摘要:背景布魯蓋達症候群(Brugada syndrome, BrS)是一種遺傳致命性成人心律不整疾病,與無法預測的心因 性猝死有關。二十多年來,有限的遺傳資訊使BrS病理生理機制難以捉摸。SCN5A基因是至今最常 見的BrS致病基因。然而,此基因只能解釋20-25 %患有BrS的白人族群、這表示有75-80 %的 BrS患者的致病基因仍是未知。在過去幾年中,由我們醫院確診或經由台灣其他醫學中心或醫院的轉 介,我們實驗室已搜集了五十五位的BrS患者。在其他醫院這樣的支持和協助下,已成為目前台灣 區最大的BrS族群。我們已發表的報告中顯示,只有8.5 %的台灣BrS患者有SCN5A基因突變。 儘管有一些與BrS相關的基因被發表(ex.CACNA1C),但僅能解釋少於5 %的患者。這表示,約85-90 %台灣BrS患者的致病基因可能位於其他基因。在我們的先導性研究,我們成功以新技術(次世代定 序(NGS}} ’於五位以Sanger定序法檢查無SCN5A基因突變的BrS病人中發現了三個嶄新的可能 致病基因(SCNN1A,KCNB2, KCNJ16)。在這個計劃中,我們將持續以次世代定序法來研究,以找 出更多的BrS致病基因,再以mass spectrometry和Sanger定序法來驗證。驗證這些基因後,我們 會在用細胞(in vitro cell model)進行電生理功能的研究,包括使用HEK 293T和HL-1細胞來考量這 些基因的重要性。轉殖老鼠常用於心臟研究,然而,老鼠的心臟電生理活性被證實和人相距太遠,不 易呈現人類心律不整的型態;而許多的證據顯示斑馬魚可作為研究人類心律不整的良好動物模型,尤 其是early repolarization如BrS。因此,我們將以基因轉殖斑馬魚來驗證這些BrS致病基因並探討 BrS致病機制。目標1.(第1年)將持續以次世代定序法來研究,找出更多的BrS可能新致病基因和用mass spectrometry 和Sanger定序法驗證BrS可能新致病基因2.(第2年)利用細胞模型進行BrS可能新致病基因的電生理功能研究:包括HEK293T和HL-1細胞 來評估及驗證這些BrS可能新致病基因以找出BrS新且重要的致病基因3.(第3年)根據第二年的研究結果,我們將以TALEN和CRISPER/Cas9嶄新技術建立組織特異 性且攜帶新且重要BrS新致病基因的轉殖斑馬魚來闡明BrS的致病機轉及藥物測試方法我們將以次世代定序法再研究10例無SCN5A突變BrS患者。利用Illumina HiSeq 2000來發現更多 BrS-致病基因,再以質譜和Sanger定序法來驗證。我們將收集五百名未患有BrS的病患基因作為對 照組,以質譜法和雙向DNA定序法來對照驗證新發現的BrS突變基因。另一方面,我們將利用細胞 模型進行新發現的BrS突變基因的電生理學功能研究,包括使用HEK 293T和HL-1細胞來評估新發 現的BrS突變基因的重要性。之後,我們將以TALEN和CRISPER/Cas9嶄新技術建立組織特異性 攜帶BrS新發現突變基因的轉殖斑馬魚。我們將執行體外心電圖記錄,斑馬魚心臟光學圖譜及藥物 測試。我們希望藉此闡明沒有SCN5A基因突變的BrS致病機制。本研究的創新及重要性流行病學研究指出,BrS在亞洲東南部的發生率比世界其他地區高,值得重視。而台灣約85-90 % 的BrS患者沒有SCN5A突變,這表示BrS致病基因可能位於其他基因。本研究將以新技術(NSG) 全面篩檢沒有SCN5A突變BrS患者的可能致病基因。目前世界上還尚未報導利用NGS找出BrS新 的可能致病基因(例如:SCN1A,KCNB2,KCNJ16...)值得我們投入研究,另外,本研究結果可作為台 灣BrS患者和他/她的家人診斷疾病和遺傳諮詢的重要資訊。科學或臨床的預期結果及貢獻因為BrS的病理生理機制致今仍不完全清楚,現行治療只能以植入體内去顫器為主。為了提高對BrS 致病機制的了解,致病基因相關資訊扮演著舉足輕重的地位。本研究中,我們希望找的新的BrS致 病基因及闡明BrS致病機制。研究結果將為全球提供BrS新的遺傳資訊,也提供國人BrS家庭成員 遺傳本土諮詢資訊及不受種族因素影響的危險分級參考。另外,也可以作為未來開發BrS抗心律不 整藥物的重要參考。
Abstract: IntroductionBrugada syndrome (BrS) is an inherited adult life-threatening disease associated with an unpredictable sudden cardiac death. Over 20 years, the pathophysiologic mechanisms of BrS still remains elusive because of limited genetic information. SCN5A gene is the most common BS-causal gene until 2013. However, it only accounts for 20-25% of this disease in Caucasian populations which means the disease-causal gene for the remained 75-80% BrS patients is still unknown. In the past few years, 55 BrS patients were diagnosed at our hospital or referred from other medical centers or hospitals in Taiwan. With support and contribution from other hospitals, this is the largest cohort of BrS in Taiwan. In our published report, only 8.5% BrS patients has SCN5A mutations in Taiwan. Although several genes were reported to be associated with BrS, all of them accounts for less 5% of cases. This implied that the disease-causal gene of approximately 85-90% BrS patients in Taiwan may locate in other genes. In our pilot study, we successfully found 3 novel possible BrS-causal genes (SCNN1A, KCNB2, KCNJ16) using a new technology (next-generation sequencing, NGS) in 3 of 5 BrS patients who were detected without SCN5A mutations by Sanger sequencing. In this project, we will extend NGS study to identify more BrS-causal genes. After validating these genes, we will perform electrophysiological studies in cell models to prioritize the importance of validated genes. Although mouse is often used for cardiologic researches, the apparent differences exist in the electrophysiological field of heart that raise doubts whether mouse should be used as an animal model for human arrhythmic diseases. Accumulating evidences have shown that the zebrafish can serve as an excellent model to study human arrhythmia, especially repolarization disorders like BrS. Therefore, we propose to generate transgenic zebrafish carrying validated BrS causal genes and elucidate the underlying mechanisms of BrS.Objectives1.(1st Year) Extend NGS study to identify more novel putative BrS-causal genes and validate novel putative BrS-causal genes using mass spectrometry and Sanger sequencing2.(2nd Year) Perform the electrophysiological functional studies of validated novel putative BrS-causal genes in cell models including HEK 293T and HL-1 to prioritize the importance of validated novel putative BrS-causal genes3.(3rd Year) Based on the results in the 2nd years, we will generate Knock-in transgenic zebrafish carrying novel and important validated BrS-causal genes to elucidate the genetic mechanisms and pathogenesis of Brugada syndrome and perform drug testMethodsWe will extend NGS study on 10 BrS patients without SCN5A mutations using Illumina HiSeq 2000 to identify more BrS-causal genes and validate these putative genes using mass spectrometry and Sanger sequencing. We will build enroll 500 patients without BrS as control to validate these mutated gene using mass spectrometry and Sanger sequencing. Afterward, we will perform the electrophysiological studies of these genes in cell models including HEK 293T and HL-1 cells to prioritize the importance of these genes. Thereafter, we will create tissue-specific transgenic zebrafish carrying mutated genes by using TALEN and CRISPER/Cas9 biotechnology. In-vitro ECG recording, optical mapping of adult zebrafish heart, and drug test will be performed. We expect to elucidate the genetic mechanisms and pathogenesis of Brugada syndrome without SCN5A mutations.What is New or Importance in this study?Epidemiologic studies reported that the prevalence of BrS in southeast of Asia is higher than other areas of the world. Approximately 85-90% of BrS patients do not have SCN5A mutations in Taiwanese, which means the BrS-causal genes may locate in other genes. This study will be the first study applying new technology (NSG) to comprehensively screen mutated genes on BrS patients without SCN5A mutations instead of using traditional method (Sanger sequencing). Several genes (SCNN1A, KCNB2, KCNJ16) identified by NGS are novel mutated genes that have not been reported for BrS in the world. They could be an important BrS-causal genes for diagnosis and genetic counseling for BrS patients and his/her family members in Taiwan and in the world.Scientific or Clinical Implication of the Expected ResultsCurrent treatment with implantable cardioverter defibrillator is mainly supportive because the pathophysiology of BrS remains unclear. To improve understanding of the pathophysiology of BrS, the genetic information play a pivotal role. With this study, we expect to identify novel BrS-causal genes and elucidate the genetic mechanisms and the pathogenesis of these novel BrS-causal genes. The results will provide new genetic information of BrS in the world and help genetic counseling for family members or risk stratification without concerning racial factors. Anti-arrhythmic medications can therefore be designed targeting the basic pathogenesis of this disease.