Authors Suvi Mäkeläinen (1), Marta Gòdia (1,†), Minas Hellsand (2), Agnese Viluma (1), Daniela Hahn (1), Karim Makdoumi (3), Caroline J. Zeiss (4), Cathryn Mellersh (5), Sally L. Ricketts (5), Kristina Narfström (6), Finn Hallböök (2), Björn Ekesten (7), Göran Andersson (1), Tomas F. Bergström (1)
Affiliations 1. SLU, Dept. of Animal Breeding and Genetics, Uppsala, 2. Uppsala University, Dept. of Neuroscience, Uppsala, 3. School of Medical Sciences, Campus USÖ, Örebro, 4. Yale University School of Medicine, New Haven, 5. Animal Health Trust, Kennel Club Genetics Centre, Newmarket, 6. University of Missouri-Columbia, College of Veterinary Medicine, Columbia, 7. SLU, Dept. of Clinical Sciences, Uppsala, †. Present address: Centre for Research in Agricultural Genomics, Dept. of Animal Genomics, Bellaterra,
Presentation Type Talk
Abstract
Inherited retinal degenerations are a clinically and genetically heterogeneous group of diseases affecting both humans and dogs. Approximately 30 genes have so far been identified in various dog breeds. One of the most widespread mutations is the p.C2Y mutation in the PRCD gene causing progressive retinal atrophy (PRA) which primarily affects rod photoreceptor cells. The PRCD mutation is currently the only known genetic cause of retinal degeneration in Labrador retrievers. There are, however, unexplained retinal degenerations in Labrador retrievers.
We diagnosed a sib-pair of Labrador retrievers, free from the PRCD mutation, with a novel form of retinal degeneration mainly affecting cone photoreceptor cells. The affected dogs were visually impaired under both daylight and dim light conditions. Ophthalmoscopic examination revealed signs of retinal vascular attenuation as well as abnormal mottling in the central and peripheral retina. Using optical coherence tomography (OCT) along the visual streak of the retina, we observed a general neuroretinal thinning, particularly in the outer nuclear layer.
The objective of this study was to find the genetic cause for this novel disease. We performed whole-genome sequencing of the affected siblings and their unaffected parents. We used conditional filtering of exonic variants assuming an autosomal recessive mode of inheritance. To further reduce the number of candidate variants, we filtered against 23 additional dog genome sequences. This resulted in 18 nonsynonymous SNVs and four INDELs which were then further evaluated using their predicted effect on the protein function and associations with human retinal diseases. The strongest candidate, a frameshift insertion leading to a premature stop codon, was validated using Sanger sequencing of additional Labrador retrievers with and without the disease. All the cases were homozygous for the insertion, whereas the unaffected dogs were either heterozygous or homozygous for the wild-type allele.
Next, we functionally evaluated the effect of the identified genetic variant, performing real-time quantitative PCR, Western blotting and fluorescence histochemistry. For this we used retinal tissue from dogs with the three different genotypes. The affected individual showed a lower relative gene expression in comparison to unaffected individuals. Using Western blotting, a full-length protein was detected in the retinas of the unaffected dogs with both genotypes. In contrast, no protein was detected in a dog affected by this disease. These results were confirmed with fluorescence histochemistry. We conclude that we have identified a loss-of-function mutation causing a novel form of retinal degeneration in Labrador retrievers. The details of these findings will be presented.