探討非症候群聽損GJB3與TMPRSS3基因突變之功能研究

Functional study of GJB3 and TMPRSS3 mutants in nonsyndromic deafness

黃瑞喜(SWEE-HEE WONG)

指導教授 ： 李宣佑

中山醫學大學/醫學院/醫學研究所/博士(2020年)

https://doi.org/10.6834/csmu202000196

摘要

遺傳性聽損一種常見的感音神經性疾病，在開發中國家大約60%由於基因突變所造成的。目前已知有59個基因的突變會導致聽損且每個基因的致病機制皆不盡相同。CONNEXIN在聽力功能上扮演的角色已被許多研究所確認，其在鉀離子再循環、耳蝸內離子平衡及聽力形成過程皆扮演重要的角色。另外，TMPRSS3 (transmembrane protease, serine 3) 已經發現牽涉在非症候群聽損中的一個新基因，當TMPRSS3 基因發生突變後已知會影響到耳蝸內Na+的再吸收因而造成聽損。TMPRSS3 (transmembrane protease, serine 3)是屬於第二型穿膜絲氨酸蛋白(type II transmembraneserine protease，TTSPs)的超級家族之一。這是第一個被提出與非症候群有關的酵素基因。本研究目的主要是著重於探討GJB3 (CX31)和TMPRSS3基因突變對其功能的影響介此來探討聽損的成因。
在之前我們實驗室針對台灣地區的非症候群聽損患者進行TMPRSS3 基因的突變分析，結果在230 位聽損患者中發現有14位聽損學童有突變的發生，所佔的比例為6.09 % (14/230)，在這10個突變點中，有3個是missense mutations： c.239G> A（p.R80H）， c.551T> C（p.L184S）和1253C> T（p.A418V）； 三個是silent mutations， 另外四個是在introns中突變，然而針對這些突變所造成的致病機轉我們並不清楚。為了了解TMPRSS3的突變對功能性的影響，我們建構了p.R80H，p.L184S和p.A418V的TMPRSS3突變的質體。再利用sGASP (secretory genetic assay of site-specific proteolysis )的研究酵素水解能力，通過sGASP系統，這三個突變點p.R80H，p.L184S和p.A418V 與正常的酵母菌生長比率分別只有19.4％，13.2％和27.6％。由sGASP結果顯示實驗室先前所找到的TMPRSS3 基因突變點 (p.R80H、p.L184S、p.A418V）確實會造成TMPRSS3 酵素水解功能活性的下降，而影響正常的功能。另外以爪蟾卵母細胞(Xenopus oocyte)表現系統，利用雙電極電壓鉗技術(Two electrode voltage clamp) 顯示這三個TMPRSS3突變蛋白均未能刺激爪蟾卵母細胞中表達的上皮鈉離子通道（ENaC），也更加的證實所發現的3 個突變點是會影響TMPRSS3 蛋白的功能。綜合以上結果，我們的計畫已經證明在病人上所發現的TMPRSS3 基因突變確實會造成蛋白功能上的喪失，進而影響到聽覺。
此外，在我們實驗室先前針對台灣地區513位非症候群聽損病患的篩檢中，已經發現在GJB3 (CX31) 基因中有六個missense mutations點，包括p.L10R、p.P18S、p.V84I、p.V174M、p.E183K及p.A194T。透過免疫螢光染色法分析，我們發現正常的CX31蛋白可以正常的表現和被運送到細胞膜，並在兩個鄰近細胞間形成間隙連接 (gap junction) 。另外，我們發現含p.L10R、p.V84I和p.A194T的CX31突變蛋白，其表現位置和正常的CX31蛋白一樣可被運送到細胞膜上。相反的，含p.P18S、p.V174M或p.E183K的CX31突變蛋白卻會堆積在細胞質中，無法正常送至細胞膜上表現。雖然p.L10R、p.V84I和p.A194T的CX31突變蛋白，其表現位置和正常的CX31蛋白一樣，但是比較Lucifer Yellow (LY)和 Propidium iodide(PI)染劑通透性實驗，發現p.L10R、p.V84I和p.A194T的CX31突變蛋白形成的間隙連接其通透性卻是大大的減少。而這三個p.L10R、p.V84I和p.A194T的CX31突變蛋白與CX31WT或CX26WT共同轉殖入HeLa細胞後，再以Lucifer Yellow (LY)和 Propidium iodide(PI)染劑檢測共同形成的間隙連接其通透性，由結果顯示這三個突變蛋白（p.L10R，p.V84I和p.A194T）雖然不會影響正常CX31或正常CX26運送到細胞膜上，但會影響和正常CX31或CX26一起形成間隙連接的通透性，顯示這三個CX31突變（p.L10R，p.V84I和p.A194T） ）對與正常CX26具有trans-dominant negative effect。這些結果讓我們初步的了解到CX31突變所造成的影響。未來我們需要更深入的針對這幾個突變點功能上的影響加以探討，以更清楚瞭解各突變點造成聽損的分子機制。

關鍵字

TMPRSS3 ； GJB3 ； 遺傳性聽損

並列摘要

Hearing loss (HL) is a common sensory disorder with an incidence of about 1 child in 1000 births in the human population. It is often caused by genetic inheritance of auditory system dysfunction; approximately 60% of HL is attributable to genetic factors . Nearly 70% of genetic HL cases are nonsyndromic. The importance of K+ recycling, Ion balance in the cochlea and hearing formation play important roles is underscored by the fact that mutations in each of connexin gene family leads to deafness in human. In addition, Recently, some studies have shown that mutations in TMPRSS3 were responsible for non-syndromic deafness (DFNB8 and DFNB10). TMPRSS3 is a member of the Type II Transmembrane Serine Protease (TTSP). This was the first description of a serine protease involved in deafness. In the proposed project, we will focus on the study of the effect on function of GJB3 gene and TMPRSS3 gene with mutation to investigate the mechanisms of hearing loss.
In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay
for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.
In addition , we identified novel heterozygous missense mutation, p.L10R, p.P18S, p.V84I, p.V174M, p.E183k and p.A194T, in the GJB3 gene encoding CX31 from 513 unrelated Taiwanese patients with non-syndromic hearing loss. Here we focused on the functional properties of six CX31 mutants derived from point mutation. Immunostaining pattern of transfected cells revealed that p.P18S, p.V174M and p.E183K mutants impaired the trafficking of CX proteins to the plasma membrane leading to accumulation of the mutant proteins in the cytoplasm, whereas p.L10R, p.V84I and p.A194T mutants showed the typical punctuate pattern of gap junction channel between neighboring expression cells as CX31 wild-type. Nevertheless, comparison of ATP release, Lucifer Yellow (LY), and Propidium iodide(PI) transfer revealed mutant channels was greatly reduced compared with wild-type junctions. The three CX31mutation( p.L10R, p.V84I and p.A194T) co-expressed with either CX31WT or CX26WT studies, suggested did not influence the trafficking of CX31WT and CX26WT. Nevertheless, dye transfer experiments revealed three CX31mutation( p.L10R, p.V84I and p.A194T) not be capable transferred Lucifer Yellow (LY) or Propidium iodide(PI) through gap junction from the injected cell to the neighboring cell. Based on these findings, we suggest that the three CX31mutation( p.L10R, p.V84I and p.A194T) mutant may have an effect on the function of the, and three CX31mutation( p.L10R, p.V84I and p.A194T) has a trans-dominant negative effect on the function of wild types CX26. These results provide a novel molecular explanation for the role that GJB3 plays in hearing loss.

並列關鍵字

hearing loss ； hair cell ； GJB3 ； TMPRSS3

參考文獻

參考文獻

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