結節硬化症：REDCap資料庫的建立以及次世代定序基因檢測

Tuberous Sclerosis Complex (TSC): REDCap Database Establishment and Next-generation Sequencing (NGS)-based Genetic Testing

李昭萱(Chao-Hsuan Li)

指導教授 ： 陳沛隆

國立臺灣大學/醫學院/分子醫學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802113

摘要

結節硬化症是一種罕見的體染色體顯性遺傳疾病，因TSC1或TSC2基因生殖細胞突變，導致全身各處器官和組織產生錯構瘤。目前有10~25%臨床上確診為結節硬化症的病人，取其周邊血液做次世代定序基因檢測，找不到致病位點。其中有部分可能是TSC1或TSC2低比例鑲嵌型，或者致病位點隱藏在較深會影響剪接的內涵子區域，於檢測時被忽略。
本研究收入了曾於臺大醫院結節硬化症整合門診看診的病人及其家屬共508位，含222個家族。根據臨床表徵，其中有168個家族先證者 (proband) 被確診為結節硬化症；10個家族臨床懷疑為結節硬化症但尚不足以確診；44個家族原本被懷疑是結節硬化症，經過轉介來臺大醫院評估後認為非結節硬化症。將這些個案的家族史、臨床表徵、基因檢測、各科追蹤檢查紀錄等資訊，整理並在臺大安全網域下建構線上TSC REDCap資料庫，呈現臺灣本土結節硬化症疾病的資料。另外，進行兩代直系親屬的基因檢測，確認一同時患有結節硬化症和多囊腎的病人身上兩個致病變異點在染色體上的相位關係；並從過去周邊血液基因檢測未找到生殖細胞突變的3位病人中，取其皮膚病灶檢體進行基因檢測，偵測到低比例的生殖細胞TSC1或TSC2突變鑲嵌型狀況；探討一患有肝細胞癌 (HCC) 的B型肝炎帶原結節硬化症病人，其肝臟腫瘤切片的基因狀況；設計minigene功能性測試系統來確認未確定致病性的剪接位變異點其轉錄出的mRNA是否有異。
在本研究臨床確診為結節硬化症的家族中，次世代定序基因檢測確診檢出率約88%，另3%為未能確定致病性的變異點 (VUS)，剩下9%沒有發現任何懷疑的變異點 (NMI)。其中有20%為TSC1基因變異，80%為TSC2基因變異；30%為家族遺傳而來，70%為新發生的變異。對於同時帶有結節硬化症和多囊腎兩個致病變異點的病人，可以透過進行直系親屬基因檢測來釐清兩變異點的相位關係；並利用皮膚病灶檢體提高低比例鑲嵌型患者，生殖細胞突變被檢測出來的比率；觀察到一位帶有TSC2致病生殖細胞變異且同時為B型肝炎帶原的結節硬化症病人，發展出肝細胞癌 (HCC) 時，則其肝腫瘤會較大且侵略性更強。針對肝腫瘤切片做基因檢測，發現除患者原本在TSC2基因上的生殖細胞錯義突變之外，另發生一個體細胞突變：約2Mb包含TSC2的大片段缺失，使此區域成為異合性丟失的狀況 (LOH)。免疫治療搭配everolimus治療對此病人肝腫瘤擴張有控制效果。最後，針對未確定致病性的剪接位變異點設計的minigene系統將繼續進行實驗確認。

關鍵字

結節硬化症 ； 線上資料庫 ； 致病性不明的剪接位變異 ； 低比例鑲嵌型 ； 兩致病變異點的相位關係 ； B肝帶原肝細胞癌病灶 ； 次世代定序

並列摘要

Tuberous Sclerosis Complex (TSC) is a rare autosomal dominant disease due to germline disease-causing variants in either TSC1 or TSC2 gene, and may develop multisystem harmartomas. However, 10%-25% of TSC patients remain genetically undiagnosed after conventional genetic testing. Part of them may carry variant(s) with low percentage mosaicism or have variants affecting splicing in deep introns, which would be missed out during interpretation of the NGS data.
We enrolled 508 subjects from 222 families from the TSC Integrated Clinic at National Taiwan University Hospital (NTUH). Among them, 168 probands were TSC-confirmed, 10 probands were TSC-suspected, and 44 probands eventually did not meet the clinical criteria of TSC. We established an online REDCap database for our TSC cohort, and collected and uploaded family histories, phenotypes, genotypes, and the follow-up examinations of these subjects into the database. Additionally, we took advantage of the next-generation sequencing (NGS)-based genetic testing to solve several critical issues. Notable examples included, but not limited to, solving the phasing issue of TSC2 and PKD1 variants in a family, detecting possible low percentage mosaic germline mutations in skin lesions of 3 TSC patients without identifiable variants in peripheral blood samples previously, and analyzing the possible second hit of from the hepatoma biopsy samples of a TSC patient with chronic hepatitis B and newly diagnosed hepatocellular carcinoma (HCC). Finally, we designed minigene experiments trying to confirm the pathogenicity of splicing variants of unknown significance (VUS).
In our study, for the 168 probands with clinically definite TSC, the detection rate of pathogenic and likely pathogenic germline variants in TSC1 or TSC2 gene was 88%; additional 3% of TSC patients had VUS, and 9% remained no mutation identified (NMI). Among TSC probands with genetic diagnosis, 20% fell in TSC1 and 80% fell in TSC2 gene. Thirty percent of the variants were inherited, while the other 70% were de novo mutations. For the patient with two variants in the TSC2 and PKD1 genes, we confirmed that the two variants were on the same chromosome 16 haplotype, based on the co-segregation observed in his son. And, we could increase the detection rate of low percentage mosaic germline mutation by analyzing the NGS data of their skin lesion samples compared with the data of peripheral blood samples. For the TSC patient with chronic hepatitis B as well as newly diagnosed HCC, we found a pathogenic TSC2 germline missense variant and a TSC2 LOH somatic mutation at least 2Mb in size in the HCC biopsy. The HCC initially grew fast and developed aggressively. Treatment combined with immunotherapy and everolimus, which is a mTOR inhibitor, seemed to be initially effective for disease control. Finally, the minigene experiments of VUS splicing variants are still underway.

並列關鍵字

Tuberous Sclerosis Complex (TSC) ； online database ； splicing variants of unknown significance (VUS) ； low percentage mosaicism ； haplotyping ； HBV+HCC biopsy ； next-generation sequencing (NGS)

參考文獻

1. O'Callaghan, F.J., et al., Prevalence of tuberous sclerosis estimated by capture-recapture analysis. Lancet, 1998. 351(9114): p. 1490.

2. Crino , P.B., K.L. Nathanson , and E.P. Henske The Tuberous Sclerosis Complex. New England Journal of Medicine, 2006. 355(13): p. 1345-1356.

3. Kwiatkowski, D.J., Tuberous Sclerosis Complex: Genes, Clinical Features and Therapeutics. Weinheim, Germany: Wiley-VCH, 2010: p. pp 29–60.

4. F, R., Die Lymphelfasse und ihre Beziehung zum Bindegewebe. [German]. Berlin: A. Hirschwald; , 1862.

5. Bourneville, D.M., Sclerose tubereuse des circonvolutions cerebrales: Idiotie et epilepsie hemiplegique. Arch Neurol (Paris), 1880. 1:81–91.