摘要:計畫之特定目標本研究之特定目標在於以全基因體複製數變異(copy number variations, CNVs)篩檢的方式,以找出無SCN5A基因突變的Brugada症候群患者的複製數變異。本研究之背景及重要性Brugada症候群是一種心因性猝死症候群,而在東南亞是好發區域,是此區域中青壯年人口猝死之重要原因之一。過去歐美的研究指出SCN5A的基因突變是Brugada症候群的致病因素,約有20-25%的Brugada症候群患者有SCN5A的基因突變。最新文獻也報導CACNA1C、CACNB2、SCN1B、KCNE3和SCN3B這五種基因突變也佔了無SCN5A基因突變Brugada症候群患者的12-13%,但仍有約70%的病人找不到致病基因。從這些已找到的突變基因,分佈的位置來看,發現這些突變基因分別散落在不同的染色體上,可見Brugada症候群致病基因絕非只在某特定染色體上,因此適合做全基因體篩檢研究。另外,這些已找到的致病基因大部分均會導致離子通道(包含鈉、鉀、鈣離子通道)的loss-of-function。近年來,複製數變異,特別是deletion,被證實會減少基因的轉錄和轉譯,所以也可能導致離子通道的loss-of-function。因此,我們計劃以全基因體複製數變異(copy number variations, CNVs)篩檢的方式,以找出無SCN5A基因突變的Brugada症候群患者的複製數變異。國際研究現況目前國際上對Brugada症候群的研究重點在於找出致病基因,過去文獻指出約有20-25%的Brugada症候群是因為SCN5A基因突變引起,但大多數病患並無SCN5A突變,這些病患往往沒有明顯的家族史,其基因背景往往較像”多基因疾病”,然而由於此疾病是罕見疾病,要作case-control的研究不易,也尚無全基因體複製數變異分析的晶片分析(GWAS)的報告,因此國際間對此疾病尚未有突破性的進展。由於台灣是此疾病的好發地區,對此疾病作深入研究在國際上有競爭力,而本實驗室多年替全國各大醫院提供免費Brugada症候群基因篩檢,因此可以集中案例,達到足以有統計power的個案數,也期待能夠對此疾病的研究能有所突破。研究方法過去幾年我們陸續收案,已從台灣南北各大醫學中心及醫院收集45位Brugada症候群者。我們將從受試者周邊血液的白血球萃取DNA,用PCR和DNA定序方式來找出是否有SCN5A基因突變。在確定病患沒有SCN5A突變後,納入本研究,並採用Illumina公司的HumanCNV370-Duo microarray進行全基因體的篩檢。在得到複製數變異基因型的資料後,將之與正常族群(control即沒有Brugada syndrome的病人)進行分析,找出與Brugada症候群相關的複製數變異分析。以上的資料將以quantitative PCR方式作最後的確認,之後,分析最具顯著性的複製數變異的鄰近基因並進行功能性的研究,例如以luciferase assay進行啟動區活動性的研究,希望能闡明無SCN5A基因突變的Brugada症候群者的複製數變異的全基因體。初步結果及預期成果限於Brugada症候群的病生理機轉還不清楚,目前此病的治療方式多為支持療法。我們初步已經收集了45名病患,這是台灣目前最大的Brugada症候群族群且具亞洲人的代表性,非白人。目前已從40位不具SCN5A基因突變的病人中挑選16個人進行Illumina HumanOmni1-Quad_v1-0_B CNV microarray晶片分析,希望能能夠找到新的CNV相關的致病基因,希望能經此計劃提高增加病例數,以確認基因之正確性。並據以了解此疾病發生的病理機轉,另外,相關的致病基因可作為重要的Brugada症候群診斷參考及家人遺傳諮詢及可做為未來在開發新的藥物治療提供新的方向。
Abstract: Introduction:Brugada syndrome is a sudden cardiac death syndrome characterized by RSR’ with ST elevation pattern in V1-V3 in the 12-lead ECG. Since 1998, SCN5A mutation has been approved of being responsible for only 20-25% of this disease in Caucasian populations. CACNA1C, CACNB2, SCN1B, KCNE3, and SCN3B genes that have been recently proposed to cause a variant of Brugada syndrome, but these gene mutations accounted only for 12-13% of Brugada syndrome. In addition, these gene mutations separately located on several different chromosomes and most of these mutations result in loss-of-function of ion channels, including sodium, potassium and calcium channels. This implied that the genetic heterogeneity exits in Brugada syndrome. Copy Number Variations (CNVs), especially deletions, have been approved to decrease the transcriptional or translational expression of genes that cause the loss-of-function of genes. We propose to elucidate the whole genome CNVs for Brugada syndrome patients without SCN5A mutation.Objective:We propose to perform genome-wide genetic CNV screening to investigate the genetic mechanisms of Brugada syndrome patients without SCN5A gene mutation.Methods:We have collected 45 patients with Brugada syndrome from our hospital or referred from other medical centers or hospitals around Taiwan. 200 healthy subjects will be enrolled as control. Total cellular DNA will be extracted from peripheral blood leukocytes. We will perform PCR and direct DNA sequencing to analyze if there is any mutation in the SCN5A gene. For those patients without SCN5A gene mutation, we will apply genome-wide association study (GWAS) using the Illumina HumanCNV370-Duo microarray. CNV intensity analysis will be performed by comparing the CNVs between Brugada syndrome patients and controls. We will use quantitative PCR to validate candidate CNV regions. With the most significantly validated genetic CNV, we will analyze the neighboring genes and functional studies will be performed if needed. For those in the coding region, in vitro expression studies will be performed. For genetic variations in the promoter region, luciferase assay will be used to investigate the promoter activity. We expect to elucidate the genetic CNV and pathogenesis of Brugada syndrome without SCN5A mutation.What is New or Innovative in this study?For Brugada syndrome patients without SCN5A gene mutation, they usually do not have family history of the disease, and the genetic mechanisms and pathophysiology remains unclear. CNV analysis on this disease has never been reported before. It could be an important disease-causal gene for diagnosing and genetic counseling for Brugada syndrome patients and his/her family members in Taiwan and in the world.Scientific or Clinical Implication of the Expected Results:Current treatment with intra-cardiac defibrillator for Brugada syndrome is mainly supportive because no medication work effectively, and the pathophysiology of Brugada syndrome remains unclear. We have already included 45 patients. With support and contribution from other hospitals, this is the largest cohort of BS in Taiwan and it could represent a cohort in Asian population, not Caucasian. Our previous study disclosed much lower mutation rate of SCN5A in BS patients in Taiwan compared to that in Caucasian populations (8% vs. 20-25%). The genetic difference among different races leads us to be not fully capable of applying Caucasian genetic information to our population. We have conducted CNV analysis on the 16 of 40 patients without SCN5A mutation. With this project, we can increase the sample size to increase the statistical accuracy, and expect to elucidate the genetic mechanisms of the disease. New therapeutic medication or modalities can therefore be designed targeting the basic pathogenesis of this disease.