- 學位論文
應用次世代定序技術建立聽損基因檢測平台
Application of Next Generation Sequencing to Establish the Genetic Testing Platform for Deafness and Associate Syndrome
摘要
聽力損失是最常見的感覺系統缺陷，其中三分之二可歸因於遺傳因素。基因檢測除可幫助精確診斷聽損發生原因，做為預後之參考外，並可作為聽損個案及家族遺傳諮詢的依據。目前已知超過100個基因與聽損有關，在過去，我們以傳統Sanger定序方法檢測族群中常見之GJB2、SLC26A4與粒線體12S RNA聽損基因變異熱點，近年來，隨著次世代定序技術的發展，對於在熱點上無法找到確診原因之聽損個案，臺大醫院耳鼻喉部與基因醫學部提供了能同時檢測159個聽損相關基因之服務，解決技術上之難題，並帶領我們了解族群中多樣的聽損原因。 本研究於臺大醫院先前研究之成果上，建立一新的次世代定序平台，針對18個較常見之非症候群性聽損基因、6個瓦登柏格症候群 (Waadenburg syndrome) 與3個BOR 症候群 (Branchio-oto-renal syndrome)基因， 共27個聽損相關基因的外顯子部分進行基因變異掃描。分別選取在26個在GJB2和12個在SLC26A4此兩體染色體隱性遺傳基因之常見變異熱點上，僅發現單一變異而未能得到確診之個案進行檢測。過程中，我們使用BWA、Picard、GATK、ANNOVA、IGV等軟體進行生物資訊分析的流程，其後資料再利用基因變異於族群中的對偶基因頻率、PolyPhen-2及SIFT等軟體預測、Sanger定序確認家族分離模式等方式，釐清個案是否在上述基因中帶有致病變異。 結果我們在26個GJB2基因上僅發現單一變異而未能確診之個案中，確認了7位所帶有的致病變異，分別是4位帶有體顯性遺傳模式的MITF p.(Arg259*)、EYA1 p.(Gly135Ser)、POU4F3 p.(Pro164Arg)、GJB3 p.(Val84Ile)，以及3位體隱性遺傳的MYO15A p.(Ser1176Valfs*14):(Ser3417del)與MYO15A c.[4143-5C>A]:p.(Tyr1392*)分別形成的複合異型合子，以及在GJB2基因上發現未曾報導於文獻上的位點c.35dupG與已知的c.35delG所形成複合異型合子。在12個SLC26A4基因上僅發現單一變異而未能確診之個案中，確認了9位皆分布於SLC26A4基因上的致病變異，形成複合異型合子而造成聽力損失。此研究中，兩族群之診斷率分別達到27%與75%。 本研究結果顯示，此次世代定序平台可應用於常見基因熱點上已發現單一變異卻無法確診之病患，幫助其找到致病原因，並可望在未來降低次世代檢測之門檻、提升次世代定序應用頻率，成為聽損基因診斷之一大助力。
並列摘要
Hereditary hearing loss is the most common inherited sensory defect, affecting about 3-10 per 1000 children. It is estimated that in developed countries, genetic causes of HL can be found in at least two-thirds of prelingual cases, i.e. hereditary hearing impairment (HHI). Recessive variants in GJB2 and SLC26A4 are the most common genetic cause of sensorineural hearing impairment (SNHI). However, a significant percentage of patients segregate only one variant in the GJB2 and SLC26A4 on conventional Sanger sequencing, thus resulting in non-confirmative genetic diagnosis. We performed Next generation sequencing (NGS) of 27 deafness genes in 26 patients with only one recessive GJB2 variant and 12 patients with only one recessive SLC26A4 variant who had mild to profound SNHI. NGS confirmed the genetic diagnosis in seven patients with a recessive GJB2 variant. Of these, four patients had autosomal dominant causative variants in other deafness genes, which are MITF p.(Arg259*), EYA1 p.(Gly135Ser), POU4F3 p.(Pro164Arg), GJB3 p.(Val84Ile); two patients had autosomal recessive variants on MYO15A p.(Ser1176Valfs*14):(Ser3417del) and MYO15A c.[4143-5C>A]:p.(Tyr1392*)； one patient had been found a novel duplication leading a frameshift variant in the second allele with GJB2 making it a compound heterozygous. For the 12 patients with one recessive SLC26A4 variant, we had confirmed nine patients had the second allele with SLC26A4 variant. The diagnosis rate reached 27% and 75% in the two groups, respectively. Our results demonstrated the utility of NGS in clarifying the genetic diagnosis in hearing-impaired patients with non-confirmative GJB2 and SLC26A4 variants detected by conventional genetic examinations. All things considered, the benefit and the cost from execute the examinations, it seems reasonable to have the NGS-based panel targets 27 deafness genes as the next step to do in patients with non-confirmative GJB2 or SLC26A4 variants.