Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China.
Sichuan Provincial Key Laboratory for Human Disease Gene Study, the Department of Medical Genetics, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan, China.
J Med Genet. 2023 Feb;60(2):174-182. doi: 10.1136/jmedgenet-2021-108259. Epub 2022 Mar 31.
Familial exudative vitreoretinopathy (FEVR) is an inheritable blinding disorder with clinical and genetic heterogeneity. Heterozygous variants in the gene have been reported to cause FEVR. However, the pathogenic basis of -associated FEVR has not been fully explored.
Whole-exome sequencing was performed on the genomic DNA of probands. Dual-luciferase reporter assay, western blotting and co-immunoprecipitation were used to characterise the impacts of variants. Quantitative real-time PCR, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and immunocytochemistry were performed on the primary human retinal microvascular endothelial cells (HRECs) to investigate the effect of depletion on the downstream genes involved in Norrin/β-catenin signalling, cell proliferation and junctional integrity, respectively. Transendothelial electrical resistance assay was applied to measure endothelial permeability. Heterozygous endothelial-specific -knockout mouse mice were generated to verify FEVR-like phenotypes in the retina.
We identified two novel heterozygous variants (p.Leu103Ter and p.Val199LeufsTer11) and one previously reported heterozygous variant (p.His369ThrfsTer2) in the gene. These variants caused truncation and degradation of β-catenin that reduced Norrin/β-catenin signalling activity. Additionally, knockdown (KD) of in HRECs led to diminished mRNA levels of Norrin/β-catenin targeted genes, reduced cell proliferation and compromised junctional integrity. The Cre-mediated heterozygous deletion of in mouse endothelial cells (ECs) resulted in FEVR-like phenotypes. Moreover, LiCl treatment partially rescued the defects in -KD HRECs and EC-specific heterozygous knockout mice.
Our findings reinforced the current pathogenesis of Norrin/β-catenin for FEVR and expanded the causative variant spectrum of for the prenatal diagnosis and genetic counselling of FEVR.
家族渗出性玻璃体视网膜病变（FEVR）是一种具有临床和遗传异质性的遗传性致盲疾病。已报道 基因中的杂合变异可导致 FEVR。然而，-相关 FEVR 的发病基础尚未完全探索。
对先证者的基因组 DNA 进行全外显子组测序。双荧光素酶报告基因检测、Western blot 和免疫共沉淀用于研究变异的影响。定量实时 PCR、EdU（5-乙炔基-2'-脱氧尿苷）掺入测定和免疫细胞化学用于研究 耗竭对 Norrin/β-catenin 信号通路、细胞增殖和连接完整性相关下游基因的影响。跨内皮电阻测定用于测量内皮通透性。生成杂合内皮特异性 -敲除小鼠以验证视网膜中类似 FEVR 的表型。
我们在 基因中发现了两个新的杂合变异（p.Leu103Ter 和 p.Val199LeufsTer11）和一个先前报道的杂合变异（p.His369ThrfsTer2）。这些变异导致 β-catenin 的截断和降解，从而降低 Norrin/β-catenin 信号活性。此外，HRECs 中的 敲低导致 Norrin/β-catenin 靶向基因的 mRNA 水平降低、细胞增殖减少和连接完整性受损。Cre 介导的内皮细胞（ECs）中 杂合缺失导致类似 FEVR 的表型。此外，LiCl 处理部分挽救了 -KD HRECs 和 EC 特异性 杂合敲除小鼠的缺陷。
我们的发现强化了 Norrin/β-catenin 对 FEVR 的当前发病机制，并扩展了 用于 FEVR 的产前诊断和遗传咨询的致病变异谱。
