A multiplex assay for detecting genetic variations in CYP2C9, VKORC1, and GGCX involved in warfarin metabolism.
BACKGROUND
Patients differ in responses to warfarin, which is commonly prescribed to treat thromboembolic events. Genetic variations in the cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), vitamin K epoxide reductase complex, subunit 1 (VKORC1), and gamma-glutamyl carboxylase (GGCX) genes have been shown to contribute to impaired metabolism of warfarin.


METHODS
We designed a custom multiplex single-nucleotide polymorphism (SNP) panel to interrogate the CYP2C9 *2, *3, VKORC1 (-1639G-->A), and GGCX (1181T-->G) alleles simultaneously in a single sample by use of single-base extension and capillary electrophoresis after genomic DNA extraction and PCR amplification.


RESULTS
Our assay successfully detected various genotypes from known controls and 24 unknown samples. It was found to be 100% concordant with sequencing results.


CONCLUSIONS
Our multiplexed SNP panel can be successfully used in genotyping of patient blood samples. Results can be combined with other clinical parameters in an algorithm for warfarin dosing. These data provide a proof-in-principle of multiplexed SNP analysis using rapid single-base extension and capillary electrophoresis, and warrant additional validation using a larger cohort of patient samples.
Rai
Alex J
AJ
Departments of Clinical Laboratories and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. raia@mskcc.org.
Udar
Nitin
N
Saad
Rana
R
Fleisher
Martin
M
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D019735:Carbon-Carbon Ligases / Q000235:genetics* / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000378:metabolism*
D014859:Warfarin; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase