建立以次世代定序為方法之結節硬化症基因檢測

Abstract

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.