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標題: | 應用次世代定序技術進行遺傳性聽力缺損之基因診斷 Genetic Diagnosis of Hereditary Hearing Impairment via Next-generation Sequencing |
作者: | Yin-Hung Lin 林盈宏 |
指導教授: | 陳沛隆 |
關鍵字: | 遺傳性聽力缺損,聽損基因,基因檢測,次世代定序,生物資訊, hereditary hearing impairment,deafness gene,genetic testing,next-generation sequencing,bioinformatics, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 感音型聽損是人類最常見的感覺缺陷。根據統計,每 1000 名兒童中約有三人患有感音型聽損。其中,百分之六十的病患可歸因於遺傳因素,稱作遺傳性聽損。遺傳性聽損是一種基因異質性非常高的疾病,目前已知有超過 100 個基因,5000多個變異點位與聽損有關。過去研究發現,利用 Sanger 定序技術,針對常見的聽損基因做檢測,只有約三分之一的聽損家族能夠達成診斷。剩下約三分之二的家族,受限於聽損基因的數量與 Sanger 定序所需耗費的大量人力與成本,造成分子診斷上的困難。近年來,次世代定序技術的興起突破了此一瓶頸,使得聽損家族能夠接受更完整的基因檢測。
本研究中,我們利用次世代定序技術,建立一個包含 159 個聽損基因的檢測平台。我們使用 Illumina MiSeq 定序儀器,產生讀長為 300 個鹼基對的雙端定序。我們建立了自動化的生物資訊分析流程並整合相關的疾病資料庫,進行定序結果的分析與判讀。利用此一次世代定序聽損基因檢測平台,我們探討了數個目前尚未解決的問題,簡述如下: (一) 診斷常見聽損基因檢測未能達成診斷之病人 我們針對 246 個未能經由常見聽損基因檢測達成診斷之家族,進行次世代定序基因檢測。我們確定了 101 個家族共 24 個致病基因,診斷率為 41%。我們在一個聽損家族中發現 GATA3 基因 c.153delT 變異。在另一個聽損家族中,我們發現 POU4F3 基因 p.Lys328Glu變異。這兩個變異皆為未曾於文獻上發表的新變異點位。同時,我們進行了功能性的研究探討這兩個新發現的基因變異之致病原因與機轉。 (二) 診斷人工耳蝸植入成效不彰之病童 人工耳蝸是目前重度與極重度聽損病童之治療方式。然而,人工耳蝸植入的效果在不同的病童間差異很大。我們選取了 12 個人工耳蝸植入成效不彰的病童與 30 個植入效果好的病童進行次世代定序基因檢測。我們發現人工耳蝸植入成效不彰的病童帶有DFNB59 基因 p.G292R 變異,可作為人工耳蝸植入成效的標記。 (三) 診斷不帶有兩個 SLC26A4 常見基因變異的大前庭導水管症候群病人 大前庭導水管症候群是常見的內耳畸形,主要由同時帶有兩個 SLC26A4 基因變異所導致。我們選取 50 個不帶有兩個 SLC26A4 常見基因變異 (c.919-2A>G 與 p.H723R) 的家族進行次世代定序基因檢測,我們在 34 個家族當中找到兩個 SLC26A4 基因變異,包括一個 SLC26A4 基因的大片段缺失。我們發現另外兩個家族帶有 EYA1 基因變異,達成了 72% 的診斷率。 (四) 診斷只帶有單一GJB2 基因變異的隱性遺傳模式聽損家族以及基因型和表現型不吻合之 GJB2 基因變異病人 大多數的GJB2 基因變異會導致隱性遺傳模式的聽損。但臨床上發現有部分病人只能找到一個 GJB2 基因變異,造成診斷上的困難,此外,部分帶有導致輕度到中度聽損的 GJB2 基因 p.V37I 變異病人呈現重度與極重度的聽損,成為診斷上的難題。我們選取了 16 個只帶有一個 GJB2 基因變異的病人與 22 個 GJB2 基因 p.V37I 變異的病人,進行次世代定序基因檢測。我們達成了七位只帶有一個 GJB2 基因變異的病人的基因診斷,包括五位病人帶有其他的聽損基因致病點位,一位病人帶有 GJB2 基因剪接位點上的變異點位,與一位為 GJB2 基因 c.235delC 變異鑲嵌型的病人。在 GJB2 基因 p.V37I 變異的病人當中,我們發現一位病人帶有 TMC1 的基因變異,解釋了其重度與極重度聽損的原因。 Sensorineural hearing impairment (SNHI) is the most common sensory deficit in humans, affecting about three per 1000 children. More than 60% of these patients have a genetic cause (i.e. hereditary hearing impairment; HHI). HHI is extremely heterogeneous, with approximately 5000 variants in more than 100 genes reported as causative variants to date. Studies across different populations revealed that hotspots screening of common deafness genes (e.g. GJB2, SLC26A4 and the mitochondrial 12S rRNA) using Sanger sequencing could only achieve genetic diagnosis in ~1/3 hearing-impaired families. Recently, the advent of next-generation sequencing (NGS) has revolutionized the clinical evaluation of hearing-impaired patients by making comprehensive genetic testing possible. In this study, we established an NGS-based diagnostic platform for HHI. A capture-based panel was designed to target 159 deafness genes. Sequencing was performed by Illumina MiSeq which generated paired-end reads of 300 nucleotides. We developed an automated bioinformatics pipeline for analyzing the sequencing data. Disease and gene-specific databases have been curated and built to enable automatic matching. We have explored several unsolved problems of genetic basis of HHI as follows: (1) Diagnosis of patients without common deafness-causing variants We subjected 246 hearing-impaired families to our NGS-based diagnostic platform. We identified causative variants in 24 genes in 101 families, yielding a diagnostic rate of 41%. We identified a novel GATA3 pathogenic variant (c.153delT, p.Phe51Leufs*144) in one family. In another family, we identified a novel missense variant of POU4F3 (c.982A>G, p.Lys328Glu) which co-segregated with the deafness phenotype. We performed function studies for these two novel variants and explored the underlying mechanism. (2) Diagnosis of hearing-impaired children with poor cochlear implantation outcomes Cochlear implantation is currently the treatment of choice for children with severe to profound hearing impairment. However, the outcomes with cochlear implants (CIs) vary significantly among recipients. Twelve children with poor CI outcomes (the “cases”) and 30 “matched controls” with good CI outcomes were subjected to the NGS-based diagnosis platform. We identified DFNB59 p.G292R which might be associated with poor CI outcomes. (3) Diagnosis of enlarged vestibular aqueduct patients without bi-allelic SLC26A4 hotspots Enlarged vestibular aqueduct (EVA) is a common inner ear malformation caused mainly by bi-allelic SLC26A4 variants. We performed the NGS-based diagnostic platform in 50 families without confirmative results by screening two SLC26A4 hotspots (c.919-2A>G and p.H723R). We identified bi-allelic SLC26A4 pathogenic variants (including a large SLC26A4 deletion) in 34 families and EYA1 causative variants in two families, yielding a diagnostic rate of 72% (36/50). (4) Diagnosis of hearing-impaired patients with non-confirmative or genotype–phenotype-mismatched GJB2 pathogenic variants A significant percentage of patients segregate only one variant causing autosomal recessive SNHI in the GJB2. In addition, some patients with mild GJB2 variants such as p.V37I develop severe-to-profound SNHI. We performed NGS in 16 patients with mono-allelic GJB2 pathogenic variant and 22 patients with homozygous GJB2 p.V37I who had SNHI of varied severity. We confirmed the genetic diagnosis in seven patients with a GJB2 pathogenic variant. Of these, five patients had causative variants in other deafness genes; one patient had a splice site variant in the second GJB2 allele; and one patient had ~69% mosaicism for GJB2 c.235delC because of mosaic uniparental disomy. One patient homozygous for p.V37I had causative variants in TMC1, which might be responsible for the severe-to-profound SNHI. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68013 |
DOI: | 10.6342/NTU201800090 |
全文授權: | 有償授權 |
顯示於系所單位: | 基因體暨蛋白體醫學研究所 |
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