PROJECT SUMMARY Genome-wide association studies (GWAS) provide an unbiased source of potential targets for the development of osteoarthritis (OA) therapies. However, there is a knowledge gap regarding the mechanisms by which identified genetic variants drive cellular and tissue dysfunction. When present in a homozygous state, a genetic variant in Chondroadherin-like (CHADL) results in a 7.7 fold increased risk of total hip replacement. Chadl is a small leucine rich proteoglycan (SLRP) with an expression pattern that is restricted to articular cartilage. The 8 bp frameshift mutation (rs532464664) makes the transcript susceptible to degradation and our data indicates that a novel protein fragment is also produced. The overall hypothesis of this proposal is that the loss of functional CHADL protein and the generation of a novel protein fragment both contribute to enhanced OA by disrupting the cartilage extra-cellular matrix. The objective of this proposal is to determine the mechanisms responsible for the association between the CHADL variant and OA risk, with the long-term goal of using these insights to develop therapies for idiopathic OA in the general (non-variant) population. The first aim is to determine the extent to which Chadl knockout and a novel “8 bp variant” mouse accelerate OA development by assessing OA at 6 and 12 months of age. The “8 bp variant” mice were generated by recapitulating the human variant in a matched, highly conserved location of the mouse genome. The second aim is to establish how CHADL disruption alters the composition, structure, and function of cartilage extra-cellular matrix. In addition to cartilage explants from Chadl knockout and 8 bp variant mice, primary human chondrocytes with over-expression of the normal or 8 bp variant forms of human CHADL will be used to generate engineered tissues for analysis. Tissues will be analyzed for the extent of collagen crosslinking, protein composition by mass spectrometry, and mechanical properties by atomic force microscopy. The proposed aims are innovative in that they use both in vivo murine and human chondrocyte systems to directly interrogate an understudied GWAS variant with high risk of OA. The outcomes of the proposed experiments will include the comprehensive analysis of OA and cartilage properties in the case of CHADL loss and expression of the specific variant. Future directions of this project are to translate the insights gained from this specific variant into a greater understanding of idiopathic OA. One example of this approach would be to investigate extra-cellular matrix cleavage products that may recapitulate the function of the protein fragment generated by the CHADL variant. These contributions are expected to have a positive impact on society by stimulating more effective strategies for the prevention and treatment of OA.