華法林用藥劑量的藥物遺傳學研究

阮相宇

doi:10.6342/NTU.2006.03096

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華法林用藥劑量的藥物遺傳學研究

Pharmacogenetics Study of Warfarin Responsiveness

阮相宇 , Masters　　Advisor：陳垣崇　　

英文 DOI： 10.6342/NTU.2006.03096

藥物遺傳學；遺傳；華法林；抗凝血劑；單核甘酸變異； Warfarin ； Pharmacogenetics ； Genetics ； SNP ； VKORC1 ； Promoter

Abstract │ Reference (55) │ Altmetrics

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Background
Warfarin is a commonly prescribed oral anticoagulant most frequently used to control and prevent thromboembolic diseases. Although warfarin is considered very efficacious for preventing thromboembolic diseases, it has a narrow therapeutic index and the risk of hemorrhage during warfarin therapy has remained high. The wide variations in inter-individual warfarin dose requirements can be only partially explained by genetic variations in CYP2C9 that affect warfarin metabolism. It has been well established that the average daily dose requirement of Asians is 40% lower than that of the Caucasians, and this inter-ethnic difference cannot be explained by genetic variations in CYP2C9 since both CYP2C9*2 and CYP2C9*3 are either rare or absent in Asians. The VKORC1 gene is a newly cloned gene encoding vitamin K epoxide reductase (VKOR), an enzyme targeted by warfarin that prevents vitamin K recycling. In this study, we propose to investigate CYP2C9 and VKORC1 variants in Chinese patients receiving low and high warfarin maintenance doses. We compared their frequencies with those in Caucasians to determine the basis for the inter-individual and inter-ethnic differences in warfarin doses.

Methods
DNA sequence variants in CYP2C9 and VKORC1 were identified in 16 Chinese patients having warfarin sensitivity (<2.0 mg/d) and resistance (>5.0 mg/d) by direct sequencing of the 5′ flanking and exonic region of CYP2C9 and VKORC1. Genotyping DNA sequence variants in 104 randomly selected Chinese patients receiving warfarin, 95 normal Chinese controls, and 92 normal Caucasians was performed by using MALDI-TOF SNP genotyping. The statistical analysis techniques used were the t-test and Wilcoxon–Mann–Whitney test; these were performed for multiple comparisons of the mean dose levels among the different genotype groups.

The VKORC1 promoter encompassing the –1639 polymorphism (from –1798 to –35) was cloned into pGL3 vector. A pRL-TK vector encoding Renilla luciferase was used as internal control to normalize firefly luciferase expression. HepG2 cells were cultured for transfection and used as host cells. Firefly and Renilla luciferase activities were measured using a luminometer.

The VKOR enzymatic assay was performed using three hepatocellular carcinoma cell lines (HCC-36, HepG2, and Hep3B) having different VKORC1–1639 genotypes. After incubation with dithiothreitol and vitamin K epoxide, vitamin K formation was determined by high performance liquid chromatography (HPLC) system using C18 reversed phase column.

Results
We identified three CYP2C9 variants—CYP2C9*3, T299A, and P382L—in four warfarin-sensitive patients. A novel VKORC1 promoter polymorphism (–1639 G>A) present in the homozygous form (genotype AA) was found in all warfarin-sensitive patients. The resistant patients were either AG or GG. Among the 104 randomly selected Chinese patients receiving warfarin, those with the AA genotype had a lower dose  requirement than those with the AG/GG genotype (P < 0.0001). The frequencies of AA, AG, and GG genotypes were comparable in Chinese patients receiving warfarin (79.7%, 17.6%, and 2.7%) and normal Chinese controls (82%, 18%, and 0%); however, these frequencies differed significantly from those observed in Caucasians (14%, 47%, and 39%) (P < 0.0001). 
The promoter polymorphism abolished the E-box consensus sequence, and a dual luciferase assay revealed that the VOKRC1 promoter with the G allele exhibited a 44% increase in activity when compared to that of the A allele. Comparison of VKOR enzymatic activity among three different human hepatoma cell lines revealed that both Hep3B and HCC-36 exhibited higher VKOR activities than HepG2.

Conclusion:
This work is the first study that provides evidence that variations within the VKORC1 gene could alter warfarin dosage requirements, both inter-individually in the Chinese and inter-ethnically between the Chinese and Caucasians. VKORC1–1639 A>G abolished the E-box consensus sequence and increased promoter activity. This led to higher VKOR activity, and hence a larger dose of warfarin was required to inhibit VKOR.

Reference ( 55 ) 〈TOP〉

1. Sadee, W. and Dai, Z. (2005) Pharmacogenetics/genomics and personalized medicine. Human Molecular Genetics, 14 Spec No. 2, R207-14.
連結：
2. Roses, A.D. (2000) Pharmacogenetics and future drug development and delivery. Lancet, 355, 1358-61.
連結：
3. Ingelman-Sundberg, M., Oscarson, M. and McLellan, R.A. (1999) Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends in Pharmacological Sciences, 20, 342-9.
連結：
4. Evans, W.E. and Relling, M.V. (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 286, 487-91.
連結：
5. Meyer, U.A. (1991) Genotype or phenotype: the definition of a pharmacogenetic polymorphism. Pharmacogenetics, 1, 66-7.
連結：

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