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B.J. Miller, G.M. Acland, G.D. Aguirre; Analysis of RPE65 Leu450Met Variation in Dogs with Inherited Retinal Degenerations . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4538.
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Excessive light may be an environmental factor which accelerates experimental retinal degeneration, or may modify the rate of the inherited diseases. The amino acid composition at codon 450 of the RPE65 protein has been shown to be associated with light damage susceptibility in mice. While leucine (wildtype) is associated with higher susceptibility, the methionine variant is protective. Similar nucleotide changes could underlie some of the variation in retinal disease severity found in non-rodent models. Purpose: To determine if the Leu450Met change is present in dogs with different allelic and non-allelic forms of inherited retinal degeneration, and, if present, determine if there is an association between disease severity and codon 450 amino acid composition. Methods: Digestion with MwoI of the 208 bp PCR product generated by primers RPE65-7F (5’-ACA CCC TGG TCA AGT AAA GCA CAT) and RPE65-6R (5’-AAA GGG TTC AGA CAC ATC CAT TG) identified the C to A transversion at nucleotide 1375. Digestion of the 208-bp product results in fragments of 140 and 68 bp in the wild type (C; leucine) while the variant (A; methionine) remains undigested. Results: 21 affected animals representing ten different breeds were screened for the codon 450 nucleotide change. Restriction analysis revealed the presence of the wildtype sequence (1375C; leucine450) in all samples. Conclusions: Preliminary results indicate the presence of leucine at amino acid 450 in all samples tested.
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