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Kentaro Ohishi, Masafumi Ohtsubo, Katsuhiro Hosono, Akira Obana, Yoshihiro Hotta, Tadahisa Hiramitsu, Shinsei Minoshima; Forward Genetic Approach for Causal Gene Identification for Rat Retinal Photic Injury. Invest. Ophthalmol. Vis. Sci. 2017;58(8):288.
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To identify genes for the susceptibility of rat retina to photic injury, eventually to elucidate an etiology for age-related macular degeneration (AMD).
White light exposure of rats at 3000 lux for 3 hrs was performed to induce the photic injury in their retinas. The susceptibility to the photic injury was evaluated by Morris water maze test and pathological analysis of the eyes. The maze test was carried out on the 4.5th day after the light exposure, in which the swimming time to reach the target was recorded. In the pathological analysis, the degeneration size of retina was observed at 4 months as an average. The genome of each individual was analyzed using polymorphic microsatellite and SNP markers. A targeted exome analysis was performed with a next generation sequencer (MiSeqTM system; illumina, Inc.).
A rat strain (WKY) susceptible to and another strain (LEW) resistant to the photic injury were crossed to produce the F1 offspring. The F1 was susceptible, meaning that the susceptibility was a Mendelian autosomal dominant trait to the resistance. We named the trait “the susceptibility to retina photic injury” and the responsible gene “Rpi1”. In order to identify the Rpi1 gene, the first backcross (BC1) offspring was produced by mating F1 with the recessive parent LEW. Using the Morris water maze test after the light exposure, susceptible individuals in the BC1 generation were selected to produce BC2 offspring. Thus, as the backcross and selection of susceptible individuals were iterated, the ratio of WKY genome was gradually decreased. After 12 generations of the backcross, an obtained BC12 offspring had a 1.6-Mb WKY genome region within chromosome 5q36 as a sole WKY component, in which the Rpi1 gene must exist. We performed the targeted exome analysis for this region to identify Rpi1 and found 15 loci with non-synonymous base changes in coding regions of 12 genes. Using additional backcross individuals and another photic injury-susceptible strain F344, recently we could narrow down to only one candidate gene (tentatively gene R) with reasonable relationship between genotype and phenotype.
We consider that the gene R is Rpi1 itself. The gene R has never been reported as the susceptibility gene of AMD. We are now extensively making efforts to give more direct evidence for the gene R as the causal gene of rat retina photic injury and possible relationship with AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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