SYBR Green-based real-time PCR assay for detection of VKORC1 and CYP2C9 polymorphisms that modulate warfarin dose requirement.
BACKGROUND
Polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are considered the major genetic factors modulating warfarin dose requirement. As a result, a rapid and economic method for genotyping patients for predicting warfarin dose could be useful in clinical applications.


METHODS
In this study, we developed and validated a melting temperature (T(m))-shift genotyping assay for detection of VKORC1 C1173T and CYP2C9 A1075C polymorphisms by using SYBR Green-based multiplex allele-specific PCR and consequent melting curve analysis. In this T(m)-shift genotyping assay, a GC-tail was incorporated into one of the two allele-specific primers to increase the melting temperature of PCR product, such that both alleles can be differentially detected simultaneously at their respective melting curve patterns. In total, 100 randomly selected samples of both VKORC1 and CYP2C9 genes from each genotype group determined by melting curve were validated by direct DNA sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis.


RESULTS
There was 100% concordance between the results of DNA sequencing and T(m)-shift genotyping, and 99% concordance between the results of DHPLC assay and T(m)-shift genotyping.


CONCLUSIONS
This allele-specific PCR assay is convenient, inexpensive and will be useful for VKORC1 and CYP2C9 genotyping in a clinic laboratory.
Huang
Sheng-Wen
SW
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, PR China.
Li
Qiang
Q
Zhu
Sheng-Yuan
SY
Li
Liang
L
Xiong
Fu
F
Jia
Yan-Kai
YK
Xu
Xiang-Min
XM
D000925:Anticoagulants / Q000008:administration & dosage*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D052160:Benzothiazoles; D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9; D003959:Diamines; D005456:Fluorescent Dyes; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D044366:Transition Temperature; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000008:administration & dosage*
D000925:Anticoagulants; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; 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