利用細胞模式探討GJB2基因突變造成非症候群聽障的機制

Gap junction 是細胞膜上的通道，他與耳蝸內細胞之間離子的平衡有關，主要是協助運送鉀離子或其他離子從毛細胞（hair cell）回到耳蝸內淋巴系統的再循環，以維持內淋巴中的鉀離子濃度。Gap junction基本組成單位是 connexin。Connexins （CXs） 基因族在許多研究已經被報導和非症候群遺傳性聽障有關。在我們實驗室先前針對台灣非症候群聽障病人中的篩檢，已經發現在 GJB2 基因（CX26）中有三個錯意突變點包括 p.G4D , p.R75Q 及 p.T123N 。然而到目前為止對於我們所發現的這些錯意突變，所造成的影響及致病機轉並不清楚。因此在本研究我們將加以探討。本論文將分兩部分來探討：第一部分我們利用 HeLa 細胞表現，先觀察細胞中正常和突變CX26 基因是否形成 gap junction 。結果發現 p.T123N 的 CX26 突變蛋白，其表現位置和正常的 CX26 蛋白一樣，可被運送到細胞膜上表現，形成 GJ 通道顆粒（plaque）。接著，透過 Scrape loading dye transfer 的方法判定形成的同質性 gap junction 功能是否正常。發現由突變蛋白 p.T123N 所形成的 GJ 通道並沒有通透染劑的能力。另外，觀察 p.G4D 和 p.R75Q 的 CX26 突變蛋白在細胞中的表現情形，發現突變蛋白的運送發生異常，導致蛋白無法正常的運送到細胞膜上形成 GJ 通道顆粒，而產生大量堆積於高基氏體及溶酶體當中。針對此觀察結果，接著我們使用藥物 Chloroquine （CQ）來抑制溶酶體中的蛋白酶，觀察是否抑制溶酶體的降解功能後，會造成更多的突變蛋白堆積，藉以確認我們的推測。利用倒立式顯微鏡觀察，結果發現不同時間點的CQ藥物作用之下，螢光並沒有因為降解功能被抑制後而增加的現象。確切變化有待之後以西方點墨法分析。第二部分我們為模擬病人中發現的 heterozygous 表現情形，利用co-transfection 的方式，探討這些突變蛋白與正常蛋白在 heterozygous 的表現情況下，是否會造成正常 CX26 蛋白的功能喪失。在共同表現蛋白的研究中，在 p.G4D或 p.R75Q 的突變和正常 CX26 蛋白共同表現我們發現正常蛋白都會影響突變蛋白，使得原本堆積的蛋白被運送到細胞膜上，而一起表現於細胞膜上。所觀察的結果與先前許多文獻發現的顯性抑制效應（dominant negative effect）現象有所不同。對於目前確切結果還不清楚仍需之後進一步的研究探討。另外，共同表現蛋白的研究中含 p.T123N 的突變和正常 CX26 蛋白共同表現，看到蛋白可以正常運送到細胞膜上表現，這與單獨轉殖的結果一樣。但是對於所形成的 GJ 是否功能正常，以及組合的 GJ 通道結構是否正常目前還不清楚，對於這個問題仍需之後進一步的研究探討。未來，為了更準確的模擬 heterozygous 的表現情形，我們將使用能夠等量 Tet-on 雙向表現系統，進一步探討這些突變蛋白在 Tet-on HeLa 細胞內的表現位置，並分析是否造成 CX26 蛋白的功能喪失。目前已完成建構带有螢光蛋白的正常或突變 GJB2 基因於同一載體上（pBI），之後進行 Tet-on HeLa 轉殖觀察其細胞內的表現位置並探討其交互作用。綜合以上所述，藉由這兩部分的觀察和研究讓我們對於GJB2 基因突變在非症候群聽障中所扮演的角色和致病機轉有進一步的了解。

Connexins (CXs) are known to be involved in human nonsyndromic genetic deafness. Gap junction intercellular communication (GJIC) has numerous functions, including K+ recycle in the inner ear for auditory function. Recently, among a cohort of patients with nonsyndromic hearing loss, we demonstrated that three heterozygous missense mutations, p.G4D, p.R75Q and p.T123N, in the GJB2 (CX26) gene is causally related to hearing loss. However, the functional change in the three mutants remains unknown. We divided this thesis into two part. First of all, we compared the mutant intracellular distribution and effect on HeLa cells. This study compared the intracellular distribution and assembly of three mutants with that of the wild-type (WT) CX26 in HeLa cell and the effect that the mutant protein had on those cells. Fluorescent localization assay of WT CX26 showed the typical punctuate pattern of gap junction channel between neighboring expression cells. In addition, our results indicted that p.T123N mutant exhibited continuous fluorescence along the apposed cell membranes by immunofluorescent assay, which is similar to the wild-type (WT). However, p.G4D and p.R75Q mutants showed impaired trafficking of the protein to the plasma membrane and accumulation in the lysosome and Golgi apparatus rather than in the cytoplasmic membrane. Therefore, p.G4D and p.R75Q mutant may lead to impairs formation of hemichannels and GJ. To confirm that we speculated that mutation protein accumulated in lysosome So that, we treated the lysosomal protease inhibitor, Chloroquine（CQ）, to stable expression p.G4D or p.R75Q HeLa cell line. We found that it did not increase accumulation of the CX26 mutant protein in the lysosome. Therefore, we suggested that p.G4D and p.R75Q mutation proteins was not degraded via lysosome degradation system. Furtherlly, we found p.G4D or p.R75Q mutants co-expressed with WT which displayed both of them were exhibited continuous fluorescence along the apposed cell membranes using co-transfection system. This result is different of only mutant transfection that the mutation protein were impairment of targeting to the plasma membrane and accumulated in cytoplasma. Moreover, p.T123N mutants co-expressed with WT which displayed both of them were exhibited continuous fluorescence along the apposed cell membranes, which is similar of only mutant transfection. However, function significance and organization ability of these mutant proteins remains unknown, requires further investigation. To confirm those result, we will compared the mutant intracellular distribution and effect on tet-on HeLa cells. We have finished the relative construct plasmid for tet-on express system. Then, those mutation protein and normal protein interaction will be confirm after the Tet-on HeLa cells in the further investigation. In summory, our results provide a novel molecular explanation for the role CX26 plays in the development of hearing loss.