a few evidence of the association between CYP2C19*17 and clopidogrel response in Chinese Han patients has been reported. The presence of CYP2C19*17 allele was linked with lower ADP-induced platelet aggregation. The frequency for CYP2C19*17 was similar to those reported previously in the Chinese Han population (1.3%), but significantly lower than those in other populations ( 3 21%). The frequency of CYP2C19*3 was 3.9%, which was similar to those reported in Asians (2 9%) and higher than those in Caucasians (<1%). But no significant relationship between CYP2C19*3 and clopidogrel response was found, which was inconsistent with previous reports in Chinese Han (Lin et al. 2015). Other than CYP2C19*2 and CYP2C19*17, we have identified the coexistence of CYP2B6*9 (rs ) and P2Y12 (rs ) and the coexistence ofcyp2b6*1b (rs ) and P2Y12 (rs ) were also associated with poor response to clopidogrel. P2Y12 (rs ) has been reported to affect the response to clopidogrel and adverse ischaemic events with the coexistence of CYP2C19*2 (Tang et al. 2013). Additionally, Sun et al. (2015) found a significant difference between P2Y12 (rs ) and lower ADP-induced platelet inhibition rate. Although in many studies CYP2C19 genetic polymorphisms have been shown to reduce clopidogrel metabolism and increase clinical outcomes, few clinical trials have validated the clinical benefit of personalizing antiplatelet therapy based on genotyping and platelet function test. Jean-Philippe Collet et al. (2012) demonstrated no significant improvements in clinical outcomes with platelet function monitoring and treatment adjustment for coronary stenting (Collet et al. 2012). A similar conclusion was identified in a metaanalysis including 19 studies, which showed that CYP2C19*2 carriers after high-dose clopidogrel treatment still had high incidence rate of clopidogrel antiplatelet resistance in patients treated with PCI (Zhang et al. 2015). Whether routine genetic testing should be performed in clinics is also not validated with little evidence (Singh et al. 2012). Hwang et al. (2010) reported adjunctive cilostazol could enhance platelet inhibition and reduce the rate of HPR irrespective of CYP2C19 genotyping, which provided evidence for genetic testing in clinics. According to evidence from published literature, ACC/AHA and FDA provided guidelines and box warnings for CYPC19 genotype-directed antiplatelet therapy, but did not recommend routine genotyping in patients on clopidogrel therapy (Amsterdam et al. 2014). In this study, we used the VerifyNow P2Y12 test to evaluate the platelet function for clopidogrel which has been used worldwide in clinical trials. There are many advantages of VerifyNow P2Y12 test compared with traditional methods for testing platelet function (such as thromboela-stogram and light turbidimetric aggregometry): easy to operate, less expensive, fast, stable and repeatable. However, VerifyNow P2Y12 test was rarely used in China until now. There are several limitations for this study. Firstly, this study was a single-centre study, which may limit the ability to make definite conclusions. Secondly, the sample size was relatively small, resulting in small numbers of CYP2C19*3 and CYP2C19*17 carriers tested (15 and 6, respectively). All CYP2C19*17 carriers were heterozygotes. Therefore, additional studies with large sample of CYP2C19*3 and CYP2C19*17 carriers are needed to further investigate the relationship between CYP2C19 and antiplatelet response of clopidogrel treatment in Chinese Han. In summary, results from this study identified that CYP2 C19*2 had significantly diminished antiplatelet responses to clopidogrel, and CYP2C19*17 was identified to be associated with an increased function to clopidogrel treatment in Chinese Han patients. The coexistence of CYP2B6*9 (rs ) and P2Y12 (rs ) and the coexistence of CYP2B6*1B (rs ) and P2Y12 (rs ) were also associated with poor response to clopidogrel. Acknowledgements Authors thank all the subjects included in this study and the staff in Department of Cardiology of EAST Hospital and Fudan Center for Genetic Epidemiology for their support. This study was supported by a grant from the Shanghai Health Bureau ( ). This study was partially supported by research funds from the Shanghai Municipal Health Bureau ( ), from School of Public Health of Fudan University (to JX) and the State Key Laboratory of Genetic Engineering of Fudan University (to SLZ). References Amsterdam E. A., Wenger N. K., Brindis R. G., Casey D. E. Jr., Ganiats T. G., Holmes Jr. D. R. et al AHA/ACC guideline for the management of patients with non-st-elevation acute coronary syndromes: a report of the american college 236 Journal of Genetics, Vol. 95, No. 2, June 2016

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