Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.
BACKGROUND
The response to the anticoagulant drug warfarin is greatly affected by genetic polymorphisms in the VKORC1 and CYP2C9 genes. Genotyping these polymorphisms has been shown to be important in reducing the time of the trial and error process for finding the maintenance dose of warfarin thus reducing the risk of adverse effects of the drug.


METHODS
We developed a real-time isothermal DNA amplification system for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. For each SNP, real-time isothermal Helicase Dependent Amplification (HDA) reactions were performed to amplify a DNA fragment containing the SNP. Amplicons were detected by fluorescently labeled allele specific probes during real-time HDA amplification.


RESULTS
Fifty clinical samples were analyzed by the HDA-based method, generating a total of 150 results. Of these, 148 were consistent between the HDA-based assays and a reference method. The two samples with unresolved HDA-based test results were repeated and found to be consistent with the reference method.


CONCLUSIONS
The HDA-based assays demonstrated a clinically acceptable performance for genotyping the VKORC1 -1639G>A SNP and two SNPs (430C>T and 1075A>C) for the CYP2C9 enzyme (CYP2C9*2 and CYP2C9*3), all of which are relevant in warfarin pharmacogenentics.
Li
Ying
Y
BioHelix Corporation, Beverly MA 01915, USA. li@biohelix.com
Jortani
Saeed A
SA
Ramey-Hartung
Bronwyn
B
Hudson
Elizabeth
E
Lemieux
Bertrand
B
Kong
Huimin
H
D000925:Anticoagulants / Q000493:pharmacokinetics / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D065729:Cytochrome P-450 CYP2C9; D004265:DNA Helicases / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology; D013997:Time Factors; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000493:pharmacokinetics / Q000494:pharmacology*
D000925:Anticoagulants; 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; D004265:DNA Helicases