WFS1突变致聋家系的鉴定及斑马鱼同源基因时空表达谱分析

Identification of deafness-causative mutations of WFS1 in two families and spatio-temporal expression profiles of its homologous genes in zebrafish

目的:对2个遗传性耳聋家系进行分子病因学鉴定,并应用斑马鱼对致聋基因WFS1进行初步功能分析。方法:利用靶向捕获测序技术,对2个家系进行外显子测序分析,确定候选致病基因。生物信息学预测人WFS1基因的功能以及模式生物斑马鱼与人类WFS1基因的同源性;采用整胚原位杂交和定量PCR法分析斑马鱼wfs1a和wfs1b的时空表达特征。结果:外显子测序及家系遗传共分离分析确定WFS1基因c.2036~2038delAGG(p.680delE)和c.1957C>T(p.653R>C)突变分别是2个家系的分子病因。定量PCR和全胚原位杂交结果显示斑马鱼wfs1a和wfs1b在胚胎发育不同时期呈现不同的时空表达特征。生物信息学分析提示wfs1b与人WFS1基因的进化距离更近,同源性更高。结论:2个遗传性耳聋家系均由WFS1突变所致,拓展了遗传性耳聋的基因突变谱。斑马鱼胚胎发育过程中wfs1a和wfs1b具有明显的时空表达特异性,wfs1b是人类WFS1的直系同源基因。研究结果既为耳聋分子诊断提供了支持,也为后续深入研究WFS1的突变致聋机制奠定了基础。

Objective:This study aims to identify the molecular etiology of two non-syndromic hearing loss families,and to use zebrafish to analyze the function of WFS1 on the occurrence of hearing loss.Methods:By using targeted capture sequencing technology,exon sequencing analysis was performed on two families to identify candidate pathogenic genes.The function of human WFS1 gene and the homology of WFS1 gene between zebrafish and human were analyzed by bioinformatics.The temporal and spatial expression characteristics of zebrafish wfs1 a and wfs1 b were analyzed by whole-embryo in situ hybridization and quantitative PCR.Results:Exon sequencing and co-segregation analysis of two families suggested that the heterozygous mutation of the WFS1 c.2036~2038 delAGG(p.680 delE)and c.1957 C>T(p.653 R>c)was the molecular pathogenesis basis of the pedigree JSNY-021 and JSNY-033,respectively.Quantitative PCR and whole-embryo in situ hybridization results showed that zebrafish wfs1 a and wfs1 b showed different spatio-temporal expression characteristics at different embryonic development stages.Bioinformatics analysis suggested that wfs1 b had a closer evolutionary distance and higher homology with human WFS1.Conclusion:This study confirmed that the molecular etiology of two autosomal dominant non-syndrome hearing loss families are WFS1 mutations,which expanded the gene mutation spectrum of hereditary hearing loss.The expression of wfs1 a/wfs1 b in the embryonic development of the zebrafish model has obvious spatio-temporal specificity.Wfs1 b is the direct homologous gene of human WFS1.The results of this study not only provide support for the molecular diagnosis of hearing loss,but also lay a foundation for further study on the mutagenesis mechanism of WFS1.