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Anastasia Marie Litke, Sarah Samuelson, Kerry Delaney, Yves Sauve, Robert L Chow; Investigating the pathogenicity of missense mutations in VSX1 and their association with corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2918.
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Two corneal dystrophies, posterior polymorphous dystrophy (PPD) and keratoconus, are associated with missense mutations in a transcription factor-encoding gene Visual System Homeobox 1 (VSX1). Despite this association, the pathogenic link between VSX1 and these diseases remains controversial. To address this issue, we utilized a variety of in vitro techniques to studying seven mutations found in disease populations spanning two highly conserved domains, the homeodomain (HD) and CVC domain. We further focused our approach with an in vivo investigation of one mutation, P247R.
Constructs expressing wild-type VSX1 or one of the seven mutations were used to investigate transcriptional activity and protein stability in vitro. Expression constructs were co-transfected into HEK 293T cells with a reporter containing VSX-specific binding sites and VSX1-dependent changes in transcriptional activity were measured. Protein stability and subcellular localization were measured through western blotting and immunolabeling, respectively. Mouse lines carrying the Vsx1 P247R mutation were generated to investigate pathogenicity in vivo. Confocal imaging was used to examine corneal morphology, curvature and thickness. Immunolabeling and electroretinogram (ERG) recordings were used to investigate retinal phenotypes compared to previously described phenotypes in Vsx1-null mice.
Similar to previous findings, our transcriptional activity assays showed that wild-type VSX1 can function as a transcriptional repressor in vitro. Five of the Vsx1 mutations trended towards an increase in transcriptional repression activity (n=5) while one, R217H, exhibited a complete loss of function phenotype (P<0.0002, n=5). No significant difference in protein stability or subcellular localization was observed in vitro (n=3). Although no changes in retinal terminal marker expression were observed in Vsx1 P247R homozygotes, ERG recordings showed a mild abnormality in the OFF response.
Vsx1 mutations spanning the HD and CVC domains altered transcriptional repression in vitro and visual signaling in vivo. These mutations therefore have the potential to be pathogenic in humans. Further investigation, however, is needed to determine whether VSX1 mutations found in disease populations are in fact causative for corneal dystrophies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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