Pharmacogenomics Study of Clopidogrel by RFLP based Genotyping of CYP2C19 in Cardiovascular Disease Patients in North-East Population of India

Prasanthi SV1, Vinayak S Jamdade2, Nityanand B Bolshette1, Ranadeep Gogoi1* and MangalaLahkar2,3 1Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Gauhati Medical College, Guwahati, Assam, India 2Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Gauhati Medical College, Guwahati, Assam, India 3Department of Pharmacology, Gauhati Medical College, Guwahati, Assam, India


Sample size
We have studied 60 patients who received clopidogrel from Gauhati medical college and hospital Assam, for period of six month from September 2012 to February 2013. 2 ml of whole blood for DNA extraction was collected using 20-gauge needle and syringe in EDTA tubes which were obtained after informed consent according to declaration of Gauhati medical college and Hospital, Guwahati.

DNA extraction
Genomic DNA was extracted by using Hipura blood genomic DNA extracting mini preparation kit by following the manufacturer's instructions. The presence of DNA was confirmed by running DNA in 0.8% agarose gel.

RFLP analysis
Resulting amplicons of CYP2C19*2; CYP2C19*3 and CYP2C19*17 were subjected for restriction digestion with SmaI, BamHI and LweI (New England Biolabs) respectively. CYP2C19*2; CYP2C19*3 PCR products were digested at 37°C for 1 hour and digestion of CYP2C19*17 amplicon was carried out with LweI for 3 hour at same temperature. Enzyme deactivation was done at 65°C for 20 min and the resulted RFLP products were analysed by 2.5% (w/v) agarose gel electrophoresis.

Discussion
This study aimed to determine the influence of genetic variations related to the cardiovascular disease patients who were on clopidogrel treatment at the time of the event. Clopidogrel is a thienopyridine prodrug that requires hepatic biotransformation to form an active metabolite that selectively and irreversibly inhibits the purinergic P2RY 12 receptor, and thereby platelet aggregation, for the platelet's life span (~10 days) [37][38][39]. Only 15% of the prodrug is available for transformation to the active agent; other 85% is hydrolyzed by esterases to inactive forms. Conversion of clopidogrel to its active metabolite requires two sequential oxidative steps involving several CYP450 enzymes (e.g., CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4/5) [40].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 super-family members, the CYP2C19 gene is highly polymorphic, having more than 25 known variant alleles (http://www.cypalleles.ki.se/cyp2c19.htm). The CYP2C19*1 allele is associated with functional CYP2C19-mediated metabolism. The most common CYP2C19 loss-of-function allele is *2 (c.681G>A; rs4244285), with allele frequencies of ~15% in Caucasians and Africans, and 29-35% in Asians [38,39,41,42].

Statistical analysis
Genotype and allele frequencies were calculated from the counts. Expected genotype numbers were calculated using the Hardy-Weinberg equation (p 2 + 2pq + q 2 = 1) using the allele numbers, where q is the number of the variant alleles (CYP2C19*2, CYP2C19*3 and CYP2C19*17) and p is the number of wild-type alleles at the corresponding CYP2C19*2, CYP2C19*3 and CYP2C19*17 loci. The χ 2 test P value (P>0.05) was consistent with the Hardy-Weinberg equilibrium (HWE). Data were recorded on a predesigned proforma  Note: The genotype reported as "no allelic variants" is suggestive of *1 alleles.     [24,[27][28][29]31,32]. Therefore, based on identified CYP2C19 genotypes, individuals can be categorized as extensive metabolizers (e.g., *1/*1), intermediate metabolizers (e.g., *1/*2), or poor metabolizers (e.g., *2/*2). In contrast, the CYP2C19*17 allele (c.-806C>T; rs12248560) results in increased activity as a consequence of enhanced transcription, with average multi-ethnic allele frequencies of ~3-21% [33,34]. Individuals who carry this allele may be categorized as ultra-rapid metabolizers (e.g., *17/*17). Some studies indicate that this allele results in enhanced platelet inhibition and clopidogrel response, and possibly an increased risk of bleeding complications [28]. However, other studies have not identified an effect of CYP2C19*17 [31,35,36], and adequate evidence for an independent effect of this allele on clinical outcomes is lacking.Probably this was the first study carried out in North East Indian population which had a higher prevalence rate of CYP2C19*2 ~40% when compared with allele frequencies of ~15% in Caucasians and Africans, and 29-35% in Asians. This study will help to make best use of benefit path and reducing harm how appears to lie in stratifying patient individuality in response to the treatment. This genetic testing aims to match treatment to an individual genetic profile. It will be helpful in designing clinical trials particularly at initial phases of drug development.It helps to reduce the number of patients needed, prove efficiency, and identify subgroups in trial design, also alternative treatment can be targeted. Over recent years, genetic testing has been increasingly used in clinical practice. Newer antiplatelet agents are failed to demonstrate superiority to clopidogrel without trade off of more bleeding [42][43][44][45][46][47]. The functional information on variants is important for justifying its clinical use. Understanding the functional meanings of CYP2C19 variants is an essential step toward shifting the current medical paradigm to highly personalised therapeutic regimen. The clinical decision strategies following CYP2C19 genotyping suggest two regimens: 1) an adjustment of drug dose according to genotype 2) an alternative drug choice [48].
To date the pharmacogenomic data on CYP2C19 clearly supports the genetic variants alter the drug response of its substrate drugs. However, clinical application of CYP2C19 pharmacogenetics is limited to certain genotypes [49]. The number of study population that would benefit from pharmacogenomics research would be greatly reduced if such studies focused on common variants for strong statistically evidence. One goal of pharmacogenomics is to provide personalised medicine and to provide an appropriate dose of the most appropriate drug to him or her. Therefore, more diversified investigations.

Conclusion
In conclusion, we have shown that loss of functional allele CYP2C19*2 had higher carriage frequency; whereas, CYP2C19*3 and *17 alleles were not found in cardiovascular patients who were taking clopidogrel, in Gauhati Medical College and Hospital, Assam, North East India. Personalized therapy to target patient's carrying these genetic variants might help to improve the clinical outcome.Our findings suggest that CYP2C19 loss of functional allele had higher carriage frequency in northeast Indian population, furthermore studies on large sample size are needed in this population so that before prescribing the medication through the detection of these alleles in a patient may help reduce the harmful adverse events and so that necessary steps should be taken like changing the dose or prescribing alternative drugs. More comprehensive and diverse research wrapping a large number of CYP2C19 variants will lay the foundation for better adapted medicine for future.