Genome-wide association analysis in dogs implicates 99 loci as risk variants for anterior cruciate ligament rupture

Abstract

Anterior cruciate ligament (ACL) rupture is a common condition that can be devastating and life changing, particularly in young adults. A non-contact mechanism is typical. Second ACL ruptures through rupture of the contralateral ACL or rupture of a graft repair is also common. Risk of rupture is increased in females. ACL rupture is also common in dogs. Disease prevalence exceeds 5% in several dog breeds, ~100 fold higher than human beings. We provide insight into the genetic etiology of ACL rupture by genome-wide association study (GWAS) in a high-risk breed using 98 case and 139 control Labrador Retrievers. We identified 129 single nucleotide polymorphisms (SNPs) within 99 risk loci. Associated loci (P<5E-04) explained approximately half of phenotypic variance in the ACL rupture trait. Two of these loci were located in uncharacterized or non-coding regions of the genome. A chromosome 24 locus containing nine genes with diverse functions met genome-wide significance (P = 3.63E-0.6). GWAS pathways were enriched for c-type lectins, a gene set that includes aggrecan, a gene set encoding antimicrobial proteins, and a gene set encoding membrane transport proteins with a variety of physiological functions. Genotypic risk estimated for each dog based on the risk contributed by each GWAS locus showed clear separation of ACL rupture cases and controls. Power analysis of the GWAS data set estimated that ~172 loci explain the genetic contribution to ACL rupture in the Labrador Retriever. Heritability was estimated at 0.48. We conclude ACL rupture is a moderately heritable highly polygenic complex trait. Our results implicate c-type lectin pathways in ACL homeostasis.

Fig 1. The Labrador Retriever is a dog breed with a relatively low amount of inbreeding.

Whiskers represent the standard error of the mean for each analysis method (n = 237 dogs).

Fig 2. Linear mixed model GWAS corrects for population structure and identifies 99 ACL associated loci explaining a large proportion of phenotypic variance.

For each linear mixed model (LMM), the QQ plots show no evidence of population stratification relative to the expected distribution. Permutation testing with each model determined genome-wide significance at (a) P<3.63E-7 for GCTA (Genome-wide Complex Trait Analysis) [33], λ = 0.987 (b) P<6.097E-7 for GEMMA (Genome-wide Efficient Mixed Model Association) [34], λ = 0.994 and (c) P<4.01E-7 for PUMA (Penalized Unified Multiple-locus Association) [35], λ = 1.012. The plots represent analysis of 118,992 SNPs from 98 cases and 139 phenotype-negative controls. (d) With GCTA, 36 loci have P<5E-4, with the most significant locus located in CFA 24, which did not meet genome-wide significance defined by minimum p-values from permutation testing. (e) With GEMMA, 47 loci have P<5E-4, with the locus on CFA 24 meeting genome-wide significance defined by minimum p-values from permutation testing. (f) With PUMA, 65 loci were significant at P<5E-4 and the locus on CFA 24 exceeded genome-wide significance defined by minimum p-values from permutation testing. The single SNP that met genome-wide significance lies within the gene PPP1R16B.

Fig 3. Phenotype variance was explained to a large degree by the associated genomic loci.

Loci identified by linear mixed model (LMM) analysis were broadly defined as SNPs with r²>0.5 within 5Mb of the peak SNP. (a) For GCTA, 36 loci in 72.7Mb of the genome explained 48.09% of the phenotypic variance. (b) For GEMMA, 47 loci in 82.7Mb of the genome explained 55.88% of the phenotypic variance. (c) For PUMA, 65 loci in 86.58Mb of the genome explained 50.28% of the phenotypic variance in the ACL rupture trait. Whiskers represent the standard error of the mean.

Fig 4. Genetic risk scoring [42] using GWAS associated loci from linear mixed model analysis with GEMMA segregates ACL rupture disease risk in case and control Labrador Retriever dogs.

(a) Distribution of the number of ACL rupture risk loci in case and control groups of Labrador Retriever dogs. The number of risk alleles in cases and controls is significantly different (P<2.2E-16) (b) ACL rupture odds ratios of weighted genetic risk scores (wGRS) relative to the first quartile. Vertical bars represent the 95% confidence intervals. * Odds ratio is significantly different from the reference first quartile.