中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2010年
3期
241-245
,共5页
杨中纯%肖自安%谢鼎华%夏昆
楊中純%肖自安%謝鼎華%夏昆
양중순%초자안%사정화%하곤
连接蛋白基因%突变%间隙连接%耳聋
連接蛋白基因%突變%間隙連接%耳聾
련접단백기인%돌변%간극련접%이롱
CX26 gene%mutation%gap junction%deafness
目的 观察连接蛋白(connexin 26,CX26)基因的一个新致聋突变c.465T→A,P.Y155X,在体外表达细胞功能的改变,以探讨其致聋的可能机理.方法 常染色体隐性遗传耳聋家系的先证者外周血抽提DNA,DNA直接测序法分析CX26基因突变.将在该家系发现的突变c.465T→A,P.Y155X和野生型CX26(wtCX26)定向克隆到pEGFP-N1质粒,构建CX26 p.Y155X-EGFP及wtCX26-EGFP融合蛋白表达载体,转染HeLa细胞,Western印迹分析蛋白的表达,共聚焦显微镜观察突变蛋白和野生型CX26在HeLa细胞的定位及有无间隙连接斑形成,染料转移实验分析间隙连接的功能.结果 在该耳聋家系发现CX26基因一个新的致聋突变:c.465T→A,P.Y155X.CX26 P.Y155X突变体在HeLa细胞表达的突变蛋白的分子量小于野生型蛋白分子量;突变蛋白在细胞质表达,不能分布到细胞膜和形成间隙连接,无染料转移.野生型表达于细胞膜并形成间隙连接斑,能转移染料.结论 CX26 P.Y155X突变体在翻译后不能从细胞内转运到细胞膜,不能形成间隙连接通道.CX26基因c.465T→A,P.Y155X导致常染色体隐性遗传性聋.
目的 觀察連接蛋白(connexin 26,CX26)基因的一箇新緻聾突變c.465T→A,P.Y155X,在體外錶達細胞功能的改變,以探討其緻聾的可能機理.方法 常染色體隱性遺傳耳聾傢繫的先證者外週血抽提DNA,DNA直接測序法分析CX26基因突變.將在該傢繫髮現的突變c.465T→A,P.Y155X和野生型CX26(wtCX26)定嚮剋隆到pEGFP-N1質粒,構建CX26 p.Y155X-EGFP及wtCX26-EGFP融閤蛋白錶達載體,轉染HeLa細胞,Western印跡分析蛋白的錶達,共聚焦顯微鏡觀察突變蛋白和野生型CX26在HeLa細胞的定位及有無間隙連接斑形成,染料轉移實驗分析間隙連接的功能.結果 在該耳聾傢繫髮現CX26基因一箇新的緻聾突變:c.465T→A,P.Y155X.CX26 P.Y155X突變體在HeLa細胞錶達的突變蛋白的分子量小于野生型蛋白分子量;突變蛋白在細胞質錶達,不能分佈到細胞膜和形成間隙連接,無染料轉移.野生型錶達于細胞膜併形成間隙連接斑,能轉移染料.結論 CX26 P.Y155X突變體在翻譯後不能從細胞內轉運到細胞膜,不能形成間隙連接通道.CX26基因c.465T→A,P.Y155X導緻常染色體隱性遺傳性聾.
목적 관찰련접단백(connexin 26,CX26)기인적일개신치롱돌변c.465T→A,P.Y155X,재체외표체세포공능적개변,이탐토기치롱적가능궤리.방법 상염색체은성유전이롱가계적선증자외주혈추제DNA,DNA직접측서법분석CX26기인돌변.장재해가계발현적돌변c.465T→A,P.Y155X화야생형CX26(wtCX26)정향극륭도pEGFP-N1질립,구건CX26 p.Y155X-EGFP급wtCX26-EGFP융합단백표체재체,전염HeLa세포,Western인적분석단백적표체,공취초현미경관찰돌변단백화야생형CX26재HeLa세포적정위급유무간극련접반형성,염료전이실험분석간극련접적공능.결과 재해이롱가계발현CX26기인일개신적치롱돌변:c.465T→A,P.Y155X.CX26 P.Y155X돌변체재HeLa세포표체적돌변단백적분자량소우야생형단백분자량;돌변단백재세포질표체,불능분포도세포막화형성간극련접,무염료전이.야생형표체우세포막병형성간극련접반,능전이염료.결론 CX26 P.Y155X돌변체재번역후불능종세포내전운도세포막,불능형성간극련접통도.CX26기인c.465T→A,P.Y155X도치상염색체은성유전성롱.
Objective To report a novel deafness-causing mutation c. 465T→A, p. Y155X in connexin 26 (CX26) (also called gap junction protein β-2, GJB2 ), and perform functional analysis of the mutated protein p. Y155X in Hela cells to explore the underlying mechanism on deafness. Methods Mutations in CX26 gene of the proband in an autosomal recessive inherited deafness family were tested by direct DNA sequencing method. Mutant p. Y155X, which was found in the deafness family, and wild type CX26 (wtCX26), were directionally subcloned into the pEGFP-N1 plasmid to construct the recombinant fusion protein expression vector of CX26 p. Y155X-EGFP and wtCX26-EGFP, followed by transfecting into HeLa cells. The expression of the mutated and wild type proteins was analyzed using Western blot analysis. The intracellular localization of proteins and the formation of gap junction-like plaques at plasma membrane were observed under confocal microscope. Gap junction coupling was tested by calcein-AM dye transfer experiment. Results A novel nonsense mutation c. 465T→A, p. Y155X in the CX26 gene was found in the autosomal recessive deafness family. The molecular weight of protein p. Y155X was smaller than that of wtCX26 in transiently expressed HeLa cells. The mutated protein failed to reach the cell surface to form gap junction plaques, and displayed cytoplasmic accumulation. Also, no calcein-AM dye was transferred from the donor cells to the recipient cells when both were transfected with CX26 p. Y155X. The wtCX26 protein localized at the cell membrane to form gap junction plaques with permeability to fluorescent dye calcein-AM. Conclusion CX26 p. Y155X could not be targeted to the plasma membrane and there was no formation of gap junction channels between the adjacent cells. The mutation c. 465T→A, p. Y155X in CX26 gene was responsible for the autosomal recessive hearing impairment in this family.
