Erregulatzaile genetiko eta epigenetikoen identifikazioa eritasun zeliakoan hurbilketa konputazional eta esperimentalen bidez

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

Erregulatzaile genetiko eta epigenetikoen identifikazioa eritasun zeliakoan hurbilketa konputazional eta esperimentalen bidez

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

From Omic Layers to Personalized Medicine in Colorectal Cancer: The Road Ahead

Colorectal cancer is a major health concern since it is a highly diagnosed cancer and the second cause of death among cancers. Thus, the most suitable biomarkers for its diagnosis, prognosis, and treatment have been studied to improve and personalize the prevention and clinical management of colorectal cancer. The emergence of omic techniques has provided a great opportunity to better study CRC and make personalized medicine feasible. In this review, we will try to summarize how the analysis of the omic layers can be useful for personalized medicine and the existing difficulties. We will discuss how single and multiple omic layer analyses have been used to improve the prediction of the risk of CRC and its outcomes and how to overcome the challenges in the use of omic layers in personalized medicine

Host Genetics and Microbiota Interactions in Colorectal Cancer: Shared or Independent Risk?

The role of microbiota in colorectal cancer has been studied since alterations in its composition were observed. In addition, there are more and more pieces of evidence that microbiota could be implicated in colorectal cancer progression. Thus, the components of the microbiota could be biomarkers for the diagnosis and prognosis of colorectal cancer. In addition, it is important to address how the microbiota interacts with the host and how the host shapes the microbiota, in order to understand the biological pathways and mechanisms involved in their relationship and the consequences of their interactions in colorectal cancer. Thereby, it could be possible to find feasible measures and treatments to prevent or better diagnose colorectal cancer. In this review, we will try to summarize the role of the microbiota in colorectal cancer and its interactions with the host and the host genetics, coming to some conclusions that could be useful to find the gaps in our knowledge and propose future steps in this field

A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation

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

Performance of the Use of Genetic Information to Assess the Risk of Colorectal Cancer in the Basque Population

The risk of developing colorectal cancer (CRC) is partially associated with genetics. Different studies have provided valuable genetic information to understand the biology behind CRC and to build models of genetic risk. However, the study of the applicability of such genetic information within the Basque population is limited. Thus, our objectives were to find out if the genetic variants associated with CRC in other populations are the same in the Basque population and to assess the performance of the use of genetic information to calculate the risk of developing CRC. We found that the available genetic information can be applied to the Basque population, although local genetic variation can affect its use. Our findings will help to refine the use of CRC genetic risk calculation in the Basque population, and we expect that our findings could be useful for other populations

Genetic Variants as Predictors of the Success of Colorectal Cancer Treatments

Some colorectal cancer (CRC) outcomes are partially associated with genetics, and different studies have proposed several genetic variants as predictors. However, analysis of their performance in other populations is limited. Thus, our objectives were to assess their use in our cohort and to find additional genetic variants associated with CRC outcomes. We found that some of the genetic variants proposed as predictors could be used in our cohort, although the addition of clinical data improved the performance. In addition, we found additional genetic variants that could be useful to predict the CRC manifestations in our population. Our findings will help to refine the use of genetic polymorphisms to predict CRC outcomes in our population, and we expect that our findings could be useful for other populations.This work was partially founded by Gipuzkoako Foru Aldundia/Diputación Foral de Gipuzkoa (Code: 111/17

Human mitochondrial DNA is extensively methylated in a non-CpG context

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.Institut National du Cancer (INCa, France); European Commission (EC) Seventh Framework Programme (FP7) Translational Cancer Research (TRANSCAN) Framework; Foundation ARC pour la Recherche sur le Cancer (France); Plan Cancer-Eva-Inserm research grant (to Z.H.); Postdoctoral Fellowship from International Agency for Research on Cancer (to V.P.), partially supported by the EC FP7 Marie Curie Actions -People -Co-funding of regional, national and international programmes (COFUND)'. Funding for open access charge: Institut National du Cancer (INCa, France); European Commission (EC) Seventh Framework Programme (FP7) Translational Cancer Research (TRANSCAN) Framework; Foundation ARC pour la Recherche sur le Cancer (France); Plan Cancer-Eva-Inserm research grant (to Z.H.)

MAGI2 Gene Region and Celiac Disease

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.This work was partially funded by the Basque Department of Education grant IT1281-19 and ISCIII Research Project PI16/00258, cofunded by the European Union ERDF, A way to make Europe to JB. AC-R is supported by an Ikerbasque Fellowship and funded by a research project grant 2017111082 from the Basque Goverment. IS was funded by a research project grant 2015111068 from the Basque Department of Health. AJ-M and AO-G are predoctoral fellows funded by FPI grants from the Basque Department of Education, Universities and Research and IR-G and MS are predoctoral fellows funded by the University of Basque Country

Transcription Factor Binding Site Enrichment Analysis In Co-Expression Modules In Celiac Disease

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.The authors thank the technical and human support provided by SGIker of the UPV/EHU. The work was funded by ISCIII Research Project Grants PI13/01201 and PI16/00258, cofunded by the European Union ERDF/ESF "A way to make Europe" and by Basque Department of Health project 2011/111034 to JRB and Basque Department of Health project 2015/111068 to I.S., N.F.-J. was supported by an IARC Postodctoral Fellowship (FP7 Marie Curie Actions-People-COFUND) and a Postdoctoral Fellowship from the Basque Department of Education. I.R.-G. and A.J.-M. are supported by predoctoral fellowship grants from the UPV/EHU and the Basque Department of Education, respectively