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研究生(外文):Hung-Lun Chiang
論文名稱(外文):Functional Significance of Intronic DNA Sequence Variants
指導教授(外文):Yuan-Tsong Chen
外文關鍵詞:SNPbranchpointsplicing enhancer silencerFabry disease
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我們使用剪接預測軟體找出位於分枝位點序列上的SNP,預測結果已經確定了位於剪接分枝位點的600個SNP,在排除不改變A鹼基的SNP後,保留了216個SNP。 然後使用條件排序和结合分枝位點定序資料,獲得影響剪接的可能具有影響SNP。 其中四個可能具有影響SNP用於進一步實驗,其中rs12769205(SNP ID)位於细胞色素P450 2C19(CYP2C19)第2内含子中。使用模擬小基因(minigene)方法實驗,顯示當A改變為鳥嘌呤(G)時,内含子將不會被剪接。 而用一般人不同基因型的B细胞所萃取mRNA進行逆轉錄聚合酶鏈式反應(RT-PCR)則有相同的剪接模式。
除了剪接分支點A,SNP改變剪接增強子或沉默子來影響剪接,這也是導致疾病的原因。過去研究指出法布瑞氏症(FD)的GLA突變(IVS4 + 919G> A: rs199473684)在台灣有較高的發生率,男性為1/875(0.11%),女性為1/399(0.25%)。 該突變與FD的晚發性心臟病有關。 然而,這種突變是否是心臟病的常見原因還不清楚。
因此我們對對照組和其他疾病組(第2型糖尿病;T2D,和心臟病組包括心臟衰竭,室性心動過速;VT,心房顫動;AF,和冠狀動脈疾病;CAD,心臟擴大)進行基因檢測。結果指出對照組中,GLA IVS4 + 919A的發生率為1,634名男性中有4名(0.245%),其中1,634名女性中有2名(0.123%); T2D中2,133名男性中有4名(0.188%)和1,816名女性中有7名(0.385%)。 在17例IVS4 + 919A攜帶者中,只有2例男性患者出現心臟異常。 此外在心臟病中,共有649名患者中的一名男性攜帶了這突變(0.002%)。建構小基因模型來研究探討不同細胞株中GLA的剪接模式。實驗顯示AGS(胃)細胞株GLA的剪接模式不同於其他細胞株(COS1,COS7,293T和心肌細胞)。這種現象可能是由pre-mRNA變異點上以組織特異性所帶來不同的增強子或沉默子引起的。
大多數攜帶GLA IVS4 + 919A的受試者並無心臟相關的疾病。 在台灣IVS4 + 919A發生率高,但是心臟病發病率並沒因此提高,表示IVS4 + 919A不是台灣造成心臟病的常見原因,顯示此一基因型和表現型相關性有待進一步研究調查。 而我們的實驗顯示GLA IVS4 + 919G> A突變會以組織特異性方式影響選擇性剪接。GLA IVS4 + 919G> A變異不是台灣地區明顯造成心臟病的常見原因。
Advances in sequence technology have improved our knowledge on human genome and nucleotide variants. Variants and SNPs are known to modulate disease susceptibility and exert different responses to pathogens, drugs, and other agents. Non-synonymous SNP leads to change in amino acid, and may be the direct cause of disease. However, effects of synonymous SNP are not as obvious. Determination and characterization of functional SNPs is therefore an important step after sequencing.
In addition, many SNPs are intronic with unknown function. SNPs occurring on intron branchpoint sites, especially at the adenine (A), would presumably affect splicing; however, this has not been systematically studied. We employed a splicing prediction tool to identify human intron branchpoint sites and screened dbSNP for SNPs located on the predicted sites.
We identified 600 SNPs located within branchpoint sites; among which, 216 showed a change in adenine (A). Then use score sorting and combining with lariat RNA-seq dataset, the potential SNPs affecting splicing are obtained. Five of the potential SNPs is selected for further experiment, one of those SNPs is rs12769205 locating in CYP2C19 intron 2. From a minigene assay, it was shown that the intron 2 was not spliced when A changes to G allele. RT-PCR done with the extracted mRNA from immortalized B cell of normal population carry of different genotype showed the same pattern.
Those selected SNPs were shown to affect RNA splicing by minigene construct or RT-PCR, and confirmed our selecting strategy has worked. However, there remains many potential SNPs (particularly with lariats sequencing data) to be further investigated, and those SNPs can serve as a reference for disease study which identified the novel SNP or variant in intron.
Apart from branchpoint A, SNP affected splicing by modifying splicing enhancers or silencers, and can be the cause of disease. For example, the GLA variant (IVS4+919G>A: rs199473684) of Fabry disease (FD) was reported to occur at high frequency in Taiwan, with 1/875 (0.11%) in males and 1/399 (0.25%) in females. This variant is linked to late-onset cardiac phenotypes of FD. However, whether this variant is a frequent cause of heart diseases is not clear.
We genotyped the GLA variant in normal controls and other disease cohorts (Type 2 diabetes, T2D and heart disease cohorts, including, heart failure, ventricular tachycardia; VT, atrial fibrillation; AF and coronary artery disease; CAD, cardiomegaly). The incidence rate of the GLA IVS4+919A was 4 in 1,634 males (0.245%) and 2 in 1,634 females (0.123%) in normal controls; 4 in 2,133 males (0.188%) and 7 in 1,816 females (0.385%) in T2D cohort. In the 17 IVS4+919A carriers detected in normal controls and T2D subjects, only 2 males have reported heart conditions abnormality. Furthermore, in heart disease cohort, one male out of a total of 649 patients (0.002%) carried the variants.
Mini-gene constructs were used to study the splicing pattern of GLA in different cell lines. Results showed the splicing pattern (included 57bp exon in intron 4) of GLA was different in AGS (stomach) cell line, compared to other cell lines (COS1, COS7, 293T and cardiomyocyte). This phenomenon maybe caused by different splicing repressors or activators sitting on the variant sites of the pre-mRNA and acting in tissue-specific manner.
Most of the subjects carrying GLA IVS4+919A were identified without cardiac phenotype. The high incidence rate of IVS4+919A in Taiwan without matching trend in heart disease indicated that IVS4+919A was not a frequent cause of heart diseases in Taiwan and suggests the genotype-phenotype correlation is in need for further investigation. We showed that the GLA IVS4+919G>A mutation influence alternative splicing in a tissue specific manner. The GLA IVS4+919G>A variant is not a frequent cause of overt heart disease in Taiwan.
Content i
Abstract iii
中文摘要 vi
List of Abbreviations viii
Chapter 1、Identification of Functional Single Nucleotide Polymorphisms in the Branchpoint Site 1
1.1 Introduction 1
1.2 Materials and Methods 4
1.2.1 Creating a dataset of SNPs located within branchpoint sites 4
1.2.2 Cell lines and genotyping 4
1.2.3 Minigene constructs 5
1.2.4 Reverse transcription-PCR (RT-PCR) 5
1.3 Results 6
1.4 Discussion 9
1.5 Conclusion 11
1.6 Perspectives 11
Chapter 2、Genetic Epidemiological Study doesn't Support GLA IVS4+919G>A Variant is a Significant Mutation in Fabry Disease 13
2.1 Introduction 13
2.2 Materials and Methods 15
2.2.1 GLA IVS4 + 919 G > A genotyping 15
2.2.2 RT-PCR and real-time PCR 15
2.2.3 Cell lines 16
2.2.4 The mini-gene constructs 16
2.3 Results 17
2.3.1 Epidemiological studies of IVS4+919A 17
2.3.2 Differential expression of GLA in tissues and cell lines 18
2.3.3 Minigene construct for splicing pattern investigation 19
2.4 Discussion 20
2.4.1 Epidemiological studies ofIVS4+919A 20
2.4.2 Differential expression of GLA in tissues and cell lines 21
2.4.3 Minigene construct for splicing pattern investigation 21
2.4.4 GLA IVS4+919G>A variant may act as a fringe allele 22
2.5 Conclusion 24
2.6 Perspectives 24
References 25
Figures 30
Tables 38
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