Investigating the genetic basis of cranial cruciate ligament rupture in the Newfoundland dog

This thesis presents work to examine the genetic basis of CCL rupture in dogs. It describes research to identify causative mutations that will help to develop a genetic screening test to identify dogs that have a high risk of developing CCL rupture. Cranial Cruciate Ligament (CCL) rupture is the most common cause of hind limb lameness in dogs and is especially common in large and giant breeds such as Newfoundlands, Rottweilers and Staffordshire Bull Terriers. CCL rupture cases and from two continents (Europe n = 48 and North America n = 48) were examined using genome wide association studies (GWAS). A candidate gene study was performed using Sequenom iPlex genotyping, on cases and controls from Newfoundlands (99 cases, 172 controls) and three other susceptible breeds: Labradors (124 cases, 165 controls), Rottweilers (57 cases, 81 controls) and Staffordshire Bull Terriers (13 cases, 38 controls). One hundred and eighty-six SNPs across 26 candidate genes were investigated for association with CCL rupture. To investigate downstream events, gene expression was compared between healthy and CCL rupture tissue. An in-vitro laboratory model of CCL rupture was investigated by examining ligamentocytes induced with and without TNFα. To investigate whether there was an auto-immune component to CCL rupture, the two main loci of the dog leucocyte antigen (DLA) system were assessed for disease association. Principle component analysis of the GWAS data revealed population stratification within the Newfoundland breed, indicative of the continent of origin (Europe or North America). GWAS identified three main regions associated with CCL rupture (on chromosomes 1, 3 and 33). Significantly associated genes SORCS2 and SEMA5B function in neurological pathways; this may indicate that mechanotransduction, neurological and neuromuscular pathways play an important role in the pathogenesis and susceptibility to CCL rupture. Candidate gene analysis identified associations with two collagen genes (collagen type-V and collagen type-I) and three extracellular matrix proteins; Aggrecan (ACAN), Opticin (OPTC) and Latent transforming growth factor beta 2 (LTBP2). Gene expression analysis revealed significant differential expressions in COL1A1 and COL1A2. These results indicate that the strength and stability of the ligament is probably important in susceptibility to CCL rupture. Gene expression results also revealed that genes involved in degradation (TRAP and DIRC2) are upregulated, indicating that the cells are trying to repair themselves whilst a simultaneous degradative process is still on going. The TNFα model may be used as an in-vitro model to study CCL rupture, but may be more useful as a model for examining changes that occur after CCL rupture rather than the early stages of the disease. We showed no association with the DLA region and CCL rupture. The identified associated regions should be further investigated and refined using next generation, targeted re-sequencing and transcriptomic approaches. This could identify the specific causative mutations involved in CCL rupture susceptibility. This study confirms that there are complex multigenetic and environmental factors involved in CCL rupture susceptibility. The work has contributed to the understanding of causative factors involved in CCL rupture susceptibility and may be an important step in the development of a screening test(s) to reduce the incidence of CCL rupture in dogs and thus improve their health and welfare.