{'fraction_non_alphanumeric': 0.034282614591809056, 'fraction_numerical': 0.03569297531868728, 'mean_word_length': 5.224172856178258, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 5, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Impact of Insulin Receptor Substrate‐1 rs956115 and CYP2C19 rs4244285 Genotypes on Clinical Outcome of Patients Undergoing Percutaneous Coronary Intervention

Background Insulin receptor substrate‐1 (IRS‐1) rs956115 is associated with vascular risk in patients with coronary artery disease and concomitant diabetes. CYP2C19*2 (rs4244285) modulates clopidogrel response and predicts the outcome of coronary artery disease. This study was designed to explore the association between IRS‐1, CYP2C19*2 genotypes, platelet reactivity, and 1‐year outcome in patients with coronary artery disease undergoing percutaneous coronary intervention. Methods and Results Genotyping was performed using an improved multiplex ligation detection reaction technique. Platelet aggregation was assessed by light transmission aggregometry. Major adverse cardiovascular events were defined as a composite of cardiovascular death, myocardial infarction, and ischemic stroke. A total of 2213 consecutive patients were screened and 1614 were recruited. At 1 month, patients with IRS‐1 CG genotype had significantly lower levels of ADP‐induced platelet aggregation compared with patients with CC homozygotes. Patients with IRS‐1 CG or GG genotype had a 2.09‐fold higher risk of major adverse cardiovascular events compared with those with CC homozygotes (95% CI, 1.04–4.19; P=0.0376). By comparison, patients with CYP2C19*2 GA or AA genotype had higher ADP‐induced platelet aggregation compared with patients with GG homozygotes. Although there was no significant difference in risk of major adverse cardiovascular events between patients with GA/AA and GG genotypes, patients with GA genotype had a 2.19‐fold higher risk than those with GG homozygotes (95% CI, 1.13–4.24; P=0.0200). No interaction between IRS‐1 and CYP2C19*2 genotypes was observed. Conclusions In patients following percutaneous coronary intervention, IRS‐1 GG/CG and CYP2C19*2 GA genotypes were associated with 2.09‐ and 2.19‐fold increased cardiovascular risk, respectively, at 1‐year follow‐up. The association between IRS‐1 genotypes and major adverse cardiovascular events appeared to be independent of known clinical predictors. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01968499.


Zong et al
Impact of IRS-1 Genotypes on Post-PCI Patients I nsulin receptor substrate-1 (IRS-1), a ligand of insulin receptor tyrosine kinase, plays a central role in the insulin signal transduction system. 1,2 Dysregulation of IRS-1 has been suggested as a common mechanism underlying insulin resistance that may lead to high platelet reactivity and low response to antiplatelet treatment in patients with type 2 diabetes. 3, CYP2C19 is one of the isoenzymes of the hepatic cytochrome P450 system, which plays a key role in the bioactivation of clopidogrel. 5,6 Patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI) who are carriers of CYP2C19 loss of function *2 (rs4244285) have lower levels of the active metabolite of clopidogrel than wild-type homozygotes, which is associated with lower clopidogrel responsiveness and an increased risk of major adverse cardiovascular events (MACE). [7][8][9] This study was designed to examine the association between IRS-1 rs956115, CYP2C19*2 genotypes and platelet reactivity as well as MACE in patients with CAD who had undergone PCI and were treated with aspirin and clopidogrel.

METHODS Ethical Considerations
All protocols for this study were reviewed and approved by the ethics committee of the First Affiliated Hospital of Nanjing Medical University (approval number 2011-SRFA 099). Written informed consent was obtained from each patient. The data that support the findings of this study are available from the corresponding author on reasonable request.

Study Design
A prospective single-center cohort study was conducted in the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. The inclusion criteria were patients with CAD undergoing urgent or elective coronary stent implantation who were aged >18 years and planning to take dual antiplatelet treatment with clopidogrel 75 mg and aspirin 100 mg once daily for at least 1 year. Patients who met any of the following criteria were excluded: (1) allergic or intolerant to aspirin or clopidogrel; (2) at high risk for bleeding (eg, platelet count <80×10 9 /L, known bleeding diathesis, active peptic ulcer, or with a history of cerebral hemorrhage within 1 year); and (3) planning to take drugs that could potentially interfere with the antiplatelet effects of aspirin (eg, NSAIDs) or clopidogrel (eg, CYP3A inhibitors or inducers). Baseline demographic and clinical characteristics as well as medical treatments and procedural details were collected on prespecified case report forms.

Laboratory Sample Collection and Preparation
After receiving >5 days of aspirin and clopidogrel, blood samples were collected 2 hours after the most recent dose (≈10 am) into one 2-mL BD Vacutainer tube (Becton, Dickinson and Company) containing 3.6 mg of K2 EDTA and into two 2-mL BD Vacutainer tubes containing 0.105 mol/L of buffered sodium citrate (3.2%). Within 1 hour of collection, blood samples were transferred to the central laboratory. EDTA samples were frozen at −80°C for subsequent genotyping, whereas citrated samples were processed immediately for platelet aggregation studies. After centrifuging citrated samples at 200 g for 8 minutes at 22°C, platelet-rich plasma was carefully separated. The remaining sample was centrifuged at 2465 g for another 10 minutes to obtain platelet-poor plasma. The platelet count in platelet-rich plasma was standardized by adding platelet-poor plasma to achieve a count of

CLINICAL PERSPECTIVE
What is New?
• In patients with recent percutaneous coronary intervention, insulin receptor substrate-1 rs956115 G allele was associated with a 2.09fold higher cardiovascular risk at 1 year. • The association between the insulin receptor substrate-1 G allele and cardiovascular outcomes was independent of CYP2C19*2 genotypes and known clinical predictors.
What are the Clinical Implications?
• Insulin receptor substrate-1 genotyping provides further opportunity to improve risk stratification of individual patients undergoing percutaneous coronary intervention. • The underlying mechanism linking insulin receptor substrate-1 genotype and cardiovascular risk warrants further investigation.

Platelet Aggregation Studies
Platelet aggregation testing was performed using a Chronolog Model 700 aggregometer (Chronolog Corporation). Immediately after preparation of plateletrich plasma, 500 μL was transferred into each of the 2 test tubes, with 500 μL platelet-poor plasma as control. Platelet aggregation was induced using ADP or arachidonic acid (AA) as agonists with final concentrations of 5 µmol/L and 1 mmol/L, respectively. ADP and AA-induced platelet aggregation (PL ADP and PL AA , respectively) was recorded using the maximum platelet aggregation within 8 minutes. PL ADP >40% was defined as high on-treatment platelet reactivity. 11

Genotype Analysis
IRS-1 (rs956115, C>G) and CYP2C19*2 (rs4244285, G>A) genotyping was performed using a custom-bydesign improved multiplex ligation detection reaction technique (Genesky Biotechnologies Inc) based on highly specific double ligation and multiplex fluorescence polymerase chain reaction. 12 For quality control, repeated testing was performed randomly in 5% of samples.

Clinical Follow-up
Patients were followed for 12 months by investigators who were blinded to the results of platelet reactivity testing and genotyping. Patients were reviewed in person or by telephone if they could not attend the clinic.

Statistical Analysis
Assuming a MACE rate of 2.3%, 14 a sample size of 1052 patients was required to detect a hazard ratio (HR) of 2.88 15 with 90% power and a 2-sided α value of 0.05. Continuous variables were described as mean±SD or median and interquartile range when data did not follow a normal distribution, and the statistical significance of any differences between groups was analyzed using a t test or nonparametric test. Categorical variables were expressed as numbers and percentages, and the statistical significance of any differences between groups was analyzed using a χ 2 test or Fisher exact method. One-way ANOVA was used to compare platelet reactivity among different genotypes of IRS-1 and CYP2C19*2. Multivariable Cox proportional hazard model analysis was used to estimate the association between genotypes of IRS-1 and CYP2C19*2 and risk of MACE, reported as HRs and 95% CIs. The model was adjusted for clinical covariables including age, previous MI, hypertension, diabetes, smoking status, previous PCI, left ventricular ejection fraction, serum creatinine, low-density lipoprotein, and diagnosis. The date of PCI was set as "time zero" with censoring at the end of study follow-up.
All data analyses were performed using SAS version 9.4 (SAS Institute Inc) and figures were created using R version 3.2.0 (R Foundation for Statistical Computing). 16,17 A 2-tailed P value of <0.05 was considered statistically significant.

RESULTS
Between March 2011 and September 2016, 2213 patients were consecutively screened and 1614 patients who fulfilled the eligibility criteria were enrolled. Three patients were excluded from the final analysis because of unsatisfactory blood sample quality. All of the remaining patients completed the genotype assessment and 1-year clinical follow-up. Platelet aggregation testing was performed in 1175 patients at baseline and in 624 patients at 1 month ( Figure 1).

Patient Characteristics
Baseline characteristics are summarized in Table 1. Patients who experienced MACE compared with those who did not were older (69.00 [14.50] versus 64.00 [15.00], P=0.0069) and more commonly having reduced left ventricular ejection fraction (25.0% versus 7.66%, P<0.0001) and diagnoses of non-ST-segmentelevation acute coronary syndromes and ST-segmentelevation MI (63.63% versus 42.44%, P=0.0010). There was no significant difference in baseline characteristics between the 602 patients screened but not included and the 1611 patients who were enrolled (Table S1, Figure 1). Of the enrolled patients, 1175 had their platelet reactivities measured at baseline and 602 remeasured at 1 month. There were no significant differences in all baseline characteristics except smoking and previous PCI between patients who underwent reassessment of platelet reactivity at 1 month and those who did not (Table S2, Figure 1).

Association Between IRS-1/CYP2C19*2 Genotypes and Cardiovascular Outcomes
For CYP2C19*2, there was no significant difference in the risk of MACE between patients with GA or AA genotype and those with GG genotype (dominant model: P=0.0759) ( Table 2). However, the risk of MACE was 2.13-fold higher in patients with GA genotype than in GG homozygotes (adjusted HR, 2.13; 95% CI, 1.10-4.12 [P=0.0248]) ( Table 2). In the meantime, no significant difference in the risk of   Figure 3D), while no significant difference was observed in the risk of MACE between AA and GG genotypes (P=0.4787) ( Table 2 and Figure 3D). The entire results with categorical, dominant, additive, recessive models are presented in Table S4.

Interaction Analysis
Among patients with GG genotype of CYP2C19*2, those who had CG or GG genotypes of IRS-1 presented with a 4.85-fold higher MACE risk than those who had CC genotype (adjusted HR, 4.85; P=0.0081) ( Figure 4). By comparison, among patients with the non-GG genotype of CYP2C19*2, those with CG or GG genotypes of IRS-1 presented with a 1.40-fold higher risk than those who had CC genotype (adjusted HR, 1.40; P=0.4764) (Figure 4). The interaction between IRS-1 and CYP2C19*2 genotypes was nonstatistically significant (P=0.1453) (Figure 4).

Association of IRS-1 Genotypes With MACE in Subgroup Analysis
We performed multivariable Cox regression analysis for IRS-1 genotypes in different patient subgroups ( Figure S3). The association between IRS-1 genotypes and MACE remained statistically significant in the subgroup of normal serum creatinine (adjusted HR, 2.09; 95% CI, 1.04-4.18) ( Figure S3). Although the adjusted HR between CG or GG and CC genotypes of IRS-1 did not reach statistical significance in the diabetes subgroup ( Figure S3), the dominant model HR of MACE for patients with CG or GG genotypes of IRS-1 tended to be similar among subgroups. No significant interactions were observed in those subgroups except left ventricular ejection fraction (interaction P=0.0006) ( Figure S3).

DISCUSSION
This study examined the association between IRS-1, CYP2C19*2 genotypes and clinical outcomes of patients undergoing PCI and receiving dual antiplatelet treatment, and found that G allele carriers of IRS-1 had a 2.09-fold higher risk of MACE compared with noncarriers at 1-year follow-up. Patients with CYP2C19*2 GA genotype had a 2.19-fold higher risk compared with GG homozygotes. The association between IRS-1 genotypes and MACE was independent of known clinical covariables, while the association between CYP2C19*2 genotypes and MACE could be mediated by lower clopidogrel response. Angiolillo et al 15 examined 7 single nucleotide polymorphisms of IRS-1. They found that IRS-1 rs956115 polymorphism was associated with a hyperreactive platelet phenotype and adverse cardiovascular outcomes in White patients with type 2 diabetes who had concomitant CAD. However, uncertainty remains about the association between IRS-1 genotypes and In this study, we found that the IRS-1 G allele was an independent prognostic factor of adverse cardiovascular outcomes in patients with nonselective CAD, irrespective of CYP2C19*2 genotype, diabetes, and other known risk factors. Although the IRS-1 G allele did not show a significant correlation with MACE in the subgroup of diabetes, our results showed the consistent tendency of almost all subgroups, as shown in Figure S3.
Regarding the underlying mechanism, Angiolillo et al 15 suggested that IRS-1 rs956115 polymorphism was associated with a hyperactive platelet phenotype in White patients with type 2 diabetes. However, in a later study by Zhang et al, 18 no association was observed between IRS-1 rs956115 polymorphism and platelet function profile. Our results were consistent with that of Zhang and colleagues' in a larger Chinese population, showing no significant difference in AA or ADP-induced platelet aggregation at baseline among different IRS-1 genotypes. Moreover, ADP-induced

Zong et al
Impact of IRS-1 Genotypes on Post-PCI Patients platelet aggregation was even lower in the IRS-1 CG genotype compared with the CC genotype at 1-month follow-up. Along with the results of Zhang et al's study, we suggest that the association between IRS-1 genotypes and the risk of MACE cannot be explained by impaired platelet reactivity to either clopidogrel or aspirin. Theoretically, IRS-1 is one of the central nodes in the insulin signaling network. 19 It has been reported that IRS-1 is necessary for insulin-stimulated activation of the phosphatidylinositol 3 kinase/AKT pathway and subsequent enhanced production of nitric oxide in endothelial cells, 20 which plays a critical role in maintaining cardiovascular homeostasis. 21 Previous studies have demonstrated that functional variants of IRS-1 directly impaired insulin-regulated nitric oxide synthesis in cultured human endothelial cells. 22,23 Considering the pivotal role of IRS-1 in the phosphatidylinositol 3 kinase/AKT signaling pathway of insulin, it may be reasonable to assume that IRS-1 rs956115 polymorphism affects the same process or an unknown pathway and consequently impacts the clinical outcome of patients with CAD.
Our results were consistent with previous reports and further confirmed that CYP2C19*2 loss of function polymorphism is a strong predictor of impaired clopidogrel response and adverse clinical outcomes. [7][8][9] This consistency, in turn, enhances the credibility of our results on IRS-1. Meanwhile, we did not find a statistically significant interaction between IRS-1 and CYP2C19*2 genotypes from the interaction analysis, which proved the IRS-1 G allele to be an independent risk factor for MACE in patients with CAD after PCI. However, the apparent lack of interaction between genotypes on MACE may also be explained by low power caused by the small number of events. Regarding medication compliance, 42 (2.6%) patients permanently discontinued 1 or 2 antiplatelet agents because of major or minor bleeding events.
Our data indicate that IRS-1 genotyping provides further opportunity to improve risk stratification of individual patients undergoing PCI. We suggest that genotyping of the IRS-1 gene should be done in patients with high ischemic risks or recurrent ischemic events to predict the prognosis. Ideally, any treatment strategy that involves genotyping of the IRS-1 gene requires prospective evaluation to confirm that identification of patients at high risk using this approach can improve clinical outcomes.

Study Limitations
This study has potential limitations. First, because of limited funding, we did not evaluate CYP2C19*3 genotypes, also a determinant of clopidogrel metabolism. A potential interaction between IRS-1 and CYP2C19*3 genotypes and their impact on the clinical outcome remains to be investigated. Second, the number of However, there were no significant differences in all baseline characteristics except smoking and previous PCI between patients who underwent reassessment of platelet reactivity at 1 month and those who did not (Table S2). Furthermore, the pattern of platelet reactivity according to genotype at 1 month were consistent with those seen at baseline (Figure 2, Figure S1), which also makes it less likely that selection bias accounts for our findings.

CONCLUSIONS
IRS-1 rs956115 G allele was associated with an increased cardiovascular risk in patients post-PCI by 2.09-fold at 1-year follow-up, which was independent  Model adjusted for clinical covariates, including age, previous myocardial infarction, hypertension, diabetes, smoking status, previous percutaneous coronary intervention, left ventricular ejection fraction, serum creatinine, low-density lipoprotein, and diagnosis. * P value indicates the association between IRS-1 genotypes and major adverse cardiovascular events in all patients.