Abstract
Purpose:
The dog is an attractive model organism to study retinal degeneration, because the eye is anatomically similar to the human one, and the phenotypic characteristics of progressive retinal atrophy (PRA) highly resemble that of human rod-cone and cone-rod dystrophies in terms of onset and progression. Also, canine PRA models have shown to be instrumental in the development of gene therapy, for instance in translating RPE65 gene augmentation therapy to the clinic. In this study, we aim to identify the genetic defect underlying PRA in several dog breeds with unknown causative mutation employing next generation sequencing technology.
Methods:
In five dog breeds segregating PRA (Airdale Terrier, Berger des Pyrénées, Chihuahua, Löwchen and Saarloos Wolfshond), whole exome sequencing was performed in one affected and one unaffected dog. A completely new pipeline was built to map all reads to the canine genome and annotate all variants to the corresponding canine genes. Subsequent filtering of the variants included removing variants that were present in unaffected dogs, and prioritization of variants that were highly conserved or present in known human retinal disease genes. Candidate pathogenic variants were validated with Sanger sequencing.
Results:
Despite the poor annotation of the canine genome, by using gene prediction software, we have been able to correctly annotate ~20,000 canine genes. In each sample, exome sequencing yielded approximately 60,000 genomic variants. After filtering and prioritizing the variants, in each affected dog, on average 37 candidate pathogenic variants were remaining. For the variants that have been validated so far, the majority (>95%) was confirmed with Sanger sequencing analysis. Currently, we are assessing the potential pathogenicity of these variants in larger cohorts and/or by performing additional experiments.
Conclusions:
Our data show that exome sequencing is a powerful tool to assess genetic variation in protein-coding parts of the genome, not only in humans but also in dogs. Further characterization of candidate variants will likely result in the identification of the underlying genetic defect in canine models of PRA, and may facilitate the development of novel therapeutic interventions in these models.
Keywords: 539 genetics •
696 retinal degenerations: hereditary