Simultaneous detection of CYP2C8*2, CYP2C8*3, CYP2C8*4 and CYP2C8*5 alleles by a nested PCR method

Abstract. Cytochrome P450 (CYP) 2C8 plays a significant role in metabolizing many clinically important drugs such as paclitaxel, amiodarone and amodiaquine. A simultaneous detection of CYP2C8*2, CYP2C8*3, CYP2C8*4 and CYP2C8*5 alleles by a PCR method will be useful for detecting single nucleotide polymorphisms (SNPs) on the CYP2C8 gene that may contribute to the inter-individual variability in metabolizing these drugs. This is a blinded randomised cross over study conducted in 24 healthy normal volunteers who received the anti-malarial drug amodiaquine and artesunate both as individual agents and in a fixed combination, over two study phases to determine the safety profile of the two preparations. Genomic DNA was isolated from the volunteers using a DNA extraction kit. A nested PCR method was applied to detect all of the CYP2C8 variants. Regions from exons 3, 5 and 8 of the gene were simultaneously amplified using a first PCR step (PCR1). Products from PCR1 were then used to run the second PCR (PCR2) for detection of the variant alleles. This method was verified in 24 healthy Malaysian volun-teers where all four variant CYP2C8 alleles were successfully amplified and confirmed by DNA sequencing. We have developed and optimized a two-step multiplex nested PCR method for genotyping all of the important CYP2C8 variants found to date.