Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes Rebecca Howard a , Julian B.S. Leathart b , David J. French a , Elaina Krishan a , Hugo Kohnke c , Mia Wadelius c, 1 , Rianne van Schie d , Talitha Verhoef d , Anke-Hilse Maitland-van der Zee d, 1 , Ann K. Daly b, ⁎ , 1 , Rita Barallon a, 1 a LGC, Teddington, Middlesex, TW11 0LY, UK b Institute of Cellular Medicine, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK c Department of Medical Sciences, Clinical Pharmacology, Uppsala University, 75185 Uppsala, Sweden d Utrecht University, Faculty of Science, PO Box 80082, 3508 TB Utrecht, The Netherlands abstract article info Article history: Received 9 June 2011 Accepted 9 July 2011 Available online xxxx Keywords: Warfarin Phenprocoumon Acenocoumarol CYP2C9 VKORC1 Point of care genotyping 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Oral anticoagulants such as warfarin, acenocoumarol and phen- procoumon are prescribed widely for the treatment of thromboem- bolic disorders. Prescribing these coumarin based derivatives is difficult due to their narrow therapeutic range with patients at risk of haemorrhage if given too high a dose. Factors such as height, weight and concurrent medications all affect dose requirement. It is also now very well established that genetic factors play a role in the variability of a patient's response. Several polymorphisms in the genes encoding the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) contribute to this variability. It is estimated that these single nucleotide polymorphisms (SNPs) account for about 40% of the variability in response to coumarin dosing regimes, with patient age and height or weight contributing an additional 15% [1–4]. In 2007, the US Food and Drug Administration (FDA) Center for Drug Evaluation and Research updated the label of warfarin to include information on genetic testing [5]. This has prompted clinical trials to demonstrate the utility of pharmacogenetic testing in coumarin anticoagulant prescription. Until now, published results from clinical trials involving genotyping have related to warfarin only, and have mainly involved small numbers of patients; they are therefore underpowered to identify significant differences in outcomes be- tween cases and controls. These studies have generally not involved determination of a patient's genotype prior to start of treatment [6– 11] with the exception of one recent study involving 230 patients [12]. It has been estimated that to detect a 10% improvement in time within range for the target international normalised ratio (INR) as a result of including genotyping during initial warfarin dosing, it would be necessary to study 442 cases and 442 controls [13] The only study completed up to the present with numbers of cases in excess of these estimates [9] found that more frequent monitoring of patients based Clinica Chimica Acta xxx (2011) xxx–xxx ⁎ Corresponding author at: Institute of Cellular Medicine, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. Tel.: + 44 (0) 191 222 7031; fax: +44 (0)191 222 0723. E-mail address: a.k.daly@ncl.ac.uk (A.K. Daly). 1 On behalf of the EU-PACT group. CCA-12365; No of Pages 7 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.07.013 Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim Please cite this article as: Howard R, et al, Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes, Clin Chim Acta (2011), doi:10.1016/j.cca.2011.07.013