在語言學習前期非症候群感音神經性聽障中Cx30.3基因突變之研究

Connexin 30.3 mutation in prelingual nonsyndromic sensorineural deafness

周奎秀(Kuei- Hsiu Chou)

指導教授 ： 李宣佑

中山醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

大約有千分之一嬰兒在出生時或在小孩早期(即語言學習前時期)罹患重度聽障。導致聽障的原因有很多，包含遺傳基因突變、環境因素或兩者兼之。Connexins 為四個相似的穿膜蛋白，主要構成gap junction 通道於相鄰的兩個細胞間，並負責調控離子和小分子的交流與通透。到目前為止與聽障有關的connexin 基因有:
Cx26(GJB2)、 CX30(GJB6)、Cx31 (GJB3)，和 Cx43 (GJA1)。
而在本研究中我們分析另一個與聽障有相關性的Cx30.3基因，其最早是在EKV的皮膚性疾病中被發現。我們收集120位聽力正常人和250位聽障孩童以及26位來自醫院的病患對Cx30.3基因進行多型性比對，結果發現有7個錯意突變(missense mutation)，分別是: 64C→T/wt (R22C) 、109G→A/wt (V37I)、220G→A/wt(V74M)、 292C→T/wt (R98C)、302G→A/wt (R101H)、370C→G/wt (R124W) 和507C→G (C169W)，以及五個多型性，分別是174C→T/wt (P58P)、451C→A/wt(R151S)、507C→G/wt(C169W) 、611A→C/wt (E204A)、611A→C (E204A)。
為了實際探討Cx30.3的功能，因此我們建構正常的Cx30.3和突變的Cx30.3 507C→G(C169W)質體表現於HeLa細胞，因為HeLa細胞本身缺乏connexins所以無法產生gap junction，故可藉以了解Cx30.3突變基因的致病機轉。在免疫螢光的實驗結果證明，野生型的Cx30.3蛋白是散佈在細胞質，而野生型Cx31蛋白則表現在細胞膜且與鄰近細胞的Cx31蛋白形成gap junction。另外當野生型的Cx30.3蛋白與野生型的Cx31蛋白共同表現時，會使原本散佈在細胞質的Cx30.3蛋白運輸至細胞膜表面，並且產生gap junction。然而突變的Cx30.3 507C→G(C169W)蛋白與野生型Cx31蛋白一起表現時，此突變的Cx30.3 507C→G(C169W)蛋白亦可運輸至細胞膜表面，並且產生gap junction，雖然結果如此，但並不一定代表此gap junction蛋白就有功能，所以在未來我們必須進一步作功能的分析來探討此突變的Cx30.3 507C→G(C169W)所形成的gap junction是否仍有正常間隙連結的功能。另外這些Cx30.3(GJB4)的變異可以幫助定義Cx30.3基因在皮膚失調症與聽力障礙中所扮演的角色。

關鍵字

非症候群感音神經性聽障 ； 間隙通道 ； 耳蝸

並列摘要

Approximately 1 in 1000 children is affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness).The cause of this disease is multifactorial and includes both genetic and environmental factors. Among gentic factors, mutations in connexin genes, such as Cx26(GJB2), Cx30(GJB6), Cx31(GJB3), and Cx43(GJA1), have been shown to involve in hearing impairment.
In this study, I have investigated the association of hearing impairment with Cx30.3(GJB4), originally found to be involved in skin disease. I have analyzed Cx30.3(GJB4) from 250 Taiwanese patients with prelingual deafness and 120 unrelated normal individuals. Among the mutations of Cx30.3 gene carried by the deafness patients, seven are missense mutations of 64C→T/wt(R22C),109G→A/wt(V37I),
220G→A/wt(V74M),292C→T/wt(R98C),302G→A/wt(R101H),
370C→G/wt(R124W) and 507C→G(C169W).I have also
identified
polymorphisms of Cx30.3 gene including 174C→T/wt(P58P),
451C→A/wt(R151S), 507C→G/wt(C169W), 611A→C/wt(E204A) and 611A→C (E204A).
In addition, I have investigated the co-expression of Cx30.3 and Cx31 for the formation of gap junction,as these two connexins have previously been shown to cooperate structurally or functionally at molecular level.The result of protein localization by immunofluorescence showed that WT-Cx30.3 was diffused in the cytoplasma, whilst WT-Cx31 was localized at the plasma membrane between adjacent cells when they were expressed individually in HeLa cell deficient in connexins that do not form gap junction. However, WT-Cx30.3 formed gap junction plaque between contiguous cells with WT-Cx31 when they were co-expressed in HeLa cells. Formation of gap junction between mutant Cx30.3(C169W) and WT-Cx31 were unaffected and were comparable with that between WT-Cx30.3and WT-Cx31 proteins.
Maintenance of the function of gap junction formed between Cx30.3 and Cx31 protein requires further study. The study of Cx30.3(GJB4) variants should help in defining the role of Cx30.3 in both skin disorders and hearing loss.

並列關鍵字

nonsyndromic sensorineural deafness ； gap junction ； connexin ； cochlea.

參考文獻

Bruzzone, R., White, T.W. and Paul, D.L.: Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 238 (1996) 1-27.

Bykhovskaya, Y., Estivill, X., Taylor, K., Hang, T., Hamon, M., Casano, R.A., Yang, H., Rotter, J.I., Shohat, M. and Fischel-Ghodsian, N.: Candidate locus for a nuclear modifier gene for maternally inherited deafness. Am J Hum Genet 66 (2000) 1905-10.

Dahl, G., Werner, R., Levine, E. and Rabadan-Diehl, C.: Mutational analysis of gap junction formation. Biophys J 62 (1992) 172-80; discussion 180-2.

Diez, J.A., Ahmad, S. and Evans, W.H.: Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions. Eur J Biochem 262 (1999) 142-8.

Falk, M.M. and Gilula, N.B.: Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access. J Biol Chem 273 (1998) 7856-64.