The Genetic Basis of Canine Osteoarthritis

Osteoarthritis (OA) is a common debilitating disease ofmammalian joints. Canine OA was classically understood to arise secondary to articular diseases, such as elbow dysplasia, hip dysplasia and cranial cruciate ligament rupture which produced a mechanical dYllfunction of the affected joint. However it is now recognised that primary factors, such as genetics, govern the severity ofOA for a given articular disease. In this study, two different aspects ofthe genetic basis to OA were investigated; gene transcription in diseased tissues and gene polymorphism frequencies in populations of dogs with diseases predisposing to OA. Evaluation of the quality of extracted mRNA from canine joint tissues by rnicrofluidic electrophoresis traces revealed that there were no differences in the quality of samples extracted using either an isopropanol or ethanol precipitation method. However, a significant proportion ofRNA samples (32%) were identified as degraded, highlighting the importance of assessing RNA quality before usage. In OA canine hip cartilage, there was an increase in the gene expression of structural matrix molecules (collagens and small leucine rich proteoglycans) and proteinases (matrix metalloproteinase 13, cathepsin -B and -D), with concurrent decreased expression of selected inhibitors or protease activity (tissue inhibitors of metalloproteinase-2 and -4) when compared to normal articular cartilage using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. The general pattern of changes in matrix-associated gene expression was similar to that reported in naturally occurring human OA cartilage. Canine-specific oligonucleotide microarray gene expression profiling of a small sample set of normal and OA articular cartilage samples identified differential expression of a number of genes not previously associated with the disease. However, the high degree of heterogeneity observed in the expression profile data generated hampered subsequent data interpretation, and highlighted the limitations of expression profiling small sample sets with limited phenotype stringency. Quantification of matrix-associated gene expression in OA elbow cartilage by RT-qPCR identified changes which were consistent with those identified in end stage hip OA cartilage, and which correlated with the radiographic severity of elbow OA for a number of genes (such as type I collagen alpha two chain, type III collagen alpha one chain and tissue inhibitor of matrix metalloproteinase 2). Matrix metalloproteinase expression in OA elbow trabecular bone was also identified to be increased when compared to normal trabecular bone, when quantified by RT-qPCR. A general pattern of increased protease and extracellular structural matrix gene expression was identified in ruptured canine cranial cruciate ligaments (CCL) when compared to intact CCLs, with both RTqPCR and oligonucleotide microarray. No significant differences were identified between the gene expression profiles of normal CCLs of a breed predisposed to CCL rupture (Labrador Retriever) when compared to a breed relatively resistant to CCL rupture (Greyhound), although a degree of risk-specific clustering was observed for expression profiles of genes which were differentially expressed in CCL rupture. The expression profiles ofruptured canine CCLs were similar to those previously reported for ruptured human CCLs. A transcriptomic basis to breed specific risk for the development ofcanine CCL rupture was not identified. Microarray data sets generated from normal and OA canine articular cartilage and normal and ruptured CCL were filtered to identify new reference genes for use in RT-qPCR experiments. One of the new reference genes (Mitochondrial ribosomal protein S7 [MRPS7]) demonstrated a high degree of stability across multiple articular tissues from normal and OA canine joints, as determined by multiple, different reference gene stability assessment algorithms, making it a potential universal reference gene for use in canine OA tissue studies. Silica membrane spin columns provided the most consistent recovery of high quantities of genomic DNA (gDNA) from EDTA preserved and clotted blood samples without the co-extraction of PCR inhibitors, when compared to phenol-chloroform or modified salt precipitation methods of DNA extraction. Spectrophotometer quantification of extracted gDNA did not provide an accurate assessment of the functional gDNA quantity with phenol-chloroform extracted samples, because ofprotein contamination. Single nucleotide polymorphisrns (SNPs) were identified in twenty candidate genes and their allele frequencies evaluated in populations of Labrador Retrievers and Golden Retrievers with cruciate ligament disease, populations of Labrador Retrievers with elbow dysplasia and hip dysplasia and compared to general populations of Labradors Retrievers and Golden Retrievers. Significant associations were identified for the minor allele and haplotype frequencies of SNPs in interieukin 12B (ILI2B) in Labrador Retrievers with elbow dysplasia, interieukin 4 (IL4) and interieukin 6 (IL6) in Labrador Retrievers with hip dysplasia, IL4 and ILl2B in Labrador Retrievers with cranial cruciate ligament rupture, and interieukin 10 (ILID) and Ankyrin repeat domain 10 (ANKRDIO) in Golden Retrievers with cranial cruciate ligament rupture. A common genomic risk for the articular disease, or OA, was not identified between the two different breeds of dog evaluated, but common genomic risks were identified for different articular diseases within a single breed. A genetic basis to canine articular disease, or OA, was confirmed.

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