Forward genetic analysis combined with OCT and fundus photography identifies mutations leading to progressive outer retinal degeneration in C57BL/6J mice

Abstract

Purpose : Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Our goal was to employ a forward genetics approach to find, in an unbiased manner, genes that are essential to these processes.

Methods : We used N-ethyl-N-nitrosourea mutagenesis in C57BL/6J mice and automated meiotic mapping in combination with a retinal screening protocol (optical coherence tomography [OCT] imaging and yellow fundus spots in fundus photography) in order to identify genes associated to retinal phenotypes.

Results : We examined 5,700 G3 mice carrying 11,305 coding/splicing mutations in 7,698 genes (genome saturation is estimated at 4.9%). Eleven genes that were previously known to cause retinal degeneration were identified. Additionally, multiple statistically significant gene-phenotype associations that have not been reported before were discovered. We used CRISPR/Cas-9 technology to target a gene associated to retinal thinning on OCT, Sfxn3, and a gene associated to increased fundus spots. We demonstrate, using OCT, light microscopy, and electroretinography (ERG) that two Sfxn3 KO mouse lines with targeted disruption of the “retinal thinning gene” developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium, disruption of the external limiting membrane, and disruption of synapses and synaptic ribbons in the outer plexiform layer. Using single cell RNA sequencing and pathway analysis of retinal cells isolated from C57Bl/6J mice we demonstrate that this gene may be important in synaptic homeostasis. Early phenotyping of the “fundus spot gene” will also be presented.

Conclusions : As distinct from other mutagenesis/mapping protocols, our approach permits instantaneous identification of causative mutations as soon as screening is completed. This allows assessment of multiple alleles at loci under consideration, identifies non-causative mutations as well as causative ones, and tabulates saturation of the genome. This protocol is helpful in finding mutations affecting retinal development and homeostasis soon after phenotyping.

This is a 2020 ARVO Annual Meeting abstract.