Fast and more affordable CYP2C19 LOF testing to personalized of clopidogrel therapy

Abstract

Abstract Background Clopidogrel is an antiplatelet drug used to treat coronary artery disease (CAD) to reduce blood clotting by inhibiting platelet activity. However, many patients experience ischemic events due to changes in clopidogrel metabolism attributed to various genetic and non-genetic factors. CYP2C19 is one of the important genes responsible for the metabolism of clopidogrel, but this gene has polymorphisms in cytochrome P450 (CYP), and this CYP2C19 Loss-of-function (LoF) polymorphisms impair clopidogrel metabolism between individuals and races. Therefore, genetic testing is an effective strategy to personalized clopidogrel and reduce the incidence of ischemic events in CAD patients. Purpose We sought to develop a nested allele-specific multiplex polymerase chain reaction (PCR) method for the detection of CYP2C9 LOF alleles. Method Genomic DNA was extracted from blood taken from 7 healthy volunteers using a commercial DNA extraction kit. A two-step PCR method was developed. First, DNA was subjected to a first PCR used to amplify exons 4 and 5 simultaneously in a single reaction tube. Second, The products of 1st PCR were then used as a template in the second PCR to detect CYP2C9 polymorphisms alleles (CYP2C19*2 and *3) using allele-specific primers. The test results were validated via sequencing. Result We successfully detected clinically important CYP2C19 LOF polymorphisms alleles by the nested allele-specific multiplex PCR method (Figure 1). This method is fast and doesn't require high-quality and expensive laboratory equipment. It was reproducible and specific and can reliably detect CYP2C19 variants. Direct sequencing of target variants has been used to validate this strategy further, and the amplified sequences were 100% identical to the CYP2C9 sequence. Conclusion The developed methods were specific to identify polymorphisms in CYP2C19 (rs4244285 and rs4986893). However, to understand ethnic differences and their potential implications for clopidogrel therapy, we highly recommend identifying relevant genetic polymorphisms before clinical manifestations arise. Funding Acknowledgement Type of funding sources: None.