NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE: COPY NUMBER VARIATION, METHYLATION AND COREGULATION IN NF  B-RELATED GENES Nora Fernandez Jimenez Doctoral thesis Leioa, 2/28/2014

Introduction Celiac disease (CD) is a chronic, immune-mediated enteropathy, caused by intolerance to ingested gluten from wheat (and similar proteins from rye and barley) that develops in genetically susceptible individuals.

Introduction GLUTEN Reversion of major disease symptoms with a life-long gluten-free diet (GFD) in most of cases Good autoimmune- disease model due to the availability of tissue from active (at Dx) / inactive (after GFD) forms Symptoms Intestinal features: diarrhea, abdominal distensión, vomiting… Atypical symptoms: neurological symptoms, infertility, osteoporosis… Clinical Features and Diagnosis

Introduction GLOBAL PUBLIC HEALTH PROBLEM

Introduction Abadie et al, 2011 HLA genotype explains around 40% of heritability HLA is necessary but not sufficient to develop CD

Introduction From Trynka et al, 2011 >24,000 samples UK Italy Netherlands Spain Poland India 39 non-HLA loci associated 63 genes PROPOSED Genome-Wide Association Studies

… all together, these variants explain ≈ 50% of heritability in CD. Where can we look for the missing heritability? Introduction

Ubiquitin–proteasome system: Selection of NFkB pathway for further analyses Castellanos-Rubio et al, 2010 Whole genome expression analysis in CD

Introduction Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics Da Sacco et al, 2012 Non coding RNAs Cis and trans gene expression regulators

Introduction Sun et al, 2013 Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics DNA methylation and histone modifications

Introduction Sun et al, 2013 Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics DNA methylation

Introduction Gasche et al, 2010 Yao et al, 2012 NF  B pathway hypomethylated in some cancers. Konishi et al, 2007 Genetic changes lead to a more aggressive phenotype in the gut. Epigenetic changes as good candidate modifications to have a role in CD.

NF  B coregulation and modulation in CD

First specific aim: 2. To look into the constitutive activation of the NF  B pathway in celiac disease, through an extensive expression analysis of 93 NF  B-related genes. Operative aims: a)To identify those genes whose alterations are not (constitutive) and are (inflammation-related) reverted by a gluten free diet, to depict their roles in the pathway and to scrutinize the relationships among them. b)To understand how in vitro gliadin challenge of biopsies can affect gene expression patterns and to check the effect of the modulation of the NF  B signaling route by MALT1 inhibition. Expression and modulation study

Basal biopsy experiment  16 CD active patients + 16 CD treated patients + 16 controls 93 gene expression analysis in Taqman Low Density Array (TLDA) format Modulation experiment  in vitro challenge with pepsin-trypsin digested gliadin (PTG) and the NF  B modulator (Z) T test for group comparisons and Pearson’s correlation matrixes for coexpression analyses gliadin Z-VRPR-FMK (Z) 4h- in vitro experiment Duodenal biopsy pieces from each patient: BasalØGliadinGliadin + Z

Expression and modulation study Basal biopsy experiment Confirmation of the constitutive overexpression of the route Downregulati on Upregulation

Basal biopsy experiment Constitutively upregulated genes belong to the core of the pathway whereas genes that are overexpressed in active CD are more peripheral (according to GeneMANIA). Active vs. Control Active and GFD vs. Control Active vs. GFD vs. Control Colocalizations Physical interactions Expression and modulation study

Basal biopsy experiment Coexpression matrixes GENE B no coexpression GENE A p<0.05 P<0.01 P<0.001 NF  B-related gene coexpression is a feature of health

Expression and modulation study MALT1 inhibition restores the coexpression patterns disrupted by gliadin in GFD-treated patients

- To determine whether changes in methylation in promoters and first exons of several NF  B-related genes occur in the celiac intestinal mucosa. - To check how methylation levels of several NF  B-related genes vary in the different stages of celiac disease. Methylation study

Implication in celiac disease NF  B-related gene selection criteria: -CpG-enriched promoters or first exons -Predesigned methylation assays commercially available -Regulatory and central functions in the NFkB biological route Gene symbol Methylation Assay Number MALT1 PM MAP3K7 PM MAP3K7IP1 PM MAP3K14 PM NFKBIA PM RELA PM TNFAIP3 PM TRADD PM Conventional pyrosequencing used for methylation level assessment T test for group comparisons and Pearson’s correlation for co-methylation analyses

Implication in celiac disease Active CD Treated CD Controls Methylation study in celiac disease Methylation level differences among groups (%)

Implication in celiac disease Methylation study in celiac disease Comethylation in celiac patients partially disrupts coexpression

Implication in celiac disease Expression and modulation study Methylation analysis +

Conclusions

Most of the studied genes that are constitutively upregulated in celiac disease belong to the core of the NF  B route and disruption of coexpression is a relevant feature of the active celiac gut. a) Constitutively overexpressed genes show physical interactions among them and are part of the core of the pathway, whereas genes upregulated only in active disease are more peripheral to the route. The regulatory equilibrium of the healthy gut is completely disrupted in active disease, and treated patients present intermediate coexpression patterns. b) In vitro gliadin challenge affects the tight coexpression observed in biopsies from controls and treated patients, especially disrupting the regulation in the latter, while the modulation of the route is able to considerably revert the effects of gliadin in both expression levels and coexpression patterns, proposing MALT1 inhibition as a putative therapeutic target for acute symptoms in celiac disease.

Conclusions Several NF  B-related genes present subtle but significant methylation level differences among active and treated celiac and control individuals. Several genes (MALT1, MALT3K7, RELA and TRADD) presented subtle differences in methylation levels between active celiac and control groups, while in general, GFD-treated patients showed intermediate levels, suggesting the partial reversion of the epigenetic alterations after more than two years of treatment. Correlation among methylation levels (co- methylations) occurred only in celiac patients, both active and treated, and was associated with the disruption of coexpression.