2016-01-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/681291摘要：一、研究背景及目的感音型聽損係人類最常見的感覺神經病變，也係新生兒最常見的先天缺陷。最近的研究證實，基因變異是導致感音型聽損的重要成因之一。在已開發國家，約2/3的聽損病童可歸因為遺傳因素，而目前學界亦已發現超過100個基因與遺傳性聽損的發生有關。針對重度感音型聽損而助聽器效果不彰之病人，「人工耳蝸」植入係目前治療上之最佳選擇。然而，人工耳蝸植入手術及術後聽語復健需耗費鉅額社會成本，且其成效變異頗大，若能於植入手術前即能預測病人之預後，不僅可幫助病人、家屬及醫療人員於術前建立合理期待，亦有助於醫療資源之有效分配。過去的研究雖已發現可能影響人工耳蝸植入效果之諸多因素，惟目前仍欠缺實用性指標，以在術前精準預測人工耳蝸植入之預後。近年來由於「大規模平行定序」技術的發展，突破了傳統基因檢測方法僅能掃描少數基因之技術瓶頸，而顯著改善吾人現有之基因檢測工具。本研究第一部份之目的，即在應用大規模平行定序技術建立人工耳蝸植入病人之基因檢測平台，並分析可否藉由完整之基因檢測結果，精準預測人工耳蝸植入病人之預後。另一方面，雖然吾人已知基因於聽損之發生扮演極重要的角色，卻囿於無法直接於人體內耳進行研究，乃有賴動物模式的建立。過去幾年的研究證實，同為哺乳類動物的基因轉殖鼠，適為研究遺傳性聽損極佳之動物模式。本研究之第二部分，吾人將針對第一部分所發現與不良預後有關之基因突變，應用TALEN基因體編輯技術培育基因轉殖鼠，進而研究其致病機制及治療方針，俾將來臨床上可用於改善人工耳蝸植入效果。二、研究方法本研究分為「釐清基因診斷與人工耳蝸植入預後之關連」及「利用基因轉殖鼠研究致病機制及治療方針」等兩部分：1. 釐清基因診斷與人工耳蝸植入預後之關連針對300名已接受人工耳蝸植入手術之病人，依序進行：常見耳聾基因突變檢測、大規模平行定序掃瞄132個已知耳聾基因、及全基因體exome掃瞄，以確立基因診斷。其後，分析基因診斷與人工耳蝸植入預後之關連，以釐清影響人工耳蝸植入效果之關鍵基因因素。2. 利用基因轉殖鼠研究致病機制及治療方針針對前部分研究所發現與不良預後有關之基因突變，應用TALEN基因體編輯技術培育基因轉殖鼠。其後進行一系列的內耳生理及分子生物研究，包括：聽覺及平衡功能評估、內耳形態研究、及轉譯體研究等，以釐清致病機制；並研究基因治療、siRNA治療、及神經滋養素(neurotrophins)等治療方式，是否可用於促進聽覺神經元再生，進而改善人工耳蝸植入之效果。三、預期成果1. 於第一部分「釐清基因診斷與人工耳蝸植入預後之關連」之研究，吾人將應用大規模平行定序技術建立人工耳蝸植入病人之基因檢測平台，以助於病人基因診斷之確立，釐清其病因。同時，藉由分析基因診斷與人工耳蝸植入預後之關連，可建立預測模式，根據完整的基因檢測結果，精準預測人工耳蝸植入病人之預後。所有研究人員亦可從中獲得有關大規模平行定序技術之操作與分析、遺傳性疾病診斷工具之研發等相關技術及方法。2. 於第二部分「利用基因轉殖鼠研究致病機制及治療方針」之研究，吾人將培育帶有不良預後相關突變之基因轉殖鼠，並釐清此等突變導致人工耳蝸植入預後不佳之機制，進而研發改善人工耳蝸植入效果之治療方式。同時，所有研究人員可以從中習得基因體編輯、基因轉殖鼠培育、小鼠內耳分子生物及生理研究、基因治療、及siRNA治療等相關技術及方法。關鍵詞：遺傳性聽損，人工耳蝸，大規模平行定序，基因轉殖鼠，基因體編輯<br> Abstract: Background and ObjectiveSensorineural hearing impairment (SNHI) is the most common birth defect and sensorineural disorder in humans. It is estimated that in developed countries, genetic causes of SNHI can be found in >2/3 of affected children, i.e. hereditary hearing impairment (HHI). To date, more than 100 genes have been identified to cause HHI. For children with severe to profound SNHI, cochlear implant (CI) is currently the best treatment modality. However, the performance outcomes vary significantly among CI recipients, and unfortunately, there is no method to identify poor CI performers prior to surgery. Given that a CI bypasses cochlear hair cells and directly stimulates auditory neurons, we hypothesize that CI performance may be affected by the site of the deafness-causing mutations and that genetic deafness would adversely impact CI performance if the pathogenesis involves the auditory nerve or central auditory pathway as opposed to the cochlea. The purpose of Part I of the present project is to investigate genetic factors which may influence the CI outcomes by using the massively parallel sequencing (MPS) technique, which, by sequencing a large amount of DNA fragments simultaneously, has proven to be a powerful tool in addressing the enetically heterogeneous HHI. Despite the clinical significance of genetics in HHI, the study of HHI in humans is limited by the inability to perform in vivo experiments. The formidable similarities between the human and mouse inner ears make transgenic mice an excellent model to address HHI in humans. In Part II of the present project, we plan to establish transgenic mouse models harboring mutations related to poor CI outcomes. We will investigate the pathophysiology and explore possible therapeutic strategies in these mouse models. These results will provide insight into how to improve the performance outcomes in the CI recipients.Material and MethodsPart I. Correlating genetic diagnoses to cochlear implantation outcomes in the patients Genetic diagnoses will be achieved in a prospective cohort composed of ~300 CI recipients by a series of genetic examinations, including directing sequencing of common deafness genes, MPS of 132 known deafness genes, and whole exome sequencing. The genetic diagnoses will then be correlated to the CI outcomes to identify the deterministic factors and establish the prediction model.Part II. Investigating the pathology and therapeutic strategies in transgenic mouse models After generating transgenic mouse models harboring genetic mutations related to poor CI outcomes, we will investigate the pathophysiology by characterizing the audiovestibular phenotypes and performing transcriptomic studies in the animals. Then we will explore the utility of therapeutic strategies, including gene therapy, siRNA therapy and neurotrophin therapy, in restoring the auditory physiology and histology which may contribute to more favorable CI outcomes.Anticipated resultsPart I. Correlating genetic diagnoses to cochlear implantation outcomes in the patients A comprehensive genetic examination platform for CI patients will be established and applied to develop a prediction model for CI outcomes. The pathogenetic mechanisms underlying the genetic diagnoses, as well as their associations with the CI outcomes, will be clarified. All the staff will learn the techniques and study designs of MPS in addressing genetic disorders.Part II. Investigating the pathology and therapeutic strategies in transgenic mouse models Transgenic mouse models segregating genetic mutations with poor CI outcomes will be generated. The pathogenetic mechanisms how certain genetic mutations lead to poor CI outcomes will be elucidated. These mouse models will become powerful tools in the future for developing potential strategies which may improve the CI outcomes. All the staff will learn the skills of the TALEN genome editing technique, molecular genetic studies in mouse inner ears, functional genetic studies, as well as gene therapy and siRNA therapy.(Keywords): hereditary hearing impairment, cochlear implant, massively parallel sequencing, transgenic mouse, genome editing遺傳性聽損人工耳蝸大規模平行定序耳聾基因hearing impairmentcochlear implantationdeafness genesmassively parallel sequencing