CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry |
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| Authors: | Beate Beer Robert Erb Florian Pitterl Harald Niederstätter Olalla Maroñas Alejandro Gesteira Angel Carracedo Irina Piatkov Herbert Oberacher |
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| Institution: | 1.Institute of Legal Medicine,Innsbruck Medical University,Innsbruck,Austria;2.Institute of Forensic Science-CIBERER,University of Santiago de Compostela,Santiago de Compostela,Spain;3.Fundación Pública Galega de Medicina Xenómica-SERGAS,Santiago de Compostela,Spain;4.Diversity Health Institute,Westmead Hospital,Sydney,Australia |
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| Abstract: | Genetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6;
OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing
status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry
(ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A
two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within
the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography.
High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution
of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For
validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS
assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy. |
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