Novel protective and risk loci in hip dysplasia in German Shepherds

Abstract

Canine hip dysplasia is a common, non-congenital, complex and hereditary disorder. It can inflict severe pain via secondary osteoarthritis and lead to euthanasia. An analogous disorder exists in humans. The genetic background of hip dysplasia in both species has remained ambiguous despite rigorous studies. We aimed to investigate the genetic causes of this disorder in one of the high-risk breeds, the German Shepherd. We performed genetic analyses with carefully phenotyped case-control cohorts comprising 525 German Shepherds. In our genome-wide association studies we identified four suggestive loci on chromosomes 1 and 9. Targeted resequencing of the two loci on chromosome 9 from 24 affected and 24 control German Shepherds revealed deletions of variable sizes in a putative enhancer element of the NOG gene. NOG encodes for noggin, a well-described bone morphogenetic protein inhibitor affecting multiple developmental processes, including joint development. The deletion was associated with the healthy controls and mildly dysplastic dogs suggesting a protective role against canine hip dysplasia. Two enhancer variants displayed a decreased activity in a dual luciferase reporter assay. Our study identifies novel loci and candidate genes for canine hip dysplasia, with potential regulatory variants in the NOG gene. Further research is warranted to elucidate how the identified variants affect the expression of noggin in canine hips, and what the potential effects of the other identified loci are.

Fig 1. Genome-wide association for canine hip dysplasia: FASTA and QTSCORE, N(cases) = 132, N(controls) = 160.

For the upper two figure segments the red horizontal lines are the thresholds for Bonferroni correction for significance. The blue horizontal line is the threshold for significance for independent tests. In the undermost segment, where the permuted P-values are shown, the single red line represents the threshold for genome-wide significance level of 0.05.

Fig 2. Genome-wide association for canine hip dysplasia: First meta-analysis, N(cases) = 132, N(controls) = 277.

For the upper two figure segments the red horizontal line is the threshold for Bonferroni correction for significance. The blue horizontal line is the threshold for significance for independent tests. In the undermost segment, where the permuted P-values are shown, the single red line represents the threshold for genome-wide significance level of 0.05.

Fig 3. Genome-wide association for canine hip dysplasia: Second meta-analysis, N(cases) = 247, N(controls) = 277.

For the upper figure segment the red horizontal line is the threshold for Bonferroni correction for significance. The blue horizontal line is the threshold for significance for independent tests. In the undermost segment, where the permuted P-values are shown, the single red line represents the threshold for genome-wide significance level of 0.05.

Fig 4. Human NOG enhancer region compared to the related region in the dog genome.

At the top of the image there is a sequence comparison between human and dog. hs = human genomic (GRCh38.p7) region at chr17:56592775–56592815. cf = canine genomic (CanFam3.1) region at chr9:31453829–31453899. d1-d4 = the corresponding sequences of Dog1-Dog4. Below the sequence comparison is first the canine genomic region including the deletion (blue triangle), gap region marked with Ns, and the complete coding sequence for NOG from a Beagle (GenBank: AB544074.1). At the bottom of the image are the corresponding human genomic regions with a TF binding site (orange box) and TFs that can bind to this site as predicted by the ENCODE ChIP-seq experiments. Green boxes are H3K4Me1 histone mark (commonly associated with enhancers) peaks, and the blue box is a H3K4Me3 histone mark (commonly associated with active promoters) peak from the ENCODE data.

Fig 5. The differential effects of three NOG regulatory alleles in a dual luciferase reporter assay.

Relative luminisence (NanoLuc/firefly luminisence expressed as median value ± standard deviation, three biological replicates with four technical replicates each per every construct). Left: HEK293 cells transfected with 50 ng plasmid DNA + 50 ng carrier DNA. Right: U-2 OS cells transfected with 10 ng plasmid DNA + 10 ng carrier DNA. pNL: empty control vector. A: construct A, B: construct B, C: construct C. *: P<0.05 relative to construct A. The difference between relative luminescence of the control plasmid and each of the constructs was always statistically significant.