Multiplex PCR-pyrosequencing assay for genotyping CYP3A5 polymorphisms

Background: The cytochrome P450 (CYP) 3A5 enzyme contributes to the metabolism of many drugs. Single nucleotide polymorphisms in the CYP3A5 gene (CYP3A5⁎3C and CYP3A5⁎6) are associated with decreased CYP3A5 expression in the liver. We designed a multiplex genotyping assay to detect the CYP3A5⁎3C and CYP3A5⁎6 polymorphisms in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction. Methods: A multiplex PCR assay was designed to simultaneously amplify 2 fragments, one containing the CYP3A5⁎3C polymorphism and the other containing the CYP3A5⁎6 polymorphism. Following PCR, multiplex genotyping was performed with pyrosequencing analysis. Results: Patient samples (n = 69) were analyzed for the CYP3A5⁎3C and CYP3A5⁎6 polymorphisms using the multiplex PCR-pyrosequencing assay. Genotypes obtained by the multiplex reaction were in 100% concordance with genotypes obtained using simplex PCR-pyrosequencing (n = 69) and direct DNA sequencing (n = 29). Conclusions: The advantage of this method is that the CYP3A5⁎3C and CYP3A5⁎6 polymorphism can be amplified in a single PCR reaction and genotyped in a single pyrosequencing reaction. This combined approach improves the time-efficiency and decreases the cost of CYP3A5 genotyping.