title: Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes.
abstract: BACKGROUND
  Coumarin anticoagulants such as warfarin are used to treat and prevent thromboembolic events in patients. The required dosage is difficult to predict and the risk of over or under anticoagulation are dependent on several environmental and clinical factors, such as concurrent medication, diet, age and genotype for polymorphisms in two genes CYP2C9 and VKORC1.
  
  
  METHODS
  A novel fluorescent PCR genotyping assay using HyBeacon® probes, was developed to enable clinical staff to genotype the CYP2C9*2 and CYP2C9*3 alleles and the VKORC1 G-1639A polymorphism directly from unextracted blood samples. A prototype PCR instrument, Genie 1, suitable for point of care use was developed to carry out the assays. The panel of tests was validated by analysing blood samples from 156 individuals and comparing genotypes with data obtained using DNA samples from the same individuals. The accuracy of genotypes obtained with the Genie 1 was compared against results from well validated real time PCR and PCR-restriction fragment length polymorphism analysis.
  
  
  RESULTS
  Identical results were obtained for the newly developed HyBeacon® method and the validation method in all cases except for one where no result was obtained for the VKORC1 polymorphism on the Genie instrument. The samples used for validation represented all six possible *2 and *3 allele-related CYP2C9 genotypes and all three VKORC1 G-1639A genotypes.
  
  
  CONCLUSIONS
  We observed excellent accuracy for the newly developed method which can determine genotype in less than 2 h.
authors.affiliation: LGC, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Howard
authors.forename: Julian B S
authors.initials: JB
authors.lastname: Leathart
authors.forename: David J
authors.initials: DJ
authors.lastname: French
authors.forename: Elaina
authors.initials: E
authors.lastname: Krishan
authors.forename: Hugo
authors.initials: H
authors.lastname: Kohnke
authors.forename: Mia
authors.initials: M
authors.lastname: Wadelius
authors.forename: Rianne
authors.initials: R
authors.lastname: van Schie
authors.forename: Talitha
authors.initials: T
authors.lastname: Verhoef
authors.forename: Anke-Hilse
authors.initials: AH
authors.lastname: Maitland-van der Zee
authors.forename: Ann K
authors.initials: AK
authors.lastname: Daly
authors.forename: Rita
authors.initials: R
authors.lastname: Barallon
mesh_terms: D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D065729:Cytochrome P-450 CYP2C9; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D064417:Vitamin K Epoxide Reductases
chemical_list: D017931:DNA Primers; 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