摘要:背景布魯蓋達症候群(Brugada syndrome, BrS)不但是一種遺傳性心律不整疾病,也是一種無法預測的猝 死症。二十多年來,有限的遺傳資訊使BrS病理生理機制難以捉摸。布魯蓋達症候群目前被認為是 一種離子通道病變的疾病,其中鈉離子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其他醫學中心或醫院轉介,我們實驗室已搜集了八十五位的BrS患 者。在其他醫院這樣的支持和協助下,已成為目前華人區最大的BrS族群。我們已發表的報告中顯 示,約只有10 %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。在去年一年期科技部補助的計劃’我們成功以新技術(次世代定序,NGS)於以Sanger 定序法檢查無SCN5A基因突變的BrS病人中發現了四個嶄新的致病基因(SCWW1A, KCNB2, KCNJ16,KCN71)這4個新發現的致病基因都是離子通道基因(一個鈉離子及三個鉀離子)和BrS 的致病生理機轉吻合並發表在Nature的系列期刊Scientific尺印orts (該領域<10%)。我們希望在這個三年計劃中,用細胞模式(in vitro cell model)及動物模式(Animal model)進行 電生理功能的研究來闡明這4個新發現的BrS離子通道致病基因的致病機轉。在動物模式實驗,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;而近年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探討BrS致病機制及進行藥物測試。目標第1年利用HEK 293T細胞模型進行新發現BrS離子通道致病基因的電生理功能研究來闡明BrS的致病機轉第2年:以TALEN和CRISPER/Cas9嶄新技術建立組織特異性且攜帶BrS新致病基因的轉殖斑 馬魚(Knock-in transgenic fish)第3年:利用攜帶BrS新致病基因的轉殖斑馬魚來闡明BrS的致病機轉及進行藥物測試方法我們將利用HEK 293T細胞模型及patch clamp技術來進行新發現的BrS突變基因的電生理學功能 研究。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。去年一年期科技部補助的 計劃,我們以新技術(NGS)全面篩檢沒有SCN5A突變BrS患者,成功找出四個BrS新的致病基因並 發表,值得我們投入研究BrS致病機制,本研究結果可作為未來開發BrS抗心律不整藥物的重要參 考。科學或臨床的預期結果及貢獻因為BrS的病理生理機制至今仍不完全清楚,現行無有效的藥物治療,只能消極地以植入體内去顫 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, 我們希望闡明我們新找出的BrS致病基因的致病機轉。研究結果將為全球提供BrS新的致病機轉資 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,更重 要的是可以作為未來開發BrS抗心律不整藥物的重要參考。
Abstract: IntroductionBrugada syndrome (BrS) is an inherited life-threatening arrhythmia associated with an unpredictable sudden cardiac death. Over 20 years, the pathophysiologic mechanisms of BrS still remain elusive because of limited genetic information. SCN5A gene is the most common BS-causal gene until 2014. 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. Although several genes were reported to be associated with BrS, all of them accounts for less 5% of cases. In the past few years, 85 BrS patients were diagnosed at our hospital or referred from other medical centers or hospitals in Taiwan. With the support and contribution from other hospitals, this is the largest cohort of BrS in Chinese Han population. In our previous published report, less than 10 % BrS patients has SCN5A mutations in Taiwan. This implied that the disease-causal gene of approximately 85-90% BrS patients in Taiwan may locate in other genes. In our last year MOST-funded project, we successfully identified 4 ion channel-related novel BrS-causal genes (SCNN1A, KCNB2, KCNJ16, KCNT1) using a new technology (Next-Generation Sequencing, NGS) in BrS patients who were detected without SCN5A mutations by Sanger sequencing and published the results in the Nature serial journals (Scientific Reports, <10% in that field) in 2014. In this 3-year project, we aim to perform electrophysiological studies not only in cell models but also in transgenic fish model to elucidate the underlying mechanisms of the 4 our published novel BrS-causal genes in BrS. Although mouse is often used for cardiovascular 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. Lately, 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 our published BrS causal genes to elucidate the underlying mechanisms of BrS and perform drug test. Objectives1st Year: Investigate the underlying mechanisms of the 4 ion channel-related novel BrS-causal genes inHEK 293T cell model2nd Year: Using TALEN and CRISPER/Cas9 biotechnology to generate Knock-in transgenic zebrafish carrying our published novel BrS-causal genes3rd Year: Elucidate the genetic mechanisms and pathogenesis of Brugada syndrome and perform drug test in the transgenic fish modelMethodsWe will perform the electrophysiological studies of these genes in HEK 293T cells using patch clamp technique to investigate the underlying mechanisms of the published novel BrS-causal 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 BrS-causal genes may locate in other genes. Our last-year MOST-funded study was the first study applying new technology (NGS) to comprehensively screen mutated genes on BrS patients instead of using traditional method (Sanger sequencing). We identified and published 4 novel BrS-causal genes in 2014, which were not reported for BrS in the world. We expect that our study results in this project could be an important reference for the development of anti-arrhythmic medications for BrS in the future.Scientific or Clinical Implication of the Expected Results No effective medications for BrS are available in clinical practice. Current treatment with implantable cardioverter defibrillator is mainly supportive for BrS because the pathophysiology of BrS remains unclear. To improve understanding of the pathophysiology of BrS, the genetic information plays a pivotal role. With this study, we expect to elucidate the genetic mechanisms and the pathogenesis of these novel BrS-causal genes. The results could provide new genetic information of BrS in the world and help physicians perform risk stratification or genetic counseling for family members. In addition, anti-arrhythmic medications can therefore be designed targeting the basic pathogenesis of this disease based on the results of this study.