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Objectives: In agreement with other autoimmune diseases, systemic sclerosis (SSc) is associated with a strong sex bias. However, unlike lupus, the effects of sex on disease phenotype and prognosis are poorly known. Therefore, we aimed to determine sex effects on outcomes., Method: We performed a prospective observational study using the latest 2013 data extract from the EULAR scleroderma trials and research (EUSTAR) cohort. We looked at (i) sex influence on disease characteristics at baseline and (ii) then focused on patients with at least 2 years of follow-up to estimate the effects of sex on disease progression and survival., Results: 9182 patients with SSc were available (1321 men) for the baseline analyses. In multivariate analysis, male sex was independently associated with a higher risk of diffuse cutaneous subtype (OR: 1.68, (1.45 to 1.94); p<0.001), a higher frequency of digital ulcers (OR: 1.28 (1.11 to 1.47); p<0.001) and pulmonary hypertension (OR: 3.01 (1.47 to 6.20); p<0.003). In the longitudinal analysis (n=4499), after a mean follow-up of 4.9 (±2.7) years, male sex was predictive of new onset of pulmonary hypertension (HR: 2.66 (1.32 to 5.36); p=0.006) and heart failure (HR: 2.22 (1.06 to 4.63); p=0.035). 908 deaths were recorded, male sex predicted deaths of all origins (HR: 1.48 (1.19 to 1.84); p<0.001), but did not significantly account for SSc-related deaths., Conclusions: Although more common in women, SSc appears as strikingly more severe in men. Our results obtained through the largest worldwide database demonstrate a higher risk of severe cardiovascular involvement in men. These results raise the point of including sex in the management and the decision-making process., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Introduction: HLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics., Methods: PBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression., Results: ER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs., Conclusions: Our study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients.

Background: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon., Methods: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo., Results: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis., Conclusions: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Background: To determine the usefulness of Ca 15.3 as a candidate biomarker in systemic sclerosis (SSc) patients with interstitial lung disease (ILD)., Methods: Two-hundred-twenty-one SSc patients with Ca 15.3 determinations were considered; 168 had evidence of interstitial lung involvement on high-resolution computed tomography (HRCT); digitalized scans were available for scoring in 84 subjects. Discrimination between patients with or without ILD, was assessed by receiving operating characteristics (ROC) analysis; correlations between HRCT scores and Ca 15.3 were performed. Survival and serial pulmonary function tasting (PFT) data were used for prognostication., Results: Ca 15.3 serum levels strongly correlated with HRCT scores (r=0.734, p<0.0001) which were predictors of survival at the 20% threshold (p=3.1∗10(-4)). Ca 15.3 had an area under ROC to detect the meaningful 20% fibrosis extent equal to 0.927 and abnormal Ca 15.3 values were capable of differentiating between patients at hi- or low-risk for progression in the group with undetermined disease extent (HR=3.209, confidence interval [CI95]=1.56-6.602, p=0.002). Ca 15.3 outperformed other PFT measures in providing a separation of survival estimates where HRCT scans are unavailable. The combined use of HRCT scores and Ca 15.3 in SSc-ILD patients was more discriminatory (HR=4.824, CI95=2.612-8.912, p<0.0001) than the staging system based on HRCT scores plus FVC (HR=2.657, CI95=1.703-4.147, p<0.0001) and characterized by lower prediction errors (0.2134 vs 0.2234)., Conclusion: Ca 15.3 is a rapid and inexpensive candidate biomarker for SSc-ILD being proportional to the extent of lung injury and specific and sensitive in assessing meaningful extents of the disease with prognostic significance., (Copyright © 2013 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are two archetypal systemic autoimmune diseases which have been shown to share multiple genetic susceptibility loci. In order to gain insight into the genetic basis of these diseases, we performed a pan-meta-analysis of two genome-wide association studies (GWASs) together with a replication stage including additional SSc and SLE cohorts. This increased the sample size to a total of 21,109 (6835 cases and 14,274 controls). We selected for replication 19 SNPs from the GWAS data. We were able to validate KIAA0319L (P = 3.31 × 10(-11), OR = 1.49) as novel susceptibility loci for SSc and SLE. Furthermore, we also determined that the previously described SLE susceptibility loci PXK (P = 3.27 × 10(-11), OR = 1.20) and JAZF1 (P = 1.11 × 10(-8), OR = 1.13) are shared with SSc. Supporting these new discoveries, we observed that KIAA0319L was overexpressed in peripheral blood cells of SSc and SLE patients compared with healthy controls. With these, we add three (KIAA0319L, PXK and JAZF1) and one (KIAA0319L) new susceptibility loci for SSc and SLE, respectively, increasing significantly the knowledge of the genetic basis of autoimmunity.

Objective: The interleukin 2 (IL-2) and interleukin 21 (IL-21) locus at chromosome 4q27 has been associated with several autoimmune diseases, and both genes are related to immune system functions. The aim of this study was to evaluate the role of the IL-2/IL-21 locus in systemic sclerosis (SSc)., Patients and Methods: The case control study included 4493 SSc Caucasian patients and 5856 healthy controls from eight Caucasian populations (Spain, Germany, The Netherlands, USA, Italy, Sweden, UK and Norway). Four single nucleotide polymorphisms (rs2069762, rs6822844, rs6835457 and rs907715) were genotyped using TaqMan allelic discrimination assays., Results: We observed evidence of association of the rs6822844 and rs907715 variants with global SSc (pc=6.6E-4 and pc=7.2E-3, respectively). Similar statistically significant associations were observed for the limited cutaneous form of the disease. The conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs6822844 polymorphism. Consistently, the rs2069762A-rs6822844T-rs6835457G-rs907715T allelic combination showed evidence of association with SSc and limited cutaneous SSc subtype (pc=1.7E-03 and pc=8E-4, respectively)., Conclusions: These results suggested that the IL-2/IL-21 locus influences the genetic susceptibility to SSc. Moreover, this study provided further support for the IL-2/IL-21 locus as a common genetic factor in autoimmune diseases.

A case control study to evaluate the possible influence of FOXP3 polymorphisms (rs3761548 and rs2280883) in the susceptibility of systemic sclerosis in an Italian Caucasian population. Subgroup analysis was also performed to test association between these SNPs and specific disease phenotypes. The study groups consisted of 467 individuals: 228 patients (194 with limited cutaneous form and 34 with diffuse cutaneous form of the disease) and 239 healthy control subjects. Genotyping was performed by high resolution melting analysis. Genotype distribution and allele frequency of the FOXP3 polymorphisms were analyzed statistically, using χ(2) or Fisher exact test. Single-marker analysis of allelic and genotype frequencies revealed that SNP rs3761548 was not associated with systemic sclerosis susceptibility. Analysis of genotype and allele distributions of the rs2280883 genetic variant was associated, only in female subjects with systemic sclerosis, its limited subtype, and anti-centromere autoantibodies. Although these findings require replication in a larger set and other populations, FOXP3 rs2280883 may represent a novel susceptibility locus for systemic sclerosis in female subjects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: A recent genome-wide association study in European systemic sclerosis (SSc) patients identified three loci (PSORS1C1, TNIP1 and RHOB) as novel genetic risk factors for the disease. The aim of this study was to replicate the previously mentioned findings in a large multicentre independent SSc cohort of Caucasian ancestry., Methods: 4389 SSc patients and 7611 healthy controls from different European countries and the USA were included in the study. Six single nucleotide polymorphisms (SNP): rs342070, rs13021401 (RHOB), rs2233287, rs4958881, rs3792783 (TNIP1) and rs3130573 (PSORS1C1) were analysed. Overall significance was calculated by pooled analysis of all the cohorts. Haplotype analyses and conditional logistic regression analyses were carried out to explore further the genetic structure of the tested loci., Results: Pooled analyses of all the analysed SNPs in TNIP1 revealed significant association with the whole disease (rs2233287 p(MH)=1.94×10(-4), OR 1.19; rs4958881 p(MH)=3.26×10(-5), OR 1.19; rs3792783 p(MH)=2.16×10(-4), OR 1.19). These associations were maintained in all the subgroups considered. PSORS1C1 comparison showed association with the complete set of patients and all the subsets except for the anti-centromere-positive patients. However, the association was dependent on different HLA class II alleles. The variants in the RHOB gene were not associated with SSc or any of its subsets., Conclusions: These data confirmed the influence of TNIP1 on an increased susceptibility to SSc and reinforced this locus as a common autoimmunity risk factor.

Objectives: Scleroderma renal crisis (SRC) is a relative rare yet dramatic event in the history of systemic sclerosis (SSc). Several factors that may precipitate or protect from the development of SRC have been described in previous case-control studies. To date, no attempt has been made to evaluate these factors in an observational fashion., Methods: Retrospective data from 410 SSc patients with disease duration <5 years at referral were evaluated in an observational fashion for the development of hypertensive or normotensive SRC within 5 years from the first visit at our centre. Baseline characteristics as well as the use of steroids or dhiydropyridine calcium-channel blockers (CCB) were analysed via the Cox regression method with time-dependent covariates., Results: In the multivariate model the diffuse subset the disease (HR=5.728 CI(95)=2.199-14.918, p<0.001) and the use of prednisone (HR=1.015, CI(95)=1.004-1.026, p=0.006) resulted to be predictors for the development of SRC, with a risk to develop SRC increased by 1.5% for every mg of prednisone/day consumed the trimester prior SRC. Contrariwise, the risk to develop SRC was highly reduced in those who were prescribed CCBs (HR=0.094, CI(95)=0.038-0.236, p<0.001)., Conclusions: Steroids exhibits a weak effect on the risk to progress toward SRC in our case series, whilst dhyidrophyridines CCB appeared to be protective against that. Further larger prospective studies are warranted to better define the role of CCB in this setting or as a background therapy for SSc.

TREX1 (DNase III) is an exonuclease involved in response to oxidative stress and apoptosis. Heterozygous mutations in TREX1 were previously observed in patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). We performed a mutational analysis of the TREX1 gene on three autoimmune diseases: SLE (210 patients) and SS (58 patients), to confirm a TREX1 involvement in the Italian population, and systemic sclerosis (SSc, 150 patients) because it shares similarities with SLE (presence of antinuclear antibodies and connective tissue damage). We observed 7 variations; two of these are novel nonsynonymous variants (p.Glu198Lys and p.Met232Val). They were detected in one SS and in one SSc patient, respectively, and in none of the 200 healthy controls typed in this study and of the 1712 published controls. In silico analysis predicts a possibly damaging role on protein function for both variants. The other 5 variations are synonymous and only one of them is novel (p.Pro48Pro). This study contributes to the demonstration that TREX1 is involved in autoimmune diseases and proposes that the spectrum of involved autoimmune diseases can be broader and includes SSc. We do not confirm a role of TREX1 variants in SLE.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10(-8), OR  = 1.22, CI 95%  = 1.14-1.30; rs2004640: P  = 4.60×10(-7), OR  = 0.84, CI 95%  = 0.78-0.90; rs10488631: P  = 7.53×10(-20), OR  = 1.63, CI 95%  = 1.47-1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10(-22), OR  = 1.75, CI 95%  = 1.56-1.97) better explained the observed association (likelihood P-value  = 1.48×10(-4)), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.

A candidate gene for TIMP-1 gene located on the X-chromosome (rs4898) was selected for a control case study to investigate a possible association of this SNP with the susceptibility to systemic sclerosis and its digit ulcer manifestation. A total of 461 individuals of Italian Caucasian origin (228 SSc patients and 233 healthy control subjects) were genotyped for TIMP-1 +372 T/C single nucleotide polymorphism rs4898. Subgroups were analyzed according to the presence or absence of digital ulcers. The CC genotype and C allele frequencies were significantly lower in female SSc patients than in controls (OR 0.53, CI 0.29-0.96, p=0.03 and OR 0.72, CI 0.53-0.98 p=0.04, respectively). CC genotypes frequency was lower also in female patients with ulcers than those without ulcers (OR 0.37, CI 0.14-1.00, p=0.03). Furthermore, CC genotype and C allele frequencies were lower also in female patients with ulcers in comparison to female healthy control subjects (OR 0.27, CI 0.10-0.70, p=0.004; OR 0.60, CI 0.40-0.89, p=0.01, respectively). The TIMP-1 rs4898 polymorphism may play a protective role in the susceptibility to SSC in females, and in particular to digital ulcer formation., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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.

Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10(-12), odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10(-7), OR = 1.36) and NFKB1 (P-value = 1.03 × 10(-6), OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.

Objective: To assess fetal and maternal outcomes in women with systemic sclerosis (SSc)., Methods: Prospectively collected data on 99 women with SSc from 25 Italian centers were analyzed retrospectively. Women with SSc were observed during 109 pregnancies (from 2000 to 2011), and outcomes were compared to those in the general obstetric population (total of 3,939 deliveries). The maternal age at conception was a mean ± SD 31.8 ± 5.3 years, and the median disease duration at conception was 60 months (range 2-193 months)., Results: SSc patients, compared to the general obstetric population, had a significantly increased frequency of preterm deliveries (25% versus 12%) and severe preterm deliveries (<34 weeks of gestation) (10% versus 5%), intrauterine growth restriction (6% versus 1%), and babies with very-low birth weight (5% versus 1%). Results of multivariable analysis showed that corticosteroid use was associated with preterm deliveries (odds ratio [OR] 3.63, 95% confidence interval [95% CI] 1.12-11.78), whereas the use of folic acid (OR 0.30, 95% CI 0.10-0.91) and presence of anti-Scl-70 antibodies (OR 0.26, 95% CI 0.08-0.85) were protective. The disease remained stable in most SSc patients, but there were 4 cases of progression of disease within 1 year from delivery, all in anti-Scl-70 antibody-positive women, 3 of whom had a disease duration of <3 years., Conclusion: Women with SSc can have successful pregnancies, but they have a higher-than-normal risk of preterm delivery, intrauterine growth restriction, and babies with very-low birth weight. Progression of the disease during or after pregnancy is rare, but possible. High-risk multidisciplinary management should be standard for these patients, and pregnancy should be avoided in women with severe organ damage and postponed in women with SSc of recent onset, particularly if the patient is positive for anti-Scl-70 antibodies., (Copyright © 2012 by the American College of Rheumatology.)

A single-nucleotide polymorphism (SNP) at the IL12RB2 locus showed a suggestive association signal in a previously published genome-wide association study (GWAS) in systemic sclerosis (SSc). Aiming to reveal the possible implication of the IL12RB2 gene in SSc, we conducted a follow-up study of this locus in different Caucasian cohorts. We analyzed 10 GWAS-genotyped SNPs in the IL12RB2 region (2309 SSc patients and 5161 controls). We then selected three SNPs (rs3790567, rs3790566 and rs924080) based on their significance level in the GWAS, for follow-up in an independent European cohort comprising 3344 SSc and 3848 controls. The most-associated SNP (rs3790567) was further tested in an independent cohort comprising 597 SSc patients and 1139 controls from the USA. After conditional logistic regression analysis of the GWAS data, we selected rs3790567 [P(MH)= 1.92 × 10(-5) odds ratio (OR) = 1.19] as the genetic variant with the firmest independent association observed in the analyzed GWAS peak of association. After the first follow-up phase, only the association of rs3790567 was consistent (P(MH)= 4.84 × 10(-3) OR = 1.12). The second follow-up phase confirmed this finding (P(χ2) = 2.82 × 10(-4) OR = 1.34). After performing overall pooled-analysis of all the cohorts included in the present study, the association found for the rs3790567 SNP in the IL12RB2 gene region reached GWAS-level significant association (P(MH)= 2.82 × 10(-9) OR = 1.17). Our data clearly support the IL12RB2 genetic association with SSc, and suggest a relevant role of the interleukin 12 signaling pathway in SSc pathogenesis.

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., (Copyright © 2012 by the American College of Rheumatology.)

Objective: To determine the role of Class II HLAs in SSc patients from Italy and Spain and in SSc patients of Caucasian ancestry., Methods: Nine hundred and forty-four SSc patients (Italy 392 patients; Spain 452 patients) and 1320 ethnically matched healthy controls (Italy 398 patients; Spain 922 patients) were genotyped up to the fourth digit by PCR with sequence-specific oligonucleotides for HLA-DRB1, DQA1 and DQB1 loci. Patients included 390 ACA-positive and 254 anti-topo I-positive subjects. Associations between SSc or SSc-specific antibodies and HLA alleles or HLA haplotypes were sought via the chi-square test after 10 000-fold permutation testing. A meta-analysis including this study cohort and other Caucasoids samples was also conducted., Results: In both the cohorts, the strongest association was observed between the HLA-DRB1*1104 allele and SSc or anti-topo I antibodies. The HLA-DRB1*1104 -DQA1*0501 -DQB1*0301 haplotype was overrepresented in Italian [odds ratio (OR) = 2.069, 95% asymptotic CIs (CI(95)) 1.486, 2.881; P < 0.001] and in Spanish patients (OR = 6.707, CI(95) 3.974, 11.319; P < 0.001) as well as in anti-topo-positive patients: Italy (OR = 2.642, CI(95) 1.78, 3.924; P < 0.001) and Spain (OR = 20.625, CI(95) 11.536, 36.876; P < 0.001). In both the populations we also identified an additional risk allele (HLA-DQB1*03) and a protective allele (HLA-DQB1*0501) in anti-topo-positive patients. The meta-analysis showed different statistically significant associations, the most interesting being the differential association between HLA-DRB1*01 alleles and ACAs (OR = 1.724, CI(95) 1.482, 2.005; P < 0.001) or topo I antibodies (OR = 0.5, CI(95) 0.384, 0.651; P < 0.001)., Conclusions: We describe multiple robust associations between SSc and HLA Class II antigens in Caucasoids that may help to understand the genetic architecture of SSc.

Objective: Polymorphisms in the genes encoding interleukin 4 (IL4), interleukin 13 (IL13), and their corresponding receptors have been associated with multiple immune-mediated diseases. Our aim was to validate these previous observations in patients with systemic sclerosis (SSc) and scrutinize the effect of the polymorphisms on gene expression in various populations of peripheral blood leukocytes., Methods: We genotyped a cohort of 2488 patients with SSc and 2246 healthy controls from The Netherlands, Spain, United Kingdom, Italy, Germany, and France. Taqman assays were used to genotype single-nucleotide polymorphisms (SNP) in the following genes: (1) IL4 (-590C>T/rs2243250); (2) IL4 receptor alpha (IL4RA) (Q576R/rs1801275); (3) IL13 (R130Q/rs20541 and -1112C>T/rs1800925); and (4) IL13RA1 (43163G>A/rs6646259). The effect of these polymorphisms on expression of the corresponding genes was assessed using quantitative RT-PCR on RNA derived from peripheral blood B cells, T cells, plasmacytoid dendritic cells, monocytes, and myeloid dendritic cells. We investigated whether these polymorphisms influenced development of pulmonary complications over 15 years in patients with SSc., Results: None of the investigated polymorphisms was associated with SSc or any SSc clinical subtype. We did not observe any effect on transcript levels in the cell subtypes or on development of pulmonary complications., Conclusion: Our data showed that polymorphisms in IL4, IL13, and their receptors do not play a role in SSc and do not influence the expression of their corresponding transcript in peripheral blood cells.

Objectives: The aim of this study was to confirm the implication of macrophage migration inhibitory factor (MIF) gene in SSc susceptibility or clinical phenotypes in a large European population., Methods: A total of 3800 SSc patients and 4282 healthy controls of white Caucasian ancestry from eight different European countries were included in the study. The MIF -173 single nucleotide polymorphism (SNP) was selected as genetic marker and genotyped using Taqman 5' allelic discrimination assay., Results: The MIF -173 SNP showed association with SSc [P = 0.04, odds ratio (OR) = 1.10, 95% CI 1.00, 1.19]. Analysis of the MIF -173 polymorphism according to SSc clinical phenotype revealed that the frequency of the -173*C allele was significantly higher in the dcSSc group compared with controls (P = 5.30E-03, OR = 1.21, 95% CI 1.07, 1.38). Conversely, the frequency of the MIF -173*C allele was significantly underrepresented in the lcSSc group compared with dcSSc patients, supporting previous findings [(P = 0.04, OR = 0.86, 95% CI 0.75, 0.99); meta-analysis including previous results (P = 0.005, OR = 0.83, 95% CI 0.73, 0.94)]., Conclusion: Our results confirm the role of MIF -173 promoter polymorphism in SSc, and provide evidence of a strong association with the dcSSc subgroup of patients. Hence, the MIF -173 variant is confirmed as a promising clinical phenotype genetic marker.

To test the involvement of osteopontin gene (OPN) in systemic sclerosis (SSc) susceptibility, two OPN single nucleotide polymorphisms previously reported to be associated with systemic lupus erythematosus, namely -156G/GG (proximal promoter) and +1239A/C (3' untranslated region (UTR)), were tested in 357 Italian patients and 864 matched control subjects. OPN serum levels were determined by enzyme-linked immunosorbent assay in 32 patients and 116 controls. Compared with the controls, in SSc patients there was a significantly increased frequency of the alleles -156G (p = 0.0086), and +1239C (p = 0.00064), paralleling the association reported for systemic lupus erythematosus. According to logistic regression analysis, this association is primarily due to the effect of +1239 single nucleotide polymorphism. OPN serum levels were significantly higher in SSc patients than in controls (p = 0.00025). These data suggest that OPN genetic variations have a role in SSc susceptibility, reporting for the first time an involvement of this molecule in SSc pathogenesis and emphasizing that SSc shares pathogenetic mechanisms with other autoimmune diseases., (Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10(-12), OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10(-6), OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10(-7), OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10(-61), OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10(-76), OR = 8.84), and in NOTCH4 with ACA P = 8.84×10(-21), OR = 0.55) and ATA (P = 1.14×10(-8), OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc., Competing Interests: The authors have declared that no competing interests exist.

The objective of this study was to determine whether the matrix metalloproteinase-9 (MMP-9) rs3918242 single nucleotide polymorphism may confer susceptibility to systemic sclerosis (SSc) with and without ulcers in an Italian Caucasian population. The MMP-9 rs3918242 functional polymorphism was genotyped in 461 subjects of Italian Caucasian origin: 228 patients with SSc (92 with and 136 without ulcers) and 233 unrelated healthy individuals. The SNP under study was in Hardy-Weinberg equilibrium in the control population. Genotype and allele distributions between SSc patients, with or without ulcers, were not statistically significant (p>0.05). A significant increase of the genotype C/T was observed in male SSc patients without ulcers when compared to patients with ulcers (P=0.04). The MMP-9 rs3918242 functional polymorphism is not associated with susceptibility to SSc. However, the presence of the polymorphism may have a protective effect on the development of ulcers in SSc male patients.

Objectives: The aim of this study was to confirm the influence of TNFSF4 polymorphisms on systemic sclerosis (SSc) susceptibility and phenotypic features., Methods: A total of 8 European populations of Caucasian ancestry were included, comprising 3014 patients with SSc and 3125 healthy controls. Four genetic variants of TNFSF4 gene promoter (rs1234314, rs844644, rs844648 and rs12039904) were selected as genetic markers., Results: A pooled analysis revealed the association of rs1234314 and rs12039904 polymorphisms with SSc (OR 1.15, 95% CI 1.02 to 1.31; OR 1.18, 95% CI 1.08 to 1.29, respectively). Significant association of the four tested variants with patients with limited cutaneous SSc (lcSSc) was revealed (rs1234314 OR 1.22, 95% CI 1.07 to 1.38; rs844644 OR 0.91, 95% CI 0.83 to 0.99; rs844648 OR 1.10, 95% CI 1.01 to 1.20 and rs12039904 OR 1.20, 95% CI 1.09 to 1.33). Association of rs1234314, rs844648 and rs12039904 minor alleles with patients positive for anti-centromere antibodies (ACA) remained significant (OR 1.23, 95% CI 1.10 to 1.37; OR 1.12, 95% CI 1.01 to 1.25; OR 1.22, 95% CI 1.07 to 1.38, respectively). Haplotype analysis confirmed a protective haplotype associated with SSc, lcSSc and ACA positive subgroups (OR 0.88, 95% CI 0.82 to 0.96; OR 0.88, 95% CI 0.80 to 0.96; OR 0.86, 95% CI 0.77 to 0.97, respectively) and revealed a new risk haplotype associated with the same groups of patients (OR 1.14, 95% CI 1.03 to 1.26; OR 1.20, 95% CI 1.08 to 1.35; OR 1.23, 95% CI 1.07 to 1.42, respectively)., Conclusions: The data confirm the influence of TNFSF4 polymorphisms in SSc genetic susceptibility, especially in subsets of patients positive for lcSSc and ACA.

Objective: To investigate the role of the Fc(α)RI 844 A>G functional polymorphism in the genetic predisposition to rheumatoid arthritis (RA) and systemic sclerosis (SSc) susceptibility., Methods: The study population was composed of 1401 patients with SSc, 642 patients with RA, and 1317 healthy controls. The Fc(α)RI (CD89) single-nucleotide polymorphism rs16986050 was genotyped by pyrosequencing., Results: We observed no significant deviation of the genotype and allele frequencies in RA and SSc compared to controls. A metaanalysis and a recessive and dominant model yielded similar negative results., Conclusion: Our data show that the Fc(α)RI 844 A>G polymorphism is not associated with SSc or RA susceptibility.

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 (p(FDRcorrected)=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 (p(FDRcorrected)=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 (p(FDRcorrected)=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.

Background: The genetic background may predispose systemic sclerosis (SSc) patients to the development of digital ulcers (DUs)., Methods: Twenty-two functional cytokine single nucleotide polymorphisms (SNPs) and 3 HLA class I and II antigens were typed at the genomic level by polymerase chain reaction in 200 Italian SSc patients. Associations with DUs were sought by parametric models and with the Multifactor Dimensionality Reduction (MDR) algorithm to depict the presence of epistasis. Biological models consistent with MDR results were built by means of Petri nets to describe the metabolic significance of our findings., Results: On the exploratory analysis, the diffuse cutaneous subset (dcSSc) was the only single factor statistically associated with DUs (p=0.045, ns after Bonferroni correction). Gene-gene analysis showed that a 3-factor model comprising the IL-6 C-174G, the IL-2 G-330T SNPs and the HLA-B*3501 allele was predictive for the occurrence of DUs in our population (testing accuracy=66.9%; p<0.0001, permutation testing)., Conclusion: Biological interpretation via Petri net showed that IL-6 is a key factor in determining DUs occurrence and that this cytokines may synergise with HLA-B*3501 to determine DUs onset. Owing to the limited number of patients included in the study, future research are needed to replicate our statistical findings as well as to better determine their functional meaning.

Objective: To determine the prevalence of and independent factors associated with joint involvement in a large population of patients with systemic sclerosis (SSc)., Methods: This study was cross-sectional, based on data collected on patients included in the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) registry. We queried this database to extract data regarding global evaluation of patients with SSc and the presence of any clinical articular involvement: synovitis (tender and swollen joints), tendon friction rubs (rubbing sensation detected as the tendon was moved), and joint contracture (stiffness of the joints that decreased their range of motion). Overall joint involvement was defined by the occurrence of synovitis and/or joint contracture and/or tendon friction rubs., Results: We recruited 7286 patients with SSc; their mean age was 56 +/- 14 years, disease duration 10 +/- 9 years, and 4210 (58%) had a limited cutaneous disease subset. Frequencies of synovitis, tendon friction rubs, and joint contractures were 16%, 11%, and 31%, respectively. Synovitis, tendon friction rubs, and joint contracture were more prevalent in patients with the diffuse cutaneous subset and were associated together and with severe vascular, muscular, renal, and interstitial lung involvement. Moreover, synovitis had the highest strength of association with elevated acute-phase reactants taken as the dependent variable., Conclusion: Our results highlight the striking level of articular involvement in SSc, as evaluated by systematic examination in a large cohort of patients with SSc. Our data also show that synovitis, joint contracture, and tendon friction rubs are associated with a more severe disease and with systemic inflammation.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify new SSc susceptibility loci, we conducted the first genome-wide association study in a population of European ancestry including a total of 2,296 individuals with SSc and 5,171 controls. Analysis of 279,621 autosomal SNPs followed by replication testing in an independent case-control set of European ancestry (2,753 individuals with SSc (cases) and 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22-23, rs2056626, P = 2.09 x 10(-7) in the discovery samples, P = 3.39 x 10(-9) in the combined analysis). Additionally, we confirm and firmly establish the role of the MHC (P = 2.31 x 10(-18)), IRF5 (P = 1.86 x 10(-13)) and STAT4 (P = 3.37 x 10(-9)) gene regions as SSc genetic risk factors.

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.

Objective: Gene-gene interaction, or epistasis, is considered a ubiquitous component of complex human diseases such as systemic sclerosis (SSc). Epistasis is difficult to model by traditional parametric approaches; therefore, nonparametric computational algorithms, such as multifactor dimensionality reduction (MDR), have been developed., Methods: A total of 242 consecutive unrelated Italian SSc patients and an equal number of well-matched healthy controls were genotyped for 22 cytokine single-nucleotide polymorphisms (SNPs; 13 cytokine genes). The distribution of the SNPs between controls and SSc patients, controls and limited cutaneous SSc (lcSSc) patients, and controls and diffuse cutaneous SSc (dcSSc) patients was tested by the MDR constructive induction algorithm and by focused interaction testing framework (FITF), a logistic regression-based approach., Results: None of the studied SNPs had main independent effects on SSc or disease subset susceptibility, therefore no epistatic interaction was detectable by FITF. The MDR analysis showed a significant epistatic interaction among the interleukin-2 (IL-2) G-330T, IL-6 C-174G, and interferon-gamma AUTR5644T SNPs and the IL-1 receptor Cpst1970T, IL-6 Ant565G, and IL-10 C-819T SNPs in lcSSc and dcSSc susceptibility, respectively. The relevance of the single multilocus attributes constructed by the MDR inductive algorithm was then confirmed by the parametric approach (P < 0.001 for both controls versus lcSSc patients and controls versus dcSSc patients)., Conclusion: We provide evidence for gene-gene interaction among cytokine SNPs in the context of SSc. The interaction among cytokine SNPs with a profibrotic or a regulatory function on profibrotic interleukins is relevant to the susceptibility to SSc subsets and it appears to be more important than the contribution of any single cytokine SNP.

Background: Vasculopathy is one of the hallmarks of systemic sclerosis (SSc), characterized by endothelial activation and over expression of adhesion molecules. A preliminary in vitro study has suggested that aminaftone, a naphtohydrochinone used in the treatment of capillary disorders, may downregulate the expression of adhesion molecules in endothelial cells., Objective: This study investigated the ex vivo effects of aminaftone on soluble adhesion molecule concentrations in patients with SSc., Methods: This randomized, open-label pilot study was conducted in patients with SSc. Patients received baseline treatment for Raynaud's phenomenon (eg, calcium channel blockers and IV cyclic iloprost) with (test) or without (control) aminaftone 75 mg or placebo TID for 12 weeks. Standard treatment for Raynaud's phenomenon was allowed as long as the dose was stable for >or=3 months prior to randomization. Concentrations of soluble E-selectin adhesion molecule 1 (sELAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble intracellular adhesion molecule 1 (sICAM-1) were measured at baseline and 12 weeks, and their variation was tested using the analysis of variance for repeated measures with statistical correction. Laboratory analyses were performed by experienced personnel blinded to treatment assignment., Results: A total of 24 patients were enrolled (21 women, 3 men; mean age, 53.4 years; aminaftone, 12 patients; control, 12 patients). Decreases in mean (SD) sELAM-1 and sVCAM-1 concentrations were significantly greater in treated patients (sELAM-1, from 17.0 [7.8] to 11.9 [9.0] pg/mL; sVCAM-1, from 51.2 [12.9] to 40.8 [13.8] ng/mL) compared with controls (sELAM-1, from 20.3 [9.9] to 20.4 [10.5] pg/mL; sVCAM-1, from 56.8 [49.6] to 62.7 [40.6] ng/mL) (both, P < 0.05 [analysis of variance or repeated measures after Bonferroni correction]). No significant changes in sICAM-1 concentrations versus controls were observed., Conclusions: In this small pilot study in this select group of patients with SSc, aminaftone was associated with downregulation of sELAM-1 and sVCAM-1 concentrations. Studies evaluating the potential role of aminaftone in the treatment of vascular sclerodermal disease and SSc are warranted.

Gene-gene and gene-environment interactions are difficult to detect by traditional parametric computational approaches. Novel nonparametric and model-free strategies, such as the multifactor dimensionality reduction (MDR) algorithm, are thus emerging as practical and feasible methods of analysis to model high-order epistatic interactions, integrating and complementing traditional logistic approaches. With traditional methods of analysis we showed that the interleukin-1 beta (IL-1 beta) C+3962T single nucleotide polymorphism (SNP), along with the Sc70 antibody and the diffuse cutaneous subset of systemic sclerosis, are important risk factors for the development of a severe ventilatory restriction in patients with systemic sclerosis (SSc); however the interactions among these and other genetic and environmental attributes were difficult to model. On the contrary, the MDR analysis detected significant two- or three-way interactions in the presence of nonlinearity. The best model identified by the multifactor dimensionality reduction algorithm included the antibody subset, the IL-1 beta C-511T and the interferon-gamma AUTR5644T SNPs, with a testing accuracy of 85% (p < 0.001) and a cross-validation consistency of 10/10. This model outperformed any one- to-three-way model constructed by considering the three factors with main independent effects identified by traditional computational approaches. Epistatic interactions among IL-1 gene complex SNPs and clinical or environmental factors are more important than the singe attributes in the development of severe ventilatory restriction in SSc patients.

Introduction: Platelet aggregation may contribute to the pathogenesis of systemic sclerosis: following activation, platelets release significant amounts of serotonin - which promotes vasoconstriction and fibrosis, and further enhances aggregation. The C+1354T polymorphism in the exonic region of the serotonin 2A receptor gene determining the His452Tyr substitution was associated with blunted intracellular responses after serotonin stimulation, and may have a role in susceptibility to scleroderma., Methods: One hundred and fifteen consecutive systemic sclerosis patients and 140 well-matched healthy control individuals were genotyped by sequence-specific primer-PCR for the His452Tyr substitution of the serotonin 2A receptor gene, and associations were sought with scleroderma and its main clinical features. The functional relevance of the His452Tyr substitution was also assessed by evaluating the aggregation of platelet-rich plasma from His452/His452 and His452/Tyr452 healthy individuals after stimulation with adenosine diphosphate +/- serotonin., Results: The T allele of the C+1354T polymorphism was underrepresented in scleroderma patients compared with control individuals (5.2% versus 12.4%, P < 0.001, chi-square test and 1,000-fold permutation test) and its carriage reduced the risk for systemic sclerosis (odds ratio = 0.39, 95% confidence interval = 0.19 to 0.85, P < 0.01). Platelets from His452/Tyr452 healthy subjects more weakly responded to serotonin stimulation compared with platelets from His452/His452 individuals (3.2 +/- 2.6-fold versus 9.6 +/- 8.6-fold increase in aggregation, P = 0.017 by Kolmogorov-Smirnov test and P = 0.003 after correction for baseline adenosine diphosphate-induced aggregation values)., Conclusion: The His452Tyr substitution may influence susceptibility to systemic sclerosis by altering platelet aggregation in response to serotonin.

Increased concentrations of interleukin-1 (IL-1) were observed in bronchoalveolar lavage fluids from patients with systemic sclerosis (SSc), and their levels were correlated with the patients' forced vital capacity (FVC). Because IL-1 production is regulated at the genetic level, we hypothesized that IL-1 gene complex single nucleotide polymorphisms (SNPs) might be relevant to the progression of ventilatory restriction in SSc. Two-hundred four Italian SSc patients were genotyped for the following IL-1 gene complex SNPs: IL-1alpha C-889T, IL-1beta C+3962T, IL-1beta C-511T, IL-1R Cpst1970T, and IL-1Ra Cmspal11100T, as well as for the following SNPs of cytokines with regulatory functions on IL-1 production: IFNgamma AUTR5644T, TNFalpha A-308G, and IL-10 A-1082G. The SNPs were inserted in a Cox regression model with disease subset, gender, autoantibodies, age at onset of disease, and prior use of immunosoppresants as covariates and the presence of FVC<55% of predicted values as outcome measure; p values were corrected for the number of pairwise comparisons. Twenty-five patients (12.3%) developed a severe ventilatory restriction after 6.8+/-6.6 years (mean+/-standard deviation) from diagnosis. In our model, the relative risk to develop a severe ventilatory restriction was increased by the antitopoisomerase I antibody (p=0.01; HR=14.67, CI95=1.87-114.92), the dcSSc subset (p=0.007; HR=3.14, CI95=1.36-7.21) and the IL-1beta C+3962T SNP (p=0.003 TT vs CC; HR=6.61, CI95=2.28-19.15). The IL-1beta C+3962T SNP is associated with the presence of severe restrictive lung physiology in Italian SSc patients.

Interleukin-10 (IL-10) can favour the development of fibrosis by promoting a relative shift towards T helper 2 responses. Three single base pair substitutions in the 5' flanking region of the IL-10 gene (G/A -1082, C/T -819 and C/A -592) influence the amount of IL-10 secreted in cell cultures: the GCC haplotype is associated with an increased production, while the ACC and the ATA haplotypes are associated with intermediate and decreased production. Accordingly, three phenotypes have been individuated: high producers (GCC+/GCC+), medium producers (GCC+/GCC-) and low producers (GCC-/GCC-). We hypothesised that IL-10 haplotypes and genotypes are differently expressed in patients with systemic sclerosis (SSc) with the limited cutaneous SSc (lcSSc) subset or the diffuse cutaneous SSc (dcSSc) subset. One hundred and sixty-one unrelated Italian patients with SSc and 94 controls have been included. Their DNA was extracted and stored before being analysed by polymerase chain reaction with sequence-specific primers. The GCC haplotype is overrepresented in patients with SSc; subjects with dcSSc were the primary contributors to these results (dcSSc: 52.2% vs controls: 37.2%; chi2= 8.519, 2 d.f., corrected P= 0.04). In Scl70-positive patients, the GCC haplotype increased the likelihood of presenting the dcSSc subset [chi2= 12.56, P < 0.0005; odds ratio (OR) = 3.89, 95% confidence interval (CI(95)) = 1.69-9.08]; these results were confirmed at the phenotypic level (chi2= 11.67, 2 d.f., P= 0.003). In Scl70-positive patients, the high-producing phenotype was associated with poor survival, independently from disease subset and gender (hazard ratio = 9.9, CI(95)= 1.6-61.27, P < 0.05). The IL-10 haplotype and genotype associated with high IL-10 production may alter the susceptibility to SSc and/or its expression, increasing the prognostic value of other well-known markers of disease severity.

Lung involvement constitutes nowadays the major cause of morbidity and mortality in scleroderma patients. Pulmonary fibrosis in systemic sclerosis (SSc) is thought to be the consequence of interstitial inflammation. Early diagnosis and treatment of active alveolitis is essential to prevent the deterioration of pulmonary function, improving outcome in SSc patients. The aim of the study was to investigate the effect of 1-year treatment with oral cyclophosphamide (CYC) on the evolution of interstitial lung disease in scleroderma patients with a diagnosis of active alveolitis. An open-label one-arm monocenteric study was conducted on 33 scleroderma patients with active alveolitis--defined as the presence of areas of 'ground-glass attenuation' on high-resolution computed tomography and a recent deterioration in lung function-treated with oral CYC 2 mg kg-1 day-1 for 1 year and medium-low dose steroids (prednisone 25 mg for 3 months and then tapered to 5 mg/day). Results showed that diffusing capacity for carbon monoxide (DLco) values remained stable after 6 months of treatment and significantly increased after 12 months (2.06+/-1.38, 2.21+/-1.62 and 2.39+/-1.64 mmol/min/kPa, at baseline/6/12 months, respectively; p<0.001 12th month vs baseline) vital capacity (VC) values slightly increased (i.e. stabilised) in the same time frame (2.46+/-0.71, 2.41+/-0.76 and 2.56+/-0.75 l). Accordingly, the vast majority of our patients (n=29, 87.9%) presented a DLco and/or a VC improvement or stabilisation with respect to baseline. Favourable results were more likely to be observed in patients with a lower Wells' radiological grade (grade I). In 25 patients followed up for further 12 months after the interruption of therapy, VC and DLco remained stable. Thus, long-term therapy with oral CYC is effective in ameliorating and/or stabilising lung function in scleroderma patients with active alveolitis, with beneficial effects lasting up to 1 year after interruption. The higher efficacy in those patients with an early pulmonary disease stage and a lower radiological grade underlies the importance of an early diagnosis and intervention.

Objectives: Interstitial lung disease (ILD) profoundly affects the health-related quality of life (HRQoL) in patients with systemic sclerosis (SSc). We tested the validity of the Saint George's Respiratory Questionnaire (SGRQ), a lung-specific HRQoL-evaluation tool, in a population of SSc patients with ILD., Methods: Twenty-eight consecutive SSc patients with a restrictive pulmonary involvement, defined as a forced vital capacity <80% of the predicted, with no pulmonary hypertension were considered. All the patients filled in the Medical Research Council (MRC) scale for perceived breathlessness, the SGRQ and the Disability Index of the Health Assessment Questionnaire (HAQ DI), and underwent evaluation with complete pulmonary function testing (PFT), 6-minute walk distance (6MWD) and high-resolution computed tomography (HRCT)., Results: The SGRQ 'activity' scores inversely correlated with the 6MWD (r = -0.86, P < 0.001) and forced vital capacity percentage of predicted values (r = -0.47) and directly correlated with HRCT (r = 0.41, P < 0.05), MRC (rho = 0.64, P < 0.001) or HAQ DI scores (r = 0.62, P < 0.001), independently of disease duration or subset. On the contrary, HAQ DI scores were influenced by those variables and corrected correlations with 6MWD (r = 0.56, P < 0.001) or HRCT scores (r = 0.36, P = NS) were less strong than those observed with the SGRQ., Conclusions: The SGRQ, although not specifically designed for scleroderma, is a valid respiratory-specific questionnaire for the evaluation of HRQoL in patients with SSc-related ILD. The SGRQ performs better in relation to exercise capacity and lung imaging than other non-respiratory-specific questionnaires widely used in scleroderma studies. Further studies are needed to address its ability to assess changes over time or in response to therapy.

Cyclophosphamide (CYC) is the cornerstone of the treatment of systemic sclerosis (SSc)-associated fibrosing alveolitis (FAS). Despite treatment with CYC, in a not negligible proportion of SSc-FAS patients, deterioration in lung function can be observed. Interleukin-1 (IL-1) cluster gene single nucleotide polymorphisms (SNPs) were implicated in the pathogenesis of some interstitial lung diseases and may favor the progression of restrictive lung disease in SSc. The present retrospective case-control study was conducted on 18 SSc patients previously treated with oral CYC (2 mg/kg) and medium-dose steroids (prednisone 25 mg for 3 months and then tapered to 5 mg/day) for the presence of FAS-defined as the presence of areas of ground-glass attenuation on high-resolution computed tomography (HRCT) and a recent deterioration in lung function. The T/C substitution at position -889 of the IL-1alpha promoter gene (T-889C) was determined by polymerase chain reaction and restriction length fragment analysis. Patients carrying the T allele showed a significant decrease in forced vital capacity (FVC) values after 12 months of therapy (2.46+/-1.09 vs 2.59+/-1.17 l), while wild-type patients showed an increase in FVC values (2.73+/-0.54 vs 2.54+/-0.5 l) (p=0.005 between the two groups, analysis of variance for repeated measures). Patients with the T-889C polymorphism presented higher baseline erythrocyte sedimentation rates (ESR) compared to wild-type patients (50.3+/-25.4 vs 23.3+/-17.7 mm/h). Baseline ESR inversely correlated with the variation of FVC (DeltaFVC) after 12 months of therapy (r=-0.50 and p<0.05). The two groups were otherwise similar with respect to autoantibodies, age, disease duration, disease subset, radiological HRCT grade, and baseline lung physiology. The T-889C polymorphism represents a marker for worse functional responses to CYC in SSc-FAS. The mechanisms by which this SNP may negatively influence the response to CYC therapy are unknown, but might be linked to increased inflammatory responses in the lungs.

HLA-B35 is associated with an increased risk for developing isolated pulmonary hypertension (iPHT) in systemic sclerosis, but the mechanisms underlying this association have not been fully elucidated yet. Endothelin-1 (ET-1) is the main pathogenetic molecule implied in the development of iPHT; therefore, we sought to determine if ECV304 cells transfected with the HLA-B35 allele produce increased amounts of ET-1 after incubation with physiological concentrations of interleukin-1 beta (IL-1beta). ECV304 cells transfected with HLA-B*3501 and HLA-B*0801 polymorphic alpha chain or with pIRESneo2 were incubated with 100 U/ml of IL-1beta for 6, 12, 24, 36 and 48 h. ET-1 levels were determined using EIA kit (CAYMAN Chemical, Ann Arbor, MI) in supernatants from different cell cultures; the relative expression of the preproendothelin-1 (PPET-1) gene was also determined by reverse transcription-polymerase chain reaction. Cells expressing the HLA-B35 allele showed significantly increased levels of ET-1 at all the selected times compared with controls or HLA-B8-transfected cells. The relative expression of the PPET-1 gene was also increased in a proportionally direct manner. The HLA-B35 allele influences the production of ET-1 in HLA-B35-transfected ECV304 cells by promoting the expression of its precursor, PPET-1. Our results provide an explanation for the epidemiological association existing between iPHT and HLA-B35.

Background: A high prevalence of depressive symptoms has been described in systemic sclerosis (SSc), but no clear association with organ involvement or objective indices of disease severity has been depicted. To date, no effort has been made to determine the prevalence of depressive symptoms in Italian patients with SSc or to clarify their cause., Methods: One-hundred-eleven SSc patients were asked to fill in the Beck Depression Inventory (BDI) questionnaire, the scleroderma Health Assessment Questionnaire (sHAQ) and two additional questions assessing the patient's familiar support and the social consequences of the patient's change in physical appearnace., Results: Thirty-seven subjects (33.4%) presented mild to severe depressive symptoms (BDI >/=17). On univariate analysis the diffuse cutaneous form of the disease (p=0.019), higher pulmonary systolic pressures on echocardiogram (p=0.016), lower FVC percentage of predicted values (p=0.022), higher sHAQ values (p<0.001) or higher VAS values for pain (p=0.007), lung involvement (p=0.02), Raynaud's phenomenon severity (p=0.002), ulcers severity (p=0.006) or disease severity (p<0.001), were associated with the presence of pathologic depressive symptoms. On multivariate analysis only the VAS for disease severity relevant to BDI scores (p=0.016). Social behaviour changes due to SSc-related physical involvement were reported in 14 patients (38%) with depressive symptoms (p=0,006) and were more likely to be observed in younger patients (p=0.001) with a more severe Raynauds's phenomenon (p=0.013)., Conclusions: Mild to severe depressive symptoms are common in SSc patients especially in those with a worse perception of disease severity, these patients should be carefully monitored and a psychological assistance counselled whenever necessary.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin and internal organs fibrosis due to an extracellular matrix (ECM) accumulation of type I collagen. The turnover of the ECM is dependent on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). The disruption of this balance is involved in SSc because higher serum TIMP-1 levels have been demonstrated in SSc patients than in controls. On this basis, we analyzed three polymorphisms: -19A>G, +261C>T, and +372T>C of the TIMP-1 gene in SSc patients (67 females, eight males) and controls (29 females, nine males). The C allele of the +372T>C single nucleotide polymorphism (SNP) was observed at a higher frequency in male patients than in healthy individuals (P=0.02), while no differences were observed in the female subjects. Our findings suggest that the +372T>C polymorphism of the TIMP-1 gene is associated with SSc in male individuals. No association with the clinical characteristics of SSc Italian patients and TIMP-1 gene polymorphisms was observed. Thus, the role of TIMP-1 gene in predisposition to SSc remains controversial.

Background and Objectives: Autoantibodies inactivating the von Willebrand factor (VWF) cleaving protease, ADAMTS-13, are among the most frequent causes of thrombotic thrombocytopenic purpura (TTP). We evaluated whether or not ADAMTS-13 deficiency and autoantibodies inactivating the protease prevalent in patients with the prototypic autoimmune diseases systemic lupus erythematosus (SLE) and systemic sclerosis (SSc)., Design and Methods: We measured, in parallel, the protease and VWF antigen (VWF:Ag) in 123 patients, 36 of whom had SLE and 87 of whom had SSc. In 14 patients with either disease who had low plasma protease levels (below 40%) we also looked for anti-ADAMTS-13 inactivating antibodies., Results: ADAMTS-13 levels were significantly lower in SLE (p=0.0013) and in SSc (p=0.0002) than in normal controls. No anti-ADAMTS activity was measurable in patients with low ADAMTS-13 levels. VWF:Ag was high in both SLE and SSc (p=0.001)., Interpretation and Conclusions: Systemic connective tissue diseases are other conditions besides TTP that are associated in some instances with low but detectable levels of ADAMTS-13. Autoantibodies inactivating protease activity are not the cause of the low plasma levels of ADAMTS-13.

Our objective was to identify a core set of variables for the assessment of vascular involvement in scleroderma that is simple, reproducible, and reflects the presence of vascular disease in SSc. To do so we carried out an extensive literature review of published studies relating to the assessment of vascular involvement in SSc, i.e. studies dealing with clinical parameters, functional vascular studies, cold presser testing, nailfold capillary microscopy and circulating vascular markers. After extensive review of published studies and critical assessment of proposed vascular parameters, the subcommittee endorsed what it considers to be the minimal requirements for the documentation of vascular involvement in SSc. The core set variables include two parameters: Raynaud's phenomenon and digital ulcers. This set is simple, reproducible and should be included in the assessment of SSc patients in clinical investigational studies. The subcommittee also however recognizes that there exists a promising set of vascular variable that still needs further investigation.

The Subcommittee members initially agreed on the concepts of disease activity, damage and severity, defining severity as the total effect of disease on organ function. It was decided to start with the assessment of severity using the Medsger's severity scale. A revised version of this scale was constructed. The rationale for the exclusion of other variables was provided.