Rapid and comprehensive determination of cytochrome P450 CYP2D6 poor metabolizer genotypes by multiplex polymerase chain reaction.

Abstract

The liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) metabolizes numerous drugs, including many antidepressants, neuroleptics, antiarrhythmics, and antihypertensive agents. Variability in the gene that encodes this enzyme is an important factor underlying variable drug treatment responses. Some 5-10% of Caucasians lack functional CYP2D6, and the genetic basis of most of these "poor metabolizer" alleles is now well defined. As the CYP2D6 status of a patient can have profound effects on response to drug treatment, it is important to devise methods that permit rapid and economical determination of CYP2D6 genotype. We have developed a robust polymerase chain reaction method that simultaneously identifies the variants CYP2D6 *3, *4, *6, *8, *11, *12, *14, *15, *19, and *20. This constitutes most of the poor metabolizer alleles described in Caucasian and Asian populations. Separate PCR reactions or Southern blots are required for *7, the *5 deletion, and the hybrid alleles *13 and *16. The multiplex assay was validated on 100 individuals previously genotyped by specific polymerase chain reaction-restriction fragment length polymorphism analysis, and proved 100% accurate in this sample. The assay performed consistently with Taq DNA polymerases from various suppliers, within a broad range of temperatures and MgCl(2) concentrations, and using genomic DNA prepared by a range of methods including extraction from dried blood spots on card. This multiplexed, amplification refractory mutation system (ARMS) method is reliable, rapid, relatively cheap, amenable to automation, and offers the advantages of minimal sample handling with no requirement for restriction enzymes as in earlier CYP2D6 assays.