A Combined Transcriptomic and Genomic Analysis Identifies a Gene Signature Associated With the Response to Anti-TNF Therapy in Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is the most frequent autoimmune disease involving the joints. Although anti-TNF therapies have proven effective in the management of RA, approximately one third of patients do not show a significant clinical response. The objective of this study was to identify new genetic variation associated with the clinical response to anti-TNF therapy in RA. Methods: We performed a sequential multi-omic analysis integrating different sources of molecular information. First, we extracted the RNA from synovial biopsies of 11 RA patients starting anti-TNF therapy to identify gene coexpression modules (GCMs) in the RA synovium. Second, we analyzed the transcriptomic association between each GCM and the clinical response to anti-TNF therapy. The clinical response was determined at week 14 using the EULAR criteria. Third, we analyzed the association between the GCMs and anti-TNF response at the genetic level. For this objective, we used genome-wide data from a cohort of 348 anti-TNF treated patients from Spain. The GCMs that were significantly associated with the anti-TNF response were then tested for validation in an independent cohort of 2,706 anti-TNF treated patients. Finally, the functional implication of the validated GCMs was evaluated via pathway and cell type epigenetic enrichment analyses. Results: A total of 149 GCMs were identified in the RA synovium. From these, 13 GCMs were found to be significantly associated with anti-TNF response (P < 0.05). At the genetic level, we detected two of the 13 GCMs to be significantly associated with the response to adalimumab (P = 0.0015) and infliximab (P = 0.021) in the Spain cohort. Using the independent cohort of RA patients, we replicated the association of the GCM associated with the response to adalimumab (P = 0.0019). The validated module was found to be significantly enriched for genes involved in the nucleotide metabolism (P = 2.41e-5) and epigenetic marks from immune cells, including CD4+ regulatory T cells (P = 0.041). Conclusions: These findings show the existence of a drug-specific genetic basis for anti-TNF response, thereby supporting treatment stratification in the search for response biomarkers in RA.

Genome-wide association study meta-analysis identifies five new loci for systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with a complex genetic inheritance. Genome-wide association studies (GWAS) have significantly increased the number of significant loci associated with SLE risk. To date, however, established loci account for less than 30% of the disease heritability and additional risk variants have yet to be identified. Here we performed a GWAS followed by a meta-analysis to identify new genome-wide significant loci for SLE. METHODS: We genotyped a cohort of 907 patients with SLE (cases) and 1524 healthy controls from Spain and performed imputation using the 1000 Genomes reference data. We tested for association using logistic regression with correction for the principal components of variation. Meta-analysis of the association results was subsequently performed on 7,110,321 variants using genetic data from a large cohort of 4036 patients with SLE and 6959 controls of Northern European ancestry. Genetic association was also tested at the pathway level after removing the effect of known risk loci using PASCAL software. RESULTS: We identified five new loci associated with SLE at the genome-wide level of significance (p < 5 × 10- 8): GRB2, SMYD3, ST8SIA4, LAT2 and ARHGAP27. Pathway analysis revealed several biological processes significantly associated with SLE risk: B cell receptor signaling (p = 5.28 × 10- 6), CTLA4 co-stimulation during T cell activation (p = 3.06 × 10- 5), interleukin-4 signaling (p = 3.97 × 10- 5) and cell surface interactions at the vascular wall (p = 4.63 × 10- 5). CONCLUSIONS: Our results identify five novel loci for SLE susceptibility, and biologic pathways associated via multiple low-effect-size loci.

Use of IFN-γ and IP-10 detection in the diagnosis of latent tuberculosis infection in patients with inflammatory rheumatic diseases.

OBJECTIVES: Biologic agents are used against rheumatic diseases, however, they increase the risk of developing severe infections and diseases such as tuberculosis. We aimed to determine the benefits of IP-10 detection to diagnose latent tuberculosis infection (LTBI) in patients with inflammatory rheumatic diseases on different immunosuppressive drug regimens, and compare these results with IFN-γ detection. MATERIALS AND METHODS: We included 64 patients with inflammatory rheumatic diseases. We used QuantiFERON Gold In-Tube (QFN-G-IT) and T-SPOT.TB to detect IFN-γ production, and an in-house ELISA for IP-10 detection from the previous QFN-G-IT stimulated samples. We assessed the combined use of IFN-γ release assays (IGRAs) and IP-10 test, and analyzed the influence of immunotherapy on the tests performance. RESULTS: We obtained 34.9% positive results by T-SPOT.TB, 25.0% by QFN-G-IT and 31.3% by IP-10 test. The combined use of IGRAs and IP-10 detection increased significantly the amount of positive results (p < 0.0001). Treatment intake had no significant effect on in vitro tests (p > 0.05). CONCLUSIONS: IP-10 and IFN-γ detection is comparable and their combined use could increase the number of positive results in the diagnosis of LTBI in rheumatic patients. The tested assays were not influenced by rheumatoid immunosuppressive therapy. Thus, IP-10 could be of use in the development of new and improved LTBI diagnostic tools.

Genome-wide pathway analysis identifies VEGF pathway association with oral ulceration in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a genetically complex rheumatic disease characterized by heterogeneous clinical manifestations of unknown etiology. Recent studies have suggested the existence of a genetic basis for SLE heterogeneity. The objective of the present study was to identify new genetic variation associated with the clinically relevant phenotypes in SLE. METHODS: A two-stage pathway-based approach was used to identify the genetic variation associated with the main clinical phenotypes in SLE. In the discovery stage, 482 SLE patients were genotyped using Illumina Human Quad610 microarrays. Association between 798 reference genetic pathways from the Molecular Signatures Database and 11 SLE phenotypes was tested using the set-based method implemented in PLINK software. Pathways significantly associated after multiple test correction were subsequently tested for replication in an independent cohort of 425 SLE patients. Using an in silico approach, we analyzed the functional effects of common SLE therapies on the replicated genetic pathways. The association of known SLE risk variants with the development of the clinical phenotypes was also analyzed. RESULTS: In the discovery stage, we found a significant association between the vascular endothelial growth factor (VEGF) pathway and oral ulceration (P value for false discovery rate (P FDR) < 0.05), and between the negative regulation signaling pathway of retinoic acid inducible gene-I/melanoma differentiation associated gene 5 and the production of antinuclear antibodies (P FDR < 0.05). In the replication stage, we validated the association between the VEGF pathway and oral ulceration. Therapies commonly used to treat mucocutaneous phenotypes in SLE were found to strongly influence VEGF pathway gene expression (P = 4.60e-4 to 5.38e-14). Analysis of known SLE risk loci identified a strong association between PTPN22 and the risk of hematologic disorder and with the development of antinuclear antibodies. CONCLUSIONS: The present study has identified VEGF genetic pathway association with the risk of oral ulceration in SLE. New therapies targeting the VEGF pathway could be more effective in reducing the severity of this phenotype. These findings represent a first step towards the understanding of the genetic basis of phenotype heterogeneity in SLE.

Identification of IRX1 as a Risk Locus for Rheumatoid Factor Positivity in Rheumatoid Arthritis in a Genome-Wide Association Study.

OBJECTIVE: Rheumatoid factor (RF) is a well-established diagnostic and prognostic biomarker in rheumatoid arthritis (RA). However, ∼20% of RA patients are negative for this anti-IgG antibody. To date, only variation at the HLA-DRB1 gene has been associated with the presence of RF. This study was undertaken to identify additional genetic variants associated with RF positivity. METHODS: A genome-wide association study (GWAS) for RF positivity was performed using an Illumina Quad610 genotyping platform. A total of 937 RF-positive and 323 RF-negative RA patients were genotyped for >550,000 single-nucleotide polymorphisms (SNPs). Association testing was performed using an allelic chi-square test implemented in Plink software. An independent cohort of 472 RF-positive and 190 RF-negative RA patients was used to validate the most significant findings. RESULTS: In the discovery stage, a SNP in the IRX1 locus on chromosome 5p15.3 (SNP rs1502644) showed a genome-wide significant association with RF positivity (P = 4.13 × 10(-8) , odds ratio [OR] 0.37 [95% confidence interval (95% CI) 0.26-0.53]). In the validation stage, the association of IRX1 with RF was replicated in an independent group of RA patients (P = 0.034, OR 0.58 [95% CI 0.35-0.97] and combined P = 1.14 × 10(-8) , OR 0.43 [95% CI 0.32-0.58]). CONCLUSION: To our knowledge, this is the first GWAS of RF positivity in RA. Variation at the IRX1 locus on chromosome 5p15.3 is associated with the presence of RF. Our findings indicate that IRX1 and HLA-DRB1 are the strongest genetic factors for RF production in RA.

Nailfold capillaroscopic findings in primary Sjögren's syndrome with and without Raynaud's phenomenon and/or positive anti-SSA/Ro and anti-SSB/La antibodies.

The aim of this study was to assess nailfold capillaroscopic (NC) findings in patients with primary Sjögren's syndrome (PSS) with and without Raynaud's phenomenon (RP) as well as in the presence of positive anti-SSA/Ro and anti-SSB/La antibodies. Videocapillaroscopy was performed in 150 patients with PSS. Data collected included demographics, presence of RP, PSS symptoms, antinuclear antibodies, rheumatoid factor, anti-Ro, anti-La, anti-CCP, salivary scintigraphy, labial biopsy, and NC findings. RP was present in 32% of PSS, keratoconjunctivitis sicca in 91%, oral xerosis in 93%, and skin or genital xerosis in 53%. In patients with positive anti-SSA/Ro (75%) and positive anti-SSB/La (40%), NC showed normal findings in 53% of cases and non-specific in 36%. In patients with PSS, NC was normal in 51% of cases and non-specific in 34%. Scleroderma pattern was found in 14 patients. RP associated with PSS had non-specific capillaroscopy in 40% of cases (p = 0.1). Pericapillary haemorrhages (p = 0.06) and capillary thrombosis (p = 0.2) were not increased, but more dilated capillaries were detected in 48% of cases. Patients with positive anti-Ro and/or anti-La have not a distinct NC profile. Patients with RP associated with PSS had more dilated capillaries, but neither pericapillary haemorrhages nor capillary thrombosis was observed.

Variation at interleukin-6 receptor gene is associated to joint damage in rheumatoid arthritis.

INTRODUCTION: Interleukin-6 (IL-6) cytokine signaling is key in Rheumatoid Arthritis (RA) pathophysiology. Blocking IL-6 receptor (IL6R) has proven to be a highly effective treatment to prevent joint damage. This study was performed to investigate the association between the genetic variation at IL6R gene and the severity of joint damage in RA. METHODS: IL6R gene tagging SNPs (n = 5) were genotyped in a discovery group of 527 RA patients from 5 different university hospitals from Spain. For each marker, a linear regression analysis was performed using an additive model and adjusting for the years of evolution of the disease, autoantibody status, gender and age. Haplotypes combining the SNPs were also estimated and tested for association with the level of joint destruction. Using an independent cohort of 705 RA patients from 6 university hospitals we performed a validation study of the SNPs associated in the discovery phase. RESULTS: In the discovery group we found a highly significant association between IL6R SNP rs4845618 and the level of joint destruction in RA (P = 0.0058, P corrected = 0.026), and a moderate association with SNP rs4453032 (P = 0.02, P corrected = 0.05). The resulting haplotype from both SNPs was more significantly associated with joint damage (P = 0.0037, P corrected = 0.011). Using the validation cohort, we replicated the association between the two IL-6R SNPs with the degree of joint destruction in RA (P = 0.007 and P = 0.04, meta-analysis P = 0.00011 and P = 0.0021, respectively), and the haplotype association (P = 0.0058, meta-analysis P = 6.64 e-5). CONCLUSIONS: Genetic variation at IL6R gene is associated with joint damage in RA.

Variation at FCGR2A and functionally related genes is associated with the response to anti-TNF therapy in rheumatoid arthritis.

OBJECTIVE: Anti-TNF therapies have been highly efficacious in the management of rheumatoid arthritis (RA), but 25-30% of patients do not show a significant clinical response. There is increasing evidence that genetic variation at the Fc receptor FCGR2A is associated with the response to anti-TNF therapy. We aimed to validate this genetic association in a patient cohort from the Spanish population, and also to identify new genes functionally related to FCGR2A that are also associated with anti-TNF response. METHODS: A total of 348 RA patients treated with an anti-TNF therapy were included and genotyped for FCGR2A polymorphism rs1081274. Response to therapy was determined at 12 weeks, and was tested for association globally and independently for each anti-TNF drug (infliximab, etanercept and adalimumab). Using gene expression profiles from macrophages obtained from synovial fluid of RA patients, we searched for genes highly correlated with FCGR2A expression. Tag SNPs were selected from each candidate gene and tested for association with the response to therapy. RESULTS: We found a significant association between FCGR2A and the response to adalimumab (P=0.022). Analyzing the subset of anti-CCP positive RA patients (78%), we also found a significant association between FCGR2A and the response to infliximab (P=0.035). DHX32 and RGS12 were the most consistently correlated genes with FCGR2A expression in RA synovial fluid macrophages (P<0.001). We found a significant association between the genetic variation at DHX32 (rs12356233, corrected P=0.019) and a nominally significant association between RGS12 and the response to adalimumab (rs4690093, uncorrected P=0.040). In the anti-CCP positive group of patients, we also found a nominally significant association between RGS12 and the response to infliximab (rs2857859, uncorrected P=0.042). CONCLUSIONS: In the present study we have validated the FCGR2A association in an independent population, and we have identified new genes associated with the response to anti-TNF therapy in RA.

GWAS replication study confirms the association of PDE3A-SLCO1C1 with anti-TNF therapy response in rheumatoid arthritis.

AIM: The present study was undertaken to replicate the association of candidate genes for anti-TNF response in rheumatoid arthritis. Candidate genes were selected from a recent genome-wide association study on anti-TNF response performed in a population from Denmark. MATERIALS & METHODS: Genomic DNA was obtained from 315 Spanish rheumatoid arthritis patients having received an anti-TNF agent as their first biological therapy. SNPs from NR2FR2, MAP2K6, CBLN2 and PDE3A-SLCO1C1 candidate loci were genotyped. RESULTS: The PDE3A-SLCO1C1 locus rs3794271 SNP showed a highly significant association with anti-TNF treatment response (p = 1.74 × 10⁻5). Combining the statistical evidence from the Spanish and Danish rheumatoid arthritis cohorts, the associated rs3794271 SNP reached a genome-wide significance level of association (p = 3.3 × 10⁻¹°). CONCLUSION: The present findings establish the PDE3A-SLCO1C1 locus as a strong genetic marker of anti-TNF therapy response.