Investigating the role of GNL3 in osteoarthritis
Ricketts, Michelle Antoinette
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Osteoarthritis (OA) is a common disease with a strong genetic component. Despite this, previous attempts to identify genetic variants that predispose to OA have met with limited success. Recently, the results of a large genome wide association study in OA has identified a novel susceptibility locus on chromosome 3 tagged by two SNPs, rs11177 (p=1.25x10-10) which lies within the coding region of GNL3 and rs6976 (p=7.24x10-11) situated in the 3’UTR of GLT8D1. The GNL3 gene encodes the protein nucleostemin which is found within the nucleolus of stem cells and tumour cells. It functions to regulate cell cycle progression, embryogenesis, tumorigenesis, tissue regeneration and ribosome biogenesis but its role in the joint is unknown. In an attempt to identify the causal variant(s) at locus 3p21.1 I conducted a mutation screen of GNL3 which identified a common non-synonymous coding variant, rs2289247, which was in strong LD (r2=0.92) with rs11177, as well as several other variants. Localisation studies showed that GNL3 was expressed at the mRNA and protein level in several joint tissues. While levels of mRNA expression were found to be significantly higher in human articular chondrocytes from OA patients as compared with controls, levels of GNL3 protein were significantly lower in OA chondrocytes than controls. Further studies showed that cytokines which have been implicated in the pathogenesis of OA such as IL1β, IL13, TNFα and FGF2 had no effect on GNL3 mRNA in cartilage. Knockdown of GNL3 using siRNA in articular chondrocytes and the chondrosarcoma cell line, JJ012, did not alter the mRNA expression of chondrogenic markers; COL2A, ACAN, MMP3, MMP13, RUNX2 and SOX9. Cultures of mesenchymal stem cells and articular chondrocytes from patients of different rs11177 genotypes, showed no difference in chondrogenic potential. Furthermore, genotypes at rs11177 and rs2289247 did not influence the expression of p53, MDM2 or GNL3 in response to stressful stimuli, including cisplatin and hypoxia, when cloned into a melanoma cell line. Studies of zebrafish carrying a loss of function mutation in gnl3 revealed a significant reduction in cartilage volume and an alteration in cartilage structure, as evident by a reduced number of chondrocytes, disorganised stacking and an increase in cartilage extracellular matrix in the mutant fish. This research has shown that gnl3 plays a vital role in chondrogenesis in zebrafish and has shown evidence of dysregulation of GNL3 expression in OA human articular chondrocytes. The in vitro studies failed to identify any specific effects of the variants rs11177 and rs2289247 on GNL3 expression, chondrogenesis or p53 stress response although, it remains possible that the variants may have modest effects that were not detected by the assays used. The zebrafish studies illustrate that gnl3 plays a critical role in normal cartilage development however further studies on GNL3 in OA would be of interest.