An Intronic SINE Insertion in FAM161A that Causes Exon-skipping is Associated with Progressive Retinal Atrophy in Tibetan Spaniels and Tibetan Terriers

Abstract

Purpose: Progressive retinal atrophy (PRA) in dogs, like its human counterpart retinitis pigmentosa (RP), is characterized by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds, and is known to be genetically heterogeneous between breeds. Approximately 19 mutations have now been identified that are associated with PRA in 49+ breeds; for the majority of breeds, the responsible mutation(s) have yet to be identified. We undertook an investigation to characterize the mutation(s) responsible for PRA in the Tibetan Spaniel breed, and subsequently developed a DNA assay to facilitate the elimination of the mutant allele from the breed.

Methods: Genome-wide association mapping of 15,674 informative SNP markers with 22 Tibetan Spaniel PRA cases and 10 controls was undertaken to identify a disease-associated locus. This locus was further examined using targeted resequencing and putative disease-causing variants were further investigated using Sanger sequencing and qRT-PCR.

Results: A novel disease locus, PRA3, on CFA10 (p_raw = 2.01x10^-5, p_genome = 0.014) was identified, where a 3.8 Mb region was homozygous within 12 cases. A short interspersed nuclear element (SINE) insertion was identified near a splice acceptor site in an intron of a functional positional candidate gene, FAM161A. Comparison of mRNA of an affected and an unaffected dog revealed that the SINE causes exon-skipping, resulting in a frame shift, leading to a downstream premature termination codon and possibly a truncated protein product. This mutation segregates with the disease in 22 out of 35 cases tested (63%). Of the PRA controls (n=116 dogs) none are homozygous for the mutation, 15% carry the mutation and 85% are homozygous wildtype. This mutation was also identified in Tibetan Terriers. Our results indicate that PRA is genetically heterogeneous in both Tibetan Spaniels and Tibetan Terriers breeds.

Conclusions: PRA3 in the Tibetan Spaniel and Tibetan Terrier dog breeds is caused by a SINE insertion in FAM161A, a gene previously associated with RP28 in humans. As such PRA3 presents as a promising large animal model for further study of PRA3 and of the function of FAM161A in the retina.

Keywords: 696 retinal degenerations: hereditary • 534 gene mapping