利用Tet-on系統探討非症候群聽障Cx26與Cx30基因突變之功能研究

Gap junction是細胞膜上的離子通道，其基本組成單位為connexin(Cx)。每六個connexin可組成一個connexon(hemichannel)，每個connexon再與鄰近細胞的connexon兩者共同組合形成一個完整的gap junction。它與耳蝸內鉀離子的循環有關，其突變常會導致聽障。先前針對台灣地區非症候群聽障患者進行基因篩選，發現了Cx26 368C>A(T123N)、Cx26 551G>A(R184Q)及Cx30 119C>T(A40V)這三個錯意突變點，帶有這些突變點的患者皆為heterozygous。為瞭解這些突變點造成的影響，我們建立了 Tet-on系統進行探討。Tet-on系統利用可雙向表現的pBI載體，藉由此載體可以同時表現兩種基因，瞭解它們之間的交互作用，且避免傳統co-transfection送入兩載體不等量的疑慮。經實驗結果證實，Cx26 551G>A突變蛋白會導致Cx蛋白堆積在高基氏體。將Cx26 551G>A突變蛋白分別與Cx26或Cx30共同組成heteromeric connexon後，Cx26 551G>A突變蛋白會影響正常Cx26或Cx30蛋白而堆積在高基氏體。Cx30 119C>T突變蛋白也會導致Cx蛋白堆積在高基氏體。Cx30 119C>T突變蛋白分別與Cx26或Cx30共同組成heteromeric connexon後，Cx30 119C>T突變蛋白也會影響正常Cx26或Cx30蛋白而堆積在高基氏體。不同於上述情形，Cx26 368C>A突變蛋白則會表現在細胞膜，與正常Cx蛋白表現位置相同。與正常Cx26及Cx30共同組成heteromeric connexon後，表現的位置然仍在細胞膜上。綜合以上結果顯示，Cx26 551G>A及Cx30 119C>T這兩個突變會造成dominant negative effect。而Cx26 368C>A則不會對Cx26蛋白運輸造成影響，但對於通透能力等方面是否會改變仍需進一步實驗證實。

Gap junction is formed by end-to-end docking of two connexons, hexamers of connexin (Cx) protein, expressed on neighboring cells. It is associated with potassium cycle in cochlear. Mutations in Cx often lead to hearing loss. Three heterozygous mutations, Cx26 368C>A (T123N), Cx26 551G>A (R184Q), and Cx30 119C>T (A40V), were identified in our previous study from Taiwanese nonsyndromic deafness patients. To understand the effects on function of these mutations in gap junction, we set up Tet-on protein expression system. In this system, we constructed a bidirectional pBI vector to express two Cx genes simultaneously. By using this expressive system, we can investigate the interaction between two genes without complications due to different amounts of the two vectors expressed by co-transfection system. Our results indicated that 551G>A missense mutation of Cx26 resulted in the accumulation of Cx protein in Golgi apparatus instead of targeting to cytoplasmic membrane. When either Cx26/Cx26 551G>A or Cx30/Cx26 551G>A mutant protein are co-expressed, the heteromeric connexon accumulated at Golgi apparatus. 119C>T missense mutation of Cx30 resulted in the accumulation of Cx protein in Golgi apparatus instead of targeting to cytoplasmic membrane.When either Cx30/Cx30 119C>T or Cx26/Cx30 119C>T mutant protein are co-expressed, the heteromeric connexon also accumulated at Golgi apparatus. In contrast, Cx26 protein derived from 368C>A missense mutation gave results similar to wild-type Cx26 with junctional plaques at zones of cell-to-cell apposition. Even Cx26 368C>A in complex with either Cx26 or Cx30, the protein still express in cell membrane. In summary, Cx26 551G>A and Cx30 119C>T had dominant negative effect on both normal Cx26 and Cx30 resulting in accumulation of the Cx protein in Golgi apparatus that impaires formation of the gap junction. Cx26 368C>A did not affect the trafficking of Cx protein, but its functional significance remains unknown. In this part, we need more analysis to explain.