Establishment of next-generation sequencing (NGS)-based genetic testing for tuberous sclerosis complex (TSC)

結節硬化症(Tuberous Sclerosis Complex)為體染色體顯性遺傳疾病，約有1/3患者是家族性遺傳，而2/3的則是屬於散發型(sporadic)；據研究估計其發生率最高約為1/6,000，男女比例相等。由於TSC1和TSC2基因突變導致患者在身體各部位生成錯構瘤(hamartomas)，使得患者會出現多樣化的症狀，在外表可觀察到臉部皮膚血管纖維瘤或額頭斑塊、指甲(邊)纖維瘤、脫色斑、鯊魚皮斑、牙齦纖維瘤症狀，其餘則是內部器官腫瘤或病變生成，亦合併癲癇、智能不足、自閉症等症狀。 TSC1及TSC2基因的轉譯蛋白分別為Hamartin與Tuberin，此蛋白在mTOR pathway中形成複合物，使Rheb不活化mTORC1，進而阻斷蛋白質轉譯及細胞週期的進行，假若TSC1或TSC2基因發生突變，則Hamartin及Tuberin無法正常結合，導致無法調控細胞增生機制，因此在全身各個器官長腫瘤。 本次研究利用次世代定序做為基因檢測方式，在結節硬化症確診的94個家族中，有78個家族找到基因致病點，16個家族未能找到。而在78個家族中有50個家族屬於點突變、20個家族是小片段缺失或插入，以及8個家族是大片段缺失或重複；在性別比例上患有結節硬化症男性為(42/94)和女性(52/94)，總比例約1：1。 次世代定序的檢測方式，不論是單突變點或小片段缺失或插入皆與Sanger定序結果一致，且較Sanger定序可更敏銳的偵測到鑲嵌體的基因變異點，即便只有6%也可偵測到。大片段缺失或插入在IGV圖上可清楚地看到斷點所在位置。統計利用次世代定序做為大片段插入或缺失檢出率為10%，而TSC1或TSC2基因總檢出率有81%(76/94)。比較次世代定序與傳統的檢測方式，在讀取的序列長度、試劑的用量、所花費的時間及金錢皆優於傳統的檢測方式，因此次世代定序應用於結節硬化症的基因檢測是一項利器。 Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder. One third of patients are familial cases and two thirds are sporadic cases caused by de novo mutations. The disease is estimated to occur in 1:6000 live births worldwide with nearly equal distribution between females and males. TSC is a genetic multisystem disorder characterized by widespread hamartomas in several organs, including the brain, heart, skin, eyes, kidney, lung, and liver, etc. The clinical manifestations are highly variable, and might include epilepsy, mental retardation, behavioral problems, skin lesions and renal problems, which often cause harm to patients physically and psychologically. The only two known disease-causing genes are TSC1 and TSC2, encoding hamartin and tuberin respectively. The hamartin-tuberin complex inhibits the mammalian target of rapamycin (mTOR) pathway, which controls cell growth, cell proliferation and several other important functions. In this current study, we applied the next-generation sequencing (NGS) technology for the TSC1 and TSC2 genes. Among the 94 patients with definite TSC diagnosis, we could identify the causative genetic variants for 78 cases. There were 16 cases with no mutation identified. In 78 cases, 50 cases had point mutation, 20 cases had small deletion or insertion (indel) and 8 cases had large deletion or duplication. Our overall detection rate using NGS were 81% (76/94). Among the detected disease-causing variants, large deletion or insertion accounted for approximately 10% (8/76). NGS and Sanger sequencing are equally good at detecting point mutations and small indels. Besides, NGS could be more sensitive to detect mosaic variant. Furthermore, it is possible to clearly detect the breakpoints of large deletion or insertion through Integrative Genomics Viewer (IGV) using NGS reads. In conclusion, NGS can replace traditional genetic testing methods, and serve as cost-effective, sensitive and reliable genetic testing platform for TSC.口試委員會審定書........................................I 中文摘要 ................................................II Abstract................................................III 目 錄 ................................................V 圖目錄..................................................VII 表目錄..................................................VIII 第一章 研究背景與動機.................................. 1 第一節 結節硬化症之疾病介紹............................ 1 第二節 結節硬化症之歷史 ................................2 第三節 結節硬化症之臨床表徵............................4 第一項 皮膚表徵........................................4 第二項 眼晴表徵 ........................................4 第三項 腦部表徵 ........................................5 第四項 心臟表徵 ........................................6 第五項 腎臟表徵 ........................................6 第六項 肺部表徵 ........................................7 第七項 口腔表徵 ........................................7 第八項 其他表徵 ........................................7 第四節 結節硬化症之檢查及診斷..........................8 第一項 結節硬化症之檢查 ................................8 第二項 結節硬化症之診斷 ................................8 第五節 結節硬化症之分子遺傳學研究及基因檢測............11 第一項 分子遺傳學研究..................................11 第二項 基因檢測 ........................................12 第六節 結節硬化症之治療 ................................16 第七節 遺傳諮詢 ........................................17 第八節 研究目的 ........................................18 第二章 研究方法 ........................................19 第一節 檢體來源 ........................................19 第二節 檢體收集 ........................................19 第三節 研究方法與步驟..................................19 第一項 Genomic DNA的萃取與定量分析.....................19 第二項 次世代基因定序分析..............................20 第三項 MLPA分析 ........................................22 第四項 聚合酶連鎖反應(Polymerase Chain Reaction)與Sanger定序分析..................................................23 第四節 遺傳諮詢 ........................................25 第三章 研究結果 ........................................26 第一節 次世代定序結果統計與分析 ........................26 第二節 利用次世代定序找出結節硬化症各種形式基因變異點之例證......................................................28 第一項 錯義突變(Missense mutation).....................28 第二項 無義突變(Nonsense mutation)..................... 30 第三項 移碼突變(Frame-shift mutation)..................31 第四項 鑲嵌體(Mosaicism)............................... 32 第五項 大片段缺失(Large deletion)...................... 36 第六項 大片段的串聯重複(Tandem duplication)............42 第三節 遺傳諮詢結果....................................45 第四章 討論............................................49 第五章 結論............................................51 參考文獻 ................................................52 附錄一..................................................6