學習語言前非症候群感音神經聽障病因的探討

Study of the etiology of prelingual non-syndromic sensorineural deafness

楊建洲(Jiann-Jou Yang)

指導教授 ： 李宣佑 教授

中山醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00112

摘要

聽障可因遺傳基因突變或環境因素，或兩者兼之引起的。約有千分之一嬰兒在出生時罹患重度聽障，在已開發國家約有60%個案是遺傳因素，學習語言前聽障是由於許多單基因突變造成的，目前已知有59個基因的突變會導致聽障，各基因的致病機制不盡相同，非常複雜。然而除了我們實驗室先前的研究外，對於台灣地區學習語言前非症候群感音神經聽障成因並不是很清楚，本研究的主要目的是探討耳蝸內影響離子濃度的恆定性和毛細胞stereocilia功能之相關基因(PDS、Cx26、Cx30、Cx43和r Cx43)突變與造成聽障的致病機制。藉由分析正常聽力和聽障的人來建立一個台灣地區這些基因的多形性和遺傳性聽障成因之資料庫，以瞭解這些基因突變在學習語言前非症候群感音神經聽障所佔的比例，可作為聽障遺傳諮詢之參考；另外藉由這些基因功能的探討將可更清楚瞭解這些基因的作用機制，以利於聽障之預防或未來基因治療之發展。
PDS基因的突變會造成Pendred syndrome或伴隨著 enlargement vestibular aqueduct (EVA) 和 Mondini dysplasia的非症候群遺傳性聽障。在我們實驗，先前在10位臨床上具有前庭導管擴大及耳蝸發育不全的非症候群語言學習前感音神經性聽障兒童共發現三個PDS基因(IVS7-2A>G、IVS16-6G>A及IVS15+5G>A)的突變點，其中以IVS7-2A>G所佔的比例最高。在本研究中我們利用haplotype分析攜帶IVS7-2A>G突變的家族成員，結果我們發現在PDS基因上的這突變點是來自一個共同的祖先。
connexins家族所構成的gap junction對於耳蝸內離子的?琠w性(ionic homeostasis)有很大的影響，因此connexin在耳蝸的表現及功能的探討為相當重要的一環。首先我們利用免疫組織化學染色法、雷射細胞擷取技術和RT-PCR等技術來探討老鼠耳蝸內Cx26 (GJB2)、Cx30 (GJB6)和Cx29 (GJE1)的分布情形，結果發現此三種蛋白質都有表現在老鼠的spiral limbus、spiral ligaments、organ of Corti 和stria vascularis。同時我們在共同免疫染色的實驗中發現Cx29(GJE1)、Cx26(GJB2)或Cx30(GJB6)都有共同表現在同一個區域，這結果暗示著Cx29(GJE1)和Cx26(GJB2)及Cx26(GJB2)在聽力的形成的過程中可能扮演著重要的角色。
另外我們分析120位聽力正常人及260位語言學習前聽障患者和部分家屬的血液DNA樣本，建立了台灣地區這4個聽障相關基因(Cx26、Cx30、Cx43和r Cx43)多型性(polymorphrisms)和突變的資料庫。結果發現在這四個基因所佔的比例為21.54 % (56/260)，其中以Cx26基因所佔的比例最高為10.76% (28/260)。13個突變點被發現在這4個基因(Cx26、Cx30、Cx43和r Cx43)，包括4個先前已經被報告過的Cx26錯意突變(235delC、299-300delAT、 368C>A、 and 551G>A)、3個新的錯意突變[Cx30 (119C>T)、Cx43 (976C>T) 和 r Cx43 (205T>C)]、1個新的缺失突變[r Cx43 (932delC)]和5個silent mutations [ 261A>T and 396G>A (Cx30)、 624C>T and 717G>A (Cx43)、 873C>T(r Cx43)]。
爲了進一步了解這些Cx gene family突變點對聽障的影響，我們建立了一個細胞模式來研究gap junction的功能，我們分析了Cx26基因的三個突變點(235delC、299-300delAT和R184Q)和兩個先前被認為突變點而在我們的研究中可以在正常人中發現的變異(V37I和V27I/E114G)。同時我們也分析了Cx30基因中的兩個突變點(A40V和P87P)。結果發現Cx26基因235del C、299-300del AT及R184Q轉殖的HeLa細胞，其突變蛋白都堆積在Golgi body並無法被送到細胞膜形成gap junction。相對的V37I和V27I/E114G轉殖的HeLa細胞，其變異蛋白可以和正常的Cx26蛋白一樣運送到細胞膜形成gap junction。另外在Cx30基因的研究我們發現A40V轉殖的HeLa細胞，其突變蛋白也都堆積在Golgi body。而P87P轉殖的HeLa細胞其蛋白質則可被送到細胞膜。
在co-transfect Cx26和Cx30重組基因實驗中發現Cx26和Cx30可共同表現在相同的位置。而且在基因突變分析中我們發現Cx26R184Q和Cx30A40V都是heterozygous的突變，因此我們將探討這兩個突變點對正常的Cx26和Cx30的蛋白有何影響。結果我們發現Cx30A40V的突變，會同時影響到正常的Cx30和Cx26蛋白無法運送到細胞膜而堆積在Goligi body。而Cx26R184Q只有影響到正常的Cx26運送功能，相反的Cx26R184Q突變蛋白表現位置會被Cx30正常的蛋白改變，由Golgi body送到細胞膜上。另外我們建立Tet-On蛋白表現系統(Tet-On inducible expression system)，是利用一個質體上可同時表現兩種不同的基因來進行上述的交互作用研究。
綜合以上的結果，我們已經建立了Cx26、Cx30、Cx43和r Cx43基因在台灣地區遺傳性非症候群性聽障的突變和多型性的資料庫，並且對於Cx26和Cx30的突變在功能上的所造的影響也有進一步的了解。另外這些資料庫的建立和功能分析系統的建立將可幫助對於多種聽障基因之作用機轉的瞭解，將有助於我們釐清造成學習語言前非症候群聽障的真正致病機轉。

關鍵字

遺傳性聽障 ； 功能研究 ； 動物模式 ； 免疫細胞染色法 ； 免疫組織染色法 ； 雷射細胞擷取技術

並列摘要

Hearing loss caused by gene mutations and environmental factors, are a common sensory disorder in the human population. Environmental factors leading to hearing loss include acoustic trauma, ototoxic drugs, and bacterial and viral infections. The incidence of congenital hearing loss is estimated at 1 in 1000 births, of which approximately 60% cases are attributed to genetic factors in the developed countries. To date, 59 auditory genes have been identified, some of which are those involved in K+ recycling and maintenance. This coincides with the notion that sensory transduction in the cochlea and the vestibular labyrinth depend on the recycling and maintenance of K+. However, in Taiwan, the data of these genes are still insufficient; therefore, further research is worthy to conduct. The overall goal of this thesis is to establish the genetic basis for the screening, diagnosis, and pathogenesis studies of prelingual non-syndromic sensorineural deafness in Taiwan.
Previously study in our laboratory, one of three mutations, IVS7-2A>G, IVS16-6G>A or IVS15+5G>A, was identified in the PDS gene in each patient association with EVA and Mondini dysplasia and the IVS7-2A>G mutation was the most common of the PDS mutations. In this study, haplotype analysis showed a significant haplotype shared among the family members carrying IVS7-2A>G mutation, suggesting that they may be derived from a common ancestor.
Connexins (Cx), a large family of membrane proteins, are key components of gap junction channels. These channels are critical intercellular pathways through which ions or small molecules are passed, regulating a variety of physiological and developmental processes. Multiple types of connexins (Cxs) products are found by immunolabeling in the mature cochlea. In current study, we performed an immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) analysis to elucidate whether the gap junction protein, including Cx26 (GJB2), Cx30(GJB6) and Cx29(GJE1) are localized in the adult mouse and rat cochlea. Our results reveal Cx26(GJB2), Cx30(GJB6) and Cx29(GJE1) was detected in the spiral limbus, spiral ligament, organ of Corti and stria vascularis using immunohistochemistry (IHC) analysis and RT-PCR. Moreover, we also found Cx29 (GJE1) with either Cx26(GJB2) or Cx30(GJB6) co-localization in same region in co-immunolabeling study. These results imply Cx29(GJE1), as well as Cx26(GJB2) and Cx30(GJB6) play a significant role in the physiology of hearing, most probably by participating in the recycling of potassium to the cochlea endolymph.
Simultaneously, the contribution of Cx gene family (Cx26, Cx30, Cx43 and r Cx43) mutations to the hearing loss in 260 nonsyndromic deafness from Taiwan was determined in this study. The prevalence of Cx gene family mutations in this study was 21.54 %, and, of those, the single gene with the highest prevalence of mutation was the Cx26 gene (10.77%, 28/260). Thirteen different deafness-causing Cx gene family (Cx26, Cx30, Cx43 and r Cx43) mutations were found in this study. Four of these mutations 235delC, 299-300delAT, 368C>A, and 551G>A of Cx26 have been described before. Three novel missense mutations, 119C>T, 976C>T, and 205T>C, were identified in Cx30, Cx43 and r Cx43 gene, respectively. One novel deletion mutation, 932delC, was identified in the r Cx43 gene. Five novel silent mutations, 261A>T and 396G>A, 624C>T and 717G>A, 873C>T, were identified in the Cx30, Cx43 and r Cx43 gene.
In functional study, three mutations and two polymorphisms in Cx26 gene and two mutations in Cx30 gene associated with nonsyndromic deafness were investigated using approaches of biochemical and cell biological. Of the Cx26 gene, V37I and V27I/E114G variants resulted in a protein that was localized to the membrane. In contrast, the 235delC, 299-300delAT, and R184Q mutations resulted in proteins with impaired trafficking and localized in the Golgi body using co-immunolabeling study. Of the Cx30 gene, the product of the A40V mutation appeared entirely intracellular, clustered in large perinuclear vesicles, without evidence for membrane targeting. The immunolocalisation of the P87P silent mutation gave similar results as that of Cx30WT, with junctional plaques at zones of cell-to-cell apposition.
Here we also create a method, Tet-On indicuble expression system, to express active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells. The result reveals both Cx26 and Cx30 proteins were co-localized at points of contact between adjacent cells and were consistent with above observed in studies using either immunolabeling of antibody or fluorescent-protein-tagged. In addition, we found A40V of Cx30 missense mutation have dominant negative and trans-dominant effect on both Cx30 and Cx26. The R184Q of Cx26 has a dominant negative effect on Cx26. In contrast, R184Q of Cx26 mutation protein in localization of cell can be rescued from Goligi body to membrane by Cx30 protein.
In summary, our study provides information for understanding the importance of genetic factors in nonsyndromic deafness of Taiwanese and that may be of use in the improvement of genetic diagnosis of hearing loss in Taiwan and first provide a comprehensive and detailed pattern of Cx26, Cx30, and Cx29 gene expression in the mouse and rats cochlea. Further, it would be interesting to find out how these Cxs exert their functions in the potassium ions recycling of the cochlea and in maintaining the high concentrations of potassium ions in the endolymph.

並列關鍵字

hearing loss ； deafness ； nonsyndromic ； PDS ； connexin ； functional study ； animal model ； Tet-on system ； immunohistochemistry ； Laser Capture Microdissection

參考文獻

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