Background: Variant alleles of vitamin K epoxide reductase complex subunit 1 gene (VKORC1), the target molecule of vitamin K antagonists, and of cytochrome P450 (CYP) 2C9, an enzyme involved in coumarin metabolism, affect the anticoagulant response of coumarins, which have a narrow therapeutic window. Genotyping for these variants allows for prediction of therapeutic drug doses. The discussion of the clinical role of genotype-guided coumarin dosing is ongoing. For pharmacogenetic information to be useful, results must be available quickly.
Methods: Here we report on the establishment of an allele-specific amplification (ASA)-PCR assay for the three most relevant polymorphisms for coumarin pharmacogenetics. The assay was validated against sequencing data on 100 random samples from Caucasian blood donors, incorporating all genotypes. Divergent results were confirmed by repeating the analysis with both methods. One hundred percent congruence with DNA sequencing was determined as the ‘pass’ criterion for the assay.
Results: The ASA-PCR assay reproducibly identified the three informative single nucleotide polymorphisms. Discrepancies between ASA-PCR and sequencing were clarified by retrospective analysis as being due to erroneous analysis or documentation. In summary, the congruence of sequencing and duplex ASA-PCR was 100%.
Conclusion: ASA-PCR is significantly faster and less expensive than sequencing. We expect that pharmaco-genetics-based dosing decisions may reduce the frequency of over- and undertreatment with vitamin K antagonists, especially during drug initiation, and thus improve patient safety.
Coumarin International Normalize Ratio Pharmacogenetic Testing Therapeutic International Normalize Ratio Range Pharmacogenetic Information
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Gabriele Spohn and Christof Geisen contributed equally to this work.
The work of the technicians of the molecular hemostaseology laboratory at the German Red Cross Blood Service Baden-Württemberg/Hesse and Institute for Transfusion Medicine and Immunohematology, who performed DNA isolation and sequencing, is gratefully acknowledged.
No funding has been received for the conduct of this study or the preparation of this manuscript. The authors have no conflicts of interest directly related to the content of this study.
Takeuchi F, McGinnis R, Bourgeois S, et al. A genome-wide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal genetic determinants of warfarin dose. PLoS Genet 2009 Mar; 5(3): e1000433PubMedCrossRefGoogle Scholar
Rettie AE, Korzekwa KR, Kunze KL, et al. Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem Res Toxicol 1992 Jan; 5(1): 54–9PubMedCrossRefGoogle Scholar
Li T, Chang CY, Jin DY, et al. Identification of the gene for vitamin K epoxide reductase. Nature 2004 Feb 5; 427(6974): 541–4PubMedCrossRefGoogle Scholar
Rost S, Fregin A, Ivaskevicius V, et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004 Feb 5; 427(6974): 537–41PubMedCrossRefGoogle Scholar
Limdi NA, Wadelius M, Cavallari L, et al. Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups. Blood 2010 May 6; 115(18): 3827–34PubMedCrossRefGoogle Scholar
Rieder MJ, Reiner AP, Gage BF, et al. Effect of VKORC 1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med 2005 Jun 2; 352(22): 2285–93PubMedCrossRefGoogle Scholar
Geisen C, Watzka M, Sittinger K, et al. VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation. Thromb Haemost 2005 Oct; 94(4): 773–9PubMedGoogle Scholar
Stehle S, Kirchheiner J, Lazar A, et al. Pharmacogenetics of oral anticoagulants: a basis for dose individualization. Clin Pharmacokinet 2008; 47(9): 565–94PubMedCrossRefGoogle Scholar
Luxembourg B, Schneider K, Sittinger K, et al. Impact of pharmacokinetic (CYP2C9) and pharmacodynamic (VKORC1, F7, GGCX, CALU, EPHX1) gene variants on the initiation and maintenance phases of phenprocoumon therapy. Thromb Haemost 2011 Jan 3; 105(1): 169–80PubMedCrossRefGoogle Scholar
Geisen C, Luxembourg B, Watzka M, et al. Prediction of phenprocoumon maintenance dose and phenprocoumon plasma concentration by genetic and non-genetic parameters. Eur J Clin Pharmacol 2011 Apr; 67(4): 371–81PubMedCrossRefGoogle Scholar
Bodin L, Verstuyft C, Tregouet DA, et al. Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genotypes as determinants of acenocoumarol sensitivity. Blood 2005 Jul 1; 106(1): 135–40PubMedCrossRefGoogle Scholar
Puehringer H, Loreth RM, Klose G, et al. VKORC 1 -1639G>A and CYP2C9*3 are the major genetic predictors of phenprocoumon dose requirement. Eur J Clin Pharmacol 2010 Jun; 66(6): 591–8PubMedCrossRefGoogle Scholar
Schalekamp T, Brasse BP, Roijers JF, et al. VKORC1 and CYP2C9 genotypes and acenocoumarol anticoagulation status: interaction between both genotypes affects overanticoagulation. Clin Pharmacol Ther 2006 Jul; 80(1): 13–22PubMedCrossRefGoogle Scholar
Schalekamp T, Brasse BP, Roijers JF, et al. VKORC1 and CYP2C9 genotypes and phenprocoumon anticoagulation status: interaction between both genotypes affects dose requirement. Clin Pharmacol Ther 2007 Feb; 81(2): 185–93PubMedCrossRefGoogle Scholar
Caraco Y, Blotnick S, Muszkat M. CYP2C9 genotype-guided warfarin prescribing enhances the efficacy and safety of anticoagulation: a prospective randomized controlled study. Clin Pharmacol Ther 2008 Mar; 83(3): 460–70PubMedCrossRefGoogle Scholar
Anderson JL, Horne BD, Stevens SM, et al. Randomized trial of genotype-guided versus standard warfarin dosing in patients initiating oral anti-coagulation. Circulation 2007 Nov 27; 116(22): 2563–70PubMedCrossRefGoogle Scholar
Ferder NS, Eby CS, Deych E, et al. Ability of VKORC 1 and CYP2C9 to predict therapeutic warfarin dose during the initial weeks of therapy. J Thromb Haemost 2010 Jan; 8(1): 95–100PubMedCrossRefGoogle Scholar
Epstein RS, Moyer TP, Aubert RE, et al. Warfarin genotyping reduces hospitalization rates results from the MM-WES (Medco-Mayo Warfarin Effectiveness Study). J Am Coll Cardiol 2010 Jun 22; 55(25): 2804–12PubMedCrossRefGoogle Scholar
Eckman MH, Rosand J, Greenberg SM, et al. Cost-effectiveness of using pharmacogenetic information in warfarin dosing for patients with non-valvular atrial fibrillation. Ann Intern Med 2009 Jan 20; 150(2): 73–83PubMedGoogle Scholar
You JH, Tsui KK, Wong RS, et al. Potential clinical and economic outcomes of CYP2C9 and VKORC1 genotype-guided dosing in patients starting warfarin therapy. Clin Pharmacol Ther 2009 Nov; 86(5): 540–7PubMedCrossRefGoogle Scholar
Gustincich S, Manfioletti G, Del Sal G, et al. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques 1991 Sep; 11(3): 298–302PubMedGoogle Scholar
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988 Feb 11; 16(3): 1215PubMedCrossRefGoogle Scholar
Aithal GP, Day CP, Leathart JB, et al. Relationship of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced hepatitis. Pharmacogenetics 2000 Aug; 10(6): 511–8PubMedCrossRefGoogle Scholar
Sullivan-Klose TH, Ghanayem BI, Bell DA, et al. The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism. Pharmacogenetics 1996 Aug; 6(4): 341–9PubMedCrossRefGoogle Scholar