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標題: | 應用次世代定序技術建立遺傳性耳聾基因檢測平台 Application of Next Generation Sequencing to Establish the Genetic Testing Platform for Hereditary Hearing Impairment |
作者: | Yin-Hung Lin 林盈宏 |
指導教授: | 陳沛隆 |
關鍵字: | 聽損,耳聾基因,基因檢測,次世代定序,生物資訊,突變點偵測, hearing impairment,deafness gene,genetic testing,next generation sequencing,bioinformatics,mutation detection, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 遺傳性聽力損失是常見的遺傳性疾病,目前已知有超過 100 個基因與聽損有關。基因檢測有助於正確診斷聽損,並精確預估病人預後。然而,傳統的 Sanger 定序檢測技術,均僅能檢測已知且較常見的耳聾基因,因此聽損病人進行基因檢測時,即使是家族史非常明顯的個案,仍有三分之二的聽損家族無法確立基因診斷。近年來,由於次世代定序技術的發展,使未能檢測出常見耳聾基因變異,但家族史明顯之多病例家族,得以同時檢測大量的耳聾基因,解決此檢測技術上之難題。
本研究由台大醫院耳鼻喉部所建立之特發性聽損研究世代中,選取十個未能檢測出<i>GJB2</i> 基因、 <i>SLC26A4</i> 基因與粒線體 12S RNA 基因等常見耳聾基因變異,但家族史明顯之多病例家族,利用次世代定序技術,針對已知 129 個耳聾基因,進行基因變異掃描。我們在 Linux 作業系統 Ubuntu 的環境下,使用BWA、Picard、GATK、ANNOVA、IGV 等軟體建立生物資訊分析流程。其後進行資料過濾,包括:基因變異於族群中之對偶基因頻率須小於百分之五、PolyPhen-2 及 SIFT 等軟體所預測之胺基酸變異致病性分數大於 0.95、直接定序確認、家族譜分析及胺基酸基之演化保留分析等,以釐清所發現之基因變異是否即為導致聽損之真實突變。 我們在十個多病例聽損家族中確認三個導致聽損之基因變異,包括:在一個體染色體顯性遺傳模式的家族中發現 <i>GATA3</i> 基因序列有一個核苷酸的缺失 (c.149delT),導致 p.Phe51LeufsX144 變異、在一個體染色體顯性遺傳模式的家族中發現 <i>POU4F3</i> 基因序列一個核苷酸的改變 (c.982A>G),導致 p.Lys328Glu 變異、以及在一個 X 染色體隱性遺傳模式的家族中發現 <i>POU3F4</i> 基因序列有一個核苷酸的插入 (c.340_341insG),導致 p.Ala116GlyfsX77 變異。此三個變異皆為未曾於文獻上發表之新突變位點。 本研究之結果顯示,次世代定序技術可應用於發現較罕見、以及未知的耳聾基因變異,而可克服當前耳聾基因檢測之技術瓶頸。此一技術之成熟運用,加諸未來可預見其成本將迅速降低,應可成為未來耳聾基因診斷之有力工具,甚至有助於發現新的耳聾基因,進而更深入瞭解聽覺生理及聽損之發生機制。 Hereditary hearing impairment (HHI) is a genetically heterogeneous condition with more than 100 deafness genes identified thus far. Despite the clinical utility of genetic diagnosis to address idiopathic sensorineural hearing impairment (SNHI), the current strategy for screening mutations via Sanger sequencing suffers from the limitation that only a limited number of DNA fragments associated with common deafness mutations can be genotyped. Consequently, a definitive genetic diagnosis cannot be achieved in more than two-thirds of families with discernible family history. Recently, the development of next generation sequencing (NGS), which generates millions of DNA sequence reads in parallel during a single experimental run, offers an alternative approach to genetic testing for hereditary hearing impairment. To investigate the diagnostic utility of NGS, we applied the NGS technique to ten multiplex families with idiopathic SNHI, in which common deafness mutations had previously been tested without positive results. We used NGS to sequence 129 known deafness genes, and applied BWA, Picard, GATK, ANNOVAR, and IGV for bioinformatics analyses. Criteria for data filtering included: allele frequencies <5%, both PolyPhen-2 and SIFT scores >0.95, Sanger sequencing confirmation, segregation pattern compatibility, and evolutionary conservation of amino acid residues. Of the ten multiplex families with idiopathic SNHI, we identified the causative genes/variants for three families. In the proband of an autosomal dominant family with nine affected members, we found a novel single nucleotide deletion mutation, c.149delT, in <i>GATA3</i>. In the proband of an autosomal dominant family with five affected members, we found a novel missense mutation, p.Lys328Glu (c.982A>G), in <i>POU4F3</i>. In the proband of an X-linked recessive family with four affected members, we found a novel single nucleotide insertion mutation, c.340_341insG, in <i>POU3F4</i>. In conclusion, we identified three novel variants as the causative mutations in three families using a NGS-based genetic test. NGS allows genetic diagnosis in multiplex families with idiopathic SNHI by detecting mutations in relatively uncommon deafness genes. With constantly improved technology and drastically reduced cost, NGS can be a powerful tool for achieving molecular diagnosis of hearing impairment and identifying new deafness gene. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56709 |
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