Discovery of novel osteoarthritis susceptibility genes, common pathways, and a mouse model using the hyperinflammatory human RIPK2 disease allele

Abstract

Purpose: Lack of understanding of the biological processes conferring susceptibility to OA and the paucity of animal models are key limitations to the development of effective therapies. Our goal is to discover molecular pathways that are vulnerability points for the development of OA and generate mouse models using human disease alleles. We discover these pathways by identifying gene mutations that have a strong effect on susceptibility to OA in families. We then determine whether and how these altered gene products confer susceptibility to OA in genetically modified mouse models. Methods: To discover pathways that when modified lead to strong and unambiguous susceptibility to OA, we analyzed the genomes of families with an early-onset bilateral form of OA affecting the first metatarsophalangeal (MTP) joint. The top candidates that we found associated with first MTP joint OA in three families were very rare coding variants affecting RIPK2, NOD2, and SMAD3. We performed quantitative bioassays in the zebrafish to determine the functional consequence of these coding variants. Genome wide association studies have previously linked non-coding variants in SMAD3 to knee and hip OA. RIPK2 and NOD2 are novel OA susceptibility genes. RIPK2 and NOD2 function in a common pathway to regulate the innate immune response. Our studies of first MTP joint OA indicate that we can identify novel genes and those previously linked to more common forms of OA. Results: Loss-of-function Smad3 mutations in mouse cause OA like phenotypes. Functional analysis of the first MTP joint OA SMAD3 variant in our zebrafish bioassays indicate that the variant allele is hypomorphic, representing a partial loss-of-function of gene activity. Given our previous analyses of the OA-associated RIPK2 allele in zebrafish and that NOD2 is the upstream activator of RIPK2, we hypothesize that the NOD2 allele will be a gain-of-function allele. We are currently determining if the NOD2 variant affects the magnitude or duration of innate immune response in the zebrafish. We have previously described a gain-of-function allele of RIPK2 that appears to act with high penetrance to confer susceptibility to first MTP joint OA. The RIPK2 protein is an intrinsic component of innate immunity transducing signals from the NOD2 pattern recognition receptor to generate a proinflammatory immune response. We have shown the OA-associated RIPK2 product has increased pro-inflammatory activity relative to the wildtype protein. Based on these findings, we propose the gene variant acts dominantly as a gain-of-function allele to over-stimulate the inflammatory response to naturally occurring joint damage leading to OA. To test this hypothesis we have introduced a single amino acid change, encoded by the RIPK2 disease allele, into an isogenic mouse strain. We are currently testing the effect of the allele on susceptibility to naturally occurring and experimentally induced arthritis in mice. Preliminary results indicate that the disease allele is sufficient to confer a heightened inflammatory response to stimuli in mice carrying the disease allele. Conclusions: Our studies indicate that we can use families to identify genes and pathways that confer strong susceptibility to the early stages of OA. Our approach has led to the discovery of i) compelling novel candidate genes (RIPK2 and NOD2), ii) multiple genes that contribute to a common pathway (RIPK2 and NOD2 - innate immune response pathway), and iii) candidate genes previously identified by GWAS as associated with hip and knee OA (SMAD3). Furthermore, our data lends new support to our hypothesis that variation in the innate inflammatory response may be a key factor in susceptibility to OA. We have also begun analyses of a new mouse OA model that was generated by introducing a human OA disease allele into the mouse Ripk2 gene. The new mouse model will allow us to determine the role of and tissues modified by the innate immune response that contribute to the early stages of the disease process.