Analysis of copy number variation in dogs implicates genomic structural variation in the development of anterior cruciate ligament rupture

Anterior cruciate ligament (ACL) rupture is an important condition of the
human knee. Second ruptures are common and societal costs are substantial.
Canine cranial cruciate ligament (CCL) rupture closely models the human
disease. CCL rupture is common in the Labrador Retriever (5.79%
prevalence), ~100-fold more prevalent than in humans. Labrador Retriever
CCL rupture is a polygenic complex disease, based on genome-wide
association study (GWAS) of single nucleotide polymorphism (SNP) markers.
Dissection of genetic variation in complex traits can be enhanced by
studying structural variation, including copy number variants (CNVs). Dogs
are an ideal model for CNV research because of reduced genetic variability
within breeds and extensive phenotypic diversity across breeds. We studied
the genetic etiology of CCL rupture by association analysis of CNV regions
(CNVRs) using 110 case and 164 control Labrador Retrievers. CNVs were
called from SNPs using three different programs (PennCNV, CNVPartition,
and QuantiSNP). After quality control, CNV calls were combined to create
CNVRs using ParseCNV and an association analysis was performed. We found
no strong effect CNVRs but found 46 small effect (max(T) permutation
P<0.05) CCL rupture associated CNVRs in 22 autosomes; 25 were
deletions and 21 were duplications. Of the 46 CCL rupture associated
CNVRs, we identified 39 unique regions. Thirty four were identified by a
single calling algorithm, 3 were identified by two calling algorithms, and
2 were identified by all three algorithms. For 42 of the associated CNVRs,
frequency in the population was <10% while 4 occurred at a
frequency in the population ranging from 10-25%. Average CNVR length was
198,872bp and CNVRs covered 0.11 to 0.15% of the genome. All CNVRs were
associated with case status. CNVRs did not overlap previous canine CCL
rupture risk loci identified by GWAS. Associated CNVRs contained 152
annotated genes; 12 CNVRs did not have genes mapped to CanFam3.1. Using
pathway analysis, a cluster of 19 homeobox domain transcript regulator
genes was associated with CCL rupture (P=6.6E-13). This gene cluster
influences cranial-caudal body pattern formation during embryonic limb
development. Clustered genes were found in 3 CNVRs on chromosome 14
(HoxA), 28 (NKX6-2), and 36 (HoxD). When analysis was limited to deletion
CNVRs, the association was strengthened (P=8.7E-16). This study suggests a
component of the polygenic risk of CCL rupture in Labrador Retrievers is
associated with small effect CNVs and may include aspects of stifle
morphology regulated by homeobox domain transcript regulator genes.