Genetics of Cruciate Ligament Rupture 59candidate genes were not related to CR
in the population studied. However, the
microsatellites they identified could be possible
candidates for other collagenopathies.
More recently, a candidate gene SNP geno-
typing approach was used to evaluate several
potential CR genes in high-risk breeds includ-
ing the Newfoundland, Labrador Retriever,
Rottweiler, and Staffordshire Bull Terrier (Baird
et al. 2014a). Genes tested for association
included collagen genes: COL1A1, COL1A2,
COL3A1,COL5A1,COL5A2,COL5A3,COL6A1,
COL6A3,COL11A1,COL11A2,COL14A1,and
COL24A1; fibril/elastic fiber formation genes:
FMOD (fibromodulin), DCN (decorin), ELN
(elastin),OPTC(opticin),LTBP2(latent trans-
forming growth factor beta binding protein
2), andBGN(biglycan); extracellular matrix
genes: FBN1 (fibrillin 1), COMP (cartilage
oligomeric matrix protein), andACAN(aggre-
can); collagen-formation genes:SERPINH1(ser-
pin pertidase inhibitor clade H (heat shock pro-
tein 47) member 1 (collagen binding protein
1),PLOD1(procollagen-lysine, 2-oxoglutarate
5-dehydrogenase), andLOX(lysyl oxidase); col-
lagen cleavage genes:MMP1 (matrix metal-
lopeptidase 1) andCTSK(cathepsin K); and
ligament/tendon/limb development geneSIX1
(SIX homeobox 1).
When breeds were analyzed separately, two
significant SNPs were identified: one in the
Labrador Retriever (correctedP<0.001) and
one in the Rottweiler (correctedP = 0.02).
The SNP identified in Labrador Retrievers was
located withinCOL24A1. The SNP identified
in Rottweilers was initially considered to be
located within the elastin gene, but was re-
mapped to an intergenic region when the
data were updated to the newest build of the
canine genome. When all breeds were con-
sidered together, three significant SNPs were
identified: two were located inCOL5A1and
the third was located withinCOL1A1.These
results suggest that collagen genes, particularly
COL24A1,COL5A1,andCOL1A1, play some
role in CR pathogenesis. It is also important
to note that the associations onCOL5A1and
COL1A1were shared across all four breeds.
This provides evidence that these mutations
occurred in the canine genome before breed
formation (Karlssonet al. 2008). The discov-
eryof genetic mutations that are shared across
multiple breeds has the most potential for clini-
cal impact.Genome-wide association
Genome-wide association studies (GWAS) use
genetic markers across the entire set of DNA
for an individual (genome) to first identify a
chromosomal region that is associated with a
disorder, and then identify all genes located
in that region. The genes are then organized
by function and selected for further investi-
gation according to possible involvement in
the development of the disease or trait being
investigated. Before the advent of commercial
SNP arrays, GWAS could be performed using
microsatellites (MSATs). A microsatellite is a
variable repeating segment of DNA, usually
found in a non-coding segment. Microsatel-
lites can be highly polymorphic and very infor-
mative. However, recent advances in DNA
sequencing technology, computational hard-
ware and bioinformatics have made SNP arrays
the tool of choice for GWAS.
Using microsatellites, a GWAS for CR was
performed using 90 Newfoundlands that were
selected for the GWAS based on their degree
of inter-relatedness and the statistical likeli-
hood that they segregated into homozygous
unaffected and homozygous affected animals
(Macrossanet al.2005). Age and other potential
contributors to the cause of CR were not consid-
ered in this analysis. A total of 495 MSATs was
used to compare genotypes and allele frequen-
cies between CR-affected and unaffected dogs.
Four markers (located on four chromosomes)
were significant after the false discovery rate
was controlled at the 0.05 level using the Storey
and Tibshirani method (Storey & Tibshirani
2003; Wilkeet al.2009; Wilke 2010). The MSATs
were CPH19 located on chromosome 3, FH3702
on chromosome 5, REN147D07 located on chro-
mosome 13, and FH3750 located on chromo-
some 24. Initial validation of the four markers
confirmed significance of three; canine chromo-
somes 3, 5, and 13. Positional candidate genes
located on chromosome 3 were sequenced for
mutation identification; Versican core protein
precursor (VCAN) and aggrecan core protein
precursor (cartilage-specific protein core protein;
CSPCP). Proteoglycans are major components