探討結節性硬化症與淋巴血管平滑肌增生症之基因型與表型特徵

Abstract

結節性硬化症（Tuberous sclerosis complex , TSC）是一種罕見的自體顯性遺傳性疾病，平均每6,000到10,000個活產新生兒當中，就有一人罹病。TSC主要因TSC1以及TSC2兩個基因發生變異所致，兩個基因負責控制胞內的mTOR（mammalian target of rapamycin）路徑，一旦發生基因變異，此路徑將不正常活化，導致細胞異常增生，在各個器官當中形成錯構瘤（hamartoma）。 儘管臨床診斷出TSC的表徵，患者經過基因檢驗可確認致病原因，但仍有少數患者無法以傳統基因檢驗的方式找到相關變異，可能原因包含基因變異發生在深度內含子（intron）或是患者本身存在低比例染色體鑲嵌現象（low-level mosaicism）等，此時用聚合酶連鎖反應（polymerase chain reaction, PCR）搭配次世代定序（next generation sequencing, NGS）的分子生物技術，方能做進一步確認。 肺部的淋巴血管平滑肌增生症（lymphangioleiomyomatosis, LAM）在部分的TSC患者當中終生伴隨，造成患者呼吸困難、胸痛、肋膜積水以及乳糜胸等。然而，患有LAM的患者當中，依照基因變異的機制又可以分為偶發性淋巴血管平滑肌肉增生症（sporadic LAM, S-LAM）以及結節性硬化症相關淋巴血管平滑肌肉增生症（TSC-associated LAM, TSC-LAM），因此欲觀察在國立臺灣大學醫學院附設醫院之LAM患者當中，TSC-LAM之比例。 臺大結節硬化症整合門診迄今已有10年以上的歷史，累積近200人的臨床數據，本研究將收集LAM患者，確認是否存在低比例染色體鑲嵌現象，並進一步確認其是否由TSC基因變異（TSC1或TSC2）所致。 本研究收案胸腔科門診具LAM的病人共69位，並收集血液以及唾液檢體，利用實驗室建立的TSC次世代定序基因檢測套組（TSC next-generation sequencing panel）進行次世代定序基因檢測（next-generation sequencing, NGS），並將結果使用Integrative Genomics Viewer (IGV) 軟體來輔助初步判讀，再經由Mutect2、Gendiseak (GDK)、SpliceAI等生資工具來進行更進一步分析患者的單一核甘酸變異（single nucleotide variants, SNVs）、染色體結構變異（structure variants, SVs）以及拷貝數變異（copy number variants, CNVs），最後根據美國醫學遺傳學暨基因體學學會（American College of Medical Genetics and Genomics, ACMG）判讀之方法，試圖為個案找出致病性變異。 我們對69位具有LAM的個案進行血液以及唾液的次世代基因定序，其中包含TSC1、TSC2、FLCN三個基因，結果顯示3位FLCN的致病性變異（pathogenic）、2位TSC2的高度懷疑致病變異（likely pathogenic）、2位TSC1和1位TSC2的臨床意義尚不明確（variant of unknown significance, VUS）之變異，其中4位為低比例染色體鑲嵌現象；其餘皆未發現致病變異，這樣的結果顯示此項檢測具有價值性，將TSC與FLCN基因所導致之Birt-Hogg-Dubé症候群（Birt-Hogg-Dubé syndrome）欲進行區分，除了影像學的輔助診斷，仍須依靠基因檢測不能只依據臨床病徵做診斷；除此，低比例染色體鑲嵌現象也需要NGS技術，方能找出臨床病徵不明顯之TSC個案。 TSC是一種具有高度異質性（heterogeneity）的自體顯性遺傳疾病，其病徵因患者之間的臨床表現差異極大，而有部分的TSC患者因症狀表現輕微或不典型，特別是低比例染色體鑲嵌的現象，可能未能及時被正確診斷。本研究旨在探討LAM的個案，致病原因是否為TSC或是其他原因所致。本研究結果除了發現低比例染色體鑲嵌的個案，顯示NGS檢驗的靈敏性與精確性，對於與TSC相似病徵的鑑別診斷也十分重要，提供胸腔科醫師以及LAM的個案更明確的醫療方針，未來仍有多個後續研究方向可探索。

Tuberous Sclerosis Complex (TSC) is a rare autosomal dominant genetic disorder, with an estimated incidence of approximately one in every 6,000 to 10,000 live births. The condition is primarily attributed to pathogenic mutations in the TSC1 or TSC2 genes, both of which play a critical role in regulating the mTOR (mammalian target of rapamycin) signaling pathway. Mutations in these genes result in dysregulation and hyperactivation of the mTOR pathway, subsequently leading to abnormal cellular proliferation and the development of hamartomas—benign, disorganized growths—in multiple organ systems. Although the clinical manifestations of TSC can lead to a diagnosis, genetic testing is often employed to confirm the underlying pathogenic mutations. However, in a small subset of patients, conventional genetic testing fails to detect causative variants. Possible explanations include mutations located within deep intronic regions or the presence of low-level mosaicism in the patient. In such cases, advanced molecular techniques—such as polymerase chain reaction (PCR) in combination with next-generation sequencing (NGS)—are required for further confirmation and precise genetic characterization. Lymphangioleiomyomatosis (LAM) is a progressive disease that may persist lifelong in a subset of patients with TSC, leading to clinical manifestations such as dyspnea, chest pain, pleural effusion, and chylothorax. Among individuals diagnosed with LAM, the condition can be further classified based on the underlying genetic mechanisms into sporadic LAM (S-LAM) and TSC-associated LAM (TSC-LAM). Therefore, the objective is to investigate the proportion of TSC-LAM cases among LAM patients treated at the National Taiwan University Hospital (NTUH). The Integrated TSC Clinic at National Taiwan University Hospital (NTUH) has been established for over a decade and has accumulated clinical data from nearly 200 patients. This study aims to collect data from patients diagnosed with LAM to determine the presence of low-level mosaicism, and to further investigate whether the condition is attributable to pathogenic variants in the TSC1 or TSC2 genes. A total of 69 patients with LAM were enrolled from the pulmonology outpatient clinic for this study. Blood and saliva samples were collected from each participant, and NGS was performed using a laboratory-developed TSC next-generation sequencing panel. The sequencing results were initially interpreted using the Integrative Genomics Viewer (IGV) software, followed by further bioinformatic analysis using tools such as Mutect2, Gendiseak (GDK), and SpliceAI to identify single nucleotide variants (SNVs), structural variants (SVs), and copy number variants (CNVs). Finally, all findings were evaluated based on the interpretation guidelines of the American College of Medical Genetics and Genomics (ACMG) in an effort to identify pathogenic variants in each individual case. We performed NGS using blood and saliva samples from 69 patients with LAM, targeting three genes: TSC1, TSC2, and FLCN. The results revealed three cases with pathogenic variants in FLCN, two cases with likely pathogenic variants in TSC2, and variants of uncertain significance (VUS) in two cases involving TSC1 and one case involving TSC2. Among these, four patients exhibited low-level mosaicism. No pathogenic variants were identified in the remaining cases. These findings underscore the clinical utility of this genetic testing approach. Differentiating between TSC and Birt-Hogg-Dubé syndrome (caused by mutations in the FLCN gene) requires more than imaging studies; molecular genetic testing is essential and cannot be replaced by clinical assessment alone. Furthermore, low-level mosaicism can only be reliably detected through NGS technologies, enabling the identification of TSC cases with subtle or atypical clinical presentations. Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder characterized by marked heterogeneity, with considerable variation in clinical manifestations among affected individuals. Some patients, particularly those with low-level chromosomal mosaicism, may present with mild or atypical symptoms, leading to delayed or missed diagnoses. The aim of this study was to investigate whether the underlying etiology of LAM in enrolled cases was attributable to TSC or to other genetic causes. The identification of cases with low-level mosaicism in our findings highlights the sensitivity and precision of NGS. Moreover, this underscores the importance of genetic testing in the differential diagnosis of diseases with overlapping clinical features, thereby offering more accurate diagnostic and treatment strategies for pulmonologists managing LAM patients. These results also open multiple avenues for future research.