Rapid detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles by tetra-primer PCR and of the CYP2D6*5 allele by multiplex long PCR

Background: Interindividual differences in CYP2D6 activity range from total absence of metabolism of certain drugs to ultrafast metabolism and can pro duce adverse effects or lack of therapeutic effect under standard therapy. Several mutations have been described in the CYP2D6 gene that abolish CYP2D 6 activity. However, four mutations explain the majority of the poor metabo lizers. We describe four single-tube assays to detect these mutations. Methods: Three tetra-primer PCR assays were developed to detect the mutatio ns in the CYP2D6*3, *4,and *6 alleles. In these single-tube assays, the CYP 2D6 locus is amplified directly, followed by the allele-specific amplificat ion on this new template. In addition, a multiplex long PCR was developed t o genotype the CYP2D6*5 allele. Two long PCR amplifications for detection o f the deletion of CYP2D6 (*5) and for detection of the CYP2D6 gene region w ere combined in one tube. Results: Analysis of 114 alleles showed no CYP2D6*3 allele, and allele freq uencies of 28.1% for CYP2D6*4, 2.6% for CYP2D6*5, and 0.9% for CYP2D6*6. Re -analysis of the DNA samples by restriction fragment length polymorphism an d sequencing analysis confirmed these results. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7-11 times) always show ed identical results. Conclusions: Our set of single-tube assays allows rapid and reproducible ge notyping of the majority of CYP2D6 poor metabolizers. (C) 2000 American Ass ociation for Clinical chemistry.