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216 p., Estudios de asociación de genoma completo han detectado polimorfismos asociados con el riesgo a padecer diabetes de tipo 1 (DM1) en ARN largos no codificantes (lncRNAs). Sin embargo, todavía se desconocen los mecanismos moleculares por los que la mayoría de estos lncRNAs contribuyen al desarrollo de la DM1.Los estudios recogidos en esta tesis doctoral confirman la presencia de lncRNAs implicados en el desarrollo de DM1. Así mismo, se ha realizado la caracterización funcional de 2 de estos lncRNAs; llamados Lnc13 y ARGI. Gracias a estos estudios se demuestra la importancia de estas moléculas no sólo en el funcionamiento celular (en esta tesis centrada en el estudio de las células ß pancreáticas), sino también en el desarrollo de diversas enfermedades como son la diabetes.

IntroductionMost of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. MethodsHere, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. ResultsWe have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. ConclusionsThese findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.

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.

[EN] Type I diabetes (T1D) is an autoimmune disease in which due to the destruction of pancreatic beta cells, patients become insulin dependent for life. Both, environmental (e.g. viral infections) and genetic factors, contribute to T1D susceptibility. Accumulating scientific evidence support the implication of viral infections in T1D development. Thus, upon a viral infection in pancreatic beta cells, the immune system is activated, leading to pancreatic beta cell inflammation (insulitis). Inflammation leads to the activation of several pro-inflammatory and pro-apoptotic pathways in beta cells that ends in pancreatic beta cell destruction. In recent years, long non coding RNAs have been associated with T1D. Thus, the main objective of the present project was to characterize the function of a T1D-associated lncRNA named Lnc2 at the pancreatic beta cell level. To this aim, pancreatic beta cells (EndoC-H1) were exposed to pro-inflammatory cytokines or intracellular synthetic viral double stranded RNA (PIC) to create a pro-inflammatory environment and analyse the expression of Lnc2. Besides that, taking into account that Lnc2 overlaps with BTNL2 in the human genome, the effect of Lnc2 on BTNL2 expression was also studied. The results showed that both, pro-inflammatory cytokines and PIC, upregulate Lnc2 expression in beta cells. Moreover, these results revealed that Lnc2 participates in the regulation of BTNL2 expression at the pancreatic beta cell level. Finally, the results of this work suggested that Lnc2 might play a role in pancreatic beta cells apoptosis. In conclusion, Lnc2 could be involved in pancreatic beta cell death, probably through the regulation of a gene that plays a role in apoptosis. Furthermore, since Lnc2 overlaps with BTNL2 in the human genome and Lnc2 regulates BTNL2 expression, the effect of Lnc2 overexpression in beta cell apoptosis might be driven by BTNL2., [EUS] 1 motako diabetesa (DM1; Diabetes Mellitus 1) gaixotasun autoimmune bat da non, pankreako beta zelulen suntsipenaren ondorioz gaixoak intsulina tratamenduarekiko dependente bihurtzen diren. DM1aren garapena faktore genetiko zein ingurune faktoreen (infekzio biralak, besteak beste) menpekoa da. Ebidentzia zientifiko ugari daude birusak eta DM1aren garapena erlazionatzen dituztenak. Hauen arabera, birus batek beta zelulak infektatzean, sistema immunitarioa aktibatzen da, pankreako irletan hantura eraginez (insulitisa). Hanturak pankreako beta zeluletan zenbait bidezidor pro-inflamatorio eta pro-apoptotiko aktibatuko lituzke, pankreako beta zelulen suntsipena eraginez. Azken urteetan, RNA luze ez kodetzaileak DM1arekin asoziatu dira eta, lan honetan, DM1arekin asoziatutako polimorfismoa duen lncRNA bat izan da aztergai, Lnc2 deritzona. Horregatik, Gradu Amaierako Lan honen helburua Lnc2-aren karakterizazio molekularra egitea izan da, giza pankreako beta zeluletan bete dezakeen funtzioa aztertzeko asmoz. Horretarako, pankreako beta zelulak (EndoC-H1) hantura sortzen duten zitokina eta harizpi bikoitzeko RNA birikoarekin (PIC) tratatu dira; beta zeluletan baldintza pro-inflamatorioak sortu eta, Lnc2-aren adierazpena aztertzeko. Horretaz gain, giza genoman Lnc2-arekin gainjarrita dagoen BTNL2 genea ere aztertu da, Lnc2-ak gene horren adierazpenean izan dezakeen eragina zehaztuz. Emaitzek adierazi dute Lnc2-aren adierazpena igo egiten dela beta zelulak zitokina zein PICarekin tratatzean. Gainera, Lnc2-ak nolabait BTNL2 genearen adierazpena erregulatzen duela ikusi da. Horretaz gain, Lnc2-ak DM1ean ematen den pankreako beta zelulen apoptosian parte hartzen duela ikusi da. Ondorioz, Lnc2-ak DM1ean pankreako beta zeluletan ematen den suntsipenean parte hartzen duela esan daiteke, apoptosian parte hartzen duen gene baten erregulazioaren bidez, adibidez. Gainera, genoman BTNL2arekin gainjarrita agertzen denez eta Lnc2-ak BTNL2aren adierazpena erregulatzen duenez, gerta d

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that develops due to the interaction between genetic and environmental factors. More than 160 loci have been associated with IBD, but the functional implication of many of the associated genes remains unclear. N6-Methyladenosine (m6A) is the most abundant internal modification in mRNA. m6A methylation regulates many aspects of mRNA metabolism, playing important roles in the development of several pathologies. Interestingly, SNPs located near or within m6A motifs have been proposed as possible contributors to disease pathogenesis. We hypothesized that certain IBD-associated SNPs could regulate the function of genes involved in IBD development via m6A-dependent mechanisms. We used online available GWAS, m6A and transcriptome data to find differentially expressed genes that harbored m6A-SNPs associated with IBD. Our analysis resulted in five candidate genes corresponding to two of the major IBD subtypes: UBE2L3 and SLC22A4 for Crohn’s Disease and TCF19, C6orf47 and SNAPC4 for Ulcerative Colitis. Further analysis using in silico predictions and co-expression analyses in combination with in vitro functional studies showed that our candidate genes seem to be regulated by m6A-dependent mechanisms. These findings provide the first indication of the implication of RNA methylation events in IBD pathogenesis.

[EUS] Genomako asoziazio-ikerketen bidez I motako diabetesa (T1D) pairatzeko arriskuari loturiko polimorfismoak aurkitu dira RNA luze ez-kodetzaileetan (lncRNA). Hala ere, lncRNA horien mekanismo molekularrak ezezagunak dira I motako diabetesaren garapenean. Gradu amaierako lan honetan I motako diabetesarekin erlazioa duten eta beta zelulen berezko immunitatean parte hartzen duten lncRNA-etan ager daitezkeen aldaketa funtzionalek zeluletan duten eragina aztertu da. Hori aintzat hartuta, proiektuaren helburu nagusia I motako diabetesarekin asoziatuta dagoen polimorfismo bat duen BACH2 RNA luze ez-kodetzailearen (lncBACH2) karakterizazio molekularra egitea izan da, horretarako pankreako beta zeluletan isilarazpen genikoan oinarritutako ikerketa funtzionalak gauzatu direlarik. Lan honetan landu den hizkuntza euskara izan da.

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Aims/hypothesis: The initial stages of type 1 diabetes are characterized 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 has been considered the etiologic gene in the region. However, recent gene expression analysis indicates that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI. We presently evaluated the role of DEXI in beta cell responses to “danger signals” and determined the mechanisms involved. Methods: Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Viral double-stranded RNA-driven beta cell inflammation and apoptosis were evaluated by RT-PCR, western blot and luciferase assays. Results: DEXI-silenced beta cells exposed to double-stranded RNA (PIC; a by-product of viral replication) showed reduced STAT1 activation and lower production of pro-inflammatory chemokines that was preceded by a reduction in IFN expression. Exposure to PIC increased chromatin-bound DEXI and IFN promoter activity. This effect on IFN promoter was inhibited in DEXI-silenced betacells, suggesting that DEXI is implicated in the regulation of IFNtranscription. In a mirror image of knockdown experiments, DEXI overexpression led to increased STAT1 and pro-inflammatory chemokine expression. Conclusions: These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune response in beta cells. Moreover, our results provide initial information on the function of DEXI.

Objective: 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. Methods: 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. Results: 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. Conclusions: 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.

240 p., El objetivo principal del presente trabajo ha sido identificar las alteraciones genéticas responsables de la diabetes neonatal o de comienzo precoz en pacientes sin alteraciones potencialmente patológicas en genes clásicamente asociados a la enfermedad. Para llevar a cabo este estudio, inicialmente se realizó un estudio genético de los genes clásicamente asociados a la diabetes neonatal en nuestra cohorte de pacientes. Tras realizar la secuenciación de dichos genes y descartar mutaciones que pudieran explicar la clínica de los pacientes, se seleccionaron ocho pacientes para realizar un estudio de exoma completo con el fin de identificar nuevas mutaciones asociadas con la enfermedad. Por último, se realizó el estudio funcional de una de las alteraciones identificadas en el análisis del exoma con el fin de establecer el impacto funcional de dicha variante en la patogénesis de la enfermedad a nivel de célula ¿ pancreática.En nuestra cohorte de 42 pacientes, el estudio estructural de los genes ABCC8, KCNJ11 e INS y el estudio de la región 6q24 revelaron alteraciones en 30 de los afectos (71%). La patogenicidad de las mutaciones se demostró usando los software de predicción y la cosegregación de la mutación en la familia. En doce pacientes (29%) no se detectaron mutaciones en ninguno de los genes analizados. Estos resultados concuerdan con otros estudios realizados previamente, en los que entre un 20 y un 40% de los pacientes con diabetes neonatal no presentaron mutaciones en los genes clásicamente asociados con la diabetes neonatal. Un 43% de los pacientes presentaba alteraciones en los genes que codifican para los canales de potasio (KCNJ11 y ABCC8), mientras que el 30% portaba alteraciones en la región 6q24 y un 23% en el gen de la insulina. Además, en uno de los pacientes que además de padecer diabetes neonatal tenía un cuadro poliglandular autoinmune caracterizado por enteropatía y autoinmunidad tiroidea, detectamos una alteración en el gen FOXP3, que ha sido previa