Abstract P4-02-17: Polymerase Chain Reaction-Liquid Chromatography-Mass Spectrometry — A Convenient and Cost-Effective Strategy for CYP2D6 Genotyping
Authors: Beer B , Erb R , Plattner S , Hubalek M , Oberguggenberger A , Meraner V , Oberacher H. 
Published: April 2011

Abstract
Background: Genetic polymorphisms in the drug metabolizing enzyme Cyotchrome P450 2D6 (CYP2D6) gene can significantly influence a person's ability to mediate CYP2D6 dependent metabolism. For example, a notable influence of the CYP2D6 genotype has been described regarding the conversion of tamoxifen to its pharmacologically active metabolites. Thus, the genetic determination of the CYP2D6 metabolizer status has the potential to represent a valuable therapeutic supplement in breast cancer therapy. However, the clinical relevance of the CYP2D6 genotype regarding the treatment of breast cancer with tamoxifen still remains to be elucidated. Due to the high number of relevant polymorphisms to be screened, pharmacogenetic CYP2D6 testing is considered to be time-consuming and laborious and is therefore limited to a few clinical studies only. To enable a more widespread use, we present polymerase chain reaction (PCR) - ion-pair reversed-phase high-performance liquid chromatography — electrospray ionization time of flight mass spectrometry (ICEMS) as fast, convenient and cost-effective strategy for CYP2D6 genotyping.
Material and Methods: After DNA extraction from saliva swabs, the CYP2D6 gene was amplified by a long range PCR. Subsequently, polymorphism-specific short amplicons were generated in a multiplexed PCR. The amplicons were directly analysed by ICEMS. The information necessary to determine the allelic state of a PCR-amplified polymorphic locus were obtained from the measured molecular masses. Gene duplications or deletions were analysed by a long range PCR protocol and subsequent agarose gel detection.
Results: The developed CYP2D6 genotyping approach enables the reliable determination of the most frequent CYP2D6 alleles in Europe (*1, *2, *3, *4, *6, *7, *8, *9, *10, *11, *12, *17, *41) including whole gene duplications and deletions (*5). The assay involves the following steps: (1) collecting biological material in form of non-invasive saliva swabs; (2) DNA extraction; (3) long range PCR for a specific CYP2D6 amplification; (3) multiplexed PCR for the generation of polymorphism-specific short amplicons; (4) direct analysis of the reaction mixture with ICEMS; (4) determination of the allelic state with the measured molecular masses; (5) analysis of gene duplications or deletions by a long range PCR protocol and subsequent agarose gel detection. Genotyping of a single sample costs about 5-10 USD and can be accomplished within 2-3 days. Parallelization increases the sample throughput and reduces costs. For proof of principle, we have applied the developed method in the context of the PRO-BETh study to determine the CYP2D6 status of 106 breast cancer patients treated with tamoxifen. According to the classification of Gaedik A. et al [1], 19% of the typed breast cancer patients turned out to be “Poor Metabolizers", 51% were “Intermediate Metabolizers", 29% were “Extensive Metabolizers” and 7% turned out to be “Ultrarapid Metabolizers”.
Conclusion: PCR-ICEMS represents a cost-effective and convenient tool for pharmacogenetic testing, which enables the determination of the CYP2D6 status using DNA obtained from a non-invasive saliva swab as template.
[1] Gaedigk A. et al, J. Clin Pharmacol Ther. 2007 Jun;81(6):817-20.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-17.