Seven-chain adaptive immune receptor repertoire analysis in rheumatoid arthritis reveals novel features associated with disease and clinically relevant phenotypes.

BACKGROUND: In rheumatoid arthritis (RA), the activation of T and B cell clones specific for self-antigens leads to the chronic inflammation of the synovium. Here, we perform an in-depth quantitative analysis of the seven chains that comprise the adaptive immune receptor repertoire (AIRR) in RA. RESULTS: In comparison to controls, we show that RA patients have multiple and strong differences in the B cell receptor repertoire including reduced diversity as well as altered isotype, chain, and segment frequencies. We demonstrate that therapeutic tumor necrosis factor inhibition partially restores this alteration but find a profound difference in the underlying biochemical reactivities between responders and non-responders. Combining the AIRR with HLA typing, we identify the specific T cell receptor repertoire associated with disease risk variants. Integrating these features, we further develop a molecular classifier that shows the utility of the AIRR as a diagnostic tool. CONCLUSIONS: Simultaneous sequencing of the seven chains of the human AIRR reveals novel features associated with the disease and clinically relevant phenotypes, including response to therapy. These findings show the unique potential of AIRR to address precision medicine in immune-related diseases.

Food groups associated with immune-mediated inflammatory diseases: a Mendelian randomization and disease severity study.

BACKGROUND/OBJECTIVES: Immune-mediated inflammatory diseases (IMIDs) are prevalent diseases. There is, however, a lack of understanding of the link between diet and IMIDs, how much dietary patterns vary between them and if there are food groups associated with a worsening of the disease. SUBJECTS/METHODS: To answer these questions we analyzed a nation-wide cohort of n = 11,308 patients from six prevalent IMIDs and 2050 healthy controls. We compared their weekly intake of the major food categories, and used a Mendelian randomization approach to determine which dietary changes are caused by disease. Within each IMID, we analyzed the association between food frequency and disease severity. RESULTS: After quality control, n = 11,230 recruited individuals were used in this study. We found that diet is profoundly altered in all IMIDs: at least three food categories are significantly altered in each disease (P < 0.05). Inflammatory bowel diseases showed the largest differences compared to controls (n ≥ 8 categories, P < 0.05). Mendelian randomization analysis supported that some of these dietary changes, like vegetable reduction in Crohn's Disease (P = 2.5 × 10-10, OR(95% CI) = 0.73(0.65, 0.80)), are caused by the disease. Except for Psoriatic Arthritis and Systemic Lupus Erythematosus, we have found ≥2 food groups significantly associated with disease severity in the other IMIDs (P < 0.05). CONCLUSIONS: This cross-disease study demonstrates that prevalent IMIDs are associated to a significant change in the normal dietary patterns. This variation is highly disease-specific and, in some cases, it is caused by the disease itself. Severity in IMIDs is also associated with specific food groups. The results of this study underscore the importance of studying diet in IMIDs.

Transcriptomic identification of TMIGD1 and its relationship with the ileal epithelial cell differentiation in Crohn's disease.

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.

Functional rare variants influence the clinical response to anti-TNF therapy in Crohn's disease.

BACKGROUND: The effect of low-frequency functional variation on anti-tumor necrosis factor alpha (TNF) response in Crohn's disease (CD) patients remains unexplored. The objective of this study was to investigate the impact of functional rare variants in clinical response to anti-TNF therapy in CD. METHODS: CD anti-TNF naïve patients starting anti-TNF treatment due to active disease [Crohn's Disease Activity Index (CDAI > 150)] were included. The whole genome was sequenced using the Illumina Hiseq4000 platform. Clinical response was defined as a CDAI score <150 at week 14 of anti-TNF treatment. Low-frequency variants were annotated and classified according to their damaging potential. The whole genome of CD patients was screened to identify homozygous loss-of-function (LoF) variants. The TNF signaling pathway was tested for overabundance of damaging variants using the SKAT-O method. Functional implication of the associated rare variation was evaluated using cell-type epigenetic enrichment analyses. RESULTS: A total of 41 consecutive CD patients were included; 3250 functional rare variants were identified (2682 damaging and 568 LoF variants). Two homozygous LoF mutations were found in HLA-B and HLA-DRB1 genes associated with lack of response and remission, respectively. Genome-wide LoF variants were enriched in epigenetic marks specific for the gastrointestinal tissue (colon, p = 4.11e-4; duodenum, p = 0.011). The burden of damaging variation in the TNF signaling pathway was associated with response to anti-TNF therapy (p = 0.016); damaging variants were enriched in epigenetic marks from CD8+ (p = 6.01e-4) and CD4+ (p = 0.032) T cells. CONCLUSIONS: Functional rare variants are involved in the response to anti-TNF therapy in CD. Cell-type enrichment analysis suggests that the gut mucosa and CD8+ T cells are the main mediators of this response.

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.

Genetic association between CD96 locus and immunogenicity to anti-TNF therapy in Crohn's disease.

The production of antibodies to anti-tumor necrosis factor alpha (TNF) agents is one of the main causes of treatment failure in Crohn's disease (CD). To date, however, the contribution of genetics to anti-TNF immunogenicity in CD is still unknown. The objective of the present study was to identify genetic variation associated with anti-TNF immunogenicity in CD. We performed a two-stage genome-wide association study in a cohort of 96 and 123 adalimumab-treated patients, respectively. In the discovery stage, we identified a genome-wide significant association between the CD96 locus and the production of antibodies to anti-TNF treatment (P = 1.88e-09). This association was validated in the replication stage (P < 0.05). The risk allele for anti-TNF immunogenicity was found to be also associated with a lack of response to anti-TNF therapy (P = 0.019). These findings represent an important step toward the understanding of the immunogenicity-based mechanisms that underlie anti-TNF response in CD.

Genetic variation at the glycosaminoglycan metabolism pathway contributes to the risk of psoriatic arthritis but not psoriasis.

OBJECTIVE: Psoriatic arthritis (PsA) is a chronic inflammatory arthritis affecting up to 30% of patients with psoriasis (Ps). To date, most of the known risk loci for PsA are shared with Ps, and identifying disease-specific variation has proven very challenging. The objective of the present study was to identify genetic variation specific for PsA. METHODS: We performed a genome-wide association study in a cohort of 835 patients with PsA and 1558 controls from Spain. Genetic association was tested at the single marker level and at the pathway level. Meta-analysis was performed with a case-control cohort of 2847 individuals from North America. To confirm the specificity of the genetic associations with PsA, we tested the associated variation using a purely cutaneous psoriasis cohort (PsC, n=614) and a rheumatoid arthritis cohort (RA, n=1191). Using network and drug-repurposing analyses, we further investigated the potential of the PsA-specific associations to guide the development of new drugs in PsA. RESULTS: We identified a new PsA risk single-nucleotide polymorphism at B3GNT2 locus (p=1.10e-08). At the pathway level, we found 14 genetic pathways significantly associated with PsA (pFDR<0.05). From these, the glycosaminoglycan (GAG) metabolism pathway was confirmed to be disease-specific after comparing the PsA cohort with the cohorts of patients with PsC and RA. Finally, we identified candidate drug targets in the GAG metabolism pathway as well as new PsA indications for approved drugs. CONCLUSION: These findings provide insights into the biological mechanisms that are specific for PsA and could contribute to develop more effective therapies.

Genetic variation associated with cardiovascular risk in autoimmune diseases.

Autoimmune diseases have a higher prevalence of cardiovascular events compared to the general population. The objective of this study was to investigate the genetic basis of cardiovascular disease (CVD) risk in autoimmunity. We analyzed genome-wide genotyping data from 6,485 patients from six autoimmune diseases that are associated with a high socio-economic impact. First, for each disease, we tested the association of established CVD risk loci. Second, we analyzed the association of autoimmune disease susceptibility loci with CVD. Finally, to identify genetic patterns associated with CVD risk, we applied the cross-phenotype meta-analysis approach (CPMA) on the genome-wide data. A total of 17 established CVD risk loci were significantly associated with CVD in the autoimmune patient cohorts. From these, four loci were found to have significantly different genetic effects across autoimmune diseases. Six autoimmune susceptibility loci were also found to be associated with CVD risk. Genome-wide CPMA analysis identified 10 genetic clusters strongly associated with CVD risk across all autoimmune diseases. Two of these clusters are highly enriched in pathways previously associated with autoimmune disease etiology (TNFα and IFNγ cytokine pathways). The results of this study support the presence of specific genetic variation associated with the increase of CVD risk observed in autoimmunity.

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.

Genome-Wide Pathway Analysis Identifies Genetic Pathways Associated with Psoriasis.

Psoriasis is a chronic inflammatory disease with a complex genetic architecture. To date, the psoriasis heritability is only partially explained. However, there is increasing evidence that the missing heritability in psoriasis could be explained by multiple genetic variants of low effect size from common genetic pathways. The objective of this study was to identify new genetic variation associated with psoriasis risk at the pathway level. We genotyped 598,258 single nucleotide polymorphisms in a discovery cohort of 2,281 case-control individuals from Spain. We performed a genome-wide pathway analysis using 1,053 reference biological pathways. A total of 14 genetic pathways (PFDR ≤ 2.55 × 10(-2)) were found to be significantly associated with psoriasis risk. Using an independent validation cohort of 7,353 individuals from the UK, a total of 6 genetic pathways were significantly replicated (PFDR ≤ 3.46 × 10(-2)). We found genetic pathways that had not been previously associated with psoriasis risk such as retinol metabolism (Pcombined = 1.84 × 10(-4)), the transport of inorganic ions and amino acids (Pcombined = 1.57 × 10(-7)), and post-translational protein modification (Pcombined = 1.57 × 10(-7)). In the latter pathway, MGAT5 showed a strong network centrality, and its association with psoriasis risk was further validated in an additional case-control cohort of 3,429 individuals (P < 0.05). These findings provide insights into the biological mechanisms associated with psoriasis susceptibility.

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.

Novel insights into the regulatory architecture of CD4+ T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most frequent autoimmune chronic inflammatory disease of the joints and it is characterized by the inflammation of the synovial membrane and the subsequent destruction of the joints. In RA, CD4+ T cells are the main drivers of disease initiation and the perpetuation of the damaging inflammatory process. To date, however, the genetic regulatory mechanisms of CD4+ T cells associated with RA etiology are poorly understood. The genome-wide analysis of expression quantitative trait loci (eQTL) in disease-relevant cell types is a recent genomic integration approach that is providing significant insights into the genetic regulatory mechanisms of many human pathologies. The objective of the present study was to analyze, for the first time, the genome-wide genetic regulatory mechanisms associated with the gene expression of CD4+ T cells in RA. Whole genome gene expression profiling of CD4+ T cells and the genome-wide genotyping (598,258 SNPs) of 29 RA patients with an active disease were performed. In order to avoid the excessive burden of multiple testing associated with genome-wide trans-eQTL analysis, we developed and implemented a novel systems genetics approach. Finally, we compared the genomic regulation pattern of CD4+ T cells in RA with the genomic regulation observed in reference lymphoblastoid cell lines (LCLs). We identified a genome-wide significant cis-eQTL associated with the expression of FAM66C gene (P = 6.51e-9). Using our new systems genetics approach we identified six statistically significant trans-eQTLs associated with the expression of KIAA0101 (P<7.4e-8) and BIRC5 (P = 5.35e-8) genes. Finally, comparing the genomic regulation profiles between RA CD4+ T cells and control LCLs we found 20 genes showing differential regulatory patterns between both cell types. The present genome-wide eQTL analysis has identified new genetic regulatory elements that are key to the activity of CD4+ T cells in RA.