次世代定序資料重新分析以釐清 TSC1 及 TSC2 基因變異位點之致病性

Abstract

結節性硬化症為罕見的單一基因異常體染色體顯性遺傳疾病，目前已知是由 TSC1 或 TSC2 基因致病變異導致，造成mTOR 路徑的抑制作用喪失，使細胞過度活化而在多個器官中形成錯構瘤（hamartoma）。依錯構瘤生長的位置不同導致患者會出現多樣的臨床表徵。目前臨床上診斷結節性硬化症患者的方式，除了臨床表徵診斷外，也同時利用基因檢測方式採取患者的周邊血液檢體進行次世代定序。然而，仍有少數臨床診斷為結節性硬化症的患者其基因檢測卻無明顯確診之證據。可能的原因包含：檢測的技術方法、剪接位點變異在較深的內含子、或是患者存在低比例鑲嵌型致病變異等，都是潛在影響確診結節性硬化症的因素。隨著時間推移、基因檢測技術的發展或是對變異位點有新的註解，TSC1 或 TSC2 基因中新的致病性變異仍在不斷被發現，並導入臨床診斷。本研究將過去臨床確診結節性硬化症的患者之基因檢測重新再分析。利用比較LOVD和ClinVar國際資料庫與使用人工智慧平台GenDiseak協助分析，並依據ACMG guideline綜合判讀之策略，試圖為個案找出致病性變異。 本研究發現在基因檢測的判讀上，目前尚無法僅依賴單一個資料庫來做參考，仍需綜合參考其他資料庫以進行判讀；搭配資料庫重新分析55位TSC個案過去之基因檢測，發現有7位（13%）個案有提升一級判讀等級，顯示重新分析過去的基因檢測結果仍有其必要性。然而經由MPSA的分析方法，有機會透過高通量分析的方式，驗證variant是否會影響剪接，以協助提高變異位點被辨識的機會，針對發現的變異位點，設計minigene功能性測試系統來確認此剪接位變異位點，將繼續進行驗證。此外鑲嵌型患者血液及唾液兩種不同組織樣本中鑲嵌比例無顯著差異。 除了基因檢測的探討外，在臨床照護端製作疾病照護手冊，為TSC個案和家屬提供疾病相關之醫療與生活照護的參考資訊，讓患者能夠更瞭解與管理自己的疾病，進而減少醫病壓力並提高照護品質。本研究綜合科學基礎研究與臨床資訊蒐集分析，希望未來能對結節性硬化症的理解與治療提供新見解。

Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic disorder caused by mutations in the TSC1 or TSC2 genes. These mutations lead to a loss of inhibition in the mTOR pathway, resulting in overactivation of cells and the formation of hamartomas in multiple organs. Depending on the location of these hamartomas, patients can exhibit a wide range of clinical manifestations. Clinically, TSC is diagnosed not only through the identification of clinical symptoms but also through genetic testing using next-generation sequencing of peripheral blood samples. However, there are still a few patients clinically diagnosed with TSC who do not have clear evidence from genetic testing. Potential reasons for this include the technical methods used in testing, splicing site mutations in deep introns, or the presence of low-percentage mosaic pathogenic mutations, all of which can affect the diagnosis of TSC. Over time, as genetic testing techniques advance and new annotations of mutation sites are made, novel pathogenic variants in the TSC1 or TSC2 genes continue to be discovered and integrated into clinical diagnoses. This study aims to reanalyze the genetic testing results of patients previously clinically diagnosed with TSC. By comparing data from international databases LOVD and ClinVar and using the artificial intelligence platform GenDiseak for analysis, we seek to identify pathogenic variants according to the ACMG guidelines. This study found that in the interpretation of genetic testing, it is currently not feasible to rely on a single database alone; instead, a comprehensive reference to multiple databases is required. By reanalyzing past genetic tests of 55 TSC cases with updated databases, we found that 7 cases (13%) had an upgraded interpretation classification, highlighting the importance of reanalyzing previous genetic test results. However, through the MPSA analysis method, there is an opportunity to validate whether variants affect splicing via high-throughput analysis, thereby improving the chances of identifying variant sites. For the detected variant sites, a minigene functional test system will be designed to confirm these splicing variant sites and further validation will continue. Additionally, there is no significant difference in the mosaicism proportion between blood and saliva samples in mosaic individuals. In addition to exploring genetic testing, we have created a disease care manual for clinical care to provide TSC patients and their families with medical and lifestyle care information. This aims to help patients better understand and manage their condition, thereby reducing stress and improving the quality of care. This study integrates basic scientific research with the collection and analysis of clinical information, aiming to provide new insights into the understanding and treatment of tuberous sclerosis in the future.