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標題: | 藉由拷貝數目變異來分析可能成為具診斷性的Brugada症候群之生物標記 The Potential Diagnostic Biomarkers of Brugada Syndrome Identified by Copy Number Variation Analysis |
作者: | Yi-Yu Su 蘇怡羽 |
指導教授: | 賴亮全(Liang-Chuan Lai) |
關鍵字: | Brugada症候群,拷貝數目變異,GSTM3,NALCN,多重聚合酶,連鎖反應,生物標記, Brugada syndrome,copy number variants,GSTM3,NALCN,multiplex PCR,biomarkers, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | Brugada症候群(Brugada Syndrome, BrS)是一種心臟疾病且會因致死性的心律不整而造成高危險性的突發性死亡,尤其常好發於亞洲輕壯年男性。在先前的研究已顯示,SCN5A突變會造成病患有Brugada症候群的心電圖,但僅有20-25%的Brugada症候群病患有SCN5A突變。然而,對於Brugada症候群的基因體變異(genomic variant),仍尚未被廣泛的研究。因此,本篇論文主是萃取出未帶有SCN5A突變之Brugada症候群病患(n=16)與健康控制組(n=16)的血液中DNA,並且利用全基因性之單一核苷酸多型性 (single-nucleotide polymorphism, SNP)微陣列晶片分析出基因型變異。此群Brugada症候群病患與健康控制組的基因型(genotyping)被分析出來後,本論文利用以下標準並且使用Partekâ軟體分析出拷貝數目變異 (copy number variation, CNV)區段:(1) 樣品基因型的強度 (intensity)標準化後,晶片上每單一核甘酸多型性之拷貝數目(copy number, CN)須至少大於2.3或是小於1.7。(2)每樣品的拷貝數目變異區段需由至少大於100個連續之單一核甘酸多型性位點變異所組成。(3)相鄰拷貝數目變異區段的P-value檢定至少小於10-4。(4)至少有37.5%的樣品在同個拷貝數目變異區域有變異。本篇研究顯示出其中有502個拷貝數目變異區域,這些區域中包含447個區域為拷貝數目減少及55個區域為拷貝數目增加。再者,將這些拷貝數目變異區域與資料庫比對後,結果顯示出有138個基因 (genes)於其中,並且將這些基因利用Ingenuity Pathways Analysis (IPA) 分析出它們的功能。其中最顯著的訊息傳導路徑裡,GSTM3是具有最低的拷貝數目。另外,因Brugada症候群被猜測與離子通道失去功能有相關,故本篇論文找到NALCN是離子通道中有最低拷貝數目。而後,本篇研究藉由多重聚合酶連鎖反應(multiplex PCR)之實驗驗證出Brugada症候群病患有GSTM3和NALCN的拷貝數目減少之現象。此外,本篇論文增加樣品數目並且顯示評估靈敏度(sensitivity)和特異度(specificity) 預測表現後,實驗結果顯示GSTM3和NALCN可成為生物標記(biomarkers)做為診斷Brugada症候群。除此之外,將GSTM3和NALCN交集起來做準確度(accuracy)預測結果後,本研究顯示GSTM3和NALCN具有加成作用。 Brugada syndrome (BrS) is a cardiac disease, which results in a high risk of sudden death by lethal arrhythmia, especially in Asian young males. Previous studies reported that it is associated with SCN5A mutations. However, these mutations can only account for 20-25% of BrS patients. The genomic aberration of BrS still remains unclear. Therefore, we have conducted a genome-wide screening from blood of BrS patients without SCN5A mutations using Illumina Omni1-Quad single-nucleotide polymorphism (SNP) chips. The genotypes of BrS patients without SCN5A mutations (n=16) and healthy controls (n=16) were examined. Regions of copy number variation (CNV) were identified by Partekâ software. We have used the following criteria: First, copy number ratio of sample intensity to mean intensity for each SNP is >2.3 or <1.7. Secondly, ≥100 continuous SNP variant loci occur in each sample. Thirdly, P-value ≦ 10-4. Finally, ≥37.5% of samples have common aberration regions. We have identified 502 common aberration regions, with 447 loss and 55 gain regions. Moreover, we have found those common aberration regions containing 138 genes and their functions have been further analyzed using Ingenuity Pathways Analysis. Among these, GSTM3 had the lowest copy number compared to other genes in the top one enriched pathway. Since BrS may be caused by the abnormality of ion channel, we have also focused on NALCN, which encodes sodium channel. Next, the absence of GSTM3 and NALCN were examined by using multiplex PCR in BrS patients. This study has showed that GSTM3 and NALCN lost in BrS patients. Also, the study has evaluated the prediction outcomes in two aspects: one is sensitivity and the other is specificity, and the results have demonstrated that GSTM3 and NALCN might be used as diagnostic biomarkers of detecting BrS. Furthermore, the prediction outcomes of the intersection of GSTM3 and NALCN have indicated that GSTM3 and NALCN could be additive. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66197 |
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