title: Warfarin genotyping with hybridization-induced aggregation on a poly(ethylene terephthalate) microdevice.
abstract: Warfarin, a commonly prescribed oral anticoagulant, is burdened by a narrow therapeutic index and high inter-individual variability in response, making it the second leading cause of drug-related emergency room visits. Since genetic factors contribute significantly to warfarin sensitivity, a genotype-guided dosing strategy may reduce the occurrence of adverse events. While numerous methods have been demonstrated for warfarin genotyping, the specifications of most assays with respect to turnaround time and cost are not ideal for routine testing. Here, we present a unique method for warfarin genotyping based on multiplex PCR coupled with Hybridization-induced Aggregation (HIA), a bead-based technique for sequence-specific detection. A multiplex allele-specific PCR reaction was used to generate products corresponding to 3 genetic variants associated with warfarin sensitivity [CYP2C9 *2, CYP2C9 *3, and VKORC1 (1173C>T)] and an internal control product. The products were detected simultaneously on a poly(ethylene terephthalate) (PeT) microdevice using HIA, which provided genotyping results in approximately 15 minutes following PCR. The genotyping results of 23 patient DNA samples using this approach were in 100% concordance with the results of a validated test (WARFGENO test, ARUP laboratories). Additionally, the PCR reaction was successfully transferred to a PeT chip, which provided accurate genotyping results from patient DNA samples in under an hour. This work demonstrates a simple, rapid, and affordable approach to warfarin genotyping based on multiplex allele-specific PCR coupled with HIA detection. By demonstrating both chemistries on PeT microdevices, we show the potential for integration on a single device for sample-to-answer genotyping.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu.
authors.forename: Hillary S
authors.identifier: 0000-0002-5046-4589
authors.initials: HS
authors.lastname: Sloane
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu.
authors.forename: Morgan B
authors.initials: MB
authors.lastname: Carter
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu.
authors.forename: Alexa E C
authors.initials: AE
authors.lastname: Cecil
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu.
authors.forename: Delphine
authors.initials: D
authors.lastname: Le Roux
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Mills
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA. landers@virginia.edu and Department of Pathology, University of Virginia, Charlottesville, Virginia 22908, USA and Department of Mechanical Engineering, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Landers
mesh_terms: D065729:Cytochrome P-450 CYP2C9 / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D009693:Nucleic Acid Hybridization; D011093:Polyethylene Terephthalates / Q000737:chemistry*; D064417:Vitamin K Epoxide Reductases / Q000235:genetics; D014859:Warfarin / Q000008:administration & dosage*
chemical_list: D015342:DNA Probes; D011093:Polyethylene Terephthalates; D014859:Warfarin; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases