| 刘慧茹,叶碧珍,魏转弟.GJB2基因突变在长岛型掌跖角化病发病中的作用[J].安徽医药,2020,24(1):118-123. |
| 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 |
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| DOI：10.3969/j.issn.1009?6469.2020.01.030 |
| 中文关键词: 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 |
| 英文关键词: Keratoderma,palmoplantar Gap junction protein beta 2 Patch?clamp techniques Blotting,western Immuno? precipitation Point mutation |
| 基金项目:东莞市社会科技项目(2018507150291435) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 withconnexin 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 theexpression 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.TheCx26 mutants have the ability of modify hemichannels and gap junctions of Cx31.It is important to study the effect of Cx26 andCx31 in the NPPK. |
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