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[EN] Objectives Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nudeotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. Design The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m(6)A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. Results Individuals harbouring the risk allele had higher m(6)A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m(6)A methylation in humans as well as in in vitro and in vivo models. Conclusion We identify a novel m(6)A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m(6)A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders. This study was supported by a grant from the Spanish Ministry of Science, Universities and Innovation (PGC2018-097573-A-I00) to AC-R. JRB was funded by ISCIII Research project PI16/00258, cofinanced by the Spanish Ministry of Economy and Competitiveness and by the European Union ERDF/ESF 'A way to make Europe'. AO-G and MS-D were funded by predoctoral fellowships from the Basque Government and the University of the Basque Country respectively. DS and LH were funded by the Spanish Ministry (MINECO) (SAF2017-83813-C3-1-R) and cofunded by the ERDF, the Centro de Investigacion Biomedica en Red de Fisiopatologia de la Obesidad y la Nutricion (CIBEROBN) (Grant CB06/03/0001 to DS), the Government of Catalonia (2017SGR278 to DS), and the Fundacio La Marato de TV3 (201627-30 to DS). CH is a Howard Hughes Medical Institute Investigator and has been funded by the National Institute of Health HG008935. We would like to thank Xuechen Yu and Justin Vargas for processing the adult CD biopsy samples obtained from Columbia University. EFV is supported by a CIHR grant 168840 and holds a Canada Research Chair.

Celiac disease (CD) is an immune-mediated disease that develops in genetically susceptible individuals upon gluten exposure. Human Leukocyte Antigen (HLA) genes in the Major Histocompatibility Complex (MHC) have been described to represent the 40% of the genetic risk to develop CD. Aiming to gain understanding of the genetic involvement in CD, high throughput studies have been performed, revealing that many CD-associated variants are located in non-coding regions, hindering the study of the functional implications of these single nucleotide polymorphisms (SNPs). In the last decade, long non-coding RNAs (lncRNAs) have been described to be influenced by disease-associated SNPs and to drive many important mechanisms involved in the development of inflammatory diseases. Here we describe the lncRNAs identified and characterized in the context of celiac disease and highlight the importance of the study of these molecules in inflammatory and autoimmune disorders.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of inflammation. To further understand the interaction between inflammatory signaling pathways and lncRNAs, we characterized the function of cardiac and apoptosis-related lncRNA (Carlr), an lncRNA expressed in both mouse and human cells of diverse tissues. Carlr expression is increased following NF-κB signaling in macrophages, with concomitant translocation to, and enrichment of, the transcript in the cytoplasm. Knockdown of Carlr results in impaired expression of NF-κB pathway genes and influences the interaction between macrophages and intestinal cells in an inflammatory environment. In human celiac disease patient samples, increased levels of the Carlr transcript were detected in the cytoplasm, alongside elevated expression of NF-κB pathway genes. These findings suggest that increased Carlr expression and/or cytoplasmic localization is required for efficient NF-κB signaling and is associated with the inflamed tissue state observed in human celiac disease.

Immune-mediated diseases, such as celiac disease, type 1 diabetes or multiple sclerosis, are a clinically heterogeneous group of diseases that share many key genetic triggers. Although the pathogenic mechanisms responsible for the development of immune mediated disorders is not totally understood, high-throughput genomic studies, such as GWAS and Immunochip, performed in the past few years have provided intriguing hints about underlying mechanisms and pathways that lead to disease. More than a hundred gene variants associated with disease susceptibility have been identified through such studies, but the progress toward understanding the underlying mechanisms has been slow. The majority of the identified risk variants are located in non-coding regions of the genome making it difficult to assign a molecular function to the SNPs. However, recent studies have revealed that many of the non-coding regions bearing disease-associated SNPs generate long non-coding RNAs (lncRNAs). LncRNAs have been implicated in several inflammatory diseases, and many of them have been shown to function as regulators of gene expression. Many of the disease associated SNPs located in lncRNAs modify their secondary structure, or influence expression levels, thereby affecting their regulatory function, hence contributing to the development of disease. AC-R is funded by an Ikerbasque Research Fellowship, SG is funded by grant R01 DK102180 (NIH).

Celiac disease (CD) is a chronic, autoimmune disease of the small intestine with a strong but complex genetic component. The disease is triggered by the consumption of dietary gluten through the presentation of immunogenic gliadin peptides to T helper lymphocytes by HLA-DQ2 and DQ8 heterodimers, which are the major contributors to the genetic risk. Recent large-scale genotyping efforts have identified a large number of additional association signals, but the functional role of the underlying genes in the pathogenesis of the disease is still unclear. In the last years, several whole transcriptome analyses have been performed in different tissues, including whole duodenal biopsies, isolated gut epithelial cells or peripheral blood from gluten-consuming CD patients at diagnosis, treated patients on gluten-free diet and nonceliac controls, sometimes after in vitro challenge with gluten-derived gliadin peptides. Although the results from the different experiments are difficult to reconcile, the main findings point to an exacerbation of the immune response, together with the dysregulation of signaling and cell cycle pathways. The effect of associated SNPs on the expression of candidate genes and the role of the noncoding genome are the new territories that the CD research community has only begun to explore.

It is known that the NFκB route is constitutively upregulated in celiac disease (CD), an immune-mediated disorder of the gut caused by intolerance to ingested gluten. Our aim was to scrutinize the expression patterns of several of the most biologically relevant components of the NFκB route in intestinal biopsies from active and treated patients and after in vitro gliadin challenge, and to assess normalization of the expression using an inhibitor of the MALT1 paracaspase. The expression of 93 NFκB genes was measured by RT-PCR in a set of uncultured active and treated CD and control biopsies, and in cultured biopsy series challenged with gliadin, the NFκB modulator, both compounds and none. Methylation of eight genes involved in NFκB signaling was analyzed by conventional pyrosequencing. Groups were compared and Pearson's correlation matrixes were constructed to check for coexpression and co-methylation. Our results confirm the upregulation of the NFκB pathway and show that constitutively altered genes usually belong to the core of the pathway and have central roles, whereas genes overexpressed only in active CD are more peripheral. Additionally, this is the first work to detect methylation level changes in celiac intestinal mucosa. Coexpression is very common in controls, whereas gliadin challenge and especially chronic inflammation present in untreated CD result in the disruption of the regulatory equilibrium. In contrast, co-methylation occurs more often in active CD. Importantly, NFκB modulation partially restores coregulation, opening the door to future therapeutic possibilities and targets.

Introduction Recent genome wide association studies (GWAS) on coeliac disease (CD) have identified risk loci harbouring genes that fit the accepted pathogenic model and are considered aetiological candidates. Methods Using Taqman single nucleotide polymorphism (SNP) and expression assays, the study genotyped 11 SNPs tagging eight GWAS regions (1q31, 2q11–2q12, 3p21, 3q25–3q26, 3q28, 4q27, 6q25 and 12q24) in a Spanish cohort of 1094 CD patients and 540 controls, and performed expression analyses of candidate genes ( RGS1 , IL18R1/IL18RAP , CCR3 , IL12A/SCHIP1 , LPP , IL2/IL21-KIAA1109 , TAGAP , and SH2B3 ) in intestinal mucosa from 29 CD children and eight controls. Results Polymorphisms in 1q31, 2q11–2q12, and 3q25 showed association in our cohort, and also 3q28 and 4q27 when combined with a previous study. Expression levels of IL12A, IL18RAP , IL21, KIAA1109, LPP, SCHIP1 , and SH2B3 were affected by disease status, but the correlation between genotype and mRNA levels was observed only in IL12A, LPP, SCHIP1 , and SH2B3 . Conclusions Expression differences between treated CD patients and controls along with SNP expression associations suggest a possible primary role for these four genes and their variants in pathogenesis. The lack of SNP effect in the remaining genes is probably a consequence of arbitrary candidate gene selection within association signals that are not based on functional studies.

The objective of this study was to identify additional diabetes susceptibility markers in the MHC that could be responsible for the differential diabetogenicity of different HLA-DR3 CEHs. High-resolution SNP genotyping of the MHC was carried out in 15 type 1 diabetes (T1D) patients and 39 non-diabetic controls, homozygous for DR3-DQ2 and with one copy of the A(*)30-B(*)18-MICA(*)4-F1C30-DRB1(*)0301-DQB1(*)0201-DPB1(*)0202 HLA haplotype. Significantly associated SNPs were replicated in an independent sample of 554 T1D patients and 841 controls without HLA matching. Electrophoretic mobility shift assay was used to show a functional effect of an associated SNP. Seven SNPs showed evidence of association in the initial discovery experiment. Upon replication, only rs419434 (upstream HLA-DOA gene) remained significant. A functional variant (rs432375) in complete LD with rs419434 was shown to affect USF-1 binding and could be responsible for the association signal in the region. We have identified a new susceptibility locus within the MHC with a modest contribution to T1D (OR=1.93; CI: 1.52-2.44; P=10(-8)) that is independent of HLA-DRB1 locus.

The functional (R620W) variant of human PTPN22 (protein tyrosine phosphatase non-receptor 22) gene has been implicated in the risk to several autoimmune disorders, including type 1 diabetes, Graves' disease, rheumatoid arthritis and systemic lupus erythematosus. In an association study of this single nucleotide polymorphism with celiac disease (CD), comparison of 262 young diagnosis patients and 214 adult controls from Spain showed a higher frequency of the minor allele in the CD group (9.7% vs 5.6% in controls; P = 0.018), suggestive of an increased genetic risk to the disease (odds ratio = 1.82; 95% confidence interval 1.1-3.0). These results support the role of PTPN22 as a general autoimmunity locus involved in tolerance induction in the thymus.

Celiac disease (CD) involves disturbance of the small-bowel mucosal vascular network, and transglutaminase autoantibodies (TGA) have been related to angiogenesis disturbance, a complex phenomenon probably also influenced by common genetic variants in angiogenesis-related genes. A set of genes with "angiogenesis" GO term identified in a previous expression microarray experiment (SCG2, STAB1, TGFA, ANG, ERBB2, GNA13, PML, CASP8, ECGF1, JAG1, HIF1A, TNFSF13 and TGM2) was selected for genetic and functional studies. SNPs that showed a trend for association with CD in the first GWAS were genotyped in 555 patients and 541 controls. Gene expression of all genes was quantified in 15 pairs of intestinal biopsies (diagnosis vs. GFD) and in three-dimensional HUVEC and T84 cell cultures incubated with TGA-positive and negative serum. A regulatory SNP in TNFSF13 (rs11552708) is associated with CD (p = 0.01, OR = 0.7). Expression changes in biopsies pointed to TGM2 and PML as up-regulated antiangiogenic genes and to GNA13, TGFA, ERBB2 and SCG2 as down-regulated proangiogenic factors in CD. TGA seem to enhance TGM2 expression in both cell models, but PML expression was induced only in T84 enterocytes while GNA13 and ERBB2 were repressed in HUVEC endothelial cells, with several genes showing discordant effects in each model, highlighting the complexity of gene interactions in the pathogenesis of CD. Finally, cell culture models are useful tools to help dissect complex responses observed in human explants.

Celiac disease (CD) is an immune-mediated disorder of the gut in which innate and adaptive responses are involved. Toll-like receptor (TLR) 2 and TLR4 participate in host defense through antigen recognition, and show altered expression in CD gut mucosa. beta-defensins are inducible antimicrobial peptides, and DEFB gene copy number polymorphisms have been associated with autoimmune and inflammatory disorders. We performed copy number analysis of TLR2, TLR4, and the beta-defensin cluster (DEFB4, DEFB103 and DEFB104) by gene-specific, real-time polymerase chain reaction (PCR) in 376 CD patients and 376 controls. TLR genes did not show copy number variation, and all samples presented with two copies. beta-defensin clusters varied between 2 and 9 copies per genome, and when grouped into bins, high copy numbers (>4) were underrepresented among patients (p = 0.023; odds ratio = 0.69, 95% CI = 0.50-0.96), suggesting that increased copy numbers could protect from CD, possibly by impeding bacterial infiltration more efficiently and preserving gut epithelial integrity.

Celiac disease (CD) is a complex, immune-mediated intolerance to gliadin that develops in genetically susceptible individuals. Although the main driving force of the disease is an aberrant autoimmune response, several other pathogenic mechanisms, many still unidentified, are also involved. In order to describe at a network level the alterations provoked by a gliadin insult on the intestinal mucosa of patients, we compared the expression profiles of biopsies from 9 active and 9 treated patients (long-term effects of gliadin), and of 10 biopsies from gluten-free diet treated patients that were incubated in vitro with or without gliadin (acute effects) and integrated significantly altered transcripts into potentially pathogenic biological processes. Using information on Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms represented among the differentially expressed genes, we observed important dysfunction in several complex networks, including those related to cell-cell communication, intracellular signaling, ubiquitin-proteasome system, cell cycle/apoptosis and extracellular matrix. The reconstruction of the role of these biological networks in the development of the intestinal lesion in CD provides a comprehensive picture of key events that contribute to the disease, and could point towards novel functional candidates that might be potential therapeutic targets or responsible for genetic susceptibility.

An aberrant immune response triggered by dietary gluten is the main driving force underlying celiac disease (CD), but other biologic pathways that are dysregulated also participate in disease development. Genetic variation within these pathways might influence expression, contributing to susceptibility to CD. We have investigated the implication of ubiquitin D (UBD), a member of the ubiquitin–proteasome system that is strongly upregulated in the intestinal mucosa of active CD. Reverse transcriptase-polymerase chain reaction analysis of intestinal biopsy sample pairs (at diagnosis vs treated) from 30 CD patients confirmed overexpression of UBD in active disease tissue (fold change = 8.3; p = 0.0022). In silico prediction tools identified rs11724 as a putative regulatory single nucleotide polymorphism and association analysis of 468 CD patients and 459 controls revealed that the minor rs11724*C allele was more frequent among patients (minor allele frequency = 0.44 vs 0.39; odds ratio [OR] = 1.23; p = 0.028) and suggested a dominant allele effect (OR = 1.49; p = 0.0045). Correlation of the rs11724 genotype and UBD mRNA levels (OR = 0.76; p = 0.0021) further supports its implication in disease development.

Celiac disease (CD) is an immunological disorder caused by intolerance to ingested gliadin and other cereal prolamins that has been included in the T(H)1-dominated group of diseases, where IL-12 induced IFNgamma is the major proinflamatory signal. Recently, another linage of T cells has been described, namely T(H)17, characterized by production of IL-17, that differentiate in response to TGFbeta and IL-6 and participate in the pathogenesis of several autoimmune diseases. Using RT-PCR analysis of gene expression, we analyzed the presence of T(H)1 (IL-12 and IFNgamma) and T(H)17 (TGFbeta, IL-6, IL-17A, IL-17F and IL-23) related cytokines in intestinal biopsies from CD patients with active disease compared to remission and from treated patients after acute, in vitro re-exposure to gliadin. Potent T(H)1 and T(H)17 responses were present in the active stage of the disease, whereas short incubation of normalized biopsies with gliadin did not increase the expression of the effector cytokines, although a tendency of upregulation for both T(H)1 and T(H)17 promoting factors was observed, suggestive of a reactivation of proinflammatory pathways. These results place CD into the group of autoimmune disorders in which T(H)17 cells also participate, although the relative importance of each T cell response and their role in the initial events of the disease need further investigation.

Toll-like receptors (TLRs) participate in the first line of immune defense through antigen pattern recognition, and ligands include exogenous and host-derived molecules. Coding variants in TLR4 have been associated with autoimmune diseases like ulcerative colitis, Crohn’s disease, and rheumatoid arthritis. Our aim was to determine whether these polymorphisms are associated with celiac disease (CD). Two coding single nucleotide polymorphisms of TLR4 (Asp299Gly and Thr399Ile) were genotyped in 95 family trios with CD as well as in 186 patients and 186 unrelated controls. There were no differences in allele, genotype or haplotype distribution, or transmission between patient and control groups. Our results do not support association of these TLR4 variants with CD.

Genome-wide scans have detected linkage to celiac disease (CD) in several genomic locations, including 19q13.4. Killer immunoglobulin-like receptor (KIR) genes map to the region and encode receptors of natural killer (NK) cells and certain T cells that modulate cytolitic activity through interactions with HLA class I ligands, participating in the innate immune response. We performed KIR genotyping in a group of 70 CD patients of Basque origin and compared gene content, genotype and haplotype frequencies to ethnically matched blood-donors. The frequency of gene combination KIR2DL5B(+)/KIR2DL5A(-) was significantly higher in the disease group, and this result was confirmed in a second group of 343 CD patients and 160 controls of Spanish origin, suggesting an implication of this 'unexpressed' gene with increased susceptibility to CD (combined OR of 3.63 (95% CI: 1.76-7.51; P=0.0004)), possibly due to the lack of an efficient inhibitory signal. Our results support the role of the KIR gene cluster in celiac disease and replicate the CD-susceptibility locus at 19q13.4.