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137 p (eusk.) 130 p. (eng.), La enfermedad celíaca es una enfermedad inflamatoria crónica desarrollada en individuos con susceptibilidad genética por una respuesta inmune inadecuada al gluten de la dieta. Sin embargo, la respuesta inmunitaria inducida por el gluten derivada de la susceptibilidad genética explica menos del 50% de la heredabilidad, lo que sugiere que además del gluten podría haber factores ambientales adicionales que contribuyan al inicio de la respuesta inmunitaria. En base a esto, se han propuesto las infecciones virales como posibles agentes desencadenantes en la enfermedad celiaca.Con el fin de investigar la implicación de las infecciones virales en el desarrollo de la enfermedad celiaca, el proyecto actual se ha centrado en la búsqueda de determinantes funcionales relacionados con la respuesta inmunitaria innata contra las infecciones por virus de ARN. Más concretamente, en la presente tesis doctoral se ha analizado una nueva capa de regulación génica de IRF7 tras la combinación de infecciones virales entéricas y el consumo de gluten, y se ha estudiado la implicación de la metilación del ARN m6A en la regulación de este transcrito antiviral y su actividad aguas abajo en la vía de señalización del IFN-I.

[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.

146 p., Eritasun Zeliakoa (EZ) genetikoki sentikorrak diren pertsonetan garatzen den gaixotasun autoimmunekonplexua da. Glutenak erantzun immunea eragiten die eta gaur egun dagoen tratamendu bakarra biziosorako glutenik gabeko dieta zorrotza jarraitzea da. EZri lotutako sentikortasun genetikoa HLA geneekin eta HLAtik kanpoko hainbat eremu genomikorekin asoziaturik dagoela jakina den arren, haien ekarpena gaixotasunean oraindik ez dago guztiz deskribatua. Horrek zaildu egiten du beharrezkoak diren dietaz besteko tratamendu berrien garapena. Lan honetan XPO1en 5'UTRan kokatutako EZri lotutako nukleotido bakarreko polimorfismoaren (SNP) inplikazio funtzionala deskribatzen dugu zeliakoen hesteko epitelioan ezaugarri den hantura-ingurunean.Arrisku aleloak m6A metilazio handiagoa eragiten du XPO1en 5'UTRan, XPO1 proteina kopuru handiagoak eta ur-beherako NF-kB bidezko hantura eraginez. Gainera, glutenaren esposizioak m6A metilazio orokorra areagotzen duela erakutsi dugu gizakietan, baita in vitro eta in vivo ereduetan ere.Hala, EZ gaixoetan aktibatzen den m6A-XPO1-NF-kB bide berri bat deskribatu dugu, hurbilketa terapeutiko berriak garatzea bultzatu dezakeena.

151 p., Eritasun Zeliakoa (EZ) genetikoki sentikorrak diren pertsonetan garatzen den gaixotasun autoimmunekonplexua da. Glutenak erantzun immunea eragiten die eta gaur egun dagoen tratamendu bakarra biziosorako glutenik gabeko dieta zorrotza jarraitzea da. EZri lotutako sentikortasun genetikoa HLA geneekin eta HLAtik kanpoko hainbat eremu genomikorekin asoziaturik dagoela jakina den arren, haien ekarpena gaixotasunean oraindik ez dago guztiz deskribatua. Horrek zaildu egiten du beharrezkoak diren dietaz besteko tratamendu berrien garapena. Lan honetan XPO1en 5'UTRan kokatutako EZri lotutako nukleotido bakarreko polimorfismoaren (SNP) inplikazio funtzionala deskribatzen dugu zeliakoen hesteko epitelioan ezaugarri den hantura-ingurunean.Arrisku aleloak m6A metilazio handiagoa eragiten du XPO1en 5'UTRan, XPO1 proteina kopuru handiagoak eta ur-beherako NF-kB bidezko hantura eraginez. Gainera, glutenaren esposizioak m6A metilazio orokorra areagotzen duela erakutsi dugu gizakietan, baita in vitro eta in vivo ereduetan ere.Hala, EZ gaixoetan aktibatzen den m6A-XPO1-NF-kB bide berri bat deskribatu dugu, hurbilketa terapeutiko berriak garatzea bultzatu dezakeena.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings.

[EN]Background Quantitative, reverse transcription PCR (qRT-PCR) is currently the gold-standard for SARS-CoV-2 detection and it is also used for detection of other virus. Manual data analysis of a small number of qRT-PCR plates per day is a relatively simple task, but automated, integrative strategies are needed if a laboratory is dealing with hundreds of plates per day, as is being the case in the COVID-19 pandemic. Results Here we present shinyCurves, an online shiny-based, free software to analyze qRT-PCR amplification data from multi-plate and multi-platform formats. Our shiny application does not require any programming experience and is able to call samples Positive, Negative or Undetermined for viral infection according to a number of user-defined settings, apart from providing a complete set of melting and amplification curve plots for the visual inspection of results. Conclusions shinyCurves is a flexible, integrative and user-friendly software that speeds-up the analysis of massive qRT-PCR data from different sources, with the possibility of automatically producing and evaluating melting and amplification curve plots.

Endometriosis is a common gynecological disorder that has been associated with endometrial, breast and epithelial ovarian cancers in epidemiological studies. Since complex diseases are a result of multiple environmental and genetic factors, we hypothesized that the biological mechanism underlying their comorbidity might be explained, at least in part, by shared genetics. To assess their potential genetic relationship, we performed a two-sample mendelian randomization (2SMR) analysis on results from public genome-wide association studies (GWAS). This analysis confirmed previously reported genetic pleiotropy between endometriosis and endometrial cancer. We present robust evidence supporting a causal genetic association between endometriosis and ovarian cancer, particularly with the clear cell and endometrioid subtypes. Our study also identified genetic variants that could explain those associations, opening the door to further functional experiments. Overall, this work demonstrates the value of genomic analyses to support epidemiological data, and to identify targets of relevance in multiple disorders.

247 p., La diabetes mellitus es un complejo trastorno metabólico caracterizado por la presencia de hiperglucemiacrónica, debida a una insuficiente secreción de insulina o a la disminución de la respuesta de los tejidos a la misma. Aunque la etiología de la enfermedad es muy diversa, el 95% de los casos de diabetes en pediatría corresponden a una diabetes tipo 1, que se caracteriza por la destrucción autoinmune de las células ß pancreáticas productoras de insulina y por la generación de autoanticuerpos que sirven comomarcadores para el diagnóstico. Es un tipo de diabetes poligénica en la que el sistema HLA tiene un papel importante en la susceptibilidad a desarrollar la patología. En un pequeño porcentaje de niños y adolescentes con clínica de diabetes tipo 1, no se detecta autoinmunidad basada en los marcadoresclásicos (IAA, GADA e IA2A). El autoanticuerpo de más reciente identificación es el ZnT8A que podría considerarse un nuevo marcador predictivo y diagnóstico de autoinmunidad. Otro tipo de diabetespresente en pediatría es la diabetes monogénica. Se trata de un grupo heterogéneo poco común dediabetes no autoinmune, asociado a alteraciones en genes relacionados con el desarrollo de la célula ßpancreática, con la síntesis de insulina o con su función. La mayor parte de las características clínicas no son específicas de este tipo de diabetes y se estima que un alto porcentaje de casos permanecen sin diagnosticar o se diagnostican erróneamente como diabetes tipo 1 o tipo 2. Los casos pediátricos condiabetes tipo 2 son excepcionales en poblaciones caucásicas.En este trabajo se ha desarrollado un radioinmunoensayo para analizar ZnT8A y se ha evaluado sucontribución para mejorar el diagnóstico de autoinmunidad en pediatría, obteniéndose un incremento dela sensibilidad diagnóstica de diabetes tipo 1, especialmente en adolescentes. Asimismo, se hadesarrollado mediante secuenciación masiva un panel de genes orientado al diagnóstico de diabetesmonogénica, para establecer la preval

Although genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection, only a few functional mutations for bovine paratuberculosis (PTB) have been characterized. Expression quantitative trait loci (eQTLs) are genetic variants typically located in gene regulatory regions that alter gene expression in an allele-specific manner. eQTLs can be considered as functional links between genomic variants, gene expression, and ultimately phenotype. In the current study, peripheral blood (PB) and ileocecal valve (ICV) gene expression was quantified by RNA-Seq from fourteen Holstein cattle with no lesions and with PTB-associated histopathological lesions in gut tissues. Genotypes were generated from the Illumina LD EuroG10K BeadChip. The associations between gene expression levels (normalized read counts) and genetic variants were analyzed by a linear regression analysis using R Matrix eQTL 2.2. This approach allowed the identification of 192 and 48 cis-eQTLs associated with the expression of 145 and 43 genes in the PB and ICV samples, respectively. To investigate potential relationships between these cis-eQTLs and MAP infection, a case-control study was performed using the genotypes for all the identified cis-eQTLs and phenotypical data (histopathology, ELISA for MAP-antibodies detection, tissue PCR, and bacteriological culture) of 986 culled cows. Our results suggested that the heterozygous genotype in the cis-eQTL-rs43744169 (T/C) was associated with the up-regulation of the MDS1 and EVI1 complex (MECOM) expression, with positive ELISA, PCR, and bacteriological culture results, and with increased risk of progression to clinical PTB. As supporting evidence, the presence of the minor allele was associated with higher MECOM levels in plasma samples from infected cows and with increased MAP survival in an ex-vivo macrophage killing assay. Moreover, the presen

Endometriosis, one of the most common gynecological disorders, is a complex disease characterized by the growth of endometrial-like tissue in extra-uterine locations and is a cause of pelvic pain and infertility. Evidence from observational studies indicate that endometriosis usually appears together with several other phenotypes. These include a list of autoimmune diseases, most of them more prevalent in women, anthropometric traits associated with leanness in the adulthood, as well as female reproductive traits, including altered hormone levels and those associated with a prolonged exposure to menstruation. However, the biological mechanisms underlying their co-morbidity remains unknown. To explore whether those phenotypes and endometriosis share a common genetic origin, we performed a systematic Two-Sample Mendelian Randomization (2SMR) analysis using public GWAS data. Our results suggest potential common genetic roots between endometriosis and female anthropometric and reproductive traits. Particularly, our data suggests that reduced weight and BMI might be mediating the genetic susceptibility to suffer endometriosis. Furthermore, data on female reproductive traits strongly suggest that genetic variants that predispose to a more frequent exposure to menstruation, through earlier age at menarche and shorter menstrual cycles, might also increase the risk to suffer from endometriosis.

[EU]Endometriosia kausa ezezaguneko gaitz kroniko eta multisistemikoa da, funtzionala den endometrio-ehuna utero-barrunbetik kanpo haztean agertzen dena, besteak beste pelbiseko mina eta antzutasuna eraginez. Ugaltze-adinean dauden emakumeen %10ak pairatzen duela balioetsi da, hauengan ondoez fisiko, mental, sexual, sozial eta ekonomikoa baldintzatzen duelarik. Eritasuna, aurkezpen konplexu eta heterogeneoa duen aldetik, fenotipo ugarirekin erlazionatu da, hala nola neurri antropometriko eta ezaugarri menstrual ezberdinekin. Hala ere, azken horiek eta endometriosiak oinarri genetiko bera partekatzen ote duten ez da oraindik ezagutzen. Genetika amankomun hau aztertzeko, bi lagineko ausazkotze mendeldar (2SMR) analisia burutu genuen, GWAS ikerketetako datu publikoak erabiliz. GWASek endometriosia eta berarekin asoziazioa erakutsi duten fenotipoak bezalako ezaugarri eta gaixotasun konplexuak ikertzeko aukera ematen dute, ezaugarriok edukitzeko joera handitzen duten aldaera genetikoak identifikatzen baitituzte. Gure 2SMR analisia burutzeko, hainbat ezaugarri antropometriko (pisua, garaiera, gorputz-masa indizea, gerri-aldaka indizea) eta menstrual (menarkia eta menopausiaren adinak, hilekoaren ziklo-luzera eta hileko-fluxuaren kantitate eta iraupena) aukeratu genuen, bakoitzak endometriosiarekin izan zezakeen lotura genetikoa ezagutzeko. Gure emaitzen arabera, pisu baxuak eta hilekoaren ziklo laburrek endometriosiarekin harreman genetikoa dute. Are gehiago, badirudi erlazioa kausala izan litekeela. Era berean, asoziazio hori bultzatzen duten zenbait aldaera genetiko identifikatzea lortu genuen. Aurkitutako ebidentzia horri esker endometriosiaren oinarria ulertzeko gertuago gaude., [EN]Endometriosis, the cause of which is unknown, is a chronic and multisystemic disease, characterized by the presence of functionally active endometrial tissue outside the womb, causing pelvic pain and infertility among others. The illness approximately affects 10% of reproductive age women and compromises physical, mental, sexual, social and economic well-being of those who suffer from it. This condition, known to have a complex and heterogeneous presentation, has been associated with many phenotypes, such as anthropometric and menstrual traits. However, whether these traits and endometriosis share common genetic roots is still undetermined. To investigate this, we performed a two-sample mendelian randomization (2SMR) analysis, using public data from GWAS studies. GWASs allow the study of complex traits and diseases like endometriosis and those phenotypes associated to it, given that they identify genetic variants that increase the susceptibility to develop them. For our 2SMR analysis, we chose several anthropometric (weight, height, body-mass index, waist-to-hip ratio) and menstrual traits (ages of menarche and menopause, menstrual cycle´s length, menstrual flow´s quantity and duration), in order to know whether they had a genetic relation with endometriosis or not. Our results showed a genetic link between low weight and short menstrual cycles and endometriosis. Furthermore, it seems that this relationship could be causal. Moreover, we were able to identify some genetic variants driving this association. Thanks to the aforementioned information we are now closer to understanding the basis of endometriosis.

The genetic background of childhood body mass index (BMI), and the extent to which the well-known associations of childhood BMI with adult diseases are explained by shared genetic factors, are largely unknown. We performed a genome-wide association study meta-analysis of BMI in 61,111 children aged between 2 and 10 years. Twenty-five independent loci reached genome-wide significance in the combined discovery and replication analyses. Two of these, located nearNEDD4LandSLC45A3, have not previously been reported in relation to either childhood or adult BMI. Positive genetic correlations of childhood BMI with birth weight and adult BMI, waist-to-hip ratio, diastolic blood pressure and type 2 diabetes were detected (R(g)ranging from 0.11 to 0.76, P-values <0.002). A negative genetic correlation of childhood BMI with age at menarche was observed. Our results suggest that the biological processes underlying childhood BMI largely, but not completely, overlap with those underlying adult BMI. The well-known observational associations of BMI in childhood with cardio-metabolic diseases in adulthood may reflect partial genetic overlap, but in light of previous evidence, it is also likely that they are explained through phenotypic continuity of BMI from childhood into adulthood. Author summary Although twin studies have shown that body mass index (BMI) is highly heritable, many common genetic variants involved in the development of BMI have not yet been identified, especially in children. We studied associations of more than 40 million genetic variants with childhood BMI in 61,111 children aged between 2 and 10 years. We identified 25 genetic variants that were associated with childhood BMI. Two of these have not been implicated for BMI previously, located close to the genesNEDD4LandSLC45A3. We also show that the genetic background of childhood BMI overlaps with that of birth weight, adult BMI, waist-to-hip-ratio, diastolic blood pressure, type 2 diabetes, and age at menarche. O

317 p., Disorders of sex development (DSD) encompass a high heterogeneous range of conditionsin which the optimal clinical management of the individuals comprise clinical description, biochemicaltesting and genetic analysis. With the arrival of next-generation sequencing (NGS) new genes andpathways have been implicated in the pathogenesis of the disease. However, only 50% of the 46,XY DSDpatients will receive a definitive diagnosis. The aim of this work is the clinical and molecularcharacterization of a DSD cohort. Patients and methods: Blood samples or DNA from a total of 125independent patients with a DSD referred from several Spanish centres and one Swiss hospital werestudied. Clinical characterization was done after examination of the data sheet provided by the clinicians.The molecular study was performed either by single-gene testing or NGS using a targeted gene panel of48 DSD-related genes. Parents were also analysed when possible. Genetic variants were analysed usingin silico prediction programmes and were classified according to its potential pathogenicity. Furtherfunctional studies of the genetic changes identified in three genes were also performed. Results: Overall,we made a genetic diagnosis in 4.4% of the individuals. In 46,XY DSD, the diagnostic rate increased upto 46.5% while in 46.XX DSD it was 29.2%. Novel changes classified as pathogenic or likely pathogenicwere mostly located in the NR5A1 and LHCGR genes. Functional impact of variants in NR5A1 andLHCGR showed a significant reduced transactivation activity while the variant of unknown significancein GATA4 showed similar activity compared to wild type. Conclusions: The heterogeneous phenotypesencompassed in this condition difficult the clinical diagnosis and highlight the need of a genetic study.The molecular analysis of DSD-related genes identified a causative genetic variant in the 40.2% of thepatients analysed in this cohort. Targeted gene sequencing panel is useful for the detection of singlenucleotidev

Abnormal DNA methylation has been described in human inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD). As other complex diseases, IBD results from the balance between genetic predisposition and environmental exposures. As such, DNA methylation may be the consequence (and potential effector) of both, genetic susceptibility variants and/or environmental signals such as cytokine exposure. We attempted to discern between these two non-excluding possibilities by performing a combined analysis of published DNA methylation data in intestinal mucosal cells of IBD and control samples. We identified abnormal DNA methylation at different levels: deviation from mean methylation signals at site and region levels, and differential variability. A fraction of such changes is associated with genetic polymorphisms linked to IBD susceptibility. In addition, by comparing with another intestinal inflammatory condition (i.e., coeliac disease) we propose that aberrant DNA methylation can also be the result of unspecific processes such as chronic inflammation. Our characterization suggests that IBD methylomes combine intrinsic and extrinsic responses in intestinal mucosal cells, and could point to knowledge-based biomarkers of IBD detection and progression.

Celiac disease (CeD) is a complex immune-mediated inflammatory condition triggered by the ingestion of gluten in genetically predisposed individuals. Literature suggests that alterations in gut microbiota composition and function precede the onset of CeD. Considering that microbiota is partly determined by host genetics, we speculated that the genetic makeup of CeD patients could elicit disease development through alterations in the intestinal microbiota. To evaluate potential causal relationships between gut microbiota and CeD, we performed a two-sample Mendelian randomization analysis (2SMR). Exposure data were obtained from the raw results of a previous genome-wide association study (GWAS) of gut microbiota and outcome data from summary statistics of CeD GWAS and Immunochip studies. We identified a number of putative associations between gut microbiota single nucleotide polymorphisms (SNPs) associated with CeD. Regarding bacterial composition, most of the associated SNPs were related to Firmicutes phylum, whose relative abundance has been previously reported to be altered in CeD patients. In terms of functional units, we linked a number of SNPs to several bacterial metabolic pathways that seemed to be related to CeD. Overall, this study represented the first 2SMR approach to elucidate the relationship between microbiome and CeD.

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.

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.

By GWAS studies on celiac disease, gene expression was studied at the level of the whole intestinal mucosa, composed by two different compartments: epithelium and lamina propria. Our aim is to analyse the gene-expression and DNA methylation of candidate genes in each of these compartments. Epithelium was separated from lamina propria in biopsies of CeD patients and CTRs using magnetic beads. Gene-expression was analysed by RT-PC; methylation analysis required bisulfite conversion and NGS. Reverse modulation of gene-expression and methylation in the same cellular compartment was observed for the IL21 and SH2B3 genes in CeD patients relative to CTRs. Bioinformatics analysis highlighted the regulatory elements in the genomic region of SH2B3 that altered methylation levels. The cREL and TNFAIP3 genes showed methylation patterns that were significantly different between CeD patients and CTRs. In CeD, the genes linked to inflammatory processes are up-regulated, whereas the genes involved in the cell adhesion/ integrity of the intestinal barrier are down-regulated. These findings suggest a correlation between gene-expression and methylation profile for the IL21 and SH2B3 genes. We identified a "gene-expression phenotype" of CeD and showed that the abnormal response to dietary antigens in CeD might be related not to abnormalities of gene structure but to the regulation of molecular pathways.

Bisulfite amplicon sequencing has become the primary choice for single-base methylation quantification of multiple targets in parallel. The main limitation of this technology is a preferential amplification of an allele and strand in the PCR due to methylation state. This effect, known as PCR bias', causes inaccurate estimation of the methylation levels and calibration methods based on standard controls have been proposed to correct for it. Here, we present a Bayesian calibration tool, MethylCal, which can analyse jointly all CpGs within a CpG island (CGI) or a Differentially Methylated Region (DMR), avoiding one-at-a-time' CpG calibration. This enables more precise modeling of the methylation levels observed in the standard controls. It also provides accurate predictions of the methylation levels not considered in the controlled experiment, a feature that is paramount in the derivation of the corrected methylation degree. We tested the proposed method on eight independent assays (two CpG islands and six imprinting DMRs) and demonstrated its benefits, including the ability to detect outliers. We also evaluated MethylCal's calibration in two practical cases, a clinical diagnostic test on 18 patients potentially affected by Beckwith-Wiedemann syndrome, and 17 individuals with celiac disease. The calibration of the methylation levels obtained by MethylCal allows a clearer identification of patients undergoing loss or gain of methylation in borderline cases and could influence further clinical or treatment decisions.

Celiac disease (CeD) is an immune-mediated enteropathy with a strong genetic component where the main environmental trigger is dietary gluten, and currently a correct diagnosis of the disease is impossible if gluten-free diet (GFD) has already been started. We hypothesized that merging different levels of genomic information through Mendelian randomization (MR) could help discover genetic biomarkers useful for CeD diagnosis. MR was performed using public databases of expression quantitative trait loci (QTL) and methylation QTL as exposures and the largest CeD genome-wide association study conducted to date as the outcome, in order to identify potential causal genes. As a result, we identified UBE2L3, an ubiquitin ligase located in a CeD-associated region. We interrogated the expression of UBE2L3 in an independent data set of peripheral blood mononuclear cells (PBMCs) and found that its expression is altered in CeD patients on GFD when compared to non-celiac controls. The relative expression of UBE2L3 isoforms predicts CeD with 100% specificity and sensitivity and could be used as a diagnostic marker, especially in the absence of gluten consumption. This approach could be applicable to other diseases where diagnosis of asymptomatic patients can be complicated.

181 p. (eng.) 184 p. (eus.), La enfermedad celíaca (EC) es una enfermedad autoinmune que se desarrolla en personas con susceptibilidad genética. Los haplotipos HLA-DQ2 y HLA-DQ8 representan alrededor del 40% de la contribución genética a la EC. En total, junto con otras variaciones genéticas comunes identificadas, podemos explicar el 50% de la heritabilidad de la enfermedad. Sin embargo, otras capas de información genómica independientes de la variación de la secuencia del ADN también podrían contribuir a la patogénesis de la EC.En esta tesis, los datos públicos de diferentes capas ómicas se han utilizado para identificar los mecanismos genéticos y epigenéticos de regulación génica que podrían estar involucrados en la patogénesis de la EC. Los experimentos previos de microarrays de expresión de nuestro grupo, los resultados del proyecto Immunochip, la información publicada sobre TADs y los datos de metilación y expresión generados recientemente se han recopilado y vuelto a analizar. Esto nos ha permitido identificar nuevos elementos reguladores y regiones genómicas involucradas en el desarrollo de la CD. Así, hemos podido observar que la gliadina influye en los cambios de co-expresión observados en la ECmediante los factores de transcripción CREB1 y IRF1. Además, la estructura 3D del genoma podría regular la co-expresión local, y la gliadina provoca cambios agudos en la metilación del ADN incluso en individuos no celíacos. En conclusión, los reguladores genéticos y epigenéticos identificados podrían estar involucrados en la patogénesis de la enfermedad y constituir nuevas dianas de intervención.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.

[ES] Partiendo de los SNPs asociados a diferentes enfermedades autoinmunes e inflamatorias humanas, se seleccionó el SNP rs1033180 y el gen IRF4. Se modificó el SNP rs1033180 mediante la herramienta de edición genómica CRISPR/Cas9 para estudiar su influencia sobre IRF4, así como su implicación en el desarrollo de la enfermedad celiaca. Para ello, se diseñó el plásmido recombinante pX459::sgIRF4, con el que se transfectaron las células U937. IDIOMA: Castellano.

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.

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.

110 p. (euskera) 112 p. (inglés), La enfermedad celíaca (EC) es una enfermedad autoinmune que se desarrolla en personas con susceptibilidad genética. Durante la patogenia, se observa un aumento de la permeabilidad intestinal debido a una disrupción funcional de las uniones estrechas o Tight Junction (TJ). Diferentes estudios apuntan a una implicación genética de la vía TJ en la susceptibilidad a la EC.Los objetivos de esta tesis han sido, determinar la alteración de la vía en la EC, caracterizar la implicación funcional de polimorfismos en la disrupción de la barrera intestinal; y por último, crear un modelo celular para la caracterización funcional de genes candidato constitutivamente alterados en la EC. Los resultados han mostrado una alteración global de la vía a nivel de expresión, con alteraciones intrínsecas de los genes PPP2R3A, TJP1 y INADL. Además, hemos descubierto la necesidad de INADL para una adecuada proliferación y migración de las células intestinales. Por último, hemos replicado la asociación de rs6962966, y hemos descubierto que los genes contiguos están alterados en los pacientes, sugiriendo una posible implicación de la región en la patogenia. En conclusión, las alteraciones genéticas en la vía TJ en la EC podrían predisponer a los individuos susceptibles a una permeabilidad intestinal aumentada, el cual facilitaría el desarrollo de la enfermedad.

123 p.(eng):129 p.(eusk), La enfermedad celíaca (EC) es una enfermedad autoinmune que se desarrolla en personas con susceptibilidad genética. En los últimos años se han realizado diversos estudios de asociación genómica, en los que se han identificado nuevas regiones asociadas a EC y se han propuesto una larga lista de genes candidato.Los objetivos de esta tesis han sido, por un lado repetir los estudios de asociación en población española y por otro lado hacer un estudio funcional de los genes candidato propuestos.Debido a las diferencias genéticas entre poblaciones, no todas las regiones genéticas aparecen asociadas en cada una de las poblaciones a estudio. En el caso de la población española, hemos obtenido resultados variados. Algunas de las regiones aparecen asociadas y otras no, y además hemos sido capaces de identificar nuevas regiones asociadas en nuestra población.Para el estudio funcional de los genes candidato, hemos analizado la expresión de dichos genes en varios tipos de muestras. Por un lado biopsias intestinales de pacientes celiacos al diagnóstico y tras el tratamiento, y controles no celíacos; y por otro lado poblaciones celulares extraídas de las biopsias de dichos pacientes, con el objetivo de analizar con mayor precisión lo que ocurre en cada uno de los tipos celulares.Los resultados obtenidos demuestran una vez más la complejidad de la EC, ya que la relación entre los genes alterados y los SNPs asociados no es directa

[ES] La enfermedad celíaca (EC) es una enteropatía autoinmune de predisposición genética, producida por la ingestión en la dieta de péptidos derivados de cereales como el trigo o la cebada. Aunque se creía que afectaba casi de forma exclusiva a los individuos europeos (1%), actualmente se conocen casos en todo el mundo. El modelo patogénico se centra en los mecanismos de la inmunidad adaptativa dependientes de la estimulación de linfocitos T CD4+ reactivos, pero existe además un efecto tóxico directo del gluten sobre el epitelio intestinal, dependiente de la inmunidad innata. La participación de la Genética en la susceptibilidad a la enfermedad es conocida desde hace tiempo, siendo el locus HLA el que explica aproximadamente el 40% del componente genético de la enfermedad. Para tratar de identificar otros genes con susceptibilidad, se han venido realizando múltiples esfuerzos durante los últimos años. Uno de los últimos, llevado a cabo en 2011, fue el Proyecto Immunochip. En él, se analizaron más de 200.000 variantes y se descubrieron 13 nuevos loci de riesgo para la EC, que junto con los descubiertos en anteriores trabajos y el locus HLA, daban un total de 40 loci de riesgo. Entre ellos, se encontraba la región que ocupa el gen LPP . Localizado en el cromosoma 3, un estudio reciente lo vincula con los procesos de adhesión celular en el intestino. En el presente trabajo, se ha estudiado el efecto de la gliadina sobre la expresión del gen de interés (LPP ) y el posible efecto de un silenciamiento del mismo sobre dos genes relacionados con las uniones celulares (ACTB y TJP1). En el caso de la gliadina, no se halló un cambio significativo en la expresión del gen. Mientras, los resultados del efecto del silenciamiento fueron dispares, no siendo concluyentes para el gen ACTB, pero encontrando una posible asociación entre los genes LPP y TJP1., [EN] The celiac disease (CD) is an autoimmune enteropathy of genetic predisposition, produced by the dietary intake of peptides derived from grains like wheat or barley. Although it was believed that it affected almost exclusively to European individuals (1%), is currently known cases worldwide. The pathogenic model focuses on the mechanisms of adaptive immunity dependent of the stimulation of reactive CD4+ T lymphocytes, but there is also a direct toxic effect of gluten on the epithelium dependent of innate immunity. The participation of Genetic susceptibility to the disease is long known, with the HLA locus which accounts for about 40% of the genetic component of the disease. To try to identify other susceptibility genes, numerous efforts have been made in recent years. One of the latest, held in 2011, was the Immunochip Project. In it, more than 200,000 variants were analyzed and 13 new risk loci for the CD were discovered, together with those found in previous work and the HLA locus, gave a total of 40 risk loci were discovered. One of these, was the region with the LPP gene. Located on chromosome 3, a new study links it to the processes of cell adhesion in the intestine. In this paper, it has been studied the effect of gliadin on the expression of the gene of interest (LPP ) and the possible effect thereof on silencing two genes related to cell junctions (ACTB and TJP1). For the gliadin, a significant change was not found in gene expression. Meanwhile, the effect of silencing results were mixed and not being conclusive for the ACTB gene, but finding a possible association between LPP and TJP1 genes.

La enfermedad celíaca, también conocida como enteropatía sensible al gluten, es un trastorno autoinmune crónico causada por la intolerancia a la ingesta de gluten y desarrollada por personas genéticamente predispuestas cuyo único tratamiento consiste en una dieta estricta libre de gluten. El desarrollo de dicha enfermedad está relacionada con una gran variedad de genes entre los que se encuentra PTPRK. Este trabajo desarrolla un estudio de la expresión de dicho gen así como un protocolo de silenciamiento del mismo.

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Background: The World Gastroenterology Organization recommends developing national guidelines for the diagnosis of Celiac Disease (CD): hence a profile of the diagnosis of CD in each country is required. We aim to describe a cross-sectional picture of the clinical features and diagnostic facilities in 16 countries of the Mediterranean basin. Since a new ESPGHAN diagnostic protocol was recently published, our secondary aim is to estimate how many cases in the same area could be identified without a small intestinal biopsy. Methods: By a stratified cross-sectional retrospective study design, we examined clinical, histological and laboratory data from 749 consecutive unselected CD children diagnosed by national referral centers. Results: The vast majority of cases were diagnosed before the age of 10 (median: 5 years), affected by diarrhea, weight loss and food refusal, as expected. Only 59 cases (7.8%) did not suffer of major complaints. Tissue transglutaminase (tTG) assay was available, but one-third of centers reported financial constraints in the regular purchase of the assay kits. 252 cases (33.6%) showed tTG values over 10 times the local normal limit. Endomysial antibodies and HLA typing were routinely available in only half of the centers. CD was mainly diagnosed from small intestinal biopsy, available in all centers. Based on these data, only 154/749 cases (20.5%) would have qualified for a diagnosis of CD without a small intestinal biopsy, according to the new ESPGHAN protocol. Conclusions: This cross-sectional study of CD in the Mediterranean referral centers offers a puzzling picture of the capacities to deal with the emerging epidemic of CD in the area, giving a substantive support to the World Gastroenterology Organization guidelines.

7 p., The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.