結節硬化症：次世代定序基因檢測以及整合性臨床照護

Abstract

結節硬化症(Tuberous Sclerosis Complex; TSC)是一種因TSC1基因或TSC2基因發生變異，導致良性錯構瘤(hamartoma)的不正常增生，系統性影響全身多重器官的體染色體顯性遺傳疾病。由於結節硬化症是造成患者全面性的症狀發生，使得其衍伸而成的臨床照護問題亦日漸增長。因此，本研究針對結節硬化症患者，深入探討次世代定序(Next-Generation Sequencing; NGS)之基因診斷及整合性臨床照護問題，期許提升醫療照護品質與減輕患者及其家屬的長期苦痛。 本研究透過國立臺灣大學醫學院附設醫院之結節硬化症整合型門診，共納入532位受試者，總計為239個家族。根據結節硬化症整合門診各主治醫師評估及基因檢測結果，在239個家族中確認了180個家族中的先證者(proband)為結節硬化症；10個家族疑似為結節硬化症，但仍無法確診；49個家族則確認非結節硬化症患者。並針對同意加入研究案之患者進行臨床資料的收集、登錄、保存、彙整至結節硬化症整合型資料庫(Integrated Database of Tuberous Sclerosis Complex)，同時本研究持續協助疑似結節硬化症患者，利用次世代定序進行基因確診；並透過藥物基因體檢測(pharmacogenomics testing)，探討mTOR抑制劑在CYP3A4基因、ABCB1基因發生變異與患者藥物療效間進行探討；針對具有強烈遺傳性癌症家族史之結節硬化症家族，嘗試以全基因體定序(Whole Genome Sequencing; WGS)進行基因診斷；最後，利用案例報告的方式，深入探討結節硬化症女性患者腎血管平滑肌脂肪瘤(renal angiomyolipoma)之研究。 本研究目前透過次世代定序基因檢測之確診檢出率為89%。其中未檢出者有3%其致病性臨床意義不明(Variation of Unknown Significance; VUS)、；另8%則完全未發現懷疑變異點。而確診患者中有21%是TSC1基因發生變異；79%則是TSC2基因發生變異。以及30%是來自父母之遺傳，70%為自體新發生(de novo)的變異。最終根據上述統計，確認臺灣的結節硬化症流行病學現況，與各文獻所提出之比例未有明顯差異。 關於整合性臨床照護問題，本研究首次針對mTOR抑制劑透過藥物基因體檢測，進行CYP3A4基因、ABCB1基因變異與患者藥物療效間之探討。目前共納入64位患者，並予收集受試者之基本資料、血中藥物濃度、藥物劑量、其它用藥及症狀變化等以評估受試者之治療效果與用藥安全。最終協助兩位血中藥物濃度無法達到建議濃度範圍之患者，判定主要是因藥物交互作用所導致；而結節硬化症合併早發性(early-onsent)、多重原發性癌症(multiple primary cancers)之患者及其家族，本研究藉由周邊血液的全基因體定序，並透過共分離(co-segregation)分析方式偵測出具有致病性(pathogenic)之TRIM45基因剪接變異點。目前文獻雖提出TRIM45基因具有腫瘤抑制(tumor suppressor)功能，但相關研究仍知之甚少。因此期望藉由取得患者之病理組織，以次世代定序偵測第二致病變異(second hit)，進一步確認結節硬化症與癌症之相關性。而若證實TSC基因帶有變異時，同時帶有癌症基因之變異，將加速腫瘤的增生促使癌症病程的發展，未來便可廣泛運用於具有癌症家族史或高危險群患者，協助定期追蹤及規劃治療策略；最後透過案例報告及與多專科團隊的討論、評估，為患有腎血管平滑肌脂肪瘤之女性患者，在妊娠前與妊娠期間、產後，建立一主要因應機制。並利用此次的案例報告經驗，使結節硬化症對於腎血管平滑肌脂肪瘤的醫療照護及遺傳諮詢更深入探討。

Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by pathogenic variants of the TSC1 or TSC2 genes, which could result in hamartomas or abnormal hyperplasia in multiple organs. It shows autosomal dominant inheritance. Considering the widespread manifestations in TSC patients, medical care for the patients becomes quite challenging. Therefore, this study aimed to provide genetic diagnosis taking advantage of next-generation sequencing (NGS), and also integrated clinical care. Through the Integrated TSC Clinic of National Taiwan University Hospital, we recruited 532 subjects in 239 families into this project. After thorough evaluations based on the diagnostic crietria of TSC, 180 among the 239 probands were confirmed to be TSC patients, 10 were suspicious TSC cases, and 49 individuals were excluded as TSC patients. With the informed consents from the participants, we collected, registered, recorded, and consolidated their clinical data into the Integrated Database of Tuberous Sclerosis Complex, using a REDCap (Research Electronic Data Capture) system. For the suspected TSC patients, NGS could sometimes provide critical information for the correct diagnosis. For several patients showing unstable blood everolimus concentration, we performed a pharmacogenomics study (including the CYP3A4 and ABCB1 genes). For a TSC family carrying multiple cancers in multiple individuals, we conducted whole genome sequencing (WGS) of 4 individuals, attempting to identify the cancer-causing gene(s). We also investigated the clinical course of renal angiomyolipoma in women during pregnancy and after delivery. Of all the TSC probands receiving our NGS-based genetic testing, 89% were found to have pathogenic or likely pathogenic variants in TSC1 or TSC2, 3% with variation of unknown significance (VUS), and 8% were negative. Among the diagnosed patients, 21% of them were caused by TSC1 while the rest 79% were caused by TSC2. The disease-causing variants were inherited in 30% of probands, and were de novo mutations in 70% of the probands. According to the aforementioned statistics, this research has confirmed that the current epidemiological condition of TSC in Taiwan does not have a significant difference compared to the ratios presented in previous literature. For integrated clinic care, we investigated several aspects. First, we performed pharmacogenomic testing related to the mTOR inhibitor, everolimus, focusing on genetic variations of CYP3A4 and ABCB1 genes. Currently, there were 64 patients involved in this study with the permission to collect personal information, therapeutic drug monitoring, medicine dosage, other medication, symptom changes, and related data for evaluating the therapeutic effect and medication-use safety of the subjects. Second, for a TSC family with early-onsent and multiple primary cancers, we performed WGS in 4 individuals. After thorough analysis, we identified a splicing variation of the TRIM45 gene, which co-segregated in affected individuals. From the literature review, a handful of studies suggested that TRIM45 gene might possess a function of tumor suppressor; however, the data were not conclusive. We will continue working on the issue to clarify if TRIM45 indeed is the causing gene of those cancers in this family. This work might help to confirm TRIM45 as a genuine novel tumor suppressor gene. Third, this study presented a case series of female renal angiomyolipoma patients before, during, and after pregnancy. Through the observation, we hoped to develop an in-depth discussion about the healthcare and genetic counseling in female renal angiomyolipoma patients with TSC.