Rapid genotyping of single nucleotide polymorphisms influencing warfarin drug response by surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectrometry

Abstract

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy.

Figure 1

Illustration of the detection of VKORC1 genotype by MS. The letter in the panel on the left refers to which bases were added to the primer during the reaction of primer extension. For example, in the third reaction, the primer based extension product was primer extended by deoxyadenosine triphosphate (dATP) and then stopped after addition of 2′,3′-Dideoxyguanosine 5′-Triphosphate (ddGTP). Patient genotype at the VKORC1 in this case is, however, AA.

Figure 2

Effect of sample enrichment and washing on the detection of VKORC1 genotype. The DNA sample from a patient with GA genotype at VKORC1 3673G>A site was subjected to PCR to amplify the VKORC1 SNP. Afterward, two different volumes of PCR products were applied to Q10 Chips: one of each with no washing step, and one of each washed with 50 mmol/L Tris-HCl, pH 7. Finally, the analytes on the Q10 Chips were detected by using SELD-TOF MS.

Figure 3

A: Dose-dependent enrichment of VKORC1 PCR product by Q10 anionic Chip. PCR was used to amplify the DNA fragment containing the VKORC1 site. Different volumes of PCR products were then loaded onto the Q10 anionic Chip. After incubation and washing with 50 mmol/L of Tris, pH 7.0, the sample was analyzed by using SELDI-TOF MS. This patient has a GA genotype at VKORC1 3673G>A site. B: pH-dependent binding of PCR product to Q10 Chips. Equal amounts of PCR amplified VKORC1 DNA (20 μl) were applied to Q10 Chips. Each Chip was subject to a washing step by using a buffer with different pH value, before analysis by SELDI-TOF MS. The buffer with pH 6 was phosphate buffer, while the buffers with pH values from 7 to 10 were Tris-HCl buffers. C: Effect of salts on the detection of genotype by SELDI-TOF MS. PCR amplified VKORC1 products (20 μl each) were applied to Q10 Chips, followed by washing with 50 mmol/L of Tris-HCl, pH 7, containing various concentrations of NaCl. Afterward, the analytes were genotyped by a SELDI-TOF MS.

Figure 4

Genotyping SNPs, VKORC1 3673G>A, CYP2C9*2, and CYP2C9*3, individually by SELDI-TOF MS. The DNA sample from one patient was genotyped for SNPs at the VKORC1 3673G>A, CYP2C9*2, and CYP2C9*3 sites. Both PCR reactions and the analysis by MS were performed separately for each SNP.

Figure 5

Confirming genotyping results by DNA sequencing. The product from each PCR assay was subject to the BigDye Terminator Reaction Chemistry version 3.1 for sequence analysis on Applied Biosystems, Foster City, CA 3730 DNA Analyzers. The arrows indicate where the SNPs are.

Figure 6

Multiplexed genotyping of three SNPs, VKORC1 3673G>A, CYP2C9*2, and CYP2C9*3. PCR amplification for each SNP was performed separately on DNA from the subject shown in Figure 4. PCR products for three SNPs were pooled and applied onto the Q10 Chip, followed by washing and genotyping by using SELDI-TOF MS.