Comparison of cytochrome P4502C9 genotyping methods and implications for the clinical laboratory

Study Objective. To compare the accuracy, speed, and cost of two methodologies used for genotyping known variants in the cytochrome P450 (CYP) 2C9 metabolizing enzyme gene. Design. Comparative study. Setting. University research center. Samples. Fifteen-milliliter mouthwash samples collected from 253 subjects participating in a warfarin pharmacogenomic study. Intervention. Genotyping for the isoleucine-to-leucine change at codon 359 (Ile(359)Leu [*3] polymorphism) was performed by using the Pyrosequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods in all 253 samples. Genotyping for the arginine-to-cysteine change at codon 144 (Arg(144)Cys [*2] polymorphism) was performed by using Pyrosequencing in all samples and by PCR-RFLP in a random subset of 136 samples. Measurements and Main Results. Comparisons of genotyping success rates, time efficiency, and cost analyses were conducted for Pyrosequencing and PCR-RFLP at each variant site. Pyrosequencing and PCR-RFLP produced similar success rates on the first genotyping attempt for the Arg(144)Cys variant (93.3% vs 90.4%, respectively) and the Ile(359)Leu variant (83.8% vs 79.1%, respectively). With Pyrosequencing, genotyping 96 samples for either polymorphism could be performed in I hour. In contrast, genotyping 96 samples by RFLP took 10 hours for the Arg(144)Cys variant and 20 hours for the Ile(359)Leu variant. Total cost/sample for Arg(144)Cys genotyping was $1.90 with PCR-Pyrosequencing and $3.14 with PCR-RFLR Total cost/sample for Ile(359)Leu genotyping was $1.88 with PCR-Pyrosequencing and $10.18 with PCR-RFLP Conclusion. Compared with RFLP, genotype determination by Pyrosequencing is a more time-efficient, cost-effective, and robust method for CYP2C9 genotyping. Because of its wide applicability and ease of use, Pyrosequencing is a promising technology for future pharmacogenomic investigations.