臺灣博碩士論文加值系統

胸腔主動脈瘤剝離症候群（Thoracic Aortic Aneurysm and Dissection syndrome，TAAD）根據臨床表徵可以細分兩個主要的類別：（1）綜合症型（Syndromic），常伴隨有多組器官受損，主要包括Marfan syndrome（MFS）、Loeys-Dietz syndrome（LDS）、Ehlers-Danlos syndrome（EDS）、Aneurysms-Osteoarthritis syndrome（AOS），以及transforming growth factor-β(TGF-β) signaling pathway相關疾病；（2）非綜合症型（Non-syndromic），只在主動脈發生病症，一般分為家族性TAAD及偶發性TAAD。然而多樣化的臨床表徵或症狀不明確，常使醫師診斷上造成困難，也帶給患者及其家庭成員莫大的痛苦。
本研究利用臺大醫院「心臟內科門診」、「小兒心臟科門診」及「基因醫學部門診」臨床確診為TAAD- Aneurysm/Dissection的30位病患及19位TAAD-non Aneurysm/Dissection但臨床表徵疑似馬凡氏症候群(MFS )的病患所提供的血液，以次世代定序技術針對Syndromic TAAD與Non-syndromic TAAD相關的27個基因進行基因檢測，其檢出率為49.0%(24/49)，其中30位確診為TAAD- Aneurysm/Dissection個案的檢出率為63.3%(19/30)，19位TAAD-non Aneurysm/Dissection個案的檢出率為26.3%(5/19)，以Sanger sequencing驗證結果，正確率為100%(20/20)。依據ACMG（American College of Medical Genetics and Genomics）準則的判斷，在Pathogenic/Likely pathogenic的基因變異中有8個FBN1及1個FBN2是目前尚未被報告過的基因變異點位。
NGS與傳統基因定序方法相較之下，NGS主要是結合微製程技術以達成高通量定序，能夠同時讀取上百萬條DNA的序列，所以可以大量降低成本，且速度更快，幫助病人確立基因診斷，找出造成胸腔主動脈擴大剝離症的原因，了解本土病患的基因變異分布情形，並將進一步尋找新的致病基因，擴展我們對此疾病的理解。

Thoracic Aortic Aneurysm and Dissection syndrome (TAAD) can be divided into two main categories according to clinical features: (1) Syndromic, often accompanied by multiple groups of organ damage, including Marfan syndrome (MFS), Loeys-Dietz syndrome (LDS), Ehlers-Danlos syndrome (EDS), Aneurysms-Osteoarthritis syndrome (AOS), and transforming growth factor-β signaling related diseases; (2) Non-syndromic, only in the aortic disease, generally divided into familial TAAD and sporadic TAAD. However, the diversity of clinical features or symptoms are not clear, and it not only resulting in the difficulties of clinical diagnosis, but also suffering greater pain to patients and their families.
In this study, we enrolled patients from National Taiwan University Hospital "Cardiology Outpatient", "Pediatric Cardiology Outpatient" and "Department of Medical Genetics";30 patients with clinically diagnosed TAAD- Aneurysm/Dissection, and 19 patients with TAAD-non Aneurysm/Dissection but the clinical manifestations of suspected Marian syndrome (MFS) were included. We extracted gDNA from peripheral blood of these 49 patients, and detected 27 genes related to Syndromic TAAD and Non-syndromic TAAD by next-generation sequencing (NGS). Total detection rate was 49.0% (24/49), with 63.3% (19/30) for 30 TAAD- Aneurysm/Dissection diagnosed patients, and 26.3% (5/19) for 19 TAAD-non Aneurysm/Dissection cases. The accuracy rate was 100% (20/20) for the doubling check with Sanger sequencing. According to the ACMG (American College of Medical Genetics and Genomics) guidelines, we found 8 variants on FBN1 and 1 variant on FBN2 to be pathogenic or likely pathogenic, and they were genetic variants which not yet been reported so far.
Comparing with traditional sequencing methods, NGS is a combination of micro-technologies to do millions of DNA sequencing simultaneously; it generates high throughputs with high resolution within several days, which help us to save time and money. With this method, we can help patients to find out their causes of thoracic aorta. Furthermore, the understanding of genetic composition of Taiwan patients expands our understanding of this disease.

口試委員會審定書…………………………………………………………………….I
誌謝……………………………………………………………………………………..II
中文摘要……………………………………………………………………………….III
Abstract………………………………………………………………………………….V
第一章、 研究背景與動機………………………………………………………...1
1.1. Thoracic Aortic Aneurysm and Dissection疾病介紹……………………...1
1.2. Thoracic Aortic Aneurysm and Dissection的臨床特徵…………………...3
1.2.1 Marfan syndrome 3
1.2.2 Loeys-Dietz syndrome 4
1.2.3 Ehlers-Danlos syndrome 4
1.2.4 TAAD Syndrome與其相關的基因 5
1.3. Thoracic Aortic Aneurysm and Dissection的診斷及檢查………………...5
1.4. 研究目的…………………………………………………………………...5
第二章、 研究方法………………………………………………………………...8
2.1. 研究對象來源及條件……………………………………………………...8
2.1.1受試者來源…………………………………………………………………………………………8
2.1.2 受試者納入排除條件…………………………………………………………………..8
2.1.3 檢體來源與取得………………………………………………………………………………9
2.2 次世代定序檢測……………………………………………………………..9
2.2.1 DNA的萃取 9
2.2.2 DNA濃度測量 10
2.2.3 TAAD syndrome基因檢測平台 11
2.2.4 次世代定序（Illumina，MiSeq）原理 11
2.2.5 定序結果判讀 13
2.3 PCR驗證(Sanger定序分析)……………………………………………..14
2.3.1 DNA 引子(Primer)之製備 14
2.3.2 聚合酶連鎖反應 15
2.4 實驗器材與材料………………………………………………………….20
第三章、 結果…………………………………………………………………….22
3.1. 次世代定序分析(Illumina system)定序結果…………………………….22
3.2. Sanger sequencing定序結果……………………………………………..30
3.3. TAAD基因檢測結果判定 ……………………………………………….33
3.4 Family testing for segregation…………………………………………….35
第四章、 討論…………………………………………………………………….43
第五章、 結論…………………………………………………………………….47
參考文獻……………………………………………………………………………….49
附錄 54

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