Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays

Abstract

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.

Method: 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.

Conclusion: 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.

Fig. 1

Real-time isothermal HDA-based VKORC1 assay. Human genomic DNA (10 ng) bearing the wild-type (Coriell cat. # NA17207), variant (NA17285), or heterozygous (NA17222) allele of the VKORC1 SNP was added to 50 µL HDA reactions containing 75 nM primers, 90 nM VKORC1-probe-C (VIC labeled), and 20 nM of the VKORC1-probe-T (FAM labeled) and incubated at 65 – 66°C for 60 cycles (2 minutes per cycle). Each reaction was performed in duplicate. The real-time signal for VIC is shown in orange and that for FAM in green. A. Real-time amplification plot for the wild-type (NA17207) template. Distinct amplification signal was only observed for the VIC probe, indicating that only wild-type amplicon was generated. B. Real-time amplification plot for the variant template (NA17285). Distinct amplification signal was only observed for the FAM probe, indicating that only variant amplicon was generated. C. Real-time HDA amplification plot for the heterozygous template (NA17222). Distinct amplification signals were observed for both the VIC and FAM probe, indicating that both wild-type and variant amplicons were generated.

Fig. 2

Genotype determination for the 50 clinical samples. Only VKORC1 assay results were shown here. In A, C_t numbers (2 minutes per cycle) from the VIC signal (orange) and FAM signal (green) for each sample were plotted against the sample number. In B, Normalized fluorescence intensity changes for VIC (y-axis) and FAM (x-axis) for each sample were plotted against each other. The y=2x and y=0.5x lines are shown on the plot. Samples are grouped as wild-type (orange), variant (green), and heterozygous (magenta), respectively. The unresolved sample (BHW-17) is in grey.

Fig. 2

Genotype determination for the 50 clinical samples. Only VKORC1 assay results were shown here. In A, C_t numbers (2 minutes per cycle) from the VIC signal (orange) and FAM signal (green) for each sample were plotted against the sample number. In B, Normalized fluorescence intensity changes for VIC (y-axis) and FAM (x-axis) for each sample were plotted against each other. The y=2x and y=0.5x lines are shown on the plot. Samples are grouped as wild-type (orange), variant (green), and heterozygous (magenta), respectively. The unresolved sample (BHW-17) is in grey.