Identification of a Human SOCS1 Polymorphism That Predicts Rheumatoid Arthritis Severity

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by an autoimmune response in the joints and an exacerbation of cytokine responses. A minority of patients with RA experience spontaneous remission, but most will show moderate/high disease activity, with aggressive joint damage and multiple systemic manifestations. There is thus is a great need to identify prognostic biomarkers for disease risk to improve diagnosis and prognosis, and to inform on the most appropriate therapy. Here we focused on suppressor of cytokine signaling 1 (SOCS1), a physiological negative regulator of cytokines that modulates cell activation. Using four independent cohorts of patients with arthritis, we characterized the correlation between SOCS1 mRNA levels and clinical outcome. We found a significant inverse correlation between SOCS1 mRNA expression and disease activity throughout the follow-up of patients with RA. Lower baseline SOCS1 levels were associated with poorer disease control in response to methotrexate and other conventional synthetic disease-modifying anti-rheumatic drugs in early arthritis, and to rituximab in established (active) RA. Moreover, we identified several single nucleotide polymorphisms in the SOCS1 gene that correlated with SOCS1 mRNA expression, and that might identify those patients with early arthritis that fulfill RA classification criteria. One of them, rs4780355, is in linkage disequilibrium with a microsatellite (TTTTC)_3-5, mapped 0.9 kb downstream of the SNP, and correlated with reduced SOCS1 expression in vitro. Overall, our data support the association between SOCS1 expression and disease progression, disease severity and response to treatment in RA. These observations underlie the relevance of SOCS1 mRNA levels for stratifying patients prognostically and guiding therapeutic decisions.

Keywords: biomarkers; cytokines; disease activity; inflammation; rheumatoid arthritis.

Figure 1

Differential SOCS1 expression throughout follow-up of patients with early arthritis by final diagnosis: discovery population. (A) Evolution of disease activity by diagnosis (rheumatoid arthritis [RA]: solid dots; undifferentiated arthritis [UA]: empty dots). (B) Variation of SOCS1 expression throughout follow-up in patients with RA (solid dots) or UA (empty dots). (C,D) SOCS1 expression according to disease activity (DA) assessed by DAS28 in visits of patients with RA (C) or UA (D). Data shown for mRNA SOCS1 levels normalized to ACTB and to mean SOCS1 expression levels in healthy donors (2^−ΔΔCt). Error bars show medians and interquartile range. Statistical significance was determined with Cuzick's non-parametric trend test. RA patients, n = 70; UA patients, n = 34; healthy donors, n = 40.

Figure 2

Baseline SOCS1 expression as a severity biomarker in patients with RA (PEARL study). (A) Disease activity estimated by DAS28 score after 2 years of follow-up, relative to baseline SOCS1 expression levels. Data shown as individual values with median and interquartile range. Statistical significance was established by the Mann-Whitney U test; low SOCS1 levels (n = 25) were defined as those values below percentile 25 at the baseline of PEARL population; the remaining patients were considered to have a normal SOCS1 level (n = 67). (B) Percentage of patients classified by EULAR response criteria after 12 months follow-up in PEARL subpopulations. Statistical significance was determined by the χ2 test. low SOCS1 levels (n = 25) were defined as those values below percentile 25 at the baseline of PEARL population; the remaining patients were considered to have a normal SOCS1 level (n = 67) as described for panel (A). (C) Percentage of responder and non-responder patients after 6 months rituximab infusion. Low SOCS1 levels (n = 14) were defined as those values below percentile 25 at the baseline of the Leeds established RA population; the remaining patients were considered to have a normal SOCS1 levels (n = 48). Statistical significance was determined as in (B). (D) Baseline SOCS1 expression relative to EULAR response criteria after 6-months rituximab infusion (Leeds study). Data shown for mRNA SOCS1 levels normalized to ACTB and to mean SOCS1 expression levels in healthy donors (2^−ΔΔCt); error bars show medians and interquartile range; (n = 14 no response, n = 25 moderate response and n = 23 good response). Statistical significance was determined using Cuzick's non-parametric trend test.

Figure 3

Effects of rs4780355-linked genomic DNA features on SOCS1 expression. (A) Peripheral blood mononuclear cells from 18 patients with RA (five T/C heterozygous, seven C/C homozygous and six T/T homozygous for rs4780355) were PHA-activated for 3 h. SOCS1 levels were determined by qRT-PCR (see Methods) before and after 3 h treatment. Values (2^−ΔΔCt) are normalized to ACTB and the mean value from heterozygous samples at t = 0; p-values from unpaired t-tests are indicated. (B) Luciferase assays. Two independent constructs for each genotype, which included the SOCS1 polyadenylation site and rs4780355 SNP, were used for Jurkat cell transfection; the Renilla/Firefly luciferase ratio was measured 24 h later. (C) miR-498 or control RNA (MISSION® siRNA Universal Negative Control #2, Sigma) were co-transfected with plasmid constructs. Renilla/Firefly ratios obtained with miR-498 were normalized to control miRNA values. Results from two independent clones and triplicate measurements. (D) Two independent clones, including the polyadenylation site, rs4780355 SNP (C or T) and its associated microsatellite [[TTTTC]₃ or [TTTTC]₅, respectively], were tested for each haplotype in two experiments with triplicate samples. Statistical significance was determined by the Mann-Whitney U test.