Gilles Chiocchia

Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci

Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis–associated haplotypes at 11 loci. Two ankylosing spondylitis–associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.

Proinflammatory Th17 cells are expanded and induced by dendritic cells in spondylarthritis-prone HLA–B27–transgenic rats

OBJECTIVEnHLA-B27/human β2-microglobulin-transgenic (B27-transgenic) rats, a model of spondylarthritis (SpA), develop spontaneous colitis and arthritis under conventional conditions. CD4+ T cells are pivotal in the development of inflammation in B27-transgenic rats. This study was undertaken to characterize the phenotype of CD4+ T cells in this model and to determine whether dendritic cells (DCs) induce proinflammatory T cells.nnnMETHODSnThe phenotype of CD4+ T cells from rat lymph nodes (LNs) draining the sites of inflammation was analyzed by flow cytometry. Immunostaining was used to detect interleukin-17 (IL-17)-producing cells in the rat joints. DCs from B27-transgenic or control rats (transgenic for HLA-B7 or nontransgenic) were cocultured with control CD4+ T cells and stimulated with anti-T cell receptor α/β.nnnRESULTSnIL-17A- and tumor necrosis factor α (TNFα)-producing CD4+ T cells were expanded in mesenteric and popliteal LNs from B27-transgenic rats. The accumulation of Th17 cells correlated with disease development, in contrast to Th1 or Treg cells. IL-17-positive mononuclear cells were detected in the arthritic joints of B27-transgenic rats but not in the joints of control rats. Finally, in vitro cocultures demonstrated that Th17 cells were preferentially induced and expanded by DCs from B27-transgenic rats, by a process that may involve defective engagement of costimulatory molecules.nnnCONCLUSIONnOur findings indicate that expanded CD4+ T cells in B27-transgenic rats exhibit a proinflammatory Th17 phenotype characterized by IL-17A and TNFα production. Furthermore, this population is preferentially induced by DCs from B27-transgenic rats. These data point toward an induction of Th17 cells as a possible pathogenic mechanism in this model of SpA. However, their pathogenic role still needs to be shown.

Angiogenesis in systemic sclerosis: Impaired expression of vascular endothelial growth factor receptor 1 in endothelial progenitor–derived cells under hypoxic conditions

OBJECTIVEnTo assess angiogenesis and explore the expression and regulation of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), and VEGFR-2, the leading mediators of angiogenesis, in SSc patients and controls.nnnMETHODSnLate-outgrowth endothelial progenitor cells (EPCs), isolated from the peripheral blood of systemic sclerosis (SSc) patients and controls, and human umbilical vein endothelial cells (HUVECs) were assessed under normal and hypoxic conditions. Genomic background was evaluated in a large case-control study (including 659 patients with SSc and 511 controls) using tag single-nucleotide polymorphisms on VEGFR1 and VEGFR2 genes.nnnRESULTSnEPCs from SSc patients had the phenotype of genuine endothelial cells and displayed in vitro angiogenic properties similar to those of HUVECs and control EPCs under basal conditions, as determined by flow cytometry, tube formation, and migration assay. However, after 6 hours of hypoxic exposure, EPCs from SSc patients exhibited lower induced expression of VEGFR-1 at the messenger RNA and protein levels, but similar VEGF and VEGFR-2 expression, compared with HUVECs or EPCs from healthy controls. There was no evidence of defective expression of hypoxia-inducible factor 1alpha. These results were supported by the lower serum levels of soluble VEGFR-1 found in SSc patients (n = 187) compared with healthy controls (n = 48) (mean +/- SD 163.7 +/- 98.5 versus 210.4 +/- 109.5 pg/ml; P = 0.0042). These abnormalities did not seem to be related to genomic background.nnnCONCLUSIONnOur findings shed new light on the possible role of VEGFR-1 in the main vascular disturbances that occur in SSc and lead to more severe disease.

Brief Report: Candidate gene study in systemic sclerosis identifies a rare and functional variant of the TNFAIP3 locus as a risk factor for polyautoimmunity

OBJECTIVEnSystemic lupus erythematosus (SLE) and systemic sclerosis (SSc) share some pathophysiologic bases as evidenced by individual and familial polyautoimmunity and common susceptibility genetic factors. With regard to the latter, there has been a recent shift from the common variant to the rare variant paradigm, since rare variants of TNFAIP3 and TREX1 with large effect sizes have recently been discovered in SLE. The present study was undertaken to investigate whether rare variants of TNFAIP3 and TREX1 are also associated with SSc.nnnMETHODSnTREX1 single-nucleotide polymorphisms (SNPs) rs3135946, rs7626978, rs3135943, and rs11797 and TNFAIP3 SNPs rs9494883, rs72063345, rs5029939, rs2230926, rs117480515, and rs7749323 were genotyped in a discovery set (985 SSc patients and 1,011 controls), and replication analysis of the most relevant results was performed in a second set (622 SSc patients and 493 controls).nnnRESULTSnNo association between TREX1 variants and SSc was observed. For TNFAIP3, we first demonstrated that a low-frequency variant, rs117480515, tagged the recently identified TT>A SLE dinucleotide. In the discovery sample, we observed that all tested TNFAIP3 variants were in linkage disequilibrium and were associated with SSc and various SSc subsets, including the polyautoimmune phenotype. We subsequently genotyped rs117480515 in the replication sample and found it to be associated solely with the SSc polyautoimmune subset (odds ratio 3.51 [95% confidence interval 2.28-5.41], P = 8.58 × 10(-9) ) in the combined populations. Genotype-messenger RNA (mRNA) expression correlation analysis revealed that the TNFAIP3 rs117480515 risk allele was associated with decreased mRNA expression.nnnCONCLUSIONnThe present findings establish the TNFAIP3 locus as a susceptibility factor for the subset of SSc with a polyautoimmune phenotype. Our results support the implication of rare/low-frequency functional variants and the critical role of A20 in autoimmunity.

Angiogenic biomarkers predict the occurrence of digital ulcers in systemic sclerosis

Objective To evaluate the possible merit of endothelial markers for the prediction of ischaemic digital ulcers in patients with systemic sclerosis (SSc). Methods Circulating endothelial progenitor cells (EPC), circulating endothelial cells and serum levels of placental growth factor (PlGF), soluble vascular adhesion molecule and vascular endothelial growth factor were measured in a prospective cohort of 100 SSc patients. The primary outcome was the occurrence of one or more new ischaemic digital ulcers during a planned 3-year follow-up. Results After the follow-up period, 17 patients developed new digital ulcers. By multivariate analysis focused on biomarkers, high PlGF serum levels and low EPC counts were identified as predictors of the occurrence of at least one new digital ulcer. In a secondary model including biomarkers together with clinical SSc characteristics all predictors of digital ulcers defined by p≤0.1 in univariate analysis, high PlGF serum levels (HR 7.26, 95% CI 1.92 to 27.41) and a history of digital ulcers (HR 9.32, 95% CI 1.51 to 59.83) were identified as independent predictors of a new digital ulcer. In an alternative model excluding patients with a history of digital ulcers at baseline, high PlGF serum levels (HR 13.46, 95% CI 1.58 to 114.73) and low EPC counts (HR 7.95, 95% CI 2.09 to 30.09) remained predictive of new digital ulcer occurrence during follow-up. Conclusion This study identified high PlGF serum levels and low circulating EPC counts as predictors of new digital ulcers in SSc. It highlights the critical role of angiogenesis in this vascular outcome. These markers may improve digital ulcer risk stratification and therefore allow earlier therapeutic intervention.

Gene Expression Profile in the Salivary Glands of Primary Sjögren's Syndrome Patients Before and After Treatment With Rituximab

OBJECTIVEnPrimary Sjögrens syndrome (SS) is a complex disorder, in part due to B cell abnormalities. Although anti-B cell therapy is promising in primary SS, no treatment has yet been demonstrated to modify the disease course. This open-label study was undertaken to evaluate the efficacy of rituximab in primary SS and to investigate whether expression of specific genes is associated with efficacy of this treatment.nnnMETHODSnFifteen patients with primary SS were treated in an open-label trial. Salivary gland biopsy specimens were obtained, and total RNA was extracted and amplified. Microarray analysis with the Affymetrix Human Genome U133 Plus 2.0 Array was used to analyze >54,000 transcripts, and potential pathways were identified.nnnRESULTSnWith gene expression data obtained before treatment, patients could be correctly classified in terms of whether they would be responders or nonresponders to rituximab. Gene pathway analysis demonstrated that the B cell signaling pathway was the most profoundly differentially expressed before treatment in the responders compared with nonresponders. Subclassification of patients based on the level of infiltration also demonstrated differential expression of genes belonging to the interferon (IFN) pathway between responders and nonresponders. Furthermore, unsupervised analysis based on gene expression modification before and after treatment allowed identification of 8 genes that were differentially expressed between responders and nonresponders, with the difference remaining significant after Bonferroni correction.nnnCONCLUSIONnOur results demonstrate the ability to elaborate a set of genes predictive of rituximab efficacy and highlight the importance of studying the differential expression of B cell and IFN pathway signaling molecules in relation to the response to anti-CD20 treatment. A randomized controlled study is currently ongoing to confirm these results.

Correlations between angiogenic factors and capillaroscopic patterns in systemic sclerosis

IntroductionWe sought to assess whether nailfold videocapillaroscopy (NVC) patterns are associated with levels of angiogenic factors in systemic sclerosis (SSc).MethodsCirculating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) were measured in the peripheral blood of 60 consecutive SSc patients. Serum levels of eight endothelial markers were measured first in these 60 patients, and then in an independent replication cohort of 43 SSc patients in case of association with NVC patterns. NVC patterns were determined by four independent investigators blinded to vascular markers.ResultsPatients with the late-NVC pattern exhibited lower EPC levels (P < 0.0001) and higher VEGF levels (P = 0.03). Higher VEGF levels were confirmed to be associated with the late-NVC pattern in the replication cohort (P = 0.01). By multivariate analysis focused on biomarkers, lower EPC (P = 0.03) and higher VEGF levels (P = 0.001) were independently associated with the late-NVC pattern. In an alternate multivariate model including these two factors and SSc-related disease characteristics, lower EPC counts (P = 0.005), higher VEGF levels (P = 0.01), a history of digital ulcers (P = 0.04), and a modified Rodnan skin score > 14 (P < 0.0001) were independently associated with the late-NVC pattern.ConclusionOur data revealed decreased EPC counts and increased VEGF levels in patients with the late-NVC pattern. Further studies are now needed to determine the role of VEGF and EPCs in endothelial injury and repair in SSc.

C8orf13-BLK is a genetic risk locus for systemic sclerosis and has additive effects with BANK1: results from a large french cohort and meta-analysis.

OBJECTIVEnAccumulating evidence suggests that B cells are involved in systemic sclerosis (SSc). BANK1 has been reproducibly reported to be associated with diffuse cutaneous SSc (dcSSc). BLK encodes another B cell signal transducer, and a functional variant at the C8orf13-BLK locus has been found to be associated with SSc in Caucasians. However, no independent replication has been reported, and there are discrepancies in the genotype-phenotype correlation between these studies in Caucasians and another study performed in the Japanese population. Therefore, in a large cohort of French Caucasians and using a meta-analysis of the available data, this study was undertaken to determine whether the C8orf13-BLK locus is associated with SSc, and to assess the possibility of interaction between BLK and BANK1 in SSc.nnnMETHODSnThe C8orf13-BLK rs13277113 genotype was determined in 1,031 patients with SSc and 1,014 control subjects for whom BANK1 genotypes were available. Meta-analysis of the 3 available data sets (6,078 individuals) was also performed.nnnRESULTSnMinor allele frequencies for rs13277113 revealed an association restricted to the dcSSc subtype (P = 0.012, odds ratio [OR] 1.29) in the French sample. Meta-analysis of the combined Caucasian populations showed an association of this genotype with both SSc (P = 0.0013, OR 1.16, 95% confidence interval [95% CI] 1.06-1.26) and dcSSc (P = 0.0012, OR 1.23, 95% CI 1.08-1.39). Inclusion of the Japanese population confirmed the overall association with the disease, with the strongest association observed with dcSSc (P = 3.27 × 10⁻⁵, OR 1.27). Secondary analysis in the French sample revealed additive effects between C8orf13-BLK and BANK1, mainly in the dcSSc subset.nnnCONCLUSIONnThese results confirm C8orf13-BLK as an SSc risk locus. The strongest effects, and particularly additive effects, were observed in the interaction between C8orf13-BLK and BANK1 in the dcSSc subset.

Independent replication establishes the CD247 gene as a genetic systemic sclerosis susceptibility factor

The first large genome-wide association (GWA) study of systemic sclerosis (SSc) recently included 2296 SSc patients and 5014 healthy controls from four case–control series of Caucasian individuals and involved the analysis of about 280 000 single nucleotide polymorphisms (SNPs).1 Outside the HLA region, SNPs mapping to the known TNPO3 - IRF5 and STAT4 regions showed the strongest association but three other loci reached genome-wide significance (namely CD247 locus in 1q22–23, CDH7 in 18q22 and EXOC2 - IRF4 near 6p25) although only one ( CD247 ) could be replicated in the second set of samples. Given the major challenge of separating the many false-positive associations from the few true-positive associations with disease in GWA studies, resulting in a stratification bias, a critical strategy is replication of results in independent samples.2 Therefore, we herein provide an attempt of …

Independent Replication and Metaanalysis of Association Studies Establish TNFSF4 as a Susceptibility Gene Preferentially Associated with the Subset of Anticentromere-positive Patients with Systemic Sclerosis

Objective. Independent replication with large cohorts and metaanalysis of genetic associations are necessary to validate genetic susceptibility factors. The known tumor necrosis factor (ligand) superfamily, member 4 gene (TNFSF4) systemic lupus erythematosus (SLE) risk locus has been found to be associated with systemic sclerosis (SSc) in 2 studies, but with discrepancies between them for genotype-phenotype correlation. Our objective was to validate TNFSF4 association with SSc and determine the subset with the higher risk. Methods. Known SLE and SSc TNFSF4 susceptibility variants (rs2205960, rs1234317, rs12039904, rs10912580, and rs844648) were genotyped in 1031 patients with SSc and 1014 controls of French white ancestry. Genotype-phenotype association analysis and metaanalysis of available data were performed, providing a population study of 4989 patients with SSc and 4661 controls, all of European white ancestry. Results. Allelic and genotypic associations were observed for the 5 single-nucleotide polymorphisms (SNP) with the subset of patients with SSc who are positive for anticentromere antibodies (ACA) and only a trend for association with SSc and limited cutaneous SSc. Rs2205960 exhibited the strongest allelic association in ACA+ patients with SSc [p = 0.0015; OR 1.37 (1.12–1.66)], with significant intra-cohort association when compared to patients with SSc positive for ACA. Metaanalysis confirmed overall association with SSc but also raised preferential association with the ACA+ subset and strongest effect with rs2205960 [T allele p = 0.00013; OR 1.33 (1.15–1.54) and TT genotype p = 0.00046; OR 2.02 (1.36–2.98)]. Conclusion. We confirm TNFSF4 as an SSc susceptibility gene and rs2205960 as a putative causal variant with preferential association in the ACA+ SSc subphenotype.