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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳沛隆(Pei-Lung Chen) | |
dc.contributor.author | Chao-Hsuan Li | en |
dc.contributor.author | 李昭萱 | zh_TW |
dc.date.accessioned | 2021-05-12T09:33:18Z | - |
dc.date.available | 2020-10-03 | |
dc.date.available | 2021-05-12T09:33:18Z | - |
dc.date.copyright | 2018-10-03 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-01 | |
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An Pediatr (Barc), 2013. 79(1): p. 42-5. 32. Smulders, Y.M., et al., Large deletion causing the TSC2-PKD1 contiguous gene syndrome without infantile polycystic disease. J Med Genet, 2003. 40(2): p. E17. 33. Schmidt, L.S. and W.M. Linehan, FLCN: The causative gene for Birt-Hogg-Dube syndrome. Gene, 2018. 640: p. 28-42. 34. Asnaghi, L., et al., mTOR: a protein kinase switching between life and death. Pharmacol Res, 2004. 50(6): p. 545-9. 35. Kwiatkowski, D.J., TSC1, TSC2, TSC3? Or mosaicism? Eur J Hum Genet, 2005. 13(6): p. 695-6. 36. Bentley, D.R., et al., Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry. Nature, 2008. 456(7218): p. 53-59. 37. R., L.H.a.D., Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics, Epub. [PMID: 20080505], 2010. 38. Richards, S., et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 2015. 17(5): p. 405-24. 39. Sanger, F., S. Nicklen, and A.R. Coulson, DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A, 1977. 74(12): p. 5463-7. 40. Paul A. Harris, R.T., Robert Thielke, Jonathon Payne, Nathaniel Gonzalez, Jose G. Conde, Research electronic data capture (REDCap) – A metadata-driven methodology and workflow process for providing translational research informatics support, J, REDCap (Research Electronic Data Capture). Biomed Inform. 2009 Apr;42(2):377-81., 2009. 41. Girdea, M., et al., PhenoTips: patient phenotyping software for clinical and research use. Hum Mutat, 2013. 34(8): p. 1057-65. 42. Tyburczy, M.E., et al., Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex. Human Molecular Genetics, 2014. 23(8): p. 2023-2029. 43. Li, S., et al., Mesenchymal–epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(9): p. 3539-3544. 44. Wang, S.-J., Topical Rapamycin and Calcitriol Therapy Trial and genetic testing for Cutaneous Lesions in Tuberous Sclerosis Complex (TSC) Patients. 2017. 45. Dabora, S.L., et al., Mutational Analysis in a Cohort of 224 Tuberous Sclerosis Patients Indicates Increased Severity of TSC2, Compared with TSC1, Disease in Multiple Organs. The American Journal of Human Genetics, 2001. 68(1): p. 64-80. 46. Sue Povey , R.E., Tuberous sclerosis database-Tuberous sclerosis 2 (TSC2). Leiden University Medical Center, 2004-2014 47. Jones, A.C., et al., Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis. Hum Mol Genet, 1997. 6(12): p. 2155-61. 48. Sue Povey , R.E., Tuberous sclerosis database-Tuberous sclerosis 1 (TSC1). Leiden University Medical Center, 2004-2014 49. Hung, C.C., et al., Molecular and clinical analyses of 84 patients with tuberous sclerosis complex. BMC Med Genet, 2006. 7: p. 72. 50. Napolioni, V. and P. Curatolo, Genetics and Molecular Biology of Tuberous Sclerosis Complex. Current Genomics, 2008. 9(7): p. 475-487. 51. Ho, D.W.H., et al., TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication. Gut, 2017. 66(8): p. 1496-1506. 52. Huynh, H., et al., Loss of Tuberous Sclerosis Complex 2 (TSC2) Is Frequent in Hepatocellular Carcinoma and Predicts Response to mTORC1 Inhibitor Everolimus. Molecular cancer therapeutics, 2015. 14(5): p. 1224-1235. 53. Cho, J., et al., Loss of Tuberous Sclerosis Complex 2 (TSC2) as a Predictive Biomarker of Response to mTOR Inhibitor Treatment in Patients with Hepatocellular Carcinoma. Translational Oncology, 2016. 9(5): p. 466-471. 54. Habib, S.L., et al., Is mTOR Inhibitor Good Enough for Treatment All Tumors in TSC Patients? Journal of Cancer, 2016. 7(12): p. 1621-1631. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/handle/123456789/1147 | - |
dc.description.abstract | 結節硬化症是一種罕見的體染色體顯性遺傳疾病,因TSC1或TSC2基因生殖細胞突變,導致全身各處器官和組織產生錯構瘤。目前有10~25%臨床上確診為結節硬化症的病人,取其周邊血液做次世代定序基因檢測,找不到致病位點。其中有部分可能是TSC1或TSC2低比例鑲嵌型,或者致病位點隱藏在較深會影響剪接的內涵子區域,於檢測時被忽略。
本研究收入了曾於臺大醫院結節硬化症整合門診看診的病人及其家屬共508位,含222個家族。根據臨床表徵,其中有168個家族先證者 (proband) 被確診為結節硬化症;10個家族臨床懷疑為結節硬化症但尚不足以確診;44個家族原本被懷疑是結節硬化症,經過轉介來臺大醫院評估後認為非結節硬化症。將這些個案的家族史、臨床表徵、基因檢測、各科追蹤檢查紀錄等資訊,整理並在臺大安全網域下建構線上TSC REDCap資料庫,呈現臺灣本土結節硬化症疾病的資料。另外,進行兩代直系親屬的基因檢測,確認一同時患有結節硬化症和多囊腎的病人身上兩個致病變異點在染色體上的相位關係;並從過去周邊血液基因檢測未找到生殖細胞突變的3位病人中,取其皮膚病灶檢體進行基因檢測,偵測到低比例的生殖細胞TSC1或TSC2突變鑲嵌型狀況;探討一患有肝細胞癌 (HCC) 的B型肝炎帶原結節硬化症病人,其肝臟腫瘤切片的基因狀況;設計minigene功能性測試系統來確認未確定致病性的剪接位變異點其轉錄出的mRNA是否有異。 在本研究臨床確診為結節硬化症的家族中,次世代定序基因檢測確診檢出率約88%,另3%為未能確定致病性的變異點 (VUS),剩下9%沒有發現任何懷疑的變異點 (NMI)。其中有20%為TSC1基因變異,80%為TSC2基因變異;30%為家族遺傳而來,70%為新發生的變異。對於同時帶有結節硬化症和多囊腎兩個致病變異點的病人,可以透過進行直系親屬基因檢測來釐清兩變異點的相位關係;並利用皮膚病灶檢體提高低比例鑲嵌型患者,生殖細胞突變被檢測出來的比率;觀察到一位帶有TSC2致病生殖細胞變異且同時為B型肝炎帶原的結節硬化症病人,發展出肝細胞癌 (HCC) 時,則其肝腫瘤會較大且侵略性更強。針對肝腫瘤切片做基因檢測,發現除患者原本在TSC2基因上的生殖細胞錯義突變之外,另發生一個體細胞突變:約2Mb包含TSC2的大片段缺失,使此區域成為異合性丟失的狀況 (LOH)。免疫治療搭配everolimus治療對此病人肝腫瘤擴張有控制效果。最後,針對未確定致病性的剪接位變異點設計的minigene系統將繼續進行實驗確認。 | zh_TW |
dc.description.abstract | Tuberous Sclerosis Complex (TSC) is a rare autosomal dominant disease due to germline disease-causing variants in either TSC1 or TSC2 gene, and may develop multisystem harmartomas. However, 10%-25% of TSC patients remain genetically undiagnosed after conventional genetic testing. Part of them may carry variant(s) with low percentage mosaicism or have variants affecting splicing in deep introns, which would be missed out during interpretation of the NGS data.
We enrolled 508 subjects from 222 families from the TSC Integrated Clinic at National Taiwan University Hospital (NTUH). Among them, 168 probands were TSC-confirmed, 10 probands were TSC-suspected, and 44 probands eventually did not meet the clinical criteria of TSC. We established an online REDCap database for our TSC cohort, and collected and uploaded family histories, phenotypes, genotypes, and the follow-up examinations of these subjects into the database. Additionally, we took advantage of the next-generation sequencing (NGS)-based genetic testing to solve several critical issues. Notable examples included, but not limited to, solving the phasing issue of TSC2 and PKD1 variants in a family, detecting possible low percentage mosaic germline mutations in skin lesions of 3 TSC patients without identifiable variants in peripheral blood samples previously, and analyzing the possible second hit of from the hepatoma biopsy samples of a TSC patient with chronic hepatitis B and newly diagnosed hepatocellular carcinoma (HCC). Finally, we designed minigene experiments trying to confirm the pathogenicity of splicing variants of unknown significance (VUS). In our study, for the 168 probands with clinically definite TSC, the detection rate of pathogenic and likely pathogenic germline variants in TSC1 or TSC2 gene was 88%; additional 3% of TSC patients had VUS, and 9% remained no mutation identified (NMI). Among TSC probands with genetic diagnosis, 20% fell in TSC1 and 80% fell in TSC2 gene. Thirty percent of the variants were inherited, while the other 70% were de novo mutations. For the patient with two variants in the TSC2 and PKD1 genes, we confirmed that the two variants were on the same chromosome 16 haplotype, based on the co-segregation observed in his son. And, we could increase the detection rate of low percentage mosaic germline mutation by analyzing the NGS data of their skin lesion samples compared with the data of peripheral blood samples. For the TSC patient with chronic hepatitis B as well as newly diagnosed HCC, we found a pathogenic TSC2 germline missense variant and a TSC2 LOH somatic mutation at least 2Mb in size in the HCC biopsy. The HCC initially grew fast and developed aggressively. Treatment combined with immunotherapy and everolimus, which is a mTOR inhibitor, seemed to be initially effective for disease control. Finally, the minigene experiments of VUS splicing variants are still underway. | en |
dc.description.provenance | Made available in DSpace on 2021-05-12T09:33:18Z (GMT). No. of bitstreams: 1 ntu-107-P05448003-1.pdf: 7687680 bytes, checksum: 6628e0837d8d09d5d530d8ec3bbea82a (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 論文委員會審定書---------------------------------------------------------------------------i
致謝---------------------------------------------------------------------------------------------ii 中文摘要---------------------------------------------------------------------------------------iii-iv Abstract--------------------------------------------------------------------------------------- v-vii 第一章 研究背景與動機 1.1結節硬化症的疾病介紹 ----------------------------------------------------------------1 1.2結節硬化症的歷史演進 ----------------------------------------------------------------1 1.3結節硬化症的致病機轉 ----------------------------------------------------------------1-2 1.4結節硬化症的臨床表徵 ----------------------------------------------------------------2-3 1.5結節硬化症的診斷標準 1.5.1臨床診斷標準 ---------------------------------------------------------------------4 1.5.2基因診斷標準 ---------------------------------------------------------------------5 1.6結節硬化症資料庫 1.6.1國際上現有的結節硬化症LOVD資料庫-----------------------------------5-6 1.6.2臺灣本土結節硬化症資料 ----------------------------------------------------6 1.7結節硬化症在基因檢測診斷上遇到的困難 1.7.1找不到懷疑的致病變異點------------------------------------------------------6-7 1.7.2找到無法判讀其致病性的變異點 (VUS) -----------------------------------7 1.8 TSC1和TSC2基因變異點的功能性評估--------------------------------------------8 1.9遺傳諮詢 (genetic counseling) --------------------------------------------------------8-9 1.10研究動機 1.10.1臺大醫院TSC REDCap整合型資料庫的建立-----------------------------9 1.10.2結節硬化症患者基因檢測上難解的問題-----------------------------------10 第二章 研究方法 2.1研究對象 2.1.1 受試者來源與收案標準----------------------------------------------------11 2.1.2 同意書簽署-------------------------------------------------------------------12 2.1.3 檢體----------------------------------------------------------------------------13 2.2研究方法 2.2.1 DNA萃取---------------------------------------------------------------------13-14 2.2.2偵測DNA品質--------------------------------------------------------------14-15 2.2.3 TSC panel---------------------------------------------------------------------15 2.2.4次世代定序 (next-generation sequencing, NGS) ----------------------16-17 2.2.5變異點致病性的判讀-------------------------------------------------------17-18 2.2.6聚合酶連鎖反應 (PCR) 及傳統定序 (Sanger) -----------------------18 2.2.7 minigene assay----------------------------------------------------------------18-19 2.2.8 REDCap資料庫--------------------------------------------------------------19 2.2.9 PhenoTips家族譜圖繪製---------------------------------------------------19 第三章 研究結果 3.1 REDCap資料庫 3.1.1 REDCap表單及項目欄位--------------------------------------------------20-27 3.1.2臺大醫院結節硬化症整合門診病人的基因檢測結果統計-----------28-30 3-2結節硬化症患者基因檢測上難解的問題 3.2.1確認同一患者身上兩個致病變異點在染色體上的相位關係--------31-33 3.2.2提升低比例TSC1或TSC2鑲嵌型患者的基因檢測檢出率---------34-36 3.2.3檢測HBV+HCC的結節硬化症病人的second hit --------------------37-38 3.2.4利用minigene來確認基因剪接 (splicing) 狀況----------------------39-50 3.3遺傳諮詢 3.3.1案例一 (TSC201)------------------------------------------------------------51 3.3.2案例二 (TSC419)------------------------------------------------------------52-53 3.3.3案例三 (TSC418)------------------------------------------------------------54-55 3.3.4案例四 (TTSC00480)-------------------------------------------------------55-56 第四章 討論 4.1臺灣的結節硬化症流行病學資料----------------------------------------------------57 4.2基因檢測檢出率-------------------------------------------------------------------------57 4.3 HBV+HCC-------------------------------------------------------------------------------58 4.4遺傳諮詢----------------------------------------------------------------------------------58 第五章 結論 -------------------------------------------------------------------------------59 參考文獻---------------------------------------------------------------------------------------60-63 附錄 附錄一、TSC PCR primers (1) TSC1 PCR primers------------------------------------------------------------------64-65 (2) TSC2, PKD1 PCR primers---------------------------------------------------------65-67 (3) minigene PCR primers for insert DNA-------------------------------------------68 (4) PCR programs-----------------------------------------------------------------------68-69 (5) minigene primers for Sanger sequencing----------------------------------------70-71 附錄二、minigene assay 實驗流程----------------------------------------------------71-74 附錄三、TSC panel-----------------------------------------------------------------------75-84 附錄四、結節硬化症臨床確診家族之proband基因檢測結果------------------85-92 | |
dc.language.iso | zh-TW | |
dc.title | 結節硬化症:REDCap資料庫的建立以及次世代定序基因檢測 | zh_TW |
dc.title | Tuberous Sclerosis Complex (TSC): REDCap Database Establishment and Next-generation Sequencing (NGS)-based Genetic Testing | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 謝豐舟(Fong-Jou Hsieh),楊偉勛(Wei-Shiung Yang) | |
dc.subject.keyword | 結節硬化症,線上資料庫,致病性不明的剪接位變異,低比例鑲嵌型,兩致病變異點的相位關係,B肝帶原肝細胞癌病灶,次世代定序, | zh_TW |
dc.subject.keyword | Tuberous Sclerosis Complex (TSC),online database,splicing variants of unknown significance (VUS),low percentage mosaicism,haplotyping,HBV+HCC biopsy,next-generation sequencing (NGS), | en |
dc.relation.page | 92 | |
dc.identifier.doi | 10.6342/NTU201802113 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2018-08-01 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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