華法林用藥劑量的藥物遺傳學研究

Pharmacogenetics Study of Warfarin Responsiveness

阮相宇(Hsiang-Yu Yuan)

指導教授 ： 陳垣崇

國立臺灣大學/醫學院/微生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.03096

摘要

Warfarin (華法林) 是一種心臟科常使用的藥物,主要用來預防深部靜脈栓塞，肺栓塞，缺血性冠狀動脈疾病，人工瓣膜置換後，以及心房顫動等，還有跟阿斯匹靈合用以治療急性心肌梗塞。但使用劑量因人而異。如果劑量過高有出血致死的危險存在，過低卻沒有效果。同時亞洲人種使用的劑量普遍比高加索人種低，可是其原因不明。本研究目的在尋找遺傳因素導致華法林用藥劑量上個人間和族群間的差異。

本研究發現一個VKORC1基因啟動子調控區的單核甘酸變異(Single Nucleotide Polymorphism) VKORC1-1639A>G和其啟動子活性可以解釋華法林個人之間和族群之間用藥劑量差異。本實驗利用16個華法林用藥量極端敏感或極端抵抗的病人，找出VKORC1-1639 A>G 的單核甘酸變異。利用MALDI-TOF技術完成了104個隨機病人的基因行鑑定進行統計分析。AA 基因型的維持劑量比AG/GG 基因型的低 (P<0.0001)。本研究並利用啟動子測定，和酵素活性測定確認VKORC1-1639A>G 是一個功能性單核甘酸變異。此變異導致VKORC1基因表現量增加44%。由於VKORC1表現亮增加導致VKOR活性增加，因此需要更高劑量的華法林去抑制。

關鍵字

藥物遺傳學 ； 遺傳 ； 華法林 ； 抗凝血劑 ； 單核甘酸變異

並列摘要

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.

並列關鍵字

Warfarin ； Pharmacogenetics ； Genetics ； SNP ； VKORC1 ； Promoter

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