J Hum Genet (2006) 51:249–253 DOI 10.1007/s10038-005-0354-5 ORIGINAL ARTICLE Taisei Mushiroda Æ Yozo Ohnishi Æ Susumu Saito Atsushi Takahashi Æ Yuka Kikuchi Æ Shigeru Saito Hideki Shimomura Æ Yasuhiko Wanibuchi Takao Suzuki Æ Naoyuki Kamatani Æ Yusuke Nakamura Association of VKORC1 and CYP2C9 polymorphisms with warfarin dose requirements in Japanese patients Received: 11 October 2005 / Accepted: 28 November 2005 / Published online: 24 January 2006 Ó The Japan Society of Human Genetics and Springer-Verlag 2006 Abstract Warfarin is the most commonly used oral homozygous for the major allele (2.5 mg/day; anticoagulant for treatment of thromboembolism, but P=5.1·10À11 in the case of intron 1À136T>C SNP). adjustment of the dose appropriate to each patient is not We then genotyped the CYP2C9 gene for the Japanese so easy because of the large inter-individual variation in common genetic variant, CYP2C9*3 and, based on the dose requirement. We analyzed single nucleotide poly- genotype of these two genes, classified patients into three morphism (SNP) genotypes of the VKORC1 and categories, which we call ‘‘warfarin-responsive index.’’ CYP2C9 genes using DNA from 828 Japanese patients The median warfarin daily dose varied significantly in treated with warfarin, and investigated association be- this classification according to the warfarin-responsive tween SNP genotype and warfarin-maintenance dose. index (2.0 mg/day for index 0 group, 2.5 mg/day for Five SNPs in VKORC1,5¢ flankingÀ1413A>G, intron index 1 group, and 3.5 mg/day for index 2 group; 1À136T>C, intron 2+124C>G, intron 2+837T>C P=4.4·10À13). Thus, analysis of the combination of and exon 3 343G>A, were in absolute linkage disequi- VKORC1 and CYP2C9 genotypes should identify war- librium, and showed a significant association with daily farin-sensitive patients who require a lower dose of drug, warfarin dose of these patients. The median warfarin allowing personalized warfarin treatment. dose of patients with homozygosity for the minor allele was 4.0 mg/day, which is significantly higher than those Keywords Pharmacogenomics Æ Warfarin Æ VKORC1 Æ heterozygous for the minor allele (3.5 mg/day) or those Vitamin K epoxide reductase Æ CYP2C9 Æ Cytochrome P450 2C9 Æ SNP T. Mushiroda Æ Y. Kikuchi Æ Y. Nakamura Laboratory for Pharmacogenetics, SNP Research Center, The Institute of Physical and Chemical Research (RIKEN), Tokyo, Japan Introduction Susumu Saito Laboratory for SNP Analysis, The application of pharmacogenomic/pharmacogenetic SNP Research Center, information to clinical treatment is expected to help in The Institute of Physical and Chemical Research (RIKEN), Tokyo, Japan the prediction of efficacy and/or toxicity of drugs, leading to appropriate therapeutic regimens for indi- A. Takahashi Æ N. Kamatani vidual patients, thus contributing to improvement of our Laboratory of Statistical Analysis, SNP Research Center, medical care. In an attempt to identify genetic parame- The Institute of Physical and Chemical Research (RIKEN), Tokyo, Japan ters that predict efficacy or risk of adverse reactions for various drugs, many investigators have conducted and Y. Ohnishi Æ Y. Nakamura (&) are conducting association studies using genetic poly- Laboratory of Molecular Medicine, morphisms detected in genes encoding drug-metaboliz- Human Genome Center, Institute of Medical Science, ing enzymes, transporters, receptors, and proteins University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan involved in the drug-signaling pathway. However, only a E-mail: [email protected] few useful examples have so far been applied to routine Tel.: +81-3-54495372 clinical practice for prediction of efficacy/toxicity prior Fax: +81-3-54495433 to drug administration (Lesko and Woodcock 2004). Shigeru Saito Æ H. Shimomura Æ Y. Wanibuchi Æ T. Suzuki Warfarin is the most commonly used oral anticoag- Tokushukai Hospital Group, Tokyo, Japan ulant in the world. Although this agent is indispensable 250 for treatment of thromboembolism, it is not so easy to Table 1 Patient characteristics (n=828) adjust the appropriate dose to each patient due to the Age (years), median (range) 68 (19–92) large inter-individual variation in the requirement for Gender (%) this drug. An insufficient dose will result in failure to Male 67 prevent thrombosis, while overdose increases the risk of Female 33 unexpected bleeding. The maintenance dose of warfarin Daily warfarin dose (mg), median (range) 2.5 (0.5–10.5) CYP2C9 genotype, n (%) is usually determined by monitoring prothrombin time *1/*1 790 (95%) using an international normalization ratio (INR). An *1/*3 37 (4%) INR of 1.5–2.5 is recommended for Asian populations *3/*3 1 (<1%) (Matsuyama et al. 2002). Although clinically available warfarin is a racemic mixture, (S)-warfarin is five times more potent as an K-dependent clotting factor II (prothrombin), VII, IX anticoagulant than (R)-warfarin (Breckenridge et al. and X. This vitamin K epoxide reductase is encoded by 1974). (S)-Warfarin is primarily metabolized to the 7- vitamin K epoxide reductase complex subunit 1 hydroxylated form in humans, principally by cyto- (VKORC1) (Rost et al. 2004;Lietal.2004). Rare chrome P450, subfamily IIC, polypeptide 9 (CYP2C9) mutations that lead to amino acid changes in the (Rettie et al. 1992). To date, more than 50 variants in the VKORC1 protein have been found in familial cases in CYP2C9 gene have been described (Allele Nomenclature vitamin K-dependent clotting factor defective patients as Committee home page: http://www.imm.ki.se/CYPal- well as in those with warfarin resistance, although these leles), of which two single-nucleotide polymorphisms mutations have not been identified in the general pop- (SNPs), CYP2C9*2 (R144C) and CYP2C9*3 (I359L), ulation (Rost et al. 2004; Harrington et al. 2005). have been well verified in relation to the wild-type allele Recently, several groups have demonstrated that non- CYP2C9*1. No Asians so far studied have the coding SNPs in VKORC1 influenced warfarin sensitivity CYP2C9*2 allele (Kimura et al. 1998; Takahashi et al. (D’Andrea et al. 2005; Wadelius et al. 2005; Yuan et al. 2003; Kirchheiner and Brockmoller 2005). Previous 2005; Rieder et al. 2005; Sconce et al. 2005). These data studies have demonstrated that the warfarin 7-hydrox- strongly suggest that differences in genetic variations in ylase activity of the CYP2C9*3 variant allele is both CYP2C9 and VKORC1 could explain the diversity approximately 10-fold lower than that of CYP2C9*1 in in warfarin sensitivity and dose requirement. Interest- in vitro analysis (Lee et al. 2002) and that carriers of the ingly, recent reports indicate an additive effect on war- variant allele required a lower maintenance dose of farin dose requirement in combinations of variant forms warfarin (Takahashi et al. 2003; Kirchheiner and of VKORC1 and CYP2C9 in Caucasian patients Brockmoller 2005). Warfarin exerts its anticoagulant (D’Andrea et al. 2005; Sconce et al. 2005). effects by interfering with regeneration of vitamin K by Furthermore, it is well known that there are signifi- reduction of its 2,3-epoxide in the vitamin K cycle, cant differences in warfarin dose requirement in different leading to inhibition of gamma-carboxylation of vitamin ethnic groups; for example, Chinese patients were re- Table 2 Daily maintenance dose of warfarin in patients with different VKORC1 genotypes Genotype No. of patients Dose (mg/day) P value Median Range 5¢ FlankingÀ1413A>G GG 6 4.0 2.0–8.0 9.1·10À11 GA 133 3.5 1.0–10.5 AA 689 2.5 0.5–10.0 Intron 1À136T>C CC 6 4.0 2.0–8.0 5.1·10À11 CT 132 3.5 1.0–10.5 TT 690 2.5 0.5–10.0 Intron 2+124C>G GG 6 4.0 2.0–8.0 5.1·10À11 GC 132 3.5 1.0–10.5 CC 690 2.5 0.5–10.0 Intron 2+837T>C CC 6 4.0 2.0–8.0 5.1·10À11 CT 132 3.5 1.0–10.5 TT 690 2.5 0.5–10.0 Exon 3 343G>A AA 6 4.0 2.0–8.0 9.1·10À11 AG 133 3.5 1.0–10.5 GG 689 2.5 0.5–10.0 251 ported to require a warfarin dose nearly 40% lower than for the TaqMan assay were obtained from Applied that required by Caucasian patients (Zhao et al. 2004). Biosystems. DNA extraction and PCR primer design In addition, the therapeutic maintenance dose of war- were performed as previously described (Ohnishi et al. farin for Japanese was also 31% lower than that for 2001). Caucasians (Takahashi et al. 2003). These phenotypic differences in dose requirement cannot be explained only by SNPs in CYP2C9 (Takahashi et al. 2003), suggesting Statistical analysis that interethnic variability in VKORC1 might play an important role. We describe here the results of associa- Differences in daily maintenance dose of warfarin in the tion studies using the SNPs in VKORC1 to identify ge- different genotype groups were evaluated by Mann– netic variations that might confer sensitivity to warfarin Whitney’s U and Kruskal–Wallis tests using SPSS soft- in Japanese patients. Based on the genotypes for ware (version 12.0, SPSS, Chicago, IL). A 5% two-tailed VKORC1 SNPs significantly associated with warfarin significance level was used in all tests. sensitivity, in combination with genotypic information of CYP2C9 polymorphisms, we developed a prediction system that was able to determine the dose appropriate Results to each patient. Patient characteristics Materials and methods Table 1 summarizes clinical information for the 828 warfarin-treated patients in the present study. The Subjects median maintenance daily dose of warfarin was 2.5 mg/ day. For the CYP2C9 gene, only CYP2C9*3, a common A total of 828 patients with warfarin treatment was re- Japanese genetic variant, was evaluated.