GJB2基因突变在长岛型掌跖角化病发病中的作用  

Mechanism of GJB2 gene mutation leading to the occurrence of nagashima-type palmoplantar Keratosis

作　　者：刘慧茹 叶碧珍 魏转弟 LIU Huiru;YE Bizhen;WEI Zhuandi(Department of Dermatology,Dongguan Traditional Chinese Medicine Hospital,Dongguan,Guangdong 523000,China)

机构地区：[1]东莞市中医院皮肤科

出　　处：《安徽医药》2020年第1期118-123,共6页Anhui Medical and Pharmaceutical Journal

基　　金：东莞市社会科技项目（2018507150291435)

摘　　要：目的鉴定一家系长岛型掌跖角化症(NPPK)的突变位点,阐明NPPK的发病机制。方法将收集的2例NPPK病人外周血,提取DNA后对GJB2、GJB3、GJB4、GJB6、GJA1五个基因进行测序,鉴定突变位点;连接蛋白转录物显微注射卵母细胞,分别为:缝隙连接蛋白(Connexin,Cx)26、Cx31、Cx26+Cx31、Cx26+Cx26⁃S183F、Cx31+Cx26⁃S183F及注射水的对照组,并通过膜片钳实验记录半通道电流;蛋白质印迹检测法检测NPPK突变体及野生型Cx31的表达水平;通过免疫共沉淀检测Cx26 NPPK突变体与Cx31的互作情况。结果测序发现Cx26(GJB2)基因发生了突变(c.548C>T),第183位的丝氨酸被苯丙氨酸取代(p.Ser183Phe)导致了NPPK;当在卵母细胞单独表达Cx26⁃S183F时,不能形成间隙连接通道或半通道;突变体与野生型Cx31的共表达,显示Cx31间隙连接通道的反式显性抑制,而不降低Cx31蛋白质合成;免疫共沉淀表明,与野生型相比,突变体Cx26对Cx31蛋白更有效地下拉,说明增强了异型连接子的形成;在Cx26突变体存在下,异型连接子的形成导致Cx31半通道活性显着增加。结论Cx26⁃S183F不能单独形成半通道或间隙连接,但在与Cx31共表达时可以增强半通道活性,从而引起NPPK。Cx26突变体具有修饰Cx31半通道和缝隙连接的能力,对研究Cx26和Cx31在表皮疾病的作用具有重要的意义。Objective To identify the mutation site in a Nagashima⁃type Palmoplantar Keratosis(NPPK)and clarify the pathogene⁃sis mechanism of NPPK.Methods We collected two blood samples from two NPPK patients.After extracted the DNA from blood samples,five genes of GJB2,GJB3,GJB4,GJB6 and GJA1 were sequenced to identify the mutation site.Oocytes were injected with connexin transcripts alone or in combination:Cx26,Cx31,Cx26+Cx31,Cx26+Cx26⁃S183F,Cx31+Cx26⁃S183F and control(injected with H2O).Then patch clamp recording technique were taken to recorded hemichannel currents.Western blot was used to detect the expression of NPPK mutant and wild⁃type Cx31.Detection of interaction between Cx26 NPPK mutant and Cx31 were detected by co⁃immunoprecipitation assay.Results we found a heterozygous c.548C>T transition at codon 183 of the GJB2 gene,then a substitu⁃tion of a serine by a phenylalanine(p.Ser183Phe),whichcaused Nagashima⁃type Palmoplantar Keratosis(NPPK).When the oocyte expresses the Cx26⁃S183F alone,it failed to form gap junction channels or hemichannels.Co⁃expression of the mutants with wild⁃type Cx31 resulted a trans⁃dominant inhibition of Cx31 gap junction channels,without decreased the synthesis of Cx31 protein.Com⁃pared with the wild⁃type cells,co⁃immunoprecipitation shown Cx31 being pulled down more efficiently with Cx26 mutant,which was confirmed that Cx26 mutant could enhanced formation of heteromeric connexons.The formation of heteromeric connexons was significantly increased Cx31 hemichannel activity in the presence of Cx26 mutants.Conclusions Cx26⁃S183F,unable to form hemichannels or gap junctions alone,however,increased hemichannel activity and caused NPPK when co⁃expressed with Cx31.The Cx26 mutants have the ability of modify hemichannels and gap junctions of Cx31.It is important to study the effect of Cx26 and Cx31 in the NPPK.

关 键 词：皮肤角化病 掌跖 缝隙连接蛋白beta 2 膜片钳术 印迹法 蛋白质 免疫沉淀法 突变位点 

分 类 号：R76[医药卫生—耳鼻咽喉科]