基因體學新穎研究方法在心血管疾病中的應用

Abstract

人類基因體計畫所獲得基因體所有序列及基因資訊，使得人們對於遺傳學及醫學中有更深入的了解，舉凡開發新的診斷測定、標靶治療、預測疾病的發作、疾病的嚴重性和進程，目前已有許多高通量技術如微陣列晶片和次世代定序使得這些目標能夠成真。在本論文中應用微陣列晶片和次世代定序方法尋找兩種與血栓生成相關的心血管疾病-心房顫動和靜脈曲張之易感基因或可能的致病機轉。 本論文研究首先著重在心房顫動，這是最常見的持續性心律失調形式。先前的全基因體關聯研究已經找到與心房顫動風險相關的常見單核苷酸多型性。在人類基因體中，已知拷貝數變異 (CNV)會導致疾病易感性，但其在心房顫動風險中的作用仍屬未知。本研究是利用全基因體多階段方法尋找與心房顫動易感性的拷貝數變異，發現鉀離子通道相互作用蛋白1 (KCNIP1)的第一個內含子中的一段插入型的常見拷貝數變異與台灣族群中的心房顫動顯著相關。在斑馬魚中過度表現KCNIP1會誘導斑馬魚產生心房顫動。本研究是首次證實KCNIP1基因中的常見拷貝數變異為心房顫動風險的高遺傳預測因子，同時解釋了其對心房顫動致病機轉中的功能。 心房顫動會增加中風風險，因此本論文接著著重在尋找與心房顫動相關的血栓栓塞風險相關的拷貝數變異。針對心房顫動並且血栓栓塞性中風患者和來自台灣健康族群的對照者進行全基因體研究，我們首次發現GNB1、PRKCZ和GNG7基因中的缺失與α-腎上腺素能受體信號傳導途徑相關，其在心房顫動所引起之中風的風險中扮演可能的作用。 常見遺傳變異僅解釋了一部分與心房顫動相關的遺傳易感性。全基因體關聯性分析所遺漏的罕見變異可能也會增加心房顫動的遺傳風險。因此下一項工作就是利用極端特徵設計，對精心挑選具有極端表型的患者及其未受影響的患者親屬進行定序，以尋找罕見的新生突變。基於常見和罕見變異可能位於相同疾病易感基因的假設，針對全基因體關聯性分析中所發表的9個已知為心房顫動易感基因進行次世代定序，找到了PITX2基因中5'端非轉譯區中的新突變，功能性分析顯示，PITX2基因5'端非轉譯區的突變顯著負調控心房心肌細胞中PITX2的表現。 最後，本論文研究重點放在與血栓生成相關的靜脈異常疾病之一¬-下肢靜脈曲張。在這項研究中使用RNA-seq技術研究患者和對照組靜脈樣本中的全轉錄體差異表現。將具有差異表現的基因進行電腦模擬分析，發現了這些基因群參與不同的生理功能網絡，並且發現HAS2基因在調控這些途徑中扮演關鍵角色，當抑制HAS2表現會導致靜脈靜脈血流擴張，因此HAS2可能是負責調節靜脈組織中血管生成、細胞粘附、血管損傷和碳水化合物代謝的轉錄網絡。 整體而言，本論文在心血管疾病研究中使用了新穎的基因體學研究方法，新發現了與台灣心房顫動遺傳風險顯著相關的常見和罕見遺傳變異（包括拷貝數變異和單核苷酸變異）。此外我們使用全轉錄體差異表現分析研究靜脈曲張的分子機制，不僅如此，我們將研究的新發現利用分子生物學方法在動物和細胞模式中進一步獲得驗證。期望透過這樣的研究方法，使得研究人員對於疾病機制有更多的理解，並且尋找可能的治療方法，或是減緩疾病的進程甚至預防疾病的發生。

The Human Genome Project attempted to obtain knowledge about the whole human genome, which thereby leads to the development of novel diagnostic assays, targeted therapies and prediction of the onset, severity and progression of diseases. This has been made possible by many high throughput technologies such as microarray and next-generation sequencing (NGS). In this work, these methods were used to identify susceptibility gene(s) understand the mechanisms of two cardiovascular diseases: atrial fibrillation (AF) and varicose vein (VV). This work first focused on AF, the most common form of sustained cardiac arrhythmia. Previous genome-wide association studies (GWASs) have identified common single nucleotide polymorphisms (SNPs) associated with risk of AF. In the human genome, copy number variations (CNVs) are also known to contribute to disease susceptibility, but its role in AF risk had never been addressed before. Using a genome-wide multi-stage approach to identify AF susceptibility CNVs, a common CNV in the first intron of KCNIP1 gene was identified strongly associated with AF in the Taiwanese populations. KCNIP1 was physically associated with potassium Kv channels and modulates atrial transient outward current in atrial myocytes, and over-expression of KCNIP1 caused inducible AF in zebrafish. This is the first time that a common CNV in the human gene was associated with AF, pointing to a functional significance. AF increases the risk of thromboembolic stroke. Therefore, the next study sought to identify the CNVs determining the AF-related thromboembolic stroke. Again, using a genome-wide approach, some deletions and amplifications in chromosomal regions were identified to be significantly associated with AF-related stroke in the Taiwanese population. Specifically, deletions in GNB1, PRKCZ, and GNG7 genes related to the alpha-adrenergic receptor signaling pathway may play a role in determining the risk of an AF-related stroke. The genetic variability of common risk variants explains only a part of the genetic predisposition to AF. Rare variants missed by GWAS might also contribute to the genetic risk of AF. Hence, for the next work, an extreme-trait design was used to sequence carefully selected probands with extreme AF phenotypes and their unaffected parents to identify rare de novo variants. Based on the hypothesis that common and rare variants may co-locate in the same disease susceptibility gene, NGS was used to sequence the nine published AF susceptibility genes by GWAS. A novel mutation in the transcriptionally active enhancer region of the PITX2 gene was identified. Functional analysis showed that this mutation significantly down-regulated PITX2 expression in atrial myocytes, which might be implicated in the mechanism of AF. Lastly, this work focused on lower extremities VV. RNA-sequencing technology and pathway analysis were used to study the global mRNA expressional change in the venous samples of diseased and control patients. Among these significantly differentially expressed genes, HAS2 was found to play a pivotal role in governing various important VV related genes and pathways. Finally, HAS2 knockdown in zebrafish resulted in dilated veins with the static venous flow, implicating that downregulation of HAS2 may underlie the mechanism of VV. In conclusion, this work implemented innovative genomic procedures in the genetic studies of AF and VV, and provides the platform for future studies to better understand the mechanisms of human cardiovascular diseases.