Development and validation of a rapid and reliable real-time PCR method for CYP3A5 genotyping

Abstract

Background: A prominent example for inter-individual differences in drug-metabolizing enzymes is the cytochrome P450 family. These monooxygenases comprise enzymes responsible for metabolism of about 90% of common medications. CYP3A5 and CYP3A4 account for 50% of hepatic cytochrome P450 and conversion of about half of all their substrates. CYP3A5 is the predominant extra-hepatic CYP enzyme and shows varying inter-individual expression attributable to genetic variations in the corresponding gene. CYP3A5*2 and *3 are the most common among Caucasian populations. Among CYP3A5 substrates are cyclosporine and tacrolimus prescribed after organ transplantations. A high incidence of nephrotoxic events after administering these drugs is related to low expression of the CYP3A5 enzyme. A fast and reliable genotyping method for the CYP3A5 gene would help avoid unwanted adverse drug reactions.

Methods: Blood samples from 143 Caucasian subjects were genotyped by means of a real-time PCR multiplex assay testing the two CYP3A5 variations, CYP3A5S*2 and *3. This assay was validated against RFLP-PCR.

Results: Both mutations examined could be found in the study. No sample was homozygous for CYP3A5*2, but 2 out of 143 showed heterozygosity (allele frequency: 0.7%). For the CYP3A5*3 variant 17 samples were heterozygous and 115 were homozygous (allele frequency 86.4%). The multiplex real-time PCR yields shorter hands-on time and reduced cost compared to RFLP-PCR.

Conclusions: Establishment of a multiplex real-time PCR has been successful as could be proven by correctly identifying the desired mutations CYP3A5*2 and CYP3A5*3 against a standard reference method.