CORE Metadata, citation and similar papers at core.ac.uk Provided by Springer - Publisher Connector Hum Genet (2007) 121:23–34 DOI 10.1007/s00439-006-0260-8 ORIGINAL INVESTIGATION Association of warfarin dose with genes involved in its action and metabolism Mia Wadelius · Leslie Y. Chen · Niclas Eriksson · Suzannah Bumpstead · Jilur Ghori · Claes Wadelius · David Bentley · Ralph McGinnis · Panos Deloukas Received: 6 July 2006 / Accepted: 1 September 2006 / Published online: 18 October 2006 © Springer-Verlag 2006 Abstract We report an extensive study of variability associated with dose (P < 0.05). VKORC1, CYP2C9, in genes encoding proteins that are believed to be CYP2C18 and CYP2C19 were signiWcant after involved in the action and biotransformation of warfa- experiment-wise correction for multiple testing rin. Warfarin is a commonly prescribed anticoagulant (P < 0.000175), however, the association of CYP2C18 that is diYcult to use because of the wide interindivid- and CYP2C19 was fully explained by linkage disequilib- ual variation in dose requirements, the narrow thera- rium with CYP2C9*2 and/or *3. PROC and APOE peutic range and the risk of serious bleeding. We were both signiWcantly associated with dose after cor- genotyped 201 patients for polymorphisms in 29 genes rection within each gene. A multiple regression model in the warfarin interactive pathways and tested them for with VKORC1, CYP2C9, PROC and the non-genetic association with dose requirement. In our study, poly- predictors age, bodyweight, drug interactions and indi- morphisms in or Xanking the genes VKORC1, CYP2C9, cation for treatment jointly accounted for 62% of vari- CYP2C18, CYP2C19, PROC, APOE, EPHX1, CALU, ance in warfarin dose. Weaker associations observed GGCX and ORM1-ORM2 and haplotypes of for other genes could explain up to »10% additional VKORC1, CYP2C9, CYP2C8, CYP2C19, PROC, F7, dose variance, but require testing and validation in an GGCX, PROZ, F9, NR1I2 and ORM1-ORM2 were independent and larger data set. Translation of this knowledge into clinical guidelines for warfarin prescrip- tion will be likely to have a major impact on the safety Y Electronic supplementary material Supplementary material is and e cacy of warfarin. available in the online version of this article at http://dx.doi.org/ 10.1007/s00439-006-0260-8 and is accessible for authorized users. Abbreviations M. Wadelius ABCB1 ATP-binding cassette transporter B1 Department of Medical Sciences, Clinical Pharmacology, gene, P-glycoprotein gene or MDR1 University Hospital, 751 85 Uppsala, Sweden APOE Apolipoprotein E CALU Calumenin gene L. Y. Chen · S. Bumpstead · J. Ghori · D. Bentley · R. McGinnis · P. Deloukas (&) CAR Constitutive androstane receptor The Wellcome Trust Sanger Institute, CYP1A1 Cytochrome P450 1A1 Wellcome Trust Genome Campus, CYP1A2 Cytochrome P450 1A2 Hinxton, Cambridgeshire, CB10 1SA, UK CYP2A6 Cytochrome P 2A6 e-mail: [email protected] 450 CYP2C18 Cytochrome P450 2C18 N. Eriksson CYP2C19 Cytochrome P450 2C19 UCR—Uppsala Clinical Research Center, CYP2C8 Cytochrome P450 2C8 Uppsala Science Park, 751 83 Uppsala, Sweden CYP2C9 Cytochrome P450 2C9 P C. Wadelius CYP3A4 Cytochrome 450 3A4 Department of Genetics and Pathology, Medical Genetics, CYP3A5 Cytochrome P450 3A5 Rudbeck Laboratory, 751 85 Uppsala, Sweden EPHX1 Epoxide hydrolase 1, microsomal gene 123 24 Hum Genet (2007) 121:23–34 F2 Coagulation factor II gene or prothrom dose requirement, which varies 20-fold, is inXuenced bin gene by factors such as intake of vitamin K, ethnicity, illness, F5 Coagulation factor V gene age, gender, concurrent medication, body mass index F7 Coagulation factor VII gene and genetic factors (Loebstein et al. 2001; Takahashi F9 Coagulation factor IX gene and Echizen 2003; Wadelius et al. 2004). F10 Coagulation factor X gene Warfarin acts through interference with the recy- FII Coagulation factor II or prothrombin cling of vitamin K in the liver, which leads to secretion FIIa Coagulation factor II activated or of inactive vitamin K-dependent proteins (Bell et al. thrombin 1972; Dahlbäck 2005). Figure 1 shows the genes that FIX Coagulation factor IX are believed to be involved in the biotransformation of FIXa Coagulation factor IX activated vitamin K, warfarin and the vitamin K-dependent clot- FV Coagulation factor V ting factors, here called the warfarin interactive path- FVII Coagulation factor VII ways (Supplementary Table S1; for details see FVIIa Coagulation factor VII activated Wadelius and Pirmohamed 2006). The main protein in FX Coagulation factor X the vitamin K epoxide reductase complex is encoded FXa Coagulation factor X activated by VKORC1 (Li et al. 2004; Rost et al. 2004). Common GAS6 Growth-arrest speciWc 6 variation in human VKORC1 is one of the most impor- GGCX Gamma-glutamyl carboxylase gene tant genetic factors determining warfarin dose MDR1 Multidrug resistance gene 1, P-glycopro (D’Andrea et al. 2005; Geisen et al. 2005; Rieder et al. tein gene or ABCB1 2005; Sconce et al. 2005; Wadelius et al. 2005; Veenstra NQO1 NAD(P)H dehydrogenase, et al. 2005; Yuan et al. 2005). Another putative subunit quinone 1 gene of the vitamin K epoxide reductase is the microsomal NR1I2 Pregnane X receptor gene epoxide hydrolase 1, encoded by EPHX1, which har- NR1I3 Constitutive androstane receptor gene bours a vitamin K-epoxide binding site (Cain et al. ORM1 Orosomucoid 1 gene or Alpha-1-acid 1997; Loebstein et al. 2005; Morisseau and Hammock glycoprotein 1 gene 2005). Calumenin, encoded by CALU, binds to vitamin ORM2 Orosomucoid 2 gene or Alpha-1-acid K epoxide reductase and inhibits the eVect of warfarin glycoprotein 2 gene (Wallin et al. 2001; Wajih et al. 2004). PCR Polymerase chain reaction A high intake of the fat-soluble vitamin K1 can PROC Protein C gene reverse the action of warfarin. Vitamin K1 is absorbed PROS1 Protein S gene from the small intestine along with dietary fat, and sub- PROZ Protein Z gene sequently cleared by the liver through an apolipopro- PT INR Prothrombin time international norma tein E (APOE) receptor speciWc uptake (Berkner and lised ratio Runge 2004). The uptake of vitamin K1 into the liver PXR Pregnane X receptor varies between diVerent APOE alleles and is in the SERPINC1 Anti-thrombin III gene order of *E2 <*E3 < *E4 (Saupe et al. 1993; Kohlme- siRNA Small interfering ribonucleic acid ier et al. 1996). It has also been suggested that the anti- SNP Single nucleotide polymorphism oxidant enzyme nicotine adenine dinucleotide UTR Untranslated region phosphate (NAD(P)H) dehydrogenase, encoded by VKORC1 Vitamin K epoxide reductase NQO1, has the potential to reduce dietary vitamin K complex subunit 1 gene (Wallin and Hutson 1982; Berkner and Runge 2004). Reduced vitamin K is the essential cofactor for the activation of vitamin K-dependent proteins by gamma- Background glutamyl carboxylase, encoded by GGCX (Wu et al. 1997; Berkner 2000). The main vitamin K-dependent Warfarin is one of the most widely used coumarin anti- proteins are clotting factors II, VII, IX and X, proteins coagulants in the treatment of atrial Wbrillation, heart C, S and Z, and the growth-arrest speciWc protein 6, valve prosthesis, recurrent stroke, deep vein thrombo- encoded by F2, F7, F9, F10, PROC, PROS1, PROZ sis and pulmonary embolism (Daly and King 2003). and GAS6, respectively (Berkner and Runge 2004). A However, its use is made diYcult by the wide interindi- non-vitamin K-dependent clotting protein anti-thrombin vidual variation in dose required to achieve a therapeu- III, encoded by SERPINC1, inhibits the vitamin K- tic eVect, the narrow therapeutic range, and the risk of dependent factors II, IX and X (Dahlbäck 2005). serious bleeding (Landefeld and Beyth 1993). Warfarin Furthermore, the point mutation Arg506Gln (FV Leiden) 123 Hum Genet (2007) 121:23–34 25 Fig. 1 An overview of the interaction between warfarin and the 29 genes. This path- way illustrates genes thought to mediate the eVects of war- farin. It also depicts a simpli- Wed representation of the biotransformation of warfarin and vitamin K in F5 is a common cause of thromboembolism, and is may be involved in the metabolism of R-warfarin therefore a known cause of warfarin treatment (Larsen (Rettie et al. 1992; Zhang et al. 1995; Kaminsky and et al. 1998). Zhang 1997; Huang et al. 2004). Cytochrome P450 The molecular basis of warfarin pharmacokinetics enzymes are induced by the nuclear hormone receptors has been extensively studied (Sanderson et al. 2005). pregnane X receptor (PXR) and constitutive andro- Warfarin is administered as a racemate that consists of stane receptor (CAR), which are encoded by NR1I2 R- and S-enantiomers, the S-form being 3–5 times and NR1I3 (Lehmann et al. 1998; Chen et al. 2004). more active than the R-form (Rettie et al. 1992). War- Based on an inhibition assay, there is some evidence farin is rapidly absorbed from the stomach and the that transport of warfarin out of the liver and into the upper gastrointestinal tract (Palareti and Legnani bile may be mediated by P-glycoprotein, which 1996), and in the circulating blood it is highly bound to is encoded by ABCB1 (MDR1) (Sussman et al. 2002). albumin and alpha 1-acid glycoproteins, encoded by P-glycoprotein can be induced by the nuclear hormone ORM1 and ORM2 (Otagiri et al. 1987). Once warfarin receptor PXR (Geick et al. 2001). has entered the liver, S-warfarin is metabolised by We and others have shown that common variants in cytochrome P450 2C9 (CYP2C9) (Rettie et al. 1992; the VKORC1 and CYP2C9 genes together with a lim- Kaminsky and Zhang 1997). Patients with CYP2C9 ited subset of environmental determinants account for variant alleles *2 and *3 alleles (http://www.imm.ki.se/ around 50–60% of the variance in warfarin dose CYPalleles/) require lower mean daily warfarin doses requirement (Sconce et al. 2005; Wadelius et al. 2005; than extensive metabolisers homozygous for the *1 Veenstra et al.