Comparison of Single Nucleotide Polymorphism genotyping of CYP2C19 by Loop-mediated isothermal amplification and real-time PCR melting curve analysis.

BACKGROUND: CYP2C19, a member of cytochrome P450 enzymes, is involved in various drug metabolisms, such as Clopidogrel. Common Single Nucleotide Polymorphisms (SNPs) of CYP2C19 gene, CYP2C19*2 and CYP2C19*3, are liable for the poor metabolism of Clopidogrel. It is crucial to identify poor metabolizers for alternative treatment as poor metabolism of Clopidogrel has been shown to correlate with worse clinical outcome in acute coronary syndrome (ACS) patients.

METHOD: A genotyping method, Loop-mediated isothermal amplification (LAMP) was employed in this study. CYP2C19*2 and CYP2C19*3 were adapted from Iwasaki M. et al. with modifications in the reaction mixtures and end-point detection method where simpler visual detection using SYBR® Safe was employed instead of a more technical and equipment demanding real-time PCR. Real-time PCR melting curve analysis is a common method for SNPs analysis and hence chosen as a reference for results obtained from the LAMP assay.

RESULTS: The CYP2C19-LAMP assay successfully detected CYP2C19*2 and CYPC19*3 mutants. The typing results of CYP2C19-LAMP assay, performed in triplicates, were concordant with the real-time PCR melting curve analysis results.

CONCLUSION: CYP2C19-LAMP assay using SYBR® Safe dye for visual detection of end-point result is a simple, rapid and cost-effective method for CYP2C19 genotyping.