外胚层发育不良患者基因突变分析及功能验证

Mutation screening and functional analysis for 8 patients with ectodermal dysplasia

目的:寻找先天性外胚层发育不良(ectodermal dysplasia, ED)可能致病的基因突变并进行功能验证。方法:收集8例ED患者血液样本并进行基因组抽提,利用全外显子测序(whole-exome sequencing,WES)对患者基因组DNA进行高通量测序。对测序结果进行质量控制,确保质检合格后,从中筛选可能的致病基因突变。利用软件对筛选的基因突变致病性进行预测,利用免疫荧光实验及双荧光素酶实验对基因突变致病性进行功能验证。结果:所有样本全外显子测序有效率(effective rate)均为97.5%以上,错误率(error rate)均小于0.03%,Q20所占比例大于97.0%,目标区域的平均测序深度均为90×以上,提示测序数据质量良好。经筛选后,共发现3个可能致病的EDA基因点突变:c.959A>G,c.1073A>G,c.1001G>A,数据库比对显示其均为罕见变异,软件预测均为致病性突变。免疫荧光实验发现3个EDA突变可导致p65蛋白核移位减少,双荧光素酶实验进一步表明3个EDA突变可导致NF-κB通路活性降低。结论:本研究利用全外显子测序在ED患者基因组中发现了EDA突变,并对突变致病基因进行了功能验证,为进一步理解ED的发病机制提供了依据。

PURPOSE:To identify the potentially pathogenic mutations in patients with ectodermal dysplasia(ED) and to investigate the pathogenicity of mutations by functional studies.METHODS:Eight Chinese ED patients were included in this study.Peripheral venous blood was taken from the patients and DNA was extracted.Whole-exome sequencing(WES)was performed using DNA samples.After quality control of the sequencing data,the potentially pathogenic mutations were screened.The pathogenicity of the mutations was predicted in silico.Immunofluorescence study and dual luciferase assays were performed to investigate the pathogenicity of the mutations.RESULTS:The effective rates of all sequencing samples were above 97.5% and the error rates were less than 0.03%.The proportions of Q20 were more than 97.0%.The average sequencing depths of the target region were more than 90 ×.The sequencing data were acceptable for further analysis.After data screening,three missense mutations of EDA were detected,including c.959 A>G,c.1073 A>G and c.1001 G>A.The allele frequency was low in population database for all three mutations and in silico analysis indicated all three mutations were disease-causing.Immunofluorescence analysis showed that p65 protein nuclear translocation was compromised by EDA mutations,dual luciferase assays also showed that the activation of NF-κB pathway was decreased by EDA mutations.CONCLUSIONS:This study identified EDA mutations in Chinese ED patients and further verified the pathogenicity of the mutations by functional studies,contributing to the understanding of the pathogenesis of ED.