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研究生:蔡麗琴
研究生(外文):Li-chin Tsai
論文名稱:整合微流體PCR 與次世代定序檢測C 型肝炎病毒NS3 基因突變率
論文名稱(外文):Integrated Microfluidic PCR and Next-Generation Sequencing system for Mutated Hepatitis C Virus NS3 Gene Detection
指導教授:劉俊仁劉俊仁引用關係
口試委員:彭福佐梁有志
口試日期:2013-07-22
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:85
中文關鍵詞:整合微流體PCR次世代定序檢C 型肝炎病毒NS3 基因突變率
外文關鍵詞:Integrated Microfluidic PCRNext-Generation Sequencing systemMutated Hepatitis C Virus NS3 Gene Detection
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根據世界衛生組織(World Health Organization,WHO)統計,截至目前為止全球有超過1 億7 仟萬人感染過C 型肝炎病毒(Hepatitis C virus),而感染者往往發展成C 型肝炎,病程超過20 年的病患,其發病率和死亡率都與肝硬化和肝癌有關。因此,如何降低C 型肝炎病毒感染及有效清除病毒,以減緩肝硬化的進展及預防肝癌的發生,一直是全球關注的醫學議題。目前對於感染第1 型C 型肝炎病毒,常以長效型干擾素(pegylated IFN-alpha)與雷巴威林(Ribavirin)一起合併治療,成功率有40~50%,但因治療時間長與副作用嚴重,大幅限制了藥物的治療效果。2011 年有兩個第一代NS3/4A 蛋白酶抑製劑Telaprevir 和Boceprevir,已經被FDA批准用於治療第1 型慢性C 型肝炎,這一類新藥物被統稱為直接作用的抗病毒藥物(direct-acting antivirals DAA),對於治療慢性C 型肝炎病毒有重要的發展與進步。然而,HCV 病毒顆粒的高遺傳多樣性與突變,會降低DAA 治療的敏感性,研究證實Resistance associated variants 經常發生在非常低的頻率(<5 %)。為解決此一問題,目前臨床試驗中最常見是以Sanger 定序做抗藥性突變分析,然而由於Sanger 定序方法不夠敏感,抗藥性突變頻率在20%至25%以下是檢測不到的,因此HCV 藥物開發諮詢組織(HCV Drug Development Advisory Group; HCV DRAG)建議,對於臨床評估新的DAA 抗病毒藥物的抗藥性,需要至少80 個克隆定序(Clonal sequencing)以檢測很低頻率的突變,但該方法費時又費力。本研究試圖整合微流體PCR 與次世代定序分析(Next Generation Sequencing,NGS)雙平台,期待可快速並準確提供高覆蓋率的突變分析檢測,並應用於新初始治療患者選擇合適的DAA 抗病毒藥物,以及新的DAA 抗病毒藥物的臨床研究用於監測治療前、中後的病毒準種的變異序列分析。
本研究中我們分析了24 個C 型肝炎病人檢體,針對NS3 Protease domain 執行Pyrosequencing,實驗結果顯示每個檢體在NS3 Protease domain 的所有Amplicon得到1230 至3627 之間序列讀取片段(Reads),成功深度測序每一個相關抗藥性的variant,基因型1a 平均得到1237 倍的覆蓋率(Coverage depth),基因型1b 平均得到942 倍的覆蓋率(Coverage depth),報告顯示<1%低頻率的抗藥性突變存在,統計分析23 個感染HCV 的病患對NS3 抗藥性的突變率,在位點36 A/M 有16 個病患有突變發生突變率為69.5%;其他如 T54A/S 位點突變率為56.5%;V55A 突變率為78.2%;Q80K/R 突變率47.8%;R155K/T 突變率為39.1%;A156D/S/T/V突變率為43.4%;V170A 突變率為39.1%。並同時驗證此方法具有高精密性(High Precision)、高準確性(High Accuracy)及高特異性(High Specificity)。不僅可降低實驗成本與時間,更可以提供抗藥性variant 高準確度的深度測序分析。
According to World Health Organization (WHO), over 1.7 hundred million people so far have been infected with Hepatitis C virus, and these situations usually resulted in Hepatitis C. The morbidity and mortality of those having more than 20 years courses associated with Liver Cirrhosis and Hepatocellular Carcinoma. Thus, how to reduce the infection of Hepatitis C virus and eliminate viruses effectively in order to slow down the progression of Liver Cirrhosis and prevent Hepatocellular Carcinoma have been always the world-focusing medical issues. Currently Hepatitis C virus type 1 often treated by pegylated IFN-alpha combined with Ribavirin achieved a success rate of 40 ~ 50%, the drug treating effect, however, was largely limited due to long treatment time and serious side effects. In 2011, two NS3/4A protease inhibitors – Telaprevir and Boceprevir were approved for Hepatitis C virus type 1. These novel drugs, collectively called direct-acting antivirals DAA, contributed important development and progression to the treatment of chronic Hepatitis C virus. Even so, the high genetic diversity and mutation of HCV in cell culture would reduce DAA therapeutic sensitivity, and the research confirmed that resistance-associated variants usually occurred at a very low frequency (<5%). To solve this problem, Sanger sequencing is a common way to perform resistance mutation analysis in clinical trial, but such analysis couldn’t detect a resistance mutation frequency below 20 to 25% ascribing to its insufficient sensitivity. Therefore, HCV Drug Development Advisory Group (HCV DRAG) suggests at least 80 clonal sequencing is required to detect very low frequency mutation for new DAA antivirus drug resistance in the clinical evaluation, which was time-consuming and laborious. In order to provide rapidly and accurately high coverage mutation analysis detection, the integrates microfluidic PCR (Fluidigm Access Array System) and next-generation sequencing (NGS) analysis was developed. In this study 24 Hepatitis C patients are analysed by performing Roche GS Junior Pyrosequencing in NS3 Protease domain, and the results reveal the sequence reads between 1230 and 3627 of all Amplicon of NS3 Protease domain in each specimen are acquired and thus attain successful depth sequencing in every associated resistance variant. The genotype 1a obtain 1237 sequence coverage depth in average, whereas genotype 1b 942 sequence coverage depth. Our finding indicates the existence of resistance mutation at very low frequency. Prevalence of HCV NS3-resistant mutation among the 24 Hepatitis C patients was as follow : The V36A/M mutation resistant to Teraprevir and Boceprevir was detected in 16 of 23 cases (69.5%),Other mutations resistant to the NS3/4A protease-inhibitor were detected in 13 of 23 cases (56.5%) at T54A/S and 18 of 23 cases (78.2%) at V55A and 11 of 23 cases (47.8%) at Q80R/K and 8 of 23 cases (39.1%) at R155K/T and 10 of 23 cases (43.4%) at R156D/S/T/V and 9 of 23 cases (39.1%) at V170A ,and meanwhile verified this method having high precision, high accuracy and high specificity. Further applying for appropriate DAA antivirus drugs treatment and new DAA antivirus drugs screening need to be evaluated.
中文摘要………………………………………………………………………………………II
英文摘要……………………………………………………………………………………IV
一、 背景
1.1 C型肝炎病毒( Hepatitis C virus )……………………………………………………1
1.2 C型肝炎病毒之結構及其功能…………………………………………………………3
1.3 C型肝炎治療現況………………………………………………………………………6
1.4 新一類的抗病毒藥物……………………………………………………………………8
1.5 NS3/4A蛋白酶抑製劑Telaprevir與 Boceprevir……………………………10
1.6 病毒抗藥性檢測方法比較………………………………………………………………13
二、 研究特定目標 ………………………………………………………………………14
三、 材料與方法
3.1 實驗流程設計及原理…………………………………………………………15
3.2 檢體收集(Sample collection)…………………………………………………………17
3.3 C型肝炎病毒萃取(HCV Viral RNA extraction)…………………………18
3.4 反轉錄酶反應-Reverse Transcription Reaction (cDNA Synthesis)……………………19
3.5 NS3 protease domain聚合酶鏈鎖反應(Polymerase chain reaction, PCR)……………20
3.6 去氧核醣核酸純化(Amplicon-purification)…………………………………22
3.7 去氧核醣核酸濃度測定(DNA Concentration detection)………………………………23
3.8 微流體PCR - Fluidigm Access Array System…………………………24
3.9 乳化聚合酶鏈鎖反應(Emulsify Polymerase chain reaction, emPCR)………………36
3.10 次世代定序分析(GS Junior Sequencing)……………………………………39
四、 結果
4.1 NS3 protease domain PCR反應增幅結果…………………………………………47
4.2 Fluidigm Primer validation PCR反應增幅結果………………………49
4.3 Fluidigm Access Array System,PCR 結果………………………………50
4.4 GS Junior sequencer Sequencing 結果……………………………………52
五、 討論……………………………………………………………………………………66
六、 參考資料………………………………………………………………………………73
1. Moradpour, D., F. Penin, and C.M. Rice, Replication of hepatitis C virus. Nat Rev Microbiol, 2007. 5(6): p. 453-63.
2. Yu, M.L., et al., Changing prevalence of hepatitis C virus genotypes: molecular epidemiology and clinical implications in the hepatitis C virus hyperendemic areas and a tertiary referral center in Taiwan. J Med Virol, 2001. 65(1): p. 58-65.
3. Honda, M., et al., A phylogenetically conserved stem-loop structure at the 5'' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J Virol, 1999. 73(2): p. 1165-74.
4. Neumann, A.U., et al., Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science, 1998. 282(5386): p. 103-7.
5. Simmonds, P., Genetic diversity and evolution of hepatitis C virus--15 years on. J Gen Virol, 2004. 85(Pt 11): p. 3173-88.
6. Sy, T. and M.M. Jamal, Epidemiology of hepatitis C virus (HCV) infection. Int J Med Sci, 2006. 3(2): p. 41-6.
7. Lin, H.H., et al., Serotypes, genotypes and levels of hepatitis C viremia in pregnant women in Taiwan. J Formos Med Assoc, 1996. 95(6): p. 429-34.
8. Lee, C.M., et al., Hepatitis C virus genotypes in southern Taiwan: prevalence and clinical implications. Trans R Soc Trop Med Hyg, 2006. 100(8): p. 767-74.
9. Song, L.Y., et al., Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes. Biochim Biophys Acta, 2002. 1558(1): p. 1-13.
10. Pileri, P., et al., Binding of hepatitis C virus to CD81. Science, 1998. 282(5390): p. 938-41.
11. Kato, N., et al., Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus. Biochem Biophys Res Commun, 1992. 189(1): p. 119-27.
12. Pavlovic, D., et al., The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A, 2003. 100(10): p. 6104-8.
13. Dumoulin, F.L., et al., Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines. Virology, 2003. 305(2): p. 260-6.
14. Lorenz, I.C., et al., Structure of the catalytic domain of the hepatitis C virus NS2-3 protease. Nature, 2006. 442(7104): p. 831-5.
15. Foy, E., et al., Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science, 2003. 300(5622): p. 1145-8.
16. Meylan, E., et al., Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature, 2005. 437(7062): p. 1167-72.
17. De Francesco, R. and G. Migliaccio, Challenges and successes in developing new therapies for hepatitis C. Nature, 2005. 436(7053): p. 953-60.
18. Gretton, S.N., A.I. Taylor, and J. McLauchlan, Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol, 2005. 86(Pt 5): p. 1415-21.
19. Gale, M., Jr., et al., Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol, 1998. 18(9): p. 5208-18.
20. Di Marco, S., et al., Interdomain communication in hepatitis C virus polymerase abolished by small molecule inhibitors bound to a novel allosteric site. J Biol Chem, 2005. 280(33): p. 29765-70.
21. George, S.L., et al., Clinical, virologic, histologic, and biochemical outcomes after successful HCV therapy: a 5-year follow-up of 150 patients. Hepatology, 2009. 49(3): p. 729-38.
22. Hoofnagle, J.H. and L.B. Seeff, Peginterferon and ribavirin for chronic hepatitis C. N Engl J Med, 2006. 355(23): p. 2444-51.
23. Fried, M.W., et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, 2002. 347(13): p. 975-82.
24. Manns, M.P., et al., Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet, 2001. 358(9286): p. 958-65.
25. Thomas, D.L., et al., Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature, 2009. 461(7265): p. 798-801.
26. Ge, D., et al., Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature, 2009. 461(7262): p. 399-401.
27. Bacon, B.R., et al., Retreating chronic hepatitis C with daily interferon alfacon-1/ribavirin after nonresponse to pegylated interferon/ribavirin: DIRECT results. Hepatology, 2009. 49(6): p. 1838-46.
28. Poynard, T., et al., Peginterferon alfa-2b and ribavirin: effective in patients with hepatitis C who failed interferon alfa/ribavirin therapy. Gastroenterology, 2009. 136(5): p. 1618-28 e2.
29. Yee, H.S., et al., Update on the management and treatment of hepatitis C virus infection: recommendations from the Department of Veterans Affairs Hepatitis C Resource Center Program and the National Hepatitis C Program Office. Am J Gastroenterol, 2012. 107(5): p. 669-89; quiz 690.
30. Jacobson, I.M., et al., Telaprevir for previously untreated chronic hepatitis C virus infection. N Engl J Med, 2011. 364(25): p. 2405-16.
31. Kwo, P.Y., et al., Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial. Lancet, 2010. 376(9742): p. 705-16.
32. Poordad, F., et al., Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med, 2011. 364(13): p. 1195-206.
33. Rong, L., et al., Rapid emergence of protease inhibitor resistance in hepatitis C virus. Sci Transl Med, 2010. 2(30): p. 30ra32.
34. Sarrazin, C., et al., SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology, 2007. 132(4): p. 1270-8.
35. Kieffer, T.L., et al., Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology, 2007. 46(3): p. 631-9.
36. Pawlotsky, J.M., The results of Phase III clinical trials with telaprevir and boceprevir presented at the Liver Meeting 2010: a new standard of care for hepatitis C virus genotype 1 infection, but with issues still pending. Gastroenterology, 2011. 140(3): p. 746-54.
37. Sullivan, J.C., et al., Evolution of treatment-emergent resistant variants in telaprevir phase 3 clinical trials. Clin Infect Dis, 2013. 57(2): p. 221-9.
38. Vermehren, J., et al., Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa-2b. J Viral Hepat, 2012. 19(2): p. 120-7.
39. Susser, S., et al., Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir. J Clin Virol, 2011. 52(4): p. 321-7.
40. Vermehren, J. and C. Sarrazin, The role of resistance in HCV treatment. Best Pract Res Clin Gastroenterol, 2012. 26(4): p. 487-503.
41. Kwong, A.D., et al., Sequence and phenotypic analysis for resistance monitoring in hepatitis C virus drug development: recommendations from the HCV DRAG. Gastroenterology, 2011. 140(3): p. 755-60.
42. Beerenwinkel, N., et al., Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data. Front Microbiol, 2012. 3: p. 329.
43. Lauck, M., et al., Analysis of hepatitis C virus intrahost diversity across the coding region by ultradeep pyrosequencing. J Virol, 2012. 86(7): p. 3952-60.
44. Susser, S., et al., Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. Hepatology, 2009. 50(6): p. 1709-18.
45. Fonseca-Coronado, S., et al., Specific detection of naturally occurring hepatitis C virus mutants with resistance to telaprevir and boceprevir (protease inhibitors) among treatment-naive infected individuals. J Clin Microbiol, 2012. 50(2): p. 281-7.
46. Nasu, A., et al., Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing. PLoS One, 2011. 6(9): p. e24907.
47. Akuta, N., et al., Emergence of telaprevir-resistant variants detected by ultra-deep sequencing after triple therapy in patients infected with HCV genotype 1. J Med Virol, 2013. 85(6): p. 1028-36.
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