June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Forward genetic analysis combined with OCT and fundus photography identifies mutations leading to progressive outer retinal degeneration in C57BL/6J mice
Author Affiliations & Notes
  • Bogale Aredo
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Bo Chen
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Yi Ding
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Yuanfei Zhu
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Cynthia Zhao
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Ashwani Kumar
    McDermott Center for Human Growth and development, UT Southwestern Medical Center, Dallas, Texas, United States
  • Chao Xing
    Department of Population and Data Sciences, UT Southwestern Medical Center, Dallas, Texas, United States
    Department of Bioinformatics, UT Southwestern Medical Center, Dallas, Texas, United States
  • Stephen Lyon
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Jamie Russell
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Xiaohong Li
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Miao Tang
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Priscilla Anderton
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Sara Ludwig
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Eva Marie Moresco
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Bruce Beutler
    Center for the Genetics of Host Defense, UT Southwestern Medical Center, Texas, United States
  • Rafael Ufret-Vincenty
    Department of Ophthalmology, UT Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   Bogale Aredo, None; Bo Chen, None; Yi Ding, None; Yuanfei Zhu, None; Cynthia Zhao, None; Ashwani Kumar, None; Chao Xing, None; Stephen Lyon, None; Jamie Russell, None; Xiaohong Li, None; Miao Tang, None; Priscilla Anderton, None; Sara Ludwig, None; Eva Marie Moresco, None; Bruce Beutler, None; Rafael Ufret-Vincenty, None
  • Footnotes
    Support  NIH grants 1R01EY022652, U19AI100627, R01AI125581, and Visual Science Core Grant EY020799. An unrestricted grant from Research to Prevent Blindness, the Patricia and Col. William Massad Retina Research Fund and a grant from the David M. Crowley Foundation.
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1896. doi:
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    • Get Citation

      Bogale Aredo, Bo Chen, Yi Ding, Yuanfei Zhu, Cynthia Zhao, Ashwani Kumar, Chao Xing, Stephen Lyon, Jamie Russell, Xiaohong Li, Miao Tang, Priscilla Anderton, Sara Ludwig, Eva Marie Moresco, Bruce Beutler, Rafael Ufret-Vincenty; Forward genetic analysis combined with OCT and fundus photography identifies mutations leading to progressive outer retinal degeneration in C57BL/6J mice. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1896.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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