Background: Familial exudative vitreoretinopathy (FEVR) is an inheritable retinal vascular disease, which often leads to severe vision loss and blindness in children. However, reported mutations can only account for 50-60% of patients with FEVR. The purpose of this study was to identify novel frizzled class receptor 4 (FZD4) and Norrin cystine knot growth factor NDP (NDP) mutations in a cohort of Indian patients with FEVR by whole-exome sequencing. Methods: We performed data filtering and bioinformatic analyses. Results: Two novel heterozygous mutations in FZD4 gene were identified, each in two different families: c.1499_1500del [p.500_500del] and c.G296C [p.C99S]. One novel mutation in NDP in another family was identified: c.A256G [p.K86E]. All FZD4 mutations affected conserved amino acid residues and were absent in 1000 control individuals. To assess the effect of these FZD4 mutations on the biological activity of the protein, we introduced each FZD4 mutation into FZD4 cDNA by the site-directed mutagenesis techniques. A Norrin/beta-catenin pathway-based luciferase reporter assay revealed that the c.1499_1500del failed to activate the luciferase reporter; in contrast, compared with the wild-type FZD4 protein, the, c.G296C [p.C99S] mutation exhibited increased luciferase reporter activity. Conclusion: Our study found two novel FZD4 mutations, with opposite effects regarding functional expression levels in Indian patients with FEVR and expands on the mutational spectrum of FZD4 in Indian FEVR patients.

中文翻译：

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印度南部家族性渗出性玻璃体视网膜病变患者的FZD4和NDP基因新突变的鉴定。

背景：家族性渗出性玻璃体视网膜病变（FEVR）是一种可遗传的视网膜血管疾病，通常会导致儿童严重的视力丧失和失明。但是，报告的突变仅占FEVR患者的50-60％。这项研究的目的是通过全外显子组测序确定印度FEVR患者队列中的新型卷曲蛋白受体4（FZD4）和Norrin胱氨酸结结生长因子NDP（NDP）突变。方法：我们进行了数据过滤和生物信息学分析。结果：在FZD4基因中鉴定出两个新的杂合突变，每个都在两个不同的家族中：c.1499_1500del [p.500_500del]和c.G296C [p.C99S]。在另一个家族的NDP中发现了一个新突变：c.A256G [p.K86E]。所有FZD4突变均影响保守的氨基酸残基，并且在1000个对照个体中不存在。为了评估这些FZD4突变对蛋白质生物学活性的影响，我们通过定点诱变技术将每个FZD4突变引入FZD4 cDNA中。基于Norrin /β-catenin途径的萤光素酶报告基因检测结果表明，c.1499_1500del无法激活萤光素酶报告基因。相反，与野生型FZD4蛋白相比，c.G296C [p.C99S]突变表现出增加的荧光素酶报道分子活性。结论：我们的研究发现了两个新的FZD4突变，对印度FEVR患者的功能表达水平有相反的影响，并且扩大了印度FEVR患者FZD4的突变谱。基于Norrin /β-catenin途径的萤光素酶报告基因检测结果表明，c.1499_1500del无法激活萤光素酶报告基因。相反，与野生型FZD4蛋白相比，c.G296C [p.C99S]突变表现出增加的荧光素酶报道分子活性。结论：我们的研究发现了两个新的FZD4突变，对印度FEVR患者的功能表达水平有相反的影响，并且扩大了印度FEVR患者FZD4的突变谱。基于Norrin /β-catenin途径的萤光素酶报告基因检测结果表明，c.1499_1500del无法激活萤光素酶报告基因。相反，与野生型FZD4蛋白相比，c.G296C [p.C99S]突变表现出增加的荧光素酶报道分子活性。结论：我们的研究发现了两个新的FZD4突变，对印度FEVR患者的功能表达水平有相反的影响，并且扩大了印度FEVR患者FZD4的突变谱。