臺灣博碩士論文加值系統

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。

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.

Acknowledgements……..……..………………………...……………………...…........iii
中文摘要………………………………………………………………………………...v
Abstracts…………………………………………………………………………...……vi
Table of Contents………………………………………...….….………………….……ix
List of Figures…………………………………………………………………………xiii
List of Tables………………………………………………………………...…………xv
List of Abbreviations………………………………………………………………......xvi
Chapter 1 Introduction……………………………………………………………1
1.1 Pharmacogenetics……………………………………………………………….1
1.2 Pharmacology of Warfarin………………………………………………………2
1.3 Warfarin Therapy and Dosage………………………………………………….5
1.4 Serious Effects and Dose Variations of Warfarin Therapy…………………...6
1.5 Effects of CYP2C9 on Warfarin Metabolism…………………………………..7
1.6 Vitamin K Epoxide Reductase and Blood Coagulation.................................9
1.7 Study Objectives………………………………………………………………..10
Chapter 2 Material & Methods……………………………...……………..…...11
2.1 Association Study…………………………………….…………..…...………..11
2.1.1 Patients………………………………………………….....…...………...11
2.1.2 Inclusion Criteria for Individuals Administered with Extreme Warfarin Dose…………………………………..……………………………..........12
2.1.3 Inclusion Criteria for Randomly Selected Individuals………………....12
2.1.4 Exclusion Criteria………………………………….………..………..…..12
2.1.5 Unrelated Healthy Controls………………………………..…………….14
2.2 Genotyping……………………………………………..…………….…………15
2.2.1 SNP Discovery…………………………………………..……..………...15
2.2.2 DNA Extraction……………………………………..……....…………….15
2.2.3 Primer Design………………………….………………....………………16
2.2.4 PCR Reaction…………………………….……………………...……….19
2.2.5 SNP Genotyping…………………………………….…………………....19
2.2.6 Statistical Analysis……………………………………..…………………20
2.3 Function Analysis and Selection Tool for SNP…………………...………….21
2.4 VKORC1 Promoter Assay……………………………………..………………22
2.4.1 Plasmid Construction…………………………………....……………….22
2.4.2 Ligations Using the pGEM-T Easy Vectors……………….....………...23
2.4.3 Transformations Using the pGEM-T Easy Vector………………….….23
2.4.4 Colony Screening………………………………………........………..…24
2.4.5 Cell Culture and Dual Luciferase Reporter Assay……...............….…24
2.5 RNA isolation and real-time quantitative RT-PCR……………………....…..25
2.6 Vitamin K Epoxide Reductase Enzymatic Activity……………..…..………..27
2.6.1 Cell Culture…………………………………………………..……...……27
2.6.2 Preparation of Vitamin K Epoxide……………………………....……....27
2.6.3 Quantitation of Vitamin K1 and KO…………………………………..29
2.6.4 Preparation of Cell Extracts………………….………………..…..........29
2.6.5 Vitamin K Epoxide Reductase Activity Assay………………..……….30
2.6.6 High Performance Liquid Chromatography System……..…………...31
2.6.7 HPLC Column Selection…………………………………....………..….31
2.6.8 Construction of Standard Calibration Curve……………………..........33
2.6.9 Vitamin K1 Measurement by HPLC………………….…………….……34
2.6.10 Protein quantification by Microtiter Plate Protocols……………...……34
Chapter 3 Results………………………………………………………….…..35
3.1 Gene Sequencing and SNP Analysis……………..………...……………….35
3.1.1 CYP2C9 and VKORC1 DNA sequence variants in selected Chinese patients with warfarin sensitivity or resistance………………………...35
3.1.2 VKORC1 -1639 G＞A polymorphism in random Chinese patients receiving warfarin….……………………………………….………...…..39
3.1.3 Genotype frequencies of VKORC1 -1639 G＞A polymorphism and CYP2C9 variants in Chinese and Caucasians..……………………….40
3.1.4 General Discussion………………………..…………………..…………43
3.2 Promoter Analysis……………………………….........................……………45
3.2.1 VKORC1 Promoter SNP in-silico Analysis……………......…………...45
3.2.2 VKORC1 Promoter Activity Assay……………………………………...46
3.2.3 RNA Isolation and Real-Time Quantitative RT-PCR……..…………...47
3.2.4 General Discussion……………………………………………………....48
3.3 VKOR Enzymatic Activity Assay………………………………..….…………51
3.3.1 HPLC measurement for Standard Sample……………....………..... 51
3.3.2 Kinetics Studies of Vitamin K Epoxide Reductase……………..…......52
3.3.3 Comparison of VKOR Enzymatic Activity among Different Human Hepatoma Cell Lines…………………………….………….…………...55
3.3.4 General Discussion……………………………..…..……………………58
Chapter 4 Discussion………..…………………………………………….…….59
References…………………...………………………………………………………64
Appendix………………...…………….……………………………………………..70

List of Figures

Figure 1. The clotting pathway and vitamin K dependent clotting factors. Vitamin K dependent clotting factors are II, VII, IX, and V………………………………………...4
Figure 2. Vitamin K cycle, including vitamin K epoxide reductase and carboxylase, and the warfarin blocking step………………………………………………..…...…………4
Figure 3a. Structural formula of S-warfarin………..……………….………...………..7
Figure 3b. Structural formula of R-warfarin…..……..……………….………...………..7
Figure 4. Chemical structures of vitamin K1. Vitamin K1 contains a functional naphthoquinone ring and an aliphatic side chain. Phylloquinone has a phytyl side chain……………………………………………………………………………………...9
Figure 5. VKORC1 position and structure was shown by ESTs using Ensembl database………………………………………………………………………………...16
Figure 6. pGL3-Basic Vector map. luc+ is the cDNA encoding for firefly luciferase…23
Figure 7. The mixture of Vitamin K1 and hydrogen peroxide is kept in 75˚C...…….....28
Figure 8. Dam-Karrer test was negative for vitamin K epoxide…………...…...………28
Figure 9. The flow chart of preparation of cell extracts for VKOR assay………..…….30
Figure 10. Scatter plot of warfarin dose against the VKORC1 -1639 genotype. Warfarin doses in selected patients with warfarin sensitivity or resistance (see Table 1 for details) were plotted against different genotypes at the VKORC1 promoter -1639 locus...……36
Figure 11. A part of SNP Function Report of VKORC1-1639 SNP from FASTSNP......45
Figure 12. The relative measurements of luciferase activity levels in HepG2 cells. pGL3 luciferase reporter containing either the A (pGL3-A) or G allele (pGL3-G) at the promoter -1639 locus. Values represented the average of 9 experiments and the error bars represented the standard deviation. pGL3-basic was used as control without any promoter sequence inserted…………...………………………………………………..47
Figure 13. Real-time quantitative RT-PCR of VKORC1 mRNA in EBV-transformed B cells. Expression levels were shown as the relative amount of VKORC1 mRNA as compared to the Chinese individual having -1639 GG genotype. Values represent average of two determinations with variations < 5 %.....................................................48
Figure 14. The UV absorption spectrum of Vitamin KO and Vitamin K1 at 254 nm…51
Figure 15. The dependence of human VKOR activity of total protein on incubation time. The protein extract was incubated for various time periods at 33°C in the presence of 10 μM vitamin K epoxide and 5 mM DTT…...…………………………………...……… 52
Figure 16. The velocity of reductase at different substrate concentrations.…....….…...53
Figure 17. The double reciprocal plot of VKOR activity. Km was determined by linear regression.………………………………...………………………...…………………..54
Figure 18. The amount of vitamin K1 formed in different human hepatoma cell lines. The incubation was stopped at 15 min, 60 min, and 100 min.………...….........………56

List of Tables

Table 1. Patient demographics and DNA sequence variants identified……..………….38
Table 2 Mean doses and other clinical characteristics of randomly selected patients on warfarin stratified according to the genotypes…………………………...……………..39
Table 3. Genotype frequencies of VKORC1 promoter polymorphism (-1639 G>A) and CYP2C9 variants in Chinese and Caucasians.…………..…………..…………………41
Table 4. Comparison of the Km among different cells or tissues………………………54
Table 5. Vitamin K1 formation of different human liver cell lines…………..….……..55
Table 6. Detailed profile for vitamin K epoxide reductase activity of different human hepatoma cell lines…………………………………………………..…………………57

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