Received on January 20, 2004
Accepted on May 17, 2004

Molecular Diagnostics and Genetics

Identification of CYP2B6 Sequence Variants by Use of Multiplex PCR with Allele-Specific Genotyping

¹ Cancer Research UK General Practice Research Group, Department of Clinical Pharmacology, University of Oxford, Oxford, United Kingdom

^* To whom correspondence should be addressed. E-mail: robert.walton{at}clinpharm.ox.ac.uk.

Background: Cytochrome P450 2B6 (CYP2B6) has a role in the metabolism of many clinically important substances, but the variation within the CYP2B6 gene has not been fully characterized.

Methods: We used a two-stage procedure. An initial multiplex PCR reaction amplified the relevant gene fragments in exonic and regulatory regions to ensure isolation of CYP2B6 from its similar pseudogene (CYP2B7). This product was then genotyped by allele-specific PCR.

Results: The assay detected the following published single-nucleotide polymorphisms: C64T (Arg22Cys), C78T, G216C, G516T (Gln172His), C777A (Ser259Arg), A785G (Lys262Arg), and C1459T (Arg487Cys), as well as additional loci found within the single-nucleotide polymorphism databases: A1190G, C1268A, C1330T, A1382G, A1402T, and an A/T SNP in intron 2 (A12917T). This approach detected all common, previously reported alleles and identified a new allele (CYP2B6*4C) present in 2.2% of a Caucasian population. Genotypic frequencies obtained were consistent with previously published results.

Conclusions: This method is simple, reliable, rapid, and amenable to automation and could facilitate the large-scale genotypic analysis of CYP2B6.