Role of NDP- and FZD4-Related Novel Mutations Identified in Patients with FEVR in Norrin/β-Catenin Signaling Pathway

Abstract

Mutations in NDP and FZD4 have been closely related to a series of retinal diseases including familial exudative vitreoretinopathy (FEVR). Our study was designed to identify novel NDP and FZD4 mutations by whole exome sequencing (WES) in a cohort of patients with a definitive diagnosis of FEVR and explore the underlying molecular mechanism. During 2016, we investigated fifty nonconsanguineous families with affected individuals exhibiting FEVR phenotype and WES identified one recently reported mutation: NDP c.127C>A (p.H43N), and five novel mutations: NDP c.129_131del (p.44del), NDP c.320_353del (p.R107Pfs), NDP c.321delG (p.L108Cfs), NDP c.377G>T (p.C126F), and FZD4 c.314T>G (p.M105R) that cosegragated with the abnormal fundus vascular manifestations in six families. All the mutations were perceived to be pathogenic or likely pathogenic according to the standards and guidelines from the American College of Medical Genetics and Genomics (ACMG) and predicted to be deleterious by a series of bioinformatics analyses. We systematically performed functional analyses on the six mutations utilizing the Topflash reporter assay, where all NDP and FZD4 mutants revealed at least 50% loss of wild-type activity. Immunoprecipitation finally demonstrated that the six mutations could degrade the Norrin-Frizzled-4 pair-binding effect to varying degrees. Finally, our study underscores the correlation between the FEVR phenotype and genotype in NDP and FZD4, extending the mutation spectrum, allowing a reliable assessment of FEVR recurrence and improving genetic counseling. Further, our findings provide essential evidence for the follow-up study of animal models and drug targets by Topflash assays and immunoprecipitation.

Genotype-phenotype associations in familial exudative vitreoretinopathy: A systematic review and meta-analysis on more than 3200 individuals

“…Overall, the conclusions of our statistical analysis matched the pathogenesis explored in the current studies. Additionally, Shuai Han et al [ 55 ] found that different gene mutations have varying degrees of effects on the activity of Norrin/β-catenin Signaling Pathway. In the future, we will further explore the correlation between gene mutations sites and clinical manifestations of cases with FEVR, and study the interaction between various mutated genes.…”

Heterozygote loss‐of‐function variants in the LRP5 gene cause familial exudative vitreoretinopathy

“…All the variants were introduced into the wild‐type pCDNA3.1‐ LRP5 expression plasmid by site‐directed mutagenesis using a Q5 Site Directed Mutagenesis Kit (New England Biolabs, USA). The luciferase activity assay was performed as previously described 28 . HEK293 STF cells stably expressing the Wnt reporter SuperTOPFlash were seeded onto a 24‐well plate, and each well was co‐transfected with the following plasmids: 100 ng LRP5 (wild‐type, mutant or empty vector), 100 ng FZD4 , 100 ng NDP , and 200 ng pGL4.1‐Renilla , using EntransterTM‐H4000 transfection reagent (Engreen, China) according to the manufacturer's instructions.…”

“…The luciferase activity assay was performed as previously described. 28 HEK293 STF cells stably expressing the Wnt reporter SuperTOPFlash were seeded onto a 24-well plate, and each well was cotransfected with the following plasmids: 100 ng LRP5 (wildtype, mutant or empty vector), 100 ng FZD4, 100 ng NDP, and 200 ng pGL4.1-Renilla, using EntransterTM-H4000 transfection reagent (Engreen, China) according to the manufacturer's instructions. After 48 hours, the transfected cells were subjected to luciferase activity assays using the Promega dual-luciferase reporter assay system according to the manufacturer protocols.…”

A novel frameshift variant in the TSPAN12 gene causes autosomal dominant FEVR

“…The Norrin/β‐catenin pathway plays an important role in retinal vascular development (Chen et al, 1993 ; Park & Yamamoto, 2019 ; Poulter et al, 2010 ; Tucci et al, 2014 ; Yang et al, 2021 ; Zhang et al, 2021 ; Zhu et al, 2021 ). In this pathway, the extracellular ligand Norrin (encoded by NDP ) binds to a receptor complex composed of Low‐density lipoprotein receptor‐related protein‐5 (LRP5) and Frizzled‐4 (FZD4) to inhibit the degradation of β‐catenin, which activates the expression of downstream genes (Fei et al, 2015 ; Han et al, 2020 ; Panagiotou et al, 2017 ; Peng et al, 2022 ; Wang et al, 2021 ; Zhao et al, 2022 ). TSPAN12 plays a role in stabilizing ligand‐receptor complex in Norrin/β‐catenin signaling pathway (Ke et al, 2013 ; Panagiotou et al, 2017 ; Schatz & Khan, 2017 ; Xin et al, 2010 ).…”