Payiarat Suwannasri1, Wanna Thongnoppakhun2, Pornpen Pramyothin3, Anunchai Assawamakin4, Chanin Limwongse5. 1. Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. 2. Division of Molecular Genetics, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok 10700, Thailand. 3. Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand. Electronic address: ppornpen@chula.ac.th. 4. Biostatistics and Informatics Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Klong Luang, Pathumthani 12120, Thailand. 5. Division of Molecular Genetics, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok 10700, Thailand. Electronic address: siclw@mahidol.ac.th.
Abstract
OBJECTIVE: To develop CYP2D6 genotyping scheme for accurate allele calling and reliable estimation of functional allele dosage in Thais. DESIGN AND METHODS: We analyzed CYP2D6 copy numbers by pentaplex PCR coupled with semi-quantitative denaturing high performance liquid chromatography (DHPLC)-based technique. Ten common SNPs were genotyped from CYP2D6 gene product using single base extension (SBE) followed by DHPLC analysis. This detection scheme was compared with real-time PCR and conventional PCR-RFLP for cost-effectiveness. RESULTS: The distribution of CYP2D6 gene copy numbers in our population ranged from zero (0.69%), one (7.99%), two (60.07%), three (28.13%) and four (3.13%). The most commonly detected SNPs were related to CYP2D6*10 haplotype. CYP2D6*36 in tandem with CYP2D6*10B is the major rearrangement type in Thais (18.75%). CONCLUSIONS: Multiplex PCR coupled with DHPLC-based strategy is convenient and reliable method for CYP2D6 genotyping offering sufficient allele coverage for Asians. Both cost and analytical time saving were shown and the method could potentially be modified to accommodate CYP2D6 genotyping in other ethnics.
OBJECTIVE: To develop CYP2D6 genotyping scheme for accurate allele calling and reliable estimation of functional allele dosage in Thais. DESIGN AND METHODS: We analyzed CYP2D6 copy numbers by pentaplex PCR coupled with semi-quantitative denaturing high performance liquid chromatography (DHPLC)-based technique. Ten common SNPs were genotyped from CYP2D6 gene product using single base extension (SBE) followed by DHPLC analysis. This detection scheme was compared with real-time PCR and conventional PCR-RFLP for cost-effectiveness. RESULTS: The distribution of CYP2D6 gene copy numbers in our population ranged from zero (0.69%), one (7.99%), two (60.07%), three (28.13%) and four (3.13%). The most commonly detected SNPs were related to CYP2D6*10 haplotype. CYP2D6*36 in tandem with CYP2D6*10B is the major rearrangement type in Thais (18.75%). CONCLUSIONS: Multiplex PCR coupled with DHPLC-based strategy is convenient and reliable method for CYP2D6 genotyping offering sufficient allele coverage for Asians. Both cost and analytical time saving were shown and the method could potentially be modified to accommodate CYP2D6 genotyping in other ethnics.
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