Warfarin Genotyping in a Single PCR Reaction for Microchip Electrophoresis

Abstract

BACKGROUND Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. METHODS We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. RESULTS Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). CONCLUSIONS This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

Applications of microfluidics and microchip electrophoresis for potential clinical biomarker analysis

“…Proper dosage is vital because an incorrect dosage can have adverse effects and in extreme cases be fatal. Genotyping for warfarin dosage using a single PCR reaction and subsequent microchip electrophoresis was reported by Poe et al [45] By using microchip PCR, the amplification time was reduced to 30 minutes from 45 minutes with benchtop PCR, and the complete assay, combining PCR with microchip electrophoresis, took only 60 minutes. The assay determined the patient's genotype using a tetraprimer amplification refractory mutation system in a single reaction (Figure 7A-B) and allowed rapid typing of over 30 samples (Figure 7C).…”

“…(C) Microchip data showing the different genotypes of 35 samples that matched 100% with results obtained from other validated methods. Adapted with permission from reference [45]. …”

Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular Diseases

“…For instance, warfarin is an anticoagulant drug often prescribed to prevent thrombosis, and patients with common SNPs in the CYP2C9 and VKORC1 genes have been suggested to successfully predict a patient’s response to the anticoagulant effects of warfarin 53, 54 . Further clinical studies will be required to warrant the use of SNP data to predict warfarin treatment.…”

Primer1: Primer Design Web Service for Tetra-Primer ARMS-PCR

“…Poe et al [9] combined multiplex T-ARMS with microfluidics hardware as an inexpensive and rapid strategy to detect three single nucleotide polymorphisms (SNPs) underlying warfarin sensitivity. Lajin et al [10] employed T-ARMS for the simultaneous detection of three polymorphisms involved in the folate-homocysteine metabolic pathway.…”