An intronic LINE-1 insertion in MERTK is strongly associated with retinopathy in Swedish Vallhund dogs

The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA). In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS) of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10(-11)) that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10(-19)). In a pooled analysis of both studies (73 cases and 186 controls), the LINE-1 insertion was associated with a similar to 20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10(-27)). Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.Peer reviewe

IGV Illustration of the LINE-1 insertion in intron 1 of the <i>MERTK</i> gene.

<p><b>A</b>. The upper panel shows the Swedish Vallhund case, and the middle and lower panels represent two control WGS for comparison (a Labrador-Golden retriever cross and a Siberian Husky, respectively). The 15 bp duplicated sequence that is the LINE-1 insertion point is boxed. The CanFam 3.1 reference sequence (Boxer) is shown above and below the figure. <b>B</b>. Diagram to clarify the LINE-1 insertion in the Swedish Vallhund case. The 15 bp duplication is shown in orange, and soft-clipped bases in sequence reads at the start and end of the LINE-1 insertion (from the upper panel of <b>A</b>) are shown in dark and light blue, respectively. The line joining these two sequences represents the 6.401 kb of LINE-1 insertion sequence.</p

Genotypes for top GWAS SNP and LINE-1 insertion in Swedish Vallhunds from the UK and Finland.

<p>Genotypes for top GWAS SNP and LINE-1 insertion in Swedish Vallhunds from the UK and Finland.</p

Agarose gel electrophoresis analysis of PCR amplification of the LINE-1 insertion in UK Swedish Vallhund retinopathy cases and controls.

<p>Lanes 1 and 7 are a 2-Log DNA ladder (New England Biolabs, Hitchin, UK); Lanes 2 and 3 are controls negative for the LINE-1 insertion; Lanes 4 and 5 are cases positive for the insertion; Lane 6 is a non-template control.</p