Investigating the pathogenicity of VSX1 missense mutations and their association with corneal disease

Abstract

PURPOSE. Despite numerous studies associating Visual System Homeobox 1 (VSX1), with posterior polymorphous corneal dystrophy and keratoconus, its role in these diseases is unclear. Here we examine the pathogenicity of VSX1 missense mutations in vitro and in a mouse genetic model. METHODS. Vsx1 transcriptional repressor activity, protein stability, and subcellular localization activity, was examined using luciferase reporter-based assays, western blotting and immunolabeling, respectively, in transfected human embryonic kidney 293T cells. A genetic model for VSX1 p.P247R was generated to investigate pathogenicity of the mutation, in vivo. A wholemount confocal imaging approach on unfixed intact eyes was developed to examine corneal morphology, curvature, and thickness. Immunolabeling and electroretinography was used to examine retinal phenotype. RESULTS. A mutation corresponding to human VSX1 p.P247R led to enhanced transcriptional repressor activity, in vitro. A mouse model for VSX1 p.P247R did not have any observable corneal defect, but did exhibit an abnormal electroretinogram response characterized by a more prominent ON as opposed to OFF panretinal responsiveness. In vitro analysis of additional VSX1 missense mutations showed that they either enhanced repressor activity or did not alter activity. CONCLUSIONS. Our results indicate that although VSX1 sequence variants can alter transcriptional activity, in the context of a mouse genetic model, at least one of these changes does not lead to corneal abnormalities. While we cannot exclude a role for VSX1 as a risk factor for corneal disease, on its own, it does not appear to play a major causative role.