J. Broen

The STAT4 gene influences the genetic predisposition to systemic sclerosis phenotype

The aim of this study was to investigate the possible role of STAT4 gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype. A total of 1317 SSc patients [896 with limited cutaneous SSc (lcSSc) and 421 with diffuse cutaneous SSc (dcSSc)] and 3113 healthy controls, from an initial case-control set of Spanish Caucasian ancestry and five independent cohorts of European ancestry (The Netherlands, Germany, Sweden, Italy and USA), were included in the study. The rs7574865 polymorphism was selected as STAT4 genetic marker. We observed that the rs7574865 T allele was significantly associated with susceptibility to lcSSc in the Spanish population [P = 1.9 x 10(-5) odds ratio (OR) 1.61 95% confidence intervals (CI) 1.29-1.99], but not with dcSSc (P = 0.41 OR 0.84 95% CI 0.59-1.21). Additionally, a dosage effect was observed showing individuals with rs7574865 TT genotype higher risk for lcSSc (OR 3.34, P = 1.02 x 10(-7) 95% CI 2.11-5.31). The association of the rs7574865 T allele with lcSSc was confirmed in all the replication cohorts with different effect sizes (OR ranging between 1.15 and 1.86), as well as the lack of association of STAT4 with dcSSc. A meta-analysis to test the overall effect of the rs7574865 polymorphism showed a strong risk effect of the T allele for lcSSc susceptibility (pooled OR 1.54 95% CI 1.36-1.74; P < 0.0001). Our data show a strong and reproducible association of the STAT4 gene with the genetic predisposition to lcSSc suggesting that this gene seems to be one of the genetic markers influencing SSc phenotype.

A rare polymorphism in the gene for Toll-like receptor 2 is associated with systemic sclerosis phenotype and increases the production of inflammatory mediators

OBJECTIVE To investigate whether polymorphisms in Toll-like receptor (TLR) genes, previously reported to be associated with immune-mediated diseases, are involved in systemic sclerosis (SSc). METHODS We genotyped 14 polymorphisms in the genes for TLRs 2, 4, 7, 8, and 9 in a discovery cohort comprising 452 SSc patients and 537 controls and a replication cohort consisting of 1,170 SSc patients and 925 controls. In addition, we analyzed 15-year followup data on 964 patients to assess the potential association of TLR variants with the development of disease complications. We analyzed the functional impact of the associated polymorphism on monocyte-derived dendritic cells. RESULTS In the discovery cohort, we observed that a rare functional polymorphism in TLR2 (Pro631His) was associated with antitopoisomerase (antitopo) positivity (odds ratio 2.24 [95% confidence interval 1.24-4.04], P=0.003). This observation was validated in the replication cohort (odds ratio 2.73 [95% confidence interval 1.85-4.04], P=0.0001). In addition, in the replication cohort the TLR2 variant was associated with the diffuse subtype of the disease (P=0.02) and with the development of pulmonary arterial hypertension (PAH) (Cox proportional hazards ratio 5.61 [95% confidence interval 1.53-20.58], P=0.003 by log rank test). Functional analysis revealed that monocyte-derived dendritic cells carrying the Pro63His variant produced increased levels of inflammatory mediators (tumor necrosis factor α and interleukin-6) upon TLR-2-mediated stimulation (both P<0.0001). CONCLUSION Among patients with SSc, the rare TLR2 Pro631His variant is robustly associated with antitopoisomerase positivity, the diffuse form of the disease, and the development of PAH. In addition, this variant influences TLR-2-mediated cell responses. Further research is needed to elucidate the precise role of TLR-2 in the pathogenesis of SSc.

Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis

Objective Two functional single nucleotide polymorphisms (SNP) in the PTPN22 gene (rs24746601 and rs33996649) have been associated with autoimmunity. The aim of this study was to investigate the role of the R263Q SNP for the first time and to re-evaluate the role of the R620W SNP in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotypes. Methods 3422 SSc patients (2020 with limited cutaneous SSc and 1208 with diffuse cutaneous SSc) and 3638 healthy controls of Caucasian ancestry from an initial case--control set of Spain and seven additional independent replication cohorts were included in our study. Both rs33996649 and rs2476601 PTPN22 polymorphisms were genotyped by TaqMan allelic discrimination assay. A meta-analysis was performed to test the overall effect of these PTPN22 polymorphisms in SSc. Results The meta-analysis revealed evidence of association of the rs2476601 T allele with SSc susceptibility (pFDRcorrected=0.03 pooled, OR 1.15, 95% CI 1.03 to 1.28). In addition, the rs2476601 T allele was significantly associated with anticentromere-positive status (pFDRcorrected=0.02 pooled, OR 1.22, 95% CI 1.05 to 1.42). Although the rs33996649 A allele was significantly associated with SSc in the Spanish population (pFDRcorrected=0.04, OR 0.58, 95% CI 0.36 to 0.92), this association was not confirmed in the meta-analysis (p=0.36 pooled, OR 0.89, 95% CI 0.72 to 1.1). Conclusion The study suggests that the PTPN22 R620W polymorphism influences SSc genetic susceptibility but the novel R263Q genetic variant does not. These data strengthen evidence that the R620W mutation is a common risk factor in autoimmune diseases.

The interleukin 23 receptor gene does not confer risk to systemic sclerosis and is not associated with systemic sclerosis disease phenotype

OBJECTIVES Multiple studies indicate the role of the interleukin (IL)-17/IL-23 axis in autoimmune diseases, including systemic sclerosis (SSc). The aim of the current study was to investigate the possible implication of the IL23R gene in SSc susceptibility and/or clinical phenotype. METHODS An initial case-control study in 143 Dutch patients with SSc and geographically matched healthy individuals (n = 246) was carried out and followed by a replication study in a cohort of 365 Spanish patients with SSc and 515 healthy individuals. Seven single nucleotide polymorphisms (SNPs) spanning the IL23R gene were selected and genotyped using a Taqman assay. RESULTS Using a Dutch cohort of patients with SSc and controls we observed an association between two (rs11209032, rs1495965) of the seven tested SNPs and disease susceptibility (allelic p values: p = 0.02 and p = 0.01 respectively). However, a replication study in an independent Spanish cohort did not confirm these findings and reveal no association of any of the IL23R-tested SNP with disease susceptibility or clinical phenotype. Similarly, a meta-analysis considering both populations did not reveal any significant association. In addition, no association was observed between IL23R genetic variants and SSc clinical phenotypes. CONCLUSIONS Our results suggest that the IL23R gene is not associated with SSc susceptibility or clinical phenotype.

Skewed X chromosomal inactivation impacts T regulatory cell function in systemic sclerosis

Objectives To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). Methods 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. Results Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r=0.45, p<0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p=0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p<0.001) associated with less efficient suppressive activity (p=0.012). Conclusions Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

Evidence that deletion at FCGR3B is a risk factor for systemic sclerosis

There is increasing evidence that gene copy number (CN) variation influences clinical phenotype. The low-affinity Fc receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment of polymorphonuclear neutrophils to sites of inflammation and their activation. Given the genetic overlap between systemic lupus erythematosus and systemic sclerosis (SSc) and the strong evidence for FCGR3B CN in the pathology of SLE, we hypothesised that FCGR3B gene dosage influences susceptibility to SSc. We obtained FCGR3B deletion status in 777 European Caucasian cases and 1000 controls. There was an inverse relationship between FCGR3B CN and disease susceptibility. CN of ⩽1 was a significant risk factor for SSc (OR=1.55 (1.13–2.14), P=0.007) relative to CN⩾2. Although requiring replication, these results suggest that impaired immune complex clearance arising from FCGR3B deficiency contributes to the pathology of SSc, and FCGR3B CN variation is a common risk factor for systemic autoimmunity.

The autoimmune disease-associated IL2RA locus is involved in the clinical manifestations of systemic sclerosis

Regulatory T cells (Tregs) are crucial in the maintenance of the immune tolerance and seem to have an important role in systemic sclerosis (SSc). The interleukin 2 receptor α (IL2RA) is an important Treg marker, and polymorphisms of IL2RA gene are associated with a number of autoimmune diseases. Therefore, we aimed to investigate for the first time the association of the IL2RA locus in SSc. For this purpose, a total of 3023 SSc patients and 2735 matched healthy controls, from six European Caucasian cohorts, were genotyped for the IL2RA gene variants rs11594656, rs2104286 and rs12722495 using the TaqMan allelic discrimination technology. The overall meta-analysis reached statistical significance when the three polymorphisms were tested for association with SSc, the limited subtype (lcSSc) and anti-centromere auto-antibodies (ACAs). However, no significant P-values were obtained when the ACA-positive patients were removed from the SSc and lcSSc groups, suggesting that these associations rely on ACA positivity. The strongest association signal with ACA production was detected for rs2104286 (PFDR=2.07 × 10−4, odds ratio=1.30 (1.14–1.47)). The associations of rs11594656 and rs12722495 were lost after conditioning to rs2104286, and allelic combination tests did not evidence a combined effect, indicating that rs2104286 best described the association between IL2RA and ACA presence in SSc.

Identification of novel genetic markers associated with the clinical phenotypes of systemic sclerosis through a genome wide association strategy

Identification of novel genetic markers associated with the clinical phenotypes of systemic sclerosis through a genome wide association strategy O Gorlova, J M Martin, B Rueda, BPC Koeleman, J Ying, M Teruel, L M Diaz-Gallo, J C Broen, M C Vonk, C P Simeon, B Z Alizadeh, MJH Coenen, A E Voskuyl, A J Schuerwegh, PLCM van Riel, M Vanthuyne, R van ‘t Slot, A Italiaander, R A Ophoff, N Hunzelmann, V Fonollosa, N Ortego-Centeno, M A González-Gay, F J García-Hernández, M F González-Escribano, P Airo, J van Laar, J Worthington, R Hesselstrand, V Smith, F De Keyser, F Houssiau, M M Chee, R Madhok, P Shiels, R Westhovens, A Kreuter, E de Baere, T Witte, L Padyukov, A Nordin, R Scorza, C Lunardi, B A Lie, A M Hoffmann-Vold, P García de la Peña, P Carreira, J Varga, M Hinchcliff, A T Lee, P Gourh, C I Amos, G Riemekasten, A Herrick, L Beretta, C Fonseca, C P Denton, P K Gregersen, S Agarwal, S Assassi, F K Tan, F C Arnett, TRDJ Radstake, M D Mayes, J Martin

A replication study confirms the association of TNFSF4 (OX40L) polymorphisms with Systemic Sclerosis in a large European cohort

The TNFSF4 gene, which encodes OX40L, is considered as a potential autoimmunity candidate gene. OX40L is expressed on activated antigen presenting cells (APCs) and endothelial cells in acute inflammation [1]. Furthermore, it enhances B-cell proliferation and differentiation, and its binding to OX40 (CD134) promotes proliferation and survival of T-cells and predisposes them to a more permissive proliferative and survival condition [2]. Interestingly, four TNFSF4 promoter single-nucleotide polymorphisms (SNP) were recently implicated in susceptibility to systemic sclerosis (SSc)[3].

Biologicals in de praktijk van de reumatoloog

SamenvattingBiologicals hebben de praktijk van de reumatoloog de afgelopen vijftien jaar geleidelijk, maar drastisch veranderd. Ze bieden een uitkomst voor patiënten met verschillende reumatologische ziekten die niet reageren op de conventionele therapieën. De reumatoloog heeft een breed scala aan biologicals tot zijn beschikking, die aangrijpen op ziekterelevante cytokines of immuuncellen. Biologicals kunnen na goede voorlichting subcutaan thuis, of in andere gevallen op de dagbehandeling van de afdeling Reumatologie worden toegediend. Waakzaamheid voor infecties en vaccinaties zijn belangrijke aandachtspunten. De verwachting is dat in de toekomst meer biologicals op de markt zullen komen, dat indicatiegebieden binnen de reumatologie worden uitgebreid en dat voor biologicals waarvan de patentbescherming afloopt er steeds meer, vaak goedkopere alternatieven op de markt komen, de zogenoemde ‘biosimilars’.