Rapid and comprehensive determination of cytochrome P450CYP2D6 poor metabolizer genotypes by multiplex polymerase chain reaction

The liver enzyme cytochrome P450 CYP2D6 (debrisoquine di-hydroxylase) metab olizes numerous drugs, including many antidepressants, neuroleptics, antiar rhythmics, and antihypertensive agents. Variability in the gene that encode s this enzyme is an important factor underlying variable drug treatment res ponses. Some 5-10% of Caucasians lack functional CYP2D6, and the genetic ba sis 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 t reatment, it is important to devise methods that permit rapid and economica l determination of CYP2D6 genotype. We have developed a robust polymerase c hain 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. Sep arate 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 1 00 individuals previously genotyped by specific polymerase chain reaction-r estriction fragment length polymorphism analysis, and proved 100% accurate in this sample. The assay performed consistently with Tag DNA polymerases f rom various suppliers, within a broad range of temperatures and MgCL2 conce ntrations, and using genomic DNA prepared by a range of methods including e xtraction from dried blood spots on card. This muitiplexed, amplification r efractory mutation system (ARMS) method is reliable, rapid, relatively chea p, amenable to automation, and offers the advantages of minimal sample hand ling with no requirement for restriction enzymes as in earlier CYP2D6 assay s. Hum Mutat 16:77-85, 2000. (C) 2000 Wiley-Liss, Inc.