AXIN2罕见变异与人类神经管畸形的相关性研究

Correlation of rare mutation in AXIN2 with human neural tube defects

目的探索AXIN2基因变异与神经管畸形(neural tube defects,NTDs)发生的相关性。方法收集2004年6月-2013年12月山西省9家医院引产的100例NTDs胎儿肌肉组织样本,对其全基因组测序结果中的AXIN2基因的变异进行生物信息学分析,在相应的样本中利用Sanger测序证实变异结果。在NE-4C细胞系中转染AXIN2野生型及AXIN2变异型质粒,利用免疫荧光和蛋白免疫印迹实验进行基因功能验证,对携带AXIN2变异的人NTDs样本进行分析。结果在100例NTDs样本中发现AXIN2基因上有5种变异,对其中3种变异[c.959A>T(p.D320V)、c.1250C>T(p.A417V)和c.1634G>A(p.G545E)]进行功能验证。细胞蛋白免疫印迹实验表明,D320V(P<0.05)和G545E(P<0.001)变异下调AXIN2蛋白表达,但3种变异均不影响β-catenin蛋白表达水平;免疫荧光实验表明,3种变异不影响AXIN2蛋白的亚细胞定位。在携带D320V及G545E变异的NTDs胚胎肌肉组织中,AXIN2的蛋白表达水平显著低于对照[D320V(P<0.05),G545E(P<0.01)]。结论NTDs样本中鉴定到的AXIN2变异D320V及G545E下调AXIN2蛋白表达量,可能通过引起Wnt信号通路异常激活而导致神经管畸形的发生。

ObjectiveTo investigate the correlation between genetic variations in the AXIN2 gene and the development of neural tube defects(NTDs).MethodsBioinformatics analysis was performed on AXIN2 gene variations identified through whole-genome sequencing of muscle tissue samples from 100 fetuses with NTDs collected from June 2004 to December 2013 among 9 hospitals in Shanxi Province.The identified variations were validated using Sanger sequencing.In NE-4C cell lines,wild-type AXIN2 and mutant AXIN2 plasmids were transfected,and gene functionality was assessed through immunofluorescence and Western blot assay.Analysis was also conducted on human NTDs samples carrying AXIN2 variations.ResultsFive distinct AXIN2 gene variations were detected in the 100 NTDs samples.Functional studies were conducted on three of these variations[c.959A>T(p.D320V),c.1250C>T(p.A417V)and c.1634G>A(p.G545E)].Western blot assay revealed that,although none of the 3 variations had an impact on the levels ofβ-catenin protein expression,the D320V(P<0.05)and G545E(P<0.001)variations led to downregulation of AXIN2 protein expression.Immunofluorescence assay demonstrated that all 3 variations did not alter the subcellular localization of AXIN2 protein.In embryonic muscle tissues carrying the D320V and G545E variations,AXIN2 protein expression was significantly lower than those in the control group[D320V(P<0.05),G545E(P<0.01)].ConclusionThis study identifies AXIN2 gene variations(D320V and G545E)in NTDs samples that result in the downregulation of AXIN2 protein expression.These variations may potentially lead to abnormal activation of the Wnt signaling pathway,thus contributing to the development of neural tube defects.