Pyrosequencing™-Based Screening for Genetic Polymorphisms in Cytochrome P450 2B6 of Potential Clinical Relevance

Abstract

Objectives: Three exonic single nucleotide polymorphisms (SNPs) in the cytochrome P450 2B6 (CYP2B6) gene, 516G>T, 785A>G and 1459C>T, have been described to be associated with functional changes in the CYP2B6 catalytic activity or protein expression. They are therefore of potential clinical importance for drug efficacy and safety of CYP2B6 substrates, in particular of antiretroviral therapy regimes that include efavirenz. Therefore, we aimed at providing genetic screening assays for these three SNPs. Methods: Simplex Pyrosequencing™ assays were developed for the selected CYP2B6 SNPs. A total of 273 DNA samples from healthy volunteers of Caucasian ethnicity were genotyped. Results:The presently developed Pyrosequencing assays afford an accurate determination of the three SNPs in the 273 DNA samples. The assays were verified by routine implementation of control samples obtained by conventional sequencing. The frequencies for the variant alleles 516T, 785G and 1459T were 0.22, 0.24 and 0.13, respectively. All genotype frequencies were in agreement with the Hardy–Weinberg equilibrium. CYP2B6 alleles CYP2B6*5A (1459T), CYP2B6*6 (516T/785G) and *7B (516T/785G/1459T) were found at allelic frequencies of 0.12, 0.20 and 0.01. Conclusion:The present reliable and quick Pyrosequencing assays afford facilitating future research on the importance of CYP2B6 pharmacogenetics for personalized drug therapy with CYP2B6 substrates.

Keywords: :

• CYP2B6*4
• *5
• *6
• *7
• drug metabolism
• pharmacogenetics
• Pyrosequencing™ technique
• screening assays
• single nucleotide polymorphism

Acknowledgements

The work was supported by the Heinrich und Fritz Riese-Stiftung, Frankfurt, Germany for a project on personalized anti-HIV therapy. The authors thank Dr Ulrich Zanger, Margarete Fischer-Bosch Institute for Clinical Pharmacology, Stuttgart, Germany, for having performed control validation assays.

Additional information

Funding

The work was supported by the Heinrich und Fritz Riese-Stiftung, Frankfurt, Germany for a project on personalized anti-HIV therapy. The authors thank Dr Ulrich Zanger, Margarete Fischer-Bosch Institute for Clinical Pharmacology, Stuttgart, Germany, for having performed control validation assays.