Identification and functional analysis of novel FZD4 mutations in Han Chinese with familial exudative vitreoretinopathy.
Identification and functional analysis of novel FZD4 mutations in Han Chinese with familial exudative vitreoretinopathy.
复制标题
汉族家族性渗出性玻璃体视网膜病变FZD4新突变的鉴定及功能分析
DOI：
10.1038/srep16120
复制
发表时间：
2015-11-04
影响因子：
4.6
通讯作者：
Zhu X
中科院分区：
文献类型：
--
作者：
Fei P;Zhu X;Jiang Z;Ma S;Li J;Zhang Q;Zhou Y;Xu Y;Tai Z;Zhang L;Huang L;Yang Z;Zhao P;Zhu X
家族性渗出性玻璃体视网膜病变（FEVR）是一种以视网膜血管发育缺陷为特征的遗传性眼病。然而，已知的基因突变只能解释大约50%的FEVR患者。为了评估Frizzled 4（FZD 4）在中国患者中的突变频率，我们分析了来自中国61个家庭的FEVR患者，以确定FZD 4的突变并研究所确定的突变对FZD 4功能的影响。通过聚合酶链反应扩增FZD 4的所有编码外显子和相邻内含子区，并进行桑格测序分析。在这些家族中发现了FZD 4基因的三个突变。其中，两个是新突变：p.E134* 和p.T503fs。这两种突变都涉及高度保守的残基，并且在800名正常个体中不存在。通过定点突变将这两种新的FZD 4突变中的每一种引入野生型FZD 4 cDNA中。将野生型和突变型FZD 4 DNA引入HEK 293细胞中，以使用荧光素酶报告基因测定分析FZD 4在Norrin/β-连环蛋白途径的Norrin依赖性活化中的功能。p.E134* 和p.T503fs突变体均未能诱导荧光素酶报告基因活性以响应Norrin。我们的研究在中国FEVR患者中发现了两种新的FZD 4突变。
Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease characterized by defects in the development of retinal vessels. However, known genetic mutations can only explain approximately 50% of FEVR patients. To assess the mutation frequency of Frizzled 4 (FZD4) in Chinese patients, we analysed patients with FEVR from 61 families from China to identify mutations in FZD4 and to study the effects of identified mutations on FZD4 function. All coding exons and adjacent intronic regions of FZD4 were amplified by polymerase chain reaction and subjected to Sanger sequencing analysis. Three mutations in the FZD4 gene were identified in these families. Of these, two were novel mutations: p.E134* and p.T503fs. Both mutations involve highly conserved residues and were not present in 800 normal individuals. Each of these two novel FZD4 mutations was introduced into wild-type FZD4 cDNA by site-directed mutagenesis. Wild-type and mutant FZD4 DNAs were introduced into HEK293 cells to analyse the function of FZD4 in Norrin-dependent activation of the Norrin/β-catenin pathway using luciferase reporter assays. Both the p.E134* and p.T503fs mutants failed to induce luciferase reporter activity in response to Norrin. Our study identified two novel FZD4 mutations in Chinese patients with FEVR.