Genetic Determinants of CYP2C19 Gene *2 and *3 Loss of Function Alleles and Response to Anti Platelet Therapy (Clopidogrel) and Cardiovascular Events. (A Study in Kashmir, North India)

The hepatic CYP2C19 enzyme contributes to the metabolism of many clinically relevant drugs such as antidepressants, benzodiazepines, mephenytoin, some proton pump inhibitors, and clopidogrel. Like many other CYP450 superfamily members, the CYP2C19 gene is highly polymorphic, having more than 25 known variant alleles (http://www. cypalleles. ki.se/cyp2c 19.htm). CYP2C19*1 represents the wild-type allele. On the basis of their ability to metabolise (S)-mephenytoin or other probe drugs, individuals can be categorized as Extensive Metabolizer (EM), PM (Poor Metabolizer or Ultra rapid Metabolizer (UM) for CYP2C19. Heterozygous EMs are sometimes also referred to as Intermediate Metabolizer (IMs) [1]. The majority of the CYP2C19 PMs are carriers of the variant alleles *2 and *3, which are loss of function alleles (LOF) whereas the *17 variant is a gain of function (GOF) allele associated with increased activity [2]. Studies have shown a marked interethnic variation in the distribution of variant alleles. The allelic frequency of CYP2C19*2 has been shown to be 15% in Africans, 29–35 % in Asians, 12–15% in Caucasians and 61% in Oceanians. The CYP2C19*3 is mainly found in Asians (5–9% in Asians, less than 0.5% in Caucasians). The allelic frequency of CYP2C19*17 has been shown to be 16% in Africans, 3–6% in Asians and 16–21% in Caucasians [3].


Introduction
The hepatic CYP2C19 enzyme contributes to the metabolism of many clinically relevant drugs such as antidepressants, benzodiazepines, mephenytoin, some proton pump inhibitors, and clopidogrel. Like many other CYP450 superfamily members, the CYP2C19 gene is highly polymorphic, having more than 25 known variant alleles (http://www. cypalleles. ki.se/cyp2c 19.htm). CYP2C19*1 represents the wild-type allele. On the basis of their ability to metabolise (S)-mephenytoin or other probe drugs, individuals can be categorized as Extensive Metabolizer (EM), PM (Poor Metabolizer or Ultra rapid Metabolizer (UM) for CYP2C19. Heterozygous EMs are sometimes also referred to as Intermediate Metabolizer (IMs) [1]. The majority of the CYP2C19 PMs are carriers of the variant alleles *2 and *3, which are loss of function alleles (LOF) whereas the *17 variant is a gain of function (GOF) allele associated with increased activity [2]. Studies have shown a marked interethnic variation in the distribution of variant alleles. The allelic frequency of CYP2C19*2 has been shown to be 15% in Africans, 29-35 % in Asians, 12-15% in Caucasians and 61% in Oceanians. The CYP2C19*3 is mainly found in Asians (5-9% in Asians, less than 0.5% in Caucasians). The allelic frequency of CYP2C19*17 has been shown to be 16% in Africans, 3-6% in Asians and 16-21% in Caucasians [3].
Clopidogrel is a prodrug whose in vivo metabolite binds to the platelet P2Y12 receptor causing irreversible blockade. The pharmacodynamic response to clopidogrel has substantial inter patient variability [4][5][6][7] and patients with coronary disease with lesser degrees of platelet inhibition in response to clopidogrel appear to be at increased risk for cardiovascular events [7][8][9]. Approximately 85% of the parent drug is inactivated by human carboxylesterase 1, whereas the remainder is transformed to the intermediate, inactive oxo-clopidogrel by CYP2C19, CYP1A2 and CYP2B6 [10].
Clopidogrel is an antiplatelet drug used in atherothrombotic diseases, such as myocardial infarction and stroke, which is an inactive prodrug that needs to be bioacti vated by a liver enzyme, CYP2C19. Several loss-of-function alleles have been previously identified [11]. CY2C19*2 and CYP2C19*3 are the two most frequent variants in Occidentals and Asians, respectively. Major cardiovascular events occur two-to three-times more frequently in patients treated with clop idogrel having decreased CYP2C19 function com pared with wildtype patients after myocardial infarction, stent throm bosis [12][13][14]. A gene-dose effect seems to occur with the patients heterozygous for one CYP2C19 vari ant showing an intermediate clinical response between wild-type patients and patients homo zygous for the loss-of-function Page 2 of 6 variants [12]. Identification CYP2C19 alleles with reduced function has also been associated with reduced responsiveness to, and thus reduced therapeutic benefit of, clopidogrel in cardiovascular patients [12,[15][16][17][18][19]. The presence of CYP2C19 loss of function alleles *2 and *3 may result in lower exposure to Clopidogrel and thus decreased platelet inhibition in clopidogrel treated patients [20]. For this reason FDA issued a Boxed Warning to the clopidogrel label to stress upon the need to identify patients with CYP2C19 alleles classified as PMs who are considered at an increased risk of adverse cardiovascular outcomes due to reduced effectiveness of clopidogrel [21]. A systematic review and meta-analysis was conducted to examine the association between CYP2C19 genotype and the clinical efficacy of clopidogrel where it was shown that the presence of at least one loss of function allele was associated with an increased risk of stent thrombosis [22]. Owing to the established role of CYP2C19 genotype and its relation in anti-platelet (Clopidogrel) therapy, the guiding principles for clopidogrel dosing is still a matter of debate. Besides, ethnic difference exists in both pharmacokinetics and pharmacodynamic and they play an important role in optimization of therapy for the individual patient and drug administration. Since no such study has been conducted in this part, so our investigation will examine the CYP2C19 polymorphism in our patients on clopidogrel treatment, and its effect on the cardiovascular outcomes in patients with Acute Coronary Syndrome.

Methodology
This study was conducted in Sher-i-Kashmir Institute of Medical Sciences, (Jammu and Kashmir, India) in the Department of cardiology, Immunology and Molecular Medicine and Advanced Centre for Human Genetics for a period of 2 years. Clearance from local SKIMS ethical committee was taken prior to study.

Study subjects
A total of 100 samples were included in this study which comprised of confirmed cases of ACS which were selected from Department of Cardiology, SKIMS. Blood samples were taken from the patients after a written pre informed consent was obtained. Demographic and clinic-pathological characteristics of each patient were recorded in a Questionnaire. Patients were followed over a period of 2 years in Cardiology Out-patient Department (OPD) and also through telephonically to check the compliance of the drug and cardiovascular events like death from any cardiovascular cause, ACS, fatal and nonfatal MI. The patients were included on criteria like ACS with or without undergoing PCI, ST Elevation Myocardial Infarction (STEMI), Non ST Elevation Myocardial Infarction (NSTEMI) and those with Unstable Angina (USA). The patients who include history of bleeding diathesis, stroke less than 3 months platelet count <70000/mm 3 , hematocrit <30% were excluded from the study. Written informed consent was obtained from all the study participants. Patients recruited for the study were put on clopidogrel 300 mg after diagnosed ACS followed by 75 mg OD, and Patients undergoing PCI received 600 mg followed by 150 mg OD for atleast for 7 days and 75 mg later for a period of one year.

DNA extraction and polymerase chain reaction (PCR)
2 -5 ml of blood sample was taken from each patient and stored at -20°C for DNA analysis. Genomic DNA was isolated using standard proteinase-K digestion, phenol/chloroform extraction, and ethanol precipitation method from wholeblood samples of both cases and controls. The primers used were Cyp19*2 F-5΄AAATTGTTTCCAATCATTTAGCT-3΄ and R-5΄ACTTCAGGGCTTGGTCAATA-3΄ whereas for Cyp19*3 F-5΄-CAGAGCTT GGCA TATTGTAT C-3΄ and R-5΄GTAAACACACA ACTAGTCAA TG-3΄ producing 321 bp and 271bp PCR products respectively. PCR was carried out in a final volume of 25 uL containing 50 ng genomic DNA template, 1x PCR buffer (Biotools, B and M Labs, Madrid, Spain) with 2 mmol/L MgCl 2, 0.4 mmol/L of each primer (Genscript, Piscataway, NJ), 50 mmol/L dNTPs (Biotools, B and M Labs), and 0.5 U Taq Polymerase (Biotools, B and MLabs). For PCR amplification, the standard protocol was used as follows: one initial denaturation step at 94°C for 7 minutes, followed by 35 denaturation cycles of 30 seconds at 94°C, 30 seconds of annealing at 53°C, and 30 seconds of extension at 72°C, followed by a final elongation cycle at 72°C for 5 minutes.

Restriction Fragment Length Polymorphism (RFLP)
Two principle alleles of CYP2C19*2 (rs4244285) and CYP2C19*3 (rs4986893) were analyzed by PCR-RFLP. Exon 4 and Exon 5 of CYP2C19 were amplified by PCR that contain the CYP2C19 *2 and *3 polymorphisms respectively. 681G>A in Exon 5 of CYP2C19 (*2) creates an aberrant splice site and destroys SmaI restriction site and 636G>A in CYP2C19*3 gene, creates a premature stop codon and abolishes BamHI restriction site. 10ul of PCR amplified DNA were digested with 5 Units of each SmaI and BamHI (Fermentas Life Sciences, USA) restriction enzymes for both polymorphisms CYP2C19 *2 and *3 respectively while keeping the other conditions as per instructions of manufacturer. The digested products were electrophoresed using 2% agarose gel and visualized by Ethidium Bromide staining. The PCR products (271bp) of *3 yielded two bands of 175bp and 96bp in wild type and three products of 96, 175 and 271bp in heterozygous variants ( Figure 1A). The PCR product (321bp) encompassing allele *2 digested into two smaller bands of 109bp and 212bp in wild type individuals whereas heterozygous variants for this allele yielded three bands of 321, 212 and 109bp and in homozygous variants the PCR product would not be digested ( Figure 1B).

Statistical Analysis
All continuous variables of study have been shown in terms of Mean ± SD and the categorical variables in terms of frequency and percentage. The standard statistical tests like T-test. Chi square test and Fischer -exact test have been used to analyze the data, moreover odds ratio with 95% confidence interval have been calculated at appropriate places. All the results obtained have been discussed on 5% level of significance (p value<0.05) considered as significant, also suitable statistical charts have been used to represent the data, SPSS version 20 was used for analysis.

Results
The mean age of our patients was 61.1 ± 8 (range 43 to 75) and majority of patients belonged to age groups of 50-60(38%) and 60-70 years (39%) where 80% of patients were males and 20% females. Hypertension and diabetes mellitus was present in 84% and 39% of patients respectively. Our study included 71% patients with ST Elevation In our study 15 patients had Cardiovascular events (CV) on follow up in which there were 04 deaths (possible stent thrombosis), 06 patients had definite stent thrombosis, 02 patients had probable stent thrombosis (who presented with MI in the same territory), 02 patients had MI in the same territory who had not undergone percutaneous coronary intervention (PCI) after coronary angiography (CAG) and 01 patient had instant restenosis (ISR) and presented with ACS. The cardiovascular events were noticed over a study period mostly during the first year of follow up.
In the poor-metabolizer group of patients 20.1% of patients had CV events on follow up compared to 15.6% in the extensive metabolizer group of patients, whereas in the intermediate group only 10% of patients had CV events (Figure 1). All these figures were not statistically significant (p <0.05) ( Table 3).

Discussion
Clopidogrel is an antiplatelet drug used in atherothrombotic diseases, such as myocardial infarction and stroke, which is an inactive prodrug that needs to be bioacti vated by a liver enzyme, CYP2C19. Several loss-of-function alleles have been previously identified [11]. CY2C19*2 and CYP2C19*3 are the two most frequent variants in Occidentals and Asians, respectively. CYP2C19 alleles are associated with reduced conversion of clopidogrel to its active metabolite. Many studies support the observations regarding reduced-function of CYP2C19 polymorphisms and platelet aggregation among clopidogreltreated subjects [20,23,24]. Ethnic difference also exists and they play an important role in optimization of therapy for the individual patient and drug administration. To our knowledge this study is the first from the subcontinent, to investigate the CYP2C19 polymorphism in our patients on clopidogrel treatment, and its effect on the cardiovascular outcomes in patients with acute coronary syndrome.
In the poor-metabolizer group of patients 20.1% of patients had CV events on follow up compared to 15.6% in the extensive metabolizer group of patients, whereas in patients in the intermediate group only 10% of patients had CV events. All these figures were not statistically    significant (p > 0.05) and these results were consistent with study of Guillame [19].
The previous studies other than conducted by Guillame et al. [19] had shown attenuated benefits of clopidogrel among carriers of loss of function alleles. Our study matched the results of the study conducted by Guillame et al. [19], although poor metabolizer group of patients had increased frequency of CV events than extensive metabolizer group but could not achieve significance. All these results can be explained to some extent by potential pleotropic effects of loss of function alleles (independent of their effects on active metabolite of clopidogrel). Moreover since variation of non-CYP genes may also have effect on responsiveness to clopidogrel and the likelihood of ischemic events.
Despite increased prevalence of heterozygous and mutant allele in our population, we could not see significant CV events in our patients on the basis of CYP2C19 *2 and CYP2C19*3 alleles (loss of function alleles), which justifies us to some extent to continue clopidogrel in our patients, the majority of which belong to low economic class who may not be able to afford the antiplatelet agents like prasugrel and ticagrelor. The FDA has added boxed warning to clopidogrel which is for patients who do not effectively metabolize the drug (poor metabolizers). However this study with comparatively less sample size prompts us to enroll more patients to elucidate whether such approach can be applied to the patients of this geographical area.

Conclusion
We conclude that patients carrying CYP2C19 loss-of-function alleles had a higher rate of subsequent cardiovascular events as against those    with normal allele. These events in CYP2C19 loss-of-function alleles were particularly marked among the patients undergoing percutaneous coronary intervention. Lack of significant events even in presence of variant alleles justifies us to some extent to continue clopidogrel in our patients.