A rapid multiplex PCR assay that can reliably discriminate the cytochrome P450 2D6 whole-gene deletion allele from 2D6*10 alleles.
BACKGROUND
Genetic polymorphisms of the human CYP2D6 gene can affect the metabolism of many drugs in clinical use. As a first step toward identifying poor drug metabolizers in the clinical setting, we developed a new multiplex PCR-based genotyping method to detect CYP2D6 whole-gene deletion.


METHODS
We validated the new method by analyzing 500 genomic DNA samples from a Japanese population with the conventional long-PCR method and the new multiplex PCR method. The long-PCR system used a forward primer for CYP2D7P (a pseudogene closely related to CYP2D6) and a common reverse primer for the untranslated region. The multiplex PCR system used the same two primers as the long PCR and an additional forward primer for CYP2D6.


RESULTS
With the long-PCR system, DNA samples identified as containing CYP2D6*5 (whole-gene deletion) formed 3.5-kb PCR products. With the multiplex PCR system, many samples yielded 4.7-kb PCR products (implying the existence of normal CYP2D6) and some DNA samples yielded 6.2-kb PCR products (probably indicating CYP2D6*10D). The long-PCR assay detected 64 CYP2D6*5 alleles among 1000 Japanese alleles; however, the new multiplex PCR system identified 5 of these 64 alleles as CYP2D6*10D.


CONCLUSIONS
The new multiplex PCR method is useful for detecting CYP2D6*5. This system could reliably discriminate CYP2D6*5 from homologous pseudogene CYP2D7P and functional CYP2D6*10D.
Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashi-tamagawa Gakuen, Machida, Tokyo 194-8543, Japan.
Maho
M
Okubo
Norie
N
Murayama
Jun
J
Miura
Makiko
M
Shimizu
Hiroshi
H
Yamazaki
D000483:Alleles; D044466:Asian People / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000172:deficiency / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D017931:DNA Primers; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D007527:Isoenzymes; C102835:cytochrome P-450 CYP2D7P; D003577:Cytochrome P-450 Enzyme System; D019389:Cytochrome P-450 CYP2D6