Background Polymorphisms in the TRAF1-C5 locus have been associated with susceptibility to rheumatoid arthritis (RA). A single nucleotide polymorphism (SNP) rs3761847, located in the first intron of the TRAF1 gene, has been identified as an RA risk locus in a genome-wide association study (GWAS); the relationship of rs3761847 with joint damage are unclear. One of the key mechanisms of joint destruction is the degradation of and invasion of cartilage by RA synovial fibroblasts (RASF).
Objectives To determine whether the expression of TRAF1-C5 genes and the invasion of RASF are associated with the genotype at rs3761847.
Methods The genotype at rs3761847 was determined using TaqMan Allelic Discrimination Assay. Invasion assays were performed using Matrigel-coated invasion chambers. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation. Neutrophils were isolated from the red blood cell layer by erythrocyte lysis; CD14+ cells were separated using magnetic cell sorting. Macrophages were differentiated from plastic-adhered monocytes in M-CSF (50ng/ml) for 8 days. Cells were stimulated with lipopolysaccharide (LPS, 100ng/ml) plus interferon γ (IFNγ, 20ng/ml), interleukin 4 (IL-4, 20ng/ml) or tumour necrosis factor α (TNF-α, 10ng/ml). Gene expression was measured by SYBR Green real-time PCR using HPRT1 as a housekeeping gene.
Results rs3761847 was in Harvey-Weinberg equilibrium in RASF (n=54, p=0.98) with genotype frequencies of 0.42 AA, 0.45 AG and 0.13 GG. When grouped by genotype, the expression of TRAF1 (total), TRAF1 transcript variant 2 and complement C5 mRNA showed no significant difference (p=0.929, p=0.583, p=0.980 respectively). This was also the case for invasion of RASF (n=43, p=0.548; mean cell count 10.4 AA, 8.3 AG and 11.1 GG). There was no correlation of genotype with common RA disease markers (CRP p=0.718, ESR p=0.179, RF titre p=0.466, ACPA titre p=0.712). In contrast, analyzing TRAF1 expression in different types of blood cells, significantly higher levels of TRAF1 mRNA were detected in monocytes carrying the AA genotype compared with the GG genotype at rs3761847, both in unstimulated (2.26-fold) as well as LPS+IFNγ-activated cells (2.04-fold; p<0.05, n=3 each). No significant association of TRAF1 expression or transcript variant utilization with rs3761847 was observed in the other blood-derived cell types studied, i.e. PBMC, macrophages and neutrophils, or using other stimuli, i.e. IL-4 and TNF-α, indicating a specific function for the rs3761847 polymorphism in unstimulated and LPS+IFNγ-activated monocytes.
Conclusions Our findings suggest that there is no relationship between invasive capacity of RASF or expression of TRAF1-C5 genes and genotype at rs3761847. In contrast, we report an association of the rs3761847 genotype and TRAF1 expression in monocytes. These data underline the importance of studying genotype-phenotype associations in the different cell types relevant for RA pathogenesis.
Disclosure of Interest None declared