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.
Highlights
Familial exudative vitreoretinopathy (FEVR) is a rare inheritable retinal-vascular disorder characterized by retinal avascularity and first described by Criswick and Schepens in 1969 [1]
All mutations were proved to be cosegregated with the abnormal phenotype of FEVR probands and perceived as pathogenic or likely pathogenic in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines, where Norrie disease protein (NDP) c.127C>A (p.H43N), NDP c.129_131del (p.44del), NDP c.320_353del (p.R107Pfs), NDP c.321delG (p.L108Cfs), and FZD4 c.314T>G (p.M105R) were graded as pathogenic and NDP c.377G>T (p.C126F) was graded as likely pathogenic (Table 1) [20]
Protein sequence alignment of human Norrin/Frizzled-4 with homologues from human and other species indicated that NDP p.H43N, NDP p.C126F, and FZD4 p.M105R were nonconservative substitutions (Figure S1)
Summary
Familial exudative vitreoretinopathy (FEVR) is a rare inheritable retinal-vascular disorder characterized by retinal avascularity and first described by Criswick and Schepens in 1969 [1]. Numerous investigations have been conducted to date into the pathological mechanism of FEVR, where the canonical Wnt signaling network has been demonstrated to play a pivotal role during retinal organogenesis and angiogenesis [6, 10]. BioMed Research International elucidated to induce the Norrin/β-catenin canonical signaling pathway, one of the main drivers for retinal angiogenesis in the mammalian eye [6, 11,12,13,14,15]. As Xu et al described, Norrin with a cysteine-knot motif could bind with the cysteine-rich domain (CRD) of Frizzled-4 and potently activate the classical Wnt signaling pathway [13]. Mutations of NDP and FZD4 in cysteine-knot motif and CRD, respectively, are a good model for elucidating the high affinity of the Norrin-Frizzled-4 pair. The similar clinical phenotype presented by NDP and FZD4 mutants has led us to inquire the mechanism of the NorrinFrizzled-4 pair involved in retinal vasculogenesis
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