Investigation Into rs26232 Genotype Association with Susceptibility and Severity of Rheumatoid Arthritis
Kevin J Sheridan, Emma R Dorris, Eimear Flanagan, Eimear Linnehan, Michelle Trenkmann, Karen Creevey, Doug Veale, Ursula Fearon and Anthony G Wilson
University College Dublin
The single nucleotide variant rs26232 has been associated with both the susceptibility to, and severity of, rheumatoid arthritis (RA). An allele dose response between rs26232 and radiological damage has been observed, with the T allele being protective against disease severity. rs26232 is situated in the first intron of C5orf30, a negative regulator of tissue damage and inflammation in RA.
This study aims to elucidate the mechanism by which rs26232 may mediate disease severity and determine the genotype-phenotype association of rs26232 in rheumatoid arthritis synovial fibroblasts (RASF).
RASF were derived from knee biopsies of RA patients taken at arthroscopy (n=33). Matrigel-coated Boyden transwell chambers were used to measure invasion. Wound healing (scratch assays) were used to measure migration Proliferation was measured via crystal violet staining of fixed RASF. Intracellular cytokine staining and cell surface markers were measured via flow cytometry. Secreted cytokines were measured using ELISA. rs26232 genotype was determined by PCR genotyping assay with allelic discrimination analysis. Quantitative real-time PCR was used to measure gene expression.
rs26232 is associated with invasion of RASFs, with the CC genotype showing increased invasion (p=0.021). The CC genotype also showed higher expression of the adhesion molecules ICAM1 (p=0.001), VCAM (p=0.05) and IP10/CXCL10 (p=0.01). No association was found between rs26232 genotype and migration, proliferation, or expression of MMP3, TIMP3, MCP1 or MIP1. There was no differential expression of C5orf30 in rs26232 genotype groups. In silico analysis of the region in which rs26232 is located identified a DNase Hypersensitivity cluster. Three genes within this region (PAM, PPIP5K2 and EIF3KP1) show expression quantitative trait loci (eQTL) association with rs26232. These genes also contain the active enhancer mark H3K27Ac. GIN1, also within this region, is neither an eQTL nor contains H3K27Ac. qPCR analysis of PAM, PPIP5K2 and GIN1 gene expression showed an association between rs26232 genotype and PAM gene expression (p=0.05).
The CC genotype of rs26232 is associated with both increased invasiveness of RASFs and increased adhesion markers. rs26232 does not mediate its affect via its nearest gene, C5orf30. Gene expression analysis of nearby genes suggests that PAM may be responsible for the phenotypes associated with rs26232 genotype.