Multiple Mutations of CYP2C19, PON1 and ABCB1 Inuence Platelet Response, Bleeding and Thrombosis Risk to Clopidogrel after Percutaneous Coronary Intervention: A Retrospective Study

Background:Different combinations of multiple mutations of CYP2C19, PON1and ABCB1 for the eciency and safety of Clopidogrel in patients undergoing percutaneous coronary intervention (PCI) remain unclear. Methods: 263 Chinese Han patients receiving 75mg Clopidogrel and 100mg Aspirin every day for 12 months after PCI were enrolled in this study. ADP-induced platelet aggregation rates, thrombosis and bleeding risk are used to compare the Clopidogrel response among the combined genetic mutation. Results: Our study demonstrated that multiple genetic mutations are very common in patients receiving Clopidogrel and Aspirin after PCI.Patients who have high ADP-induced platelet aggregation rate with more mutations of CYP2C19, PON1 and ABCB1. Patients with full mutations of CYP2C19, PON1 and ABCB1 have highest risk of thrombosis and lowest risk of bleeding in patients receiving clopidogrel and aspirin with one-year follow-up duration. Conclusions:We summarized multiple genetic polymorphism inuences platelet reactivity, bleeding and thrombosis risk to Clopidogrel after percutaneous coronary intervention. wild PCI,Percutaneous Coronary Intervention;ADP,adenosine diphosphate; BMI, body mass index; AMI, myocardial Infarction;

patients with continuous drug prescription records; (4) patients who calculated the ADP dependent Platelet aggregation rate when clopidogrel treated for at least 7 days and stayed at a stable plasma concentration.The exclusion criteria: (1) patientswho experienced the major bleeding and thrombotic events in the last 6 months before PCI; (2)Patients who received anticoagulation drugs in the last 6 months before PCI; (3) patients received CYP2C19 or ABCB1inhibitors (such as Omeprazole or Clarithromycin)during 12 months after PCI; (3) patients who stopped or adjusted the dose of clopidogrel and aspirin during dual-antiplatelet therapy; (4) patients who switched to ticagrelor or prasugrel; (5)patients with severerenal and hepatic function failure.(6)patients with large surgery during the observation period.
The study was approved by the ethics committee ofThe Fifth People's Hospital of Shanghai.

Platelet aggregation assays
Blood samples for platelet aggregation ratio assays were done after 7 days with clopidogrel and aspirin in patients undergoing PCI. Blood samples (3 ml) werecollected in the tube containing 10 µmol/L sodiumcitrate. The platelet-rich plasma (PRP) and the plateletpoor plasma (PPP) were separated by centrifuged. ThePPP and PRP samples were heated to 37°C for 3 min,then 20 µmol/L of ADP was added to the samples tomeasure platelet aggregation rate (PAR).

Genotyping
Details on Genotyping technology has been published previously [3,11]. All patients enrolled in this study have undergone genetic testing for CYP2C19, ABCB1and PON1 genotypes. The DNA were extracted and the single nucleotide polymorphisms (SNPs) of each allele(CYP2C19*2, CYP2C19*3, ABCB1 and PON-1) were determined by the DNA assay method on a Real-Time PCR System according to the manufacturer's instructions. Carrier status for all alleles was available and reported as wild type, heterozygous or homozygous for the minor allele.

Outcomes and follow-up
The thrombotic and bleeding events were extracted from out-patients follow-ups for 12 months after PCI. Thrombotic eventsare composed of stent thrombus,Stroke, TIA and target vessel-related MI. The bleeding events is consisting of major bleeding (Cerebral hemorrhage, Gastrointestinal bleeding, Fundus bleeding) and minor bleeding (Subcutaneous bleeding, Gums bleeding and so on)

Statistical analysis
Variables are presented as the mean ± standard deviation (SD) or frequencies (percentage). Continuous variables were compared using Student's t-test or one-way ANOVA, and categorical variables were compared using the chisquared test or Fisher's exact test. To examine the association between the risk factors and the ADP induced platelet aggregation rate, we performed univariate and multivariate Cox regression analyses. To avoid the potential bias of covariates between high and low ADP induced platelet aggregation rate, a casecontrol matching method was used to match variables that included age, gender, hemoglobin and hepatic function. Matching tolerance was 0.02. To compare the predictability for high ADP induced platelet aggregation rate among the genotyping mutation, logistic regression analysis was performed. Thrombosis and bleeding event rates were estimated by the Kaplan Meier method according to the multiple genetic mutation groups and compared by logrank tests.

Results
Identi cation of Multiple mutations of CYP2C19, ABCB1 and PON-1 in Han Chinese Partients. CYP2C19, PON-1 and ABCB1 genotypes were assayed in 263patients undergoing PCI. The distribution of CYP2C19*2, CYP2C19*3,PON Q192R and ABCB1 3435 C allelic and genotype frequency are shown in Table  1.Approximately 46% of the population had one or two heterozygous mutation of CYP2C19*2 or CYP2C19*3 (CYP2C19 Intermediate metabolite, IM) and 14% had homozygous alleles (CYP2C19 poor metabolite, PM). In terms of the prevalence of ABCB1 3435 C > T polymorphism, 40% of the population had one T-allele (CT, heterozygous mutation) and 17% had two T-alleles (TT, homozygous mutation). In terms of the prevalence of PON1 polymorphism, 46% of the population had one or more A-allele (GA or AA) and 18% had two A-alleles (AA). Only19 patients with no mutation of CYP2C19, PON1 and ABCB1 gene; 24 patients with three heterozygous mutation of CYP2C19, PON1 and ABCB1 gene; 25 patients with single homozygous mutation alone in CYP2C19, PON1 and ABCB1 gene (Table 1). A total of 263 patients were enrolled in our study. Table 2 shows the baseline characteristics of the overall population according to the ADP dependent Platelet aggregation rate. The patients with high ADP-Platelet aggregation were older and had lowerhemoglobin. They also exhibited a higher prevalence of acute MI as an index diagnosis, chronic liver disease, CYP2C19 variants, as well as a greater incidence of higher ADP dependent Platelet aggregation compared with those who achieved a successful anti-platelet therapy ( Table 2). The values are expressed as the mean ± SD or n (%) of patients.
Genetic polymorphism is associated with high platelet aggregation rate.
To order to compare the difference distribution of genotyping mutation between high and low ADP-induced platelet aggregation rates, a 1:1 case-control matching procedure was performed to avoid the potential bias of covariates. After matching for age gender, AMI, hemoglobin and hepatic function, there were no differences in age gender, AMI, hemoglobin and hepatic functionafter matching between high and low ADP-induced platelet aggregation rates. There remained a signi cantly higher mutation of CYP2C19, PON1 and ABCB1 gene in high ADP-induced platelet aggregation rate (Table 3). Multiple genetic mutations in uenced the adverse outcomes of dual antiplatelet therapy.
Subsequently, the bleeding and thrombotic events were analyzed to explore the correlation between multiple mutations and adverse outcomes. The rate of any bleeding and major thrombosis is signi cantly different in individual CYP2C19, PON1 and ABCB1 loss-of-function allele carriers compared with noncarriers.The lower risk of bleeding and higher risk of thrombosis was observed on interaction amongcarriers with different number of variants. Besides, regardless of carrying or not carrying CYP2C19 variants, PON1 or ABCB1 variants increased the rate of thrombotic events (Table 4).

Difference between multiple heterozygous mutation and single homozygous mutation
In order to compare the difference between multiple heterozygous mutation and single homozygous mutation, we extracted the information of patients with three heterozygous and single homozygous mutation of CYP2C19, PON1 and ABCB1 polymorphism. Patients with multiple heterozygous genotype in the clopidogrel plus aspirin group had a higher risk of bleeding (33% vs 8%; P = 0.028 for Chi-square test) and lower risk of thrombosis (0% vs 24%; p = 0.01 for Chi-square test) (Table 5). While,the thrombosis risk are not different in the two groups.

Discussion
Our study evaluated the ADP-induced Platelet aggregation rate and safety outcomesin patients with the combinedmutations of the CYP2C19, PON1 and ABCB1 undergoing PCI receiving clopidogrel and aspirin. Our study demonstrated that patients with multiple mutations of CYP2C19, PON1 and ABCB1 have higher ADPinduced Platelet aggregation rate and more bleeding and thrombotic events. Patients with all mutations of CYP2C19, PON1 and ABCB1have highest risk of thrombosis and lowest risk of bleeding in patients receiving clopidogrel and aspirin with one-year follow-up.
Multiple studies have consistently shown that PCI-treated patients with impaired clopidogrel-induced platelet inhibition increased the risk for stent thrombosis and ischemic events. Low platelet reactivity is associated with an increased risk of bleeding events, while high platelet reactivity (HPR) is correlated with a high incidence of thrombotic events [12]. Different platelet function testing, such as VerifyNow, Multiplate, vasodilator-stimulated phosphoprotein, thromboelastography (TEG) with platelet mapping and light transmission aggregometry are available for the assessment of on-treatment platelet reactivity to adenosine diphosphate(ADP) [13,14].However, based on the results of platelet function testing, a series of studies were conducted to escalate the appropriate P2Y12 inhibitors on clinical therapy and failed to improve the safety outcomes [15,16].Peng and his collaborators [3] explored the relationships of the effects of CYP2C19 and PON1 Q192R polymorphism and ADPinduced platelet inhibition by thrombelastography (TEG) in ACS patients with clopidogrel. They found individual CYP2C19 and PON1 variants in uenced platelet response on clopidogrel and both LOF CYP2C19 and PON1 192R variants are independent risk factors of high platelet reactivity. However, they didn't evaluate the in uence of the multiple variants on safety outcomes. Some researchers measured P2Y12 platelet reaction units using the VerifyNow P2Y12 test to evaluate the effect of clopidogrel on platelet aggregation.Treatment with prasugrel signi cantly reduced HPR compared with clopidogrelby P2Y12 reaction unitthresholds [17].For patients with longterm DAPT (aspirin + clopidogrel) after PCI with coronary stent implantation, switching from clopidogrel to prasugrel resulted in a stable reduction in PRU, regardless of CYP2C19 polymorphism [18]. In our study, we used ADP-induced platelet aggregation rate to evaluate the platelet response on clopidogrel. After PCI, several established risk factors, such as age, gender, hepatic function, Hemoglobin and genetic polymorphism have been incorporated into a statistical model for clopidogrel response prediction. While these factors could'tbe used to fully explain future bleeding and thrombosis events. In order to exclude the potential bias of other factors, we performed case-control matching procedure and compared the consistency between the genetic polymorphism and ADP-induced platelet aggregation rate, we found patients with allelic mutations of CYP2C19, PON1 and ABCB1have high platelet aggregation rate on clopidogrel.
Besides, we also found patients with combined mutations of CYP2C19, PON1 and ABCB1 have higher risk of thrombosis and lower risk of bleeding in patients receiving clopidogrel and aspirin with one-year follow-up. In a PCI population taking clopidogrel, the interest in genetic testing for clopidogrel metabolism has grown in recent years to augment standard risk evaluation. The allelic mutant of CYP2C19 which play a signi cant role in the rst oxidation step will lead to lower ADP-induced platelet aggregation rate and higher risk of thrombosis. But the in uence on individual PON1 and ABCB1variants iscontradictory and controversial. Chen et al [10] and Hulot et al [19] found the allelic mutant of PON1 was not a major determinant on the clopidogrel pharmacokinetics, pharmacodynamics, and clinical e cacy. While Nishio et al [20], Chen et al[8]and Li et al [21]drawed the inconsistent conclusions that PON1 Q192R genotype in uenced clopidogrel responsiveness by relative platelet inhibition, especially in CYP2C19 loss-of-function carriers. Similar disputes also occurred in ABCB1 genotype [22][23][24][25]. Most studies revealed the individual CYP2C19, PON1 and ABCB1variants on the platelet reactivity and clinical outcomes on patients receiving long-term DAPT (aspirin + clopidogrel) after PCI. A multicomponent guiding approach to personalize clopidogrel treatment is needed [26]. Some studies focus on the combined status of two at-risk variantsin PCI patients on clopidogrel [3,8,9,20].In the prior studies, both the CYP2C19 poor matebolism status and ABCB1 3435 TT genotype wasconsidered to in uence the clopidogrel metabolization and transportationandcontinuously elicited clopidogrel response [5]. The individuals carrying both at-risk variants showed the highest event rate compared to those with other combined genetic subsets. They could not nd any association between adverse events and PON-1 genotypes. However,Peng et al found both mutations of CYP2C19 and PON1 192R variants are independent risk factors of high platelet reactivity except ABCB1 variants [3]. There is a paucity of data regarding the combined effect of three at-risk variants, and no data are available to evaluate the incremental prognostic value of their combined effect over conventional clinical risk factors. In our study, we evaluated the combined effect of three at-risk variants on clinical outcomes of patients receiving clopidogrel and aspirin. The lower risk of bleeding and higher risk of thrombosis was observed on interaction among carriers with different number of variants. Besides, regardless of carrying or not carrying CYP2C19 variants, both of PON1 and ABCB1 variants increased the rate of thrombotic events.
There were still some limitations in our study. Firstly, the sample size was relatively small. Secondly, we didn't use another clinically validated and standardized platelet function testing, such as P2Y12 reaction unit (PRU), vasodilator-stimulated phosphoprotein or TEGwith platelet mapping, to de ne the platelet reactivity in the follow up duration 12 months. Lastly, in this retrospective study, we only included clinical cases with genetic test and platelet aggregation rate and complete follow-up data within one year. This may lead to bias in the inclusion of cases for ignoring patients who transferred to other hospitals and lost to follow-up after adverse events. Finally, we did not exclude the effects of other metabolism enzymes on clopidogrel resistance. Despite we concluded that patients with all mutations of CYP2C19, PON1 and ABCB1 have highest risk of thrombosis and lowest risk of bleeding in patients receiving clopidogrel and aspirin with one-year follow-up duration. Further large-scale and prospective multi-center collaborative research is still essentialto draw de nite conclusions on the current issues.

Declarations
Ethics approval and consent to participate The protocol used was approved by the Ethics Committee of The Fifth people's Hospital of Shanghai, Fudan University (No. 2018-192).Due to the retrospective nature of the study, informed consent was waived by the Ethics Committee of The Fifth people's Hospital of Shanghai. All the human data have been performed in accordance with the declaration of Helsinki.All methods were carried out in accordance with the declaration of Helsinki and relevant guidelines and regulations.

Consent for publication
All the authors are consent for publication.

Availability of data and material
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Con icts of interests
All the authors have no con ict of interest to declare.

Funding
The study was supported by grants from Shanghai Minhang Health and Family Planning Commission