利用次世代基因定序方法篩選眼角膜翳狀贅肉的外顯子基因體可能之基因變異序列

Abstract

研究背景及目的：眼角膜翳狀贅肉是一種常見的眼表疾病，目前統計全球約有10.2%的患病人口，在紫外線照射強烈的低緯度台灣南部40歲以上成人盛行率更達到25.2%。眼角膜翳狀贅肉病灶發生在角膜及結膜交界處，其主要病理變化包含其表皮細胞不正常增生和異常的纖維化血管結締組織增生，一開始病灶只局限於角膜的邊緣輪部，當病灶慢慢長大時會蔓延至角膜中間，產生散光增加，嚴重者增生組織更會遮蔽視線，使視力嚴重受損。由於眼角膜翳狀贅肉本身也會有炎性反應，而且組織隆起造成淚液分布不均和堆積，會讓患者有眼部充血、刺痛、溢淚等不適感。目前眼角膜翳狀贅肉唯一治療方式只有手術切除，進行切除手術時，常會合併羊膜、結膜移植或同時使用抗癌藥物來避免手術治療後的復發。由於其有手術後復發率高的特性，加上在許多組織病理上也有一些半腫瘤組織變化，因此先前的研究都朝向和腫瘤相關的細胞學或基因變異的研究，但即使已進行許多的研究，它的致病機轉仍然不是很明確清楚。眼角膜翳狀贅肉好發於熱帶或亞熱帶紫外線照射強烈的區域，因此除了紫外線因子流行病學調查研究外，先前的研究也大都針對紫外線造成的基因變異，因而造成表現如腫瘤般特性的觀察來探討其生成機轉。在文獻上有幾個可能的基因變異已經被發現和眼角膜翳狀贅肉生成有關，例如：XRCC1、hOGG1及TP53，很不幸的是這些基因變異在後來其他研究中重複性很低，而且都是選擇性的針對某一個基因來進行研究，因此其正確性也較會被存疑。在基因體研究及次世代定序技術發展之後，開啟了一個新的研究模式。本研究的目的是希望利用次世代高通量基因體定序方法分析眼角膜翳狀贅肉的全外顯子基因體定序 (whole exomic sequencing )，搭配熱點基因資料庫 (hot-spot genes database)來建構出眼角膜翳狀贅肉可能的致病基因模式。 研究方法：在經人體試驗委員會核准，手術前經病人簽署同意書後，眼角膜翳狀贅肉的組織檢體在病人手術中被收集。檢體進行DNA萃取，經品質和量的驗證後，經過Ion AmpliSeq™ Exome solution外顯子基因體增幅 (exomic enrichment ) 後，用Ion Proton Torrent晶片定序儀進行全基因體分析，定序資料使用以下分析軟體：FastQC進行品質管制、TMAP進行序列比對組合排列、SAMtool及IGV進行定序檔案的轉換、TVC及GATK尋找篩選特異變異點、IR進行基因序列的註解，最後搭配熱點基因資料庫進行突變點基因分析。 結果：共有五個檢體符合定序品質條件，每個樣本的定序輸出量為57.7Mb，定序深度達129倍以上，變異資料比對癌症熱點基因庫後，從所有的變異位點中共找出7個錯義突變位點 (missense 基因突變 )：SYNE1、TP53、RET、STK11、CEBPA、TNFAIP3、IL7R。有三個變異位點是五個病人都有的：PDGFRA、SYNE1、NOTCH1。 結論：我們找出七個錯義突變位點的基因，為可能的驅動突變基因位點。還有三個為所有樣本都有的變異位點。然而，其細胞生物交互作用以及生物路徑的分析未來還需要更完整的證實。 關鍵字：眼角膜翳狀贅肉；次世代定序；外顯子基因體定序；熱點基因資料庫。

Purpose：Pterygium is a common ocular surface disease occurring in 10.2% population in the world and 25.2% Southern Taiwanese adults. It will cause local inflammation and local irritation around the lesion, and vision would be threatened when the pterygium encroaches over the surface of cornea. The most interesting and devastating characteristics of pterygium is its high recurrence rate after surgery. Due to this tumor-like characteristic, its pathogenesis is believed to be associated with ultraviolet irradiation and consequently genetic variations. For example, some susceptibility genes, ex. XRCC1, hOGG1 and TP53, were found to be associated with pterygial formation. Unfortunately, the causing mechanism is still unclear at present after many studies in decades because these genetic variations cannot be repeated in the subsequent studies. With the advent of modern genetic analysis method developed recently, we proposed to develop a reasonable and a novel approach to study the genetic variations of pterygium. In current study, we adopted the whole exomic sequencing resulting from next generation sequencing and analyzed the sequencing variations integrating with hot-spot genes database. Method：Pterygial specimens were collected from 5 patients during surgical excision. These 5 samples were subjected to genomic DNA extraction. After exomic enrichment by Ion AmpliSeq™ Exome solution, the DNA library was used for exomic sequencing with Ion Proton Torrent Chip platform. Raw sequencing data were analyzed with the following programs steps by steps：FastQC for quality control, TMAP for mapping and assembling/alignment, SAMtool and IGV for file conversion, TVC and GATK for calling variants, IR for annotation. The sequencing variants results were integrated with hot-spot cancer genes database and CHB samples from HapMap phase2 build 36 release 28 for evaluation of possible genetic variations. Result：There were 5 samples undergone exomic sequencing. Each sample went through a 57.7Mb output and the depth was achieved over 129x coverage. After being integrated with hot-spot genes database, we found 7 missense mutations：SYNE1、TP53、RET、STK11、CEBPA、TNFAIP3、IL7R. We also found 3 mutations owned by all samples：PDGFRA、SYNE1、NOTCH1. Conclusion：These 7 missense mutations could be driver mutations. All samples share 3 common SNPs. The biological interactions and pathway analysis should be validated in the future.