Genetic analyses of Lhasa Apso dogs with progressive retinal atrophy identifies a LINE-1 insertion in the promoter region of a retinal candidate gene.

Presenter Rebekkah Hitti
Authors Rebekkah J Hitti (1,2), Louise M Burmeister (1), Sally L Ricketts (1), Louise Pettitt (1), Mike Boursnell (1), Ellen C Schofield (1), David Sargan (2), Cathryn Mellersh (1)
Affiliations 1. Kennel Club Genetics Centre, Animal Health Trust, Lanwades Park, Newmarket, Suffolk, CB8 7UU, UK, 2. Department of Veterinary Medicine, University of Cambridge, Madingley Road Cambridge, CB3 0ES, UK.
Presentation Type Talk


Canine progressive retinal atrophy (PRA) is a degenerative retinal disease characterised by photoreceptor degeneration over time, increasing in severity and ultimately leading to vision loss. PRA affects multiple breeds and significantly impacts welfare. In the Lhasa Apso (LA) dog, PRA manifests typically as a mid-late onset form. Utilisation of whole-exome sequencing (WES) data previously generated in our laboratory from three PRA-affected LA (cases) and three PRA-unaffected LA (controls) did not reveal any obvious exonic or splice site polymorphisms segregating with the disease, indicating a non–coding mutation. This presented the opportunity for further investigations using a genome-wide association study (GWAS) and whole-genome sequencing (WGS) approach to identify the genetic cause of PRA and develop a DNA test.

A GWAS was conducted by genotyping 44 LA dogs (17 cases, 27 controls) on the Illumina Canine HD 170K chip. Allelic association statistics were adjusted for multiple testing using the PLINK Max(T) permutation procedure, and for population stratification and relatedness using Efficient Mixed-Model Association eXpedited (EMMAX). After stringent filtering and quality control, we tested 108,263 SNPs on 42 dogs, comprising 15 cases and 27 controls (call rate ≥97%; minor allele frequency ≥95%; genotype calls ≥90%). Analysis revealed a genome-wide significant association on canine chromosome 33 (-log praw = 2.2 x 10-16) which remained significant after correcting for multiple testing (pgenome = 0.9 x 10-5) and population substructure (p=raw1.6 x 10-17). A 1.3 megabase homozygous disease-associated region was defined, harbouring two candidate genes previously associated with human retinal degeneration.

WGS was undertaken on a single PRA affected LA, and manual interrogation of the critical region in identified a long interspersed nuclear element-1 (LINE-1) insertion, situated within the predicted promotor region of a retinal candidate gene. Due to the position of the LINE-1 insertion, it was not detected in the original WES data of the same case. The LINE-1 insertion was genotyped in 447 dogs across 122 breeds, including 63 LA dogs, and is private to the LA. Seventeen LA dogs (all clinically affected with PRA) were homozygous for the LINE-1 insertions, eight were heterozygous and thirty-eight were homozygous for the wildtype allele.

As a result of this study a DNA test for this form of PRA, termed PRA4, has been developed at the Animal Health Trust. To date, 457 LA from 15 countries have been tested for PRA4 (354 UK dogs; carrier frequency 17%; allele frequency 0.09). This study highlights the power of utilising several genetic approaches to identify a PRA mutation and develop a diagnostic test to help dog breeders make informed breeding choices, minimising the risk of producing PRA-affected LA dogs.