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Clinical Chemistry 50: 1372-1377, 2004. First published June 3, 2004; 10.1373/clinchem.2004.031708
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(Clinical Chemistry. 2004;50:1372-1377.)
© 2004 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

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

Robyn M. Jacob, Elaine C. Johnstone, Matt J. Neville and Robert T. Waltona

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

aAddress correspondence to this author at: General Practice Research Group, Cancer Research UK, Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, United Kingdom. Fax 44-1865-224989; 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. The aim of the present study was to develop a reliable and robust assay for determining genotypic variants.

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 (SNP) 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.




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J. K. Blievernicht, E. Schaeffeler, K. Klein, M. Eichelbaum, M. Schwab, and U. M. Zanger
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