探討非症候群聽障基因CLDN14突變之功能研究

人類 CLAUDIN 14 (CLDN14)基因的突變或缺失會造成非症候群性聽障DFNB29。已有研究指出，在Cldn 14基因剔除的老鼠中發現，老鼠耳內的毛細胞退化，會使得陽離子過度通透，而導致聽障。CLDN14基因為CLDN家族的一員，也是tight junction的組成蛋白，tight junction兼具屏障與防禦的功能，在細胞間形成選擇性的通道讓離子與分子通透。過去，我們實驗室從語言學習前期非症候群聽障患者中發現了三個CLDN14基因突變，這些突變包含了兩個錯意突變：52 A>G/WT (M18V)與424 G>A/WT (D142N)，及一個核苷酸缺失造成frameshift的突變：167-168 del GG。本論文主要是探討CLDN14基因發生突變後對CLDN14蛋白表現所造成的影響。我們將兩個錯意突變與一個缺失的突變基因和正常CLDN14基因表現的質體，分別轉殖入MDCK細胞株中，觀察突變與正常基因在細胞內表現位置的差異性。從結果中我們發現，D142N突變的CLDN14蛋白表現位置與正常的CLDN14蛋白相同，在細胞膜與細胞質內皆有表現。而M18V與167-168 del GG的突變蛋白卻無法運送到細胞膜上，而在細胞內形成堆積的現象，因此，我們認為CLDN14的M18V與167-168 del GG突變可能降低tight junction的生成與功能，進而導致聽障。

The mutations or deletions of CLAUDIN 14 (CLDN14) gene are responsible for nonsyndromic deafness DFNB29 in human. Furthermore, studies with Cldn14-null mice indicated such deafness is due to the degeneration of hair cells leading to cation overload. CLDN14 is a member of CLDN family involved in tight junction strand formation. Tight junction contains the barrier and fence functions to passage of ions and molecules through the selectively paracellular pathway. Previously, we had identified 3 mutants of CLDN14 in prelingual nonsyndromic sensorineural deafness, including 2 missense mutations (M18V and D142N) and 1 deletion mutant (with GG nucleotides at 167-168 being deleted to cause frameshift). To investigate the impact of mutations of CLDN14, the localizations of mutants and wild-type CLDN14 were studied by transfections performed on MDCK cells. Our results indicate that CLDN14-D142N protein localized at the plasma membrane and in cytoplasmic compartment, similar to that of CLDN14-WT protein, while CLDN14-M18V and GG deletion mutant proteins were remained and accumulated in the cytoplasma without being transferred to the plasma membrane. These results suggest that mutations of CLDN14, M18V and GG deletion might cause deafness by inhibitng the formation and function of tight junction.