Increase in the risk of clopidogrel resistance and consequent TIMI flow impairment by DNA hypomethylation of CYP2C19 gene in STEMI patients undergoing primary percutaneous coronary intervention (PPCI)

Abstract Clopidogrel resistance is an important risk factor of ischemic event recurrence after optimal antiplatelet therapy. This study aims to investigate the role of CYP2C19 gene DNA methylation as one of the epigenetic factors for the risk of clopidogrel resistance in STEMI patients undergoing PPCI. ST‐segment elevation myocardial infarction (STEMI) patients undergoing PPCI were pretreated with clopidogrel, and their platelet function was measured using VerifyNow™ assay. The criteria for high on‐treatment platelet reactivity (HPR) were defined according to the expert consensus criteria (PRU >208). DNA methylation of the CYP2C19 gene was performed using bisulfite genomic sequencing technology. Furthermore, clinical, laboratory, and angiographic data including TIMI flow were collected. Among 122 patients, clopidogrel resistance was found in 22%. DNA methylation level percentage was lower in the clopidogrel resistance group (76.7 vs. 88.8, p‐value .038). But, the <50% methylation group was associated with increased risk of clopidogrel resistance (OR =4.5, 95%CI =2.1–9.3, p‐value = .018). This group was also found to have suboptimal post‐PCI TIMI flow (OR =3.4 95%CI =1.3–8.7, p‐value =.045). The lower DNA methylation level of the CYP2C19 gene increases the risk of clopidogrel resistance and subsequent poorer clinical outcome.

clopidogrel, recurrent ischemic events still occur in up to 8%. 3,4 Many studies have demonstrated the role of HPR as an important risk factor for cardiovascular events during treatment with clopidogrel. 5 This effect leads to the failure of platelet inhibition by clopidogrel, a clinical phenomenon known as clopidogrel resistance. Several factors including genetic and nongenetic, potentially contribute to the variability of platelet response. 6 Some of the nongenetic factors are compliance, comorbidities, and drug-drug combinations. Based on the genetic polymorphism of CYP2C19 gene, notably CYP2C19*2 and CYP2C19*3, the loss-offunction allele leads to a poor metabolizer phenotype. 7 These factors only partially explain the occurrence of clopidogrel resistance, and current studies have now shifted to the role of epigenetic factors. DNA methylation is an important epigenetic marker including microRNA and histone modification. 8 Most studies regarding DNA methylation were conducted in the cancer field as well as autoimmune and neurodegenerative diseases. Compared with single nucleotide polymorphism, there is only limited evidence of an association between DNA methylation and clopidogrel resistance. 6 Jia et al. 6,9,10 demonstrated the correlation between clopidogrel resistance and several genes such as P2Y12 and PON1, where their methylation level was associated with the degree of clopidogrel response, depending on the function of each gene. These results underline the role of DNA methylation and clopidogrel resistance. However, the relationship between CYP2C19 gene DNA methylation and clopidogrel resistance has never been addressed.
Since CYP2C19 is the principal enzyme responsible for the clopiogrel biotransformation, it was postulated that DNA methylation of the CYP2C19 gene affects platelet responsiveness to the drug.
Hence, this study aims to investigate whether DNA methylation of the CYP2C19 gene contributes to the development of clopidogrel resistance and a worse clinical outcome in ACS patients. It is expected to give a better understanding of the vast mechanisms of clopidogrel resistance.

| Blood sample collection and platelet function test
Venous blood samples were collected in EDTA and citrate tubes.
Standard biochemical parameters such as ureum, creatinine, blood glucose, and lipid profile were obtained. Platelet function was measured using VerifyNow P2Y12 assay (Accumetrics Inc.). The results were reported in P2Y12 reaction units (PRU), and the values greater than 208 were considered as clopidogrel resistance. 11

| DNA methylation assay
Human genomic DNA was extracted from peripheral blood mononuclear cells using the QIAamp DNA Blood Mini Kit (Qiagen). The first step was bisulfite conversion of DNA which deaminates unmethylated cytosines to form uracil using EpiTech Bisulfite Kits. The second was polymerase chain reaction (PCR) primer design using Methyl Primer Express Software v1.0 and 7500 Fast Real-Time PCR System (Table 1).
The last was primer optimization and analysis of melting profiles.
Furthermore, the melting profiles of PCR products from the sample DNA were compared to those from fully methylated and unmethylated reference samples.

| Angiographic analysis
Coronary angiogram was analyzed to evaluate pre-and post-PCI Thrombolysis in Myocardial Infarction (TIMI) flow grade, pre-PCI angiographic thrombus grade, and procedural related complication such as dissection.

| Statistical analysis
Continuous data with a normal distribution were presented as means ± standard deviation, and compared using independent T-Test or Mann-Whitney. Categorical data were presented as numbers and percentages and also compared by the X 2 or Fisher's exact test. Logistic regression was utilized to determine clopidogrel resistance predictors and reduced final TIMI flow. A two-tailed p-value less than .05 indicated statistical significance, whereas the analyses were carried out by IBM SPSS Statistics 22.0 (SPSS Inc).  Table 2. Moreover, fragment GRCh38.p13 (Genome Reference Consortium Human Build 38 patch release 13) was used, which includes three CpG island sequences all located in the gene body. Only one sequence was analyzed and it gave an optimal result, whereas the median percentage of DNA methylation was 87.73%. CYP2C19

| Patients' characteristics
genotyping was performed to evaluate the potential modulating effect of *2 and *3 allelic variants of the CYP2C19 gene. Wildtype phenotype was observed in 62% of the patients, whereas heterozygous phenotype was in 36%. The least common variant was homozygous, found in less than 1%. Laboratory and echocardiography parameters are presented in Table 3.

| Correlation between DNA methylation and clopidogrel resistance
According to Table 4, DNA methylation percentage was lower in the clopidogrel resistance group compared to nonresistance (88. 8 ± 22.7 vs. 76.7 ± 32.9, p = .03). The level of DNA methylation was further divided into two groups, namely <50% and ≥50% methylation. The percentage of patients that belong to <50% methylation group was higher in the clopidogrel resistance group (22.2 vs. 8, p = .047) than in nonresistance. To overcome the effect of confounding factors, logistic regression analysis was performed, whereas genetic and nongenetic factors were included. Apart from the DNA methylation level, several variables were found related to the occurrence of clopidogrel resistance. Those were CYP2C19 genetic polymorphism, proton-pump inhibitor usage, and comorbidities (diabetes mellitus and chronic kidney disease). A separate analysis found sex as the only independent predictor of DNA methylation level, where males had a higher percentage (Table S1).

| Factors affecting post-PCI TIMI flow
Post-PCI TIMI flow was divided into two groups, namely TIMI flow 3 and <3.
a Data presented as mean ± standard deviations.
b Data presented as median (min−max). Other studies showed that DNA methylation tends to exhibit two different effects on gene expression. 8 When this occurs at pro- The role of DNA methylation in repressing gene transcription in the promoter region has been extensively studied. 15 There are three possible roles of intragenic DNA methylation, but the associated mechanism is not fully elucidated. 16 Several studies by Jia et al. 6,9,10 attempted to determine the relationship between DNA methylation of other genes and clopidogrel resistance. The genes were ATP-binding cassette subfamily B member 1 (ABCB1), P2Y12, and paraoxonase 1 (PON1). As opposed to CYP2C19, CGI islands in those genes are located in the promoter region. DNA methylation in the promoter region inhibits the binding of transcription factor, therefore the gene expression is silenced. 19 The P2Y12 study reported lower DNA methylation percentage in clopidogrel resistance group, 6 however, that of PON1 found an opposite result. 9 Lower methylation percentage of P2Y12 increases the gene's transcription, and consequently more P2Y12 receptors are available for ADP binding, which causes an increase in platelet activity. PON1 gene plays role in clopidogrel biotransformation. Hence, a higher methylation percentage decreases corresponding gene transcription, leading to less clopidogrel active metabolite. But, no significant relationship was found between ABCB1 gene DNA methylation and clopidogrel resistance.

TA B L E 3 Laboratory and echocardiographic parameters
Many factors influence DNA methylation status, such as demographics, diet, and environment. 20 Sex was found as the only variable that has a significant difference in methylation level which is higher in males compared to females. The study by Osman et al. 21 showed that males have higher levels of DNA methylation than females, even though the reason for this phenomenon is not fully explored. It is well recognized that DNA methylation is tissue-specific, and using blood samples to determine its level might not give similar results. 22 In terms of CYP2C19 (a liver enzyme), Kathryn et al.
used the liver sample to measure DNA methylation level. However, Lindner et al. stated that DNA methylation in the liver being highly invasive is strongly reflected by DNA methylation in red blood cells.
Therefore, it is possible to use a blood sample to measure CYP2C19 gene DNA methylation. Studies by Jia et al. 6,9,10 also used such measurement methods. This supports the use of a blood sample as a fine substitute to demonstrate CYP2C19 gene DNA methylation level instead of using the human liver.
Aside from epigenetic factor, potential genetic, and nongenetic influence on clopidogrel resistance status were also investigated. had been previously demonstrated. 25 Since response to clopidogrel depends on its activation by CYP2C19, patients with either intermediate or poor metabolizer profile tend to exhibit less antiplatelet effect.
The study by Mega et al. 26 found that the aforementioned reducedfunction variants are responsible for lower plasma levels of clopidogrel active metabolites. This finding is essential considering that the prevalence of CYP2C19 polymorphism is higher in the Asian population.
Drug interaction with proton pump inhibitor (PPI) in pharmacodynamics and clinical study showed contradicting results, 27 and this is owing to the inhibition of CYP2C19. Diabetes mellitus is a wellknown risk factor for inadequate response of antiplatelets. 28 The mechanisms contributing to this phenomenon possibly has multiple factors, such as higher level of oxidative stress, procoagulants, and lack of sensitivity to insulin. All these factors cause impaired platelet P2Y12 receptor blockade responses. Another independent predictor found is the presence of chronic kidney disease (CKD).
Hajimet et al. 29 and Patrik et al. 30 reported a higher prevalence of clopidogrel resistance in a population with CKD (defined as an estimated glomerular filtration rate of less than 60 ml/min/1.73 m 2 ). The decreased effectivity of antiplatelet in CKD patients is partly due to less expression of CYP2C19, higher platelet turnover, increased procoagulants, and defect on the platelet as well as in prostaglandin metabolism.

| Relationship between epigenetic, genetic, and nongenetic factors with post-PCI TIMI flow
The causal relationship between DNA methylation and clinical outcome such as post-PCI TIMI flow has not been addressed in previous studies. One possible mechanism is the increased prevalence of clopidogrel resistance. Capranazone et al. 31

| CON CLUS ION
In summary, it was indicated that DNA methylation of CYP2C19 gene plays an important role in the development of clopidogrel resistance apart from genetic influence such as polymorphism. The importance of epigenetic factors in the field of cardiovascular disease was also pointed out. Further studies with larger and more diverse populations in the genetic and epigenetic field are recommended for a better understanding.

D I SCLOS U R E
The authors declare no conflict of interests regarding the publication of this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.