Association of Human FOS Promoter Variants with the Occurrence of Knee-Osteoarthritis in a Case Control Association Study

Abstract

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2⁻3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2⁻2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.

Keywords: AP-1; FOS; JUN; knee osteoarthritis; promoter.

Figure 1

Reporter gene expression. The graph shows JUN (a,b) and FOS (c,d) promoter-dependent expression of firefly luciferase 1d and 2d following transfection of K4IM human fibroblasts, either in non-stimulated cells or in cells stimulated with 10 ng/mL PMA for 8 h (determined in biological triplicates; mean ± standard error of the mean (SEM)). Firefly luciferase expression levels were normalized to renilla luciferase expression levels in the respective samples (transfection and normalization control). Results are presented as relative values (in %) related to the expression level in the presence of the major allele at the respective time point (a,c). For better comparison, relative values related to the expression level in the presence of the major allele in unstimulated cells at 1 d are also given (b,d). ** p ≤ 0.005, * p ≤ 0.05 compared to the major allele. For JUN, the major allele is the rs4647009-deletion (minor allele: Insertion CA), for FOS, the major allele refers to the combination of rs7101-T and rs2239615-T.

Figure 2

Reporter gene expression in HeLa cells. The graph shows JUN (a,b) and FOS (c,d) promoter-dependent expression of firefly luciferase 1d and 2d following transfection of HeLa human epithelial cells (±10 ng/mL PMA for 8 h, biological triplicates; in c and d, 48 h values: Biological duplicates; mean ± SEM). Firefly luciferase expression levels were normalized to renilla luciferase expression levels. Expression levels are shown as relative values (in %; a,c). For comparison, relative values related to the expression level in the presence of the major allele in unstimulated cells at 1 d are also given (b,d). * p ≤ 0.05 compared to the major allele.

Figure 3

Meta-analysis of the German and Finnish study. A fixed effect meta-analysis of the German and Finnish knee-OA studies was performed under additive and recessive models of inheritance. Evidence was stronger for the recessive model (Additive model: Allelic OR = 1.2, 95% CI 1.01–1.5, p = 0.037, p_{heterogeneity} = 0.06; Recessive model: OR = 2.1, 95% CI 1.25–3.42, p = 0.004, p_{heterogeneity} = 0.82).