Comparison of cytochrome P450 2C9 genotyping methods and implications for the clinical laboratory.
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.


METHODS
Comparative study.


METHODS
University research center.


METHODS
Fifteen-milliliter mouthwash samples collected from 253 subjects participating in a warfarin pharmacogenomic study.


METHODS
Genotyping for the isoleucine-to-leucine change at codon 359 (Ile359Leu [*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 (Arg144Cys [*2] polymorphism) was performed by using Pyrosequencing in all samples and by PCR-RFLP in a random subset of 136 samples.


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 Arg144Cys variant (93.3% vs 90.4%, respectively) and the Ile359Leu variant (83.8% vs 79.1%, respectively). With Pyrosequencing, genotyping 96 samples for either polymorphism could be performed in 1 hour. In contrast, genotyping 96 samples by RFLP took 10 hours for the Arg144Cys variant and 20 hours for the Ile359Leu variant. Total cost/sample for Arg144Cys genotyping was dollars 1.90 with PCR-Pyrosequencing and dollars 3.14 with PCR-RFLP. Total cost/sample for Ile359Leu genotyping was dollars 1.88 with PCR-Pyrosequencing and dollars 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.
Aquilante
Christina L
CL
Department of Pharmacy Practice, College of Pharmacy, University of Florida, Gainesville 32610, USA.
Lobmeyer
Maximilian T
MT
Langaee
Taimour Y
TY
Johnson
Julie A
JA
D000483:Alleles; D003362:Cost-Benefit Analysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005838:Genotype; D006801:Humans; D007753:Laboratories; D010597:Pharmacogenetics / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000191:economics; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA / Q000191:economics; D013997:Time Factors; D014859:Warfarin / Q000494:pharmacology*
D014859:Warfarin; D003577:Cytochrome P-450 Enzyme System