A rapid multiplex PCR assay that can reliably discriminate the cytochrome P450 2D6 whole-gene deletion allele from 2D6*10 alleles

Clin Chim Acta. 2012 Oct 9;413(19-20):1675-7. doi: 10.1016/j.cca.2012.05.013. Epub 2012 May 23.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Asian People / genetics
  • Cytochrome P-450 CYP2D6 / deficiency
  • Cytochrome P-450 CYP2D6 / genetics*
  • Cytochrome P-450 Enzyme System / genetics
  • DNA Primers
  • Gene Deletion*
  • Genotype
  • Humans
  • Isoenzymes / deficiency
  • Isoenzymes / genetics
  • Multiplex Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • Isoenzymes
  • cytochrome P-450 CYP2D7P
  • Cytochrome P-450 Enzyme System
  • Cytochrome P-450 CYP2D6