Functional and protein structure analysis of a novel FZD4 mutation c.428T>C in a family with familial exudative vitreoretinopathy

Abstract

Purpose : Familial exudative vitreoretinopathy (FEVR) is an autosomal dominant disease linked to genes like FZD4. Despite progress in genotyping, a gap remains between genetic insights and clinical manifestations. This retrospective study explores FEVR's molecular mechanism in a specific familial context using comprehensive methods. The aim is to present clinical features while elucidating the functional analysis of a novel FZD4 mutation.

Methods : Records from June 2020 to June 2023 were reviewed, focusing on four family members. Collected clinical data included demographics and clinical course. Whole exome sequencing and Sanger sequencing were employed to identify the causative gene responsible for FEVR within the family. To understand the functional consequences of a novel FZD4 variant (Leu143Pro), cellular models expressing both the wild-type and variant FZD4 gene were tested.

Results : A family afflicted by FEVR was identified. The proband, exhibiting a history of nystagmus and amblyopia, presented with an acute angle closure attack in the left eye (Figure_1). Following uneventful cataract surgery, intraocular pressure of the left eye decreased markedly postoperatively. Genetic analysis and familial segregation confirmed the presence of an FZD4 (NM_012193): c.T428C (p.Leu143Pro) mutation with varying degrees of affected phenotype in affected family members. Western blot analysis revealed that FZD4 expression of FZD4-L143P was significant lower than that of FZD4-WT in CDC-HMEC1 cells, a human microvascular endothelial cells (Figure_2). Low protein level in the FZD4-L143P was reversed partially by the addition of MG132, a proteosome inhibitor. Furthermore, protein structure analysis ascertained that the L143P mutation disrupted a helical structure typically observed in the wild-type Frizzled-4 protein and potentially interferes with its binding to the Wnt ligand.

Conclusions : FZD4 variant (Leu143Pro) is associated with FZD4 protein instability which is mediated by proteasomal degradation, and it also interferes its binding with the Wnt ligand. This study offers insights into the management of acute angle closure in FEVR, and elucidates the pathogenic implications of a previously unreported missense mutation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure_1. Clinical images of the proband.

 

Figure_2. Western blot showing lower protein level in FZD4-L143P cells (A-B) is reversed by the addition of MG132 (C).