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Nanosystems that simultaneously contain fluorescent and magnetic modules can offer decisive advantages in the development of new biomedical approaches. A biomaterial that enables multimodal imaging and contains highly efficient nanoheaters together with an intrinsic temperature sensor would become an archetypical theranostic agent. In this work, we have designed a magneto-luminescent system based on Fe

Long noncoding RNAs (lncRNAs) have been identified in almost all vertebrates, but the functional characterization of these RNA molecules is being challenging, mainly due to the lack of linear sequence homology between species. In this work, we aimed to find functional evolutionary convergent lncRNAs involved in development by screening of k-mer content (non linear similarity) and secondary structure-based approaches combined within silico, in vitroandin vivovalidation analysis. From the currently identified Madagascar gecko genes, we found a lncRNA with a similar k-mer content and structurally concordant with the human lncRNAEVX1AS. Analysis of function related characteristics together with locus-specific targeting of human and geckoEVX1AS(i.e. CRISPR Display) in human neuroepithelial cells and chicken mesencephalon confirmed that geckoEvx1as-likelncRNA mimics humanEVX1ASfunction and inducesEVX1expression independently of the target species. Our data show functional conservation of non-homologous lncRNAs and presents a useful approach for the definition and manipulation of lncRNA function within different model organisms.

Local heat generation from magnetic nanoparticles (MNPs) exposed to alternating magnetic fields can revolutionize cancer treatment. However, the application of MNPs as anticancer agents is limited by serious drawbacks. Foremost among these are the fast uptake and biodegradation of MNPs by cells and the unpredictable magnetic behavior of the MNPs when they accumulate within or around cells and tissues. In fact, several studies have reported that the heating power of MNPs is severely reduced in the cellular environment, probably due to a combination of increased viscosity and strong NP agglomeration. Herein, we present an optimized protocol to coat magnetite (Fe3O4) NPs larger than 20 nm (FM-NPs) with high molecular weight PEG molecules that avoid collective coatings, prevent the formation of large clusters of NPs and keep constant their high heating performance in environments with very different ionic strengths and viscosities (distilled water, physiological solutions, agar and cell culture media). The great reproducibility and reliability of the heating capacity of this FM-NP@PEG system in such different environments has been confirmed by AC magnetometry and by more conventional calorimetric measurements. The explanation of this behavior has been shown to lie in preserving as much as possible the magnetic single domain-type behavior of nearly isolated NPs. In vitro endocytosis experiments in a colon cancer-derived cell line indicate that FM-NP@PEG formulations with PEGs of higher molecular weight (20 kDa) are more resistant to endocytosis than formulations with smaller PEGs (5 kDa), showing quite large uptake mean-life (τ > 5 h) in comparison with other NP systems. The in vitro magnetic hyperthermia was performed at 21 mT and 650 kHz during 1 h in a pre-endocytosis stage and complete cell death was achieved 48 h posthyperthermia. These optimal FM-NP@PEG formulations with high resistance to endocytosis and predictable magnetic response will aid the progress and accuracy of the emerging era of theranostics.

Genome wide association studies (GWAS) have identified many loci contributing to genetic variation of complex traits. Immune mediated disorders are complex diseases for which hundreds of risk alleles have been identified by GWAS. However, the intergenic location of most of the signals has make it difficult to decipher their implication in disease pathogenesis. A significant number of immune disease-associated SNPs are located within long noncoding RNAs (lncRNAs). LncRNAs have gained importance due to their involvement in the regulation of a wide range of biological processes, including immune responses. GWAS SNPs located within lncRNAs can affect their regulatory capacity by modifying their secondary structure, altering their expression levels or regulating the transcription of different isoforms. In this review we discuss the functional implications of immune-related lncRNAs harboring disease associated SNPs on various disease conditions.

The main objective of the preparation of the Fe

[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.

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m6A are still technically challenging. Here, we describe a simple RT-QPCR-based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues.

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.

mRNA stability influences gene expression and translation in almost all living organisms, and the levels of mRNA molecules in the cell are determined by a balance between production and decay. Maintaining an accurate balance is crucial for the correct function of a wide variety of biological processes and to maintain an appropriate cellular homeostasis. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of gene expression through different molecular mechanisms, including mRNA stabilization. In this review we provide an overview on the molecular mechanisms by which lncRNAs modulate mRNA stability and decay. We focus on how lncRNAs interact with RNA binding proteins and microRNAs to avoid mRNA degradation, and also on how lncRNAs modulate epitranscriptomic marks that directly impact on mRNA stability. LncRNA work in author’s laboratory is supported by European Foundation for the Study of Diabetes (EFSD)-EFSD/JDRF/Lilly Programme on Type 1 Diabetes Research and the Spanish Ministry of Science, Innovation and Universities (PID2019-104475GA-I00) to I.S; and Spanish Ministry of Science, Innovation and Universities (SAF2017-91873-EXP and PGC2018-097573-A-I00) to ACR. M.S.D. and I.G.M. are supported by a Predoctoral Fellowship Grant from the UPV/EHU (Universidad del País Vasco/Euskal Herriko Unibertsitatea) and A.O.G. is supported by a Predoctoral Fellowship Grant from the Education Department of Basque Government.

The currently existing magnetic hyperthermia treatments usually need to employ very large doses of magnetic nanoparticles MNPs and or excessively high excitation conditions H f gt; 1010 A m s to reach the therapeutic temperature range that triggers cancer cell death. To make this anticancer therapy truly minimally invasive, it is crucial the development of improved chemical routes that give rise to monodisperse MNPs with high saturation magnetization and negligible dipolar interactions. Herein, we present an innovative chemical route to synthesize Zn doped magnetite NPs based on the thermolysis of two kinds of organometallic precursors i a mixture of two monometallic oleates FeOl ZnOl , and ii a bimetallic ironzinc oleate Fe3 amp; 8722;yZnyOl . These approaches have allowed tailoring the size 10 amp; 8722;50 nm , morphology spherical, cubic, and cuboctahedral , and zinc content ZnxFe3 amp; 8722;xO4, 0.05 lt; x lt; 0.25 of MNPs with high saturation magnetization amp; 8805;90 Am2 kg at RT . The oxidation state and the local symmetry of Zn2 and Fe2 3 cations have been investigated by means of X ray absorption near edge structure XANES spectroscopy, while the Fe center distribution and vacancies within the ferrite lattice have been examined in detail through Mo amp; 776;ssbauer spectroscopy, which has led to an accurate determination of the stoichiometry in each sample. To achieve good biocompatibility and colloidal stability in physiological conditions, the ZnxFe3 amp; 8722;xO4 NPs have been coated with high molecular weight poly ethylene glycol PEG . The magnetothermal efficiency of ZnxFe3 amp; 8722;xO4 PEG samples has been systematically analyzed in terms of composition, size, and morphology, making use of the latest generation AC magnetometer that is able to reach 90 mT. The heating capacity of Zn0.06Fe2.94O4 cuboctahedrons of 25 nm reaches a maximum value of 3652 W g at 40 kA m and 605 kHz , but most importantly, they reach a highly satisfactory value 600 W g under strict safety excitation conditions at 36 kA m and 125 kHz . Additionally, the excellent heating power of the system is kept identical both immobilized in agar and in the cellular environment, proving the great potential and reliability of this platform for magnetic hyperthermia therapies

The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting., This work was supported by a Research Project Grant from the Basque Department of Health (2015111068) and a Research Grant from Fundación de la Sociedad Española de Diabetes (FSED) to I.S. I.G.-M. and A.O.-G. were supported by Predoctoral Fellowship Grants from the UPV/EHU (Universidad del Pais Vasco/Euskal Herriko Unibertsitatea) and the Basque Department of Education, respectively. A.C.-R. was funded by a Grant from the Spanish Ministry of Science, Innovation and Universities (PGC2018-097573-A-I00). D.L.E. received support from the Fonds National de la Recherche 420 Scientifique (FNRS), Welbio Grant CR-2015A-06. B.R.G. was funded by the Spanish Ministry of Science, Innovation and Universities; the European Fund for Regional Development (FEDER) (Grant BFU2017-83588-P); and the Juvenile Diabetes Research Foundation (Grants 17-2013-372 and 2-SRA-2019-837-S-B).

The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level.We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84.MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells.We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.

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.

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3′UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic β-cells exposed to inflammatory stimuli. J.R.B. is funded by Project ISCIII-PI16/00258 and co-funded by the European Union ERDF/ESF “A way to make Europe”. I.S. is funded by a research project grant 2015111068 of the Basque Department of Health. A.C.R. was funded by a Juan de la Cierva reincorporation and an Ikerbasque fellowships and a research project grant from Asociación Celiacos Madrid. I.R.G., A.O.G. and A.J.M. are supported by predoctoral fellowship grants from the UPV/EHU and the Basque Department of Education.

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). Upon stimulation, lnc13 levels are reduced, thereby allowing increased expression of the repressed genes. Lnc13 levels are significantly decreased in small intestinal biopsy samples from patients with celiac disease, which suggests that down-regulation of lnc13 may contribute to the inflammation seen in this disease. Furthermore, the lnc13 disease-associated variant binds hnRNPD less efficiently than its wild-type counterpart, thus helping to explain how these single-nucleotide polymorphisms contribute to celiac disease.

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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.