Department of Ophthalmology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Sichuan Provincial Key Laboratory for Human Disease Gene Study, Center for Medical Genetics and Department of Laboratory Medicine, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, China.
Invest Ophthalmol Vis Sci. 2024 Apr 1;65(4):1. doi: 10.1167/iovs.65.4.1.
The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4.
Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/β-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells.
All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/β-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment.
We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.
本研究旨在报告五个在家族性渗出性玻璃体视网膜病变（FEVR）中发现的新型 FZD4 突变，并分析和总结 96 个报告的 FZD4 错义突变中的 34 个的致病机制。
纳入 5 名被诊断为 FEVR 的先证者及其家庭成员进行研究。对所有参与者进行眼部检查和靶向基因 panel 测序。基于从 FZD4 的每个结构域中选择突变，构建了携带 29 个先前报道的 FZD4 错义突变和 5 个新突变的质粒。在 HEK293T、HEK293STF 和 HeLa 细胞中，研究这些质粒突变对蛋白表达水平、Norrin/β-catenin 激活能力、膜定位、Norrin 结合能力和 DVL2 募集能力的影响。
导致 FEVR 的五个新突变（S91F、V103E、C145S、E160K、C377F）均发现破坏 FZD4 蛋白的 Norrin/β-catenin 激活。在总共审查了 34 个报告的错义突变后，我们根据它们的功能变化对所有突变进行了分类：信号肽突变、影响二硫键的半胱氨酸突变、影响 Norrin 结合的细胞外结构域突变、影响膜定位的跨膜结构域（TM）1 和 TM7 突变、影响 DVL2 募集的细胞内结构域突变。
我们扩展了与 FEVR 相关的 FZD4 突变谱，并通过实验证明，FZD4 中的错义突变可以根据不同的功能变化分为五类。
