Background A recent genome wide association study identified the variant rs26232 in the first intron of the uncharacterized gene, C5orf30, as a rheumatoid arthritis (RA) susceptibility variant1. In addition, it has been associated with severity of radiological joint damage suggesting a role in tissue breakdown2. To date there is no function assigned for C5orf30 and neither the gene or protein show homology to any known functional sequences. However, C5orf30 is highly conserved in chimpanzee, dog, cow, mouse, chicken, and zebrafish (orthologs).
Objectives The aim of this study is to determine the biological roles of C5orf30 in rheumatoid arthritis.
Methods Immunohistochemistry on synovial samples was used to determine expression of C5orf30 including co-localisation using antibodies to macrophages (CD68), fibroblasts (5B5), T (CD3) & B (CD19) cells. Real time PCR and western blotting were used to examine C5orf30 transcript and protein levels in fibroblast-like synovial cells (FLS treated with TNF & hypoxia). To investigate gene function siRNA was used to knockdown (KD) either C5orf30 or a non-targeting control (NTC) in synovial FLS in vitro. After knockdown cell viability, proliferation, invasion and migration were all assessed and an Illumina BeadChip was used to analyse effects of siRNA-mediated C5orf30 repression on global gene expression.
Results Confocal microscopy revealed C5orf30 to be strongly expressed in both the cytoplasmic compartment of RA synovial lining cells including macrophages and fibroblasts, but not T & B cells. C5orf30 was undetectable in arthroscopy sections obtained from osteoarthritis or control synovium. C5orf30 was expressed in FLS and was found to be up-regulated by hypoxia (8-fold) and down-regulated by TNF treatment (0.5-fold). We found that C5orf30KD did not affect cell viability and proliferation but increased the invasiveness of FLS assayed using Matrigel (p=0.01) and increased FLS migration in a scratch wound assay (p=0.02) (n=6). In support of this gene profiling studies revealed upregulation of cell migration, adhesion, angiogenesis, and immune and inflammatory pathways as a result of C5orf30KD.
Conclusions C5orf30 is expressed in synovial cells and to a much lesser extent in circulating peripheral blood leukocytes obtained from RA patients. C5orf30 knockdown increased FLS migration and invasion into matrigel confirming that C5orf30 is a negative regulator of tissue breakdown.
Stahl EA, Raychaudhuri S, Remmers EF, et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet 2010;42:508-14.
Teare MD, Knevel R, Morgan MD, et al. Allele-Dose Association of the C5orf30 rs26232 Variant With Joint Damage in Rheumatoid Arthritis. Arthritis Rheum. 2013;65:2555-61.
Disclosure of Interest : None declared