結節硬化症之次世代定序基因檢測 - 強調局部病灶檢體定序及臨床研究資料庫

Abstract

結節硬化症（Tuberous sclerosis complex）是一種自體顯性遺傳，因TSC1或TSC2基因發生致病變異點而造成。能夠引起疾病的基因變異型態多樣，且有10-20%無法從以傳統分子檢驗診斷方法找到致病變異點。台大醫院結節硬化症基因研究案，以次世代定序方法檢驗110個結節硬化症之首位確診病患（proband）及其家族位於TSC1或TSC2基因變異點。共有96（87%）個家族於TSC1或TSC2基因找到致病變異點（pathogenic variant）或高度懷疑之致病變異點（likely pathogenic variant），14（13%）個家族並沒有找到致病變異點（no mutation identified）。在有找到致病變異點的 96 個家族中，57（59%）個家族屬於點突變（point mutation），28（29%）個家族為小片段插入、重複、或缺失（small indel），以及11（11%）個家族是大片斷缺失或重複（Large deletion/duplication）。十四（15%）個家族是TSC1基因變異造成的，而82（85%）個家族是TSC2基因變異造成。有5個家族首位確診患者（Proband）帶有鑲嵌型的致病變異點，另有2個帶有異型合子致病變異點之患病個案之其中一親代帶有鑲嵌型致病變異點，皆位於TSC2基因。另外，本研究亦於一家族發現一剪接位點變異位於TSC2, c.138+5G>T，其中2個患病之家族成員皆帶有此變異點，無患病者則無。 針對未能於正規次世代定序基因檢測直接找到致病變異點之患者，後續亦使用局部病灶來檢測可能之變異點，共有10個病灶檢體，從3個沒有找到變異點的個案及2個已發現致病點的個案（陽性對照）取得。經由此方式在一個原本沒有找到變異點個案的局部病灶檢體中檢測出一個位於TSC2基因的非常低對偶基因比率（Allele percentage）（0.6%）的變異點，推測此變異點可能為一個Second Hit變異點，並暗示可能此名個案的Germline變異也同樣位於TSC2基因。 根據以上的結果，針對少數基因檢測無法找出致病變異點之臨床確診結節硬化症患者，推測有以下幾個原因，提供未來研究之用：（1）落在TSC1或TSC2基因但非常低對偶基因比率的鑲嵌型變異點；（2）變異點發生在TSC1或TSC2基因非靠近外顯子的剪接位點、啟動子（Promoter）或強化子（enhancer）區域；（3）技術問題而導致沒有成功找到變異點；（4）發生在另外第三個結節硬化症目前未知的致病基因。 另外，為管理結節硬化基因研究所收集的臨床資料及基因檢測結果，設立了線上臨床研究資料庫 – 臨床研究資訊系統（Clinical Study Information System, CSIS），根據結節硬化症基因研究設計了客製化之資料建置表單，且此系統是以授權研究人員之個人帳號密碼來做為保護資料。所有收集資料包含了病人之臨床資料及基因檢測結果目前都已經分別輸入系統並妥善管理。

Tuberous sclerosis complex (TSC) is an autosomal dominant disease due to causative variants at either TSC1 or TSC2. The disease-causating mutations are highly variable and 10–20% of TSC individuals have no causative variants identified after thorough conventional molecular diagnostic assessment. In this study, 110 definite TSC probands were studied using next-generation sequencing (NGS) to search for pathogenic variants at TSC1 and TSC2 genes. “Pathogenic” or “likely pathogenic” variants were identified in 96 (87%) probands; however, 14 (13%) probands could not be assigned causative variants. Among the 96 identified causative variants, 58 (60%) were single nucleotide substitutions, 28 (29%) were small indels, and 10 (10%) were large deletions/duplications. Fourteen (15%) probands were found to have variants at TSC1 and 82 (85%) were found to have variants at TSC2. Seven individuals were mosaic regarding to their causative variants, including 5 probands and parents of other two non-mosaic probands. A splicing site variant “TSC2, c.138+5G>T” was identified in one TSC family co-segregated in 2 affected family members. With the intent to investigate the genetic etiology in those TSC patients without identifiable causative variants at TSC1 or TSC2, we conducted sequencing using focal lesion DNA. A total of 10 focal lesions obtained from 3 no-mutation-identified probands and 2 probands with one germline mutation (positive control) were studied. One pathogenic mutation at TSC2 with 0.6% of allele percentage was detected in the focal lesion DNA of one no-mutation-identified proband. This variant was considered as the second-hit mutation and indicated that the germline mutation in this patient is located in TSC2 gene as well. However, no potential second-hit mutation was identified in the positive control samples. These findings concluded and suggested that there were several possible reasons for no-mutation-identified status in TSC patients: 1) mosaicism with very low allele percentage for variants at TSC1 or TSC2; 2) variants in introns that affect splicing, or in promoter and enhancer regions of TSC1 and TSC2; 3) variants detection failure due to technical issues; 4) variants at a yet-unknown third TSC gene. Furthermore, to manage the clinical research data collected from TSC Genetic Research, a user-friendly online clinical research database - Clinical Study Information System (CSIS) was established. The study-specified data entry forms were designed for TSC Genetic Research and the system was under security protection by personal ID and password for researchers who were delegated to this study. All the available data including the clinical information from subjects and genetic test results were entered and managed by our study team accordingly.