NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE: COPY NUMBER VARIATION, METHYLATION AND

1. NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE: COPY NUMBER VARIATION, METHYLATION AND COREGULATION IN NFkB-RELATED GENES Nora FernandezJimenez Doctoral thesis Leioa, 2/28/2014

2. 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

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

4. Introduction GLOBAL PUBLIC HEALTH PROBLEM

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

6. Introduction Genome-WideAssociationStudies 39 non-HLA loci associated 63 genes PROPOSED >24,000 samples UK Italy Netherlands Spain Poland India FromTrynkaet al, 2011

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

8. Introduction Wholegenomeexpressionanalysis in CD Ubiquitin–proteasome system: Selection of NFkB pathway for further analyses Castellanos-Rubio et al, 2010

9. Introduction Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics Non codingRNAs Cisandtrans gene expressionregulators Da Sacco et al, 2012

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

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

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

13. NFkBcoregulation and modulation in CD

14. First specific aim: 2. To look into the constitutive activation of the NFkBpathway in celiac disease, through an extensive expression analysis of 93 NFkB-related genes. • Operativeaims: • 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. • To understand how in vitro gliadin challenge of biopsies can affect gene expression patterns and to check the effect of the modulation of the NFkBsignaling route by MALT1 inhibition. Expressionandmodulationstudy

15. Expression and modulation study Basal biopsy experiment16 CD active patients + 16 CD treated patients + 16 controls Modulation experiment in vitro challenge with pepsin-trypsin digested gliadin (PTG) and the NFkB modulator (Z) gliadin Z-VRPR-FMK (Z) 93 gene expressionanalysisin TaqmanLowDensity Array (TLDA) format 4h- in vitro experiment T test for group comparisons and Pearson’s correlation matrixes for coexpression analyses Duodenal biopsy pieces from each patient: Basal Ø Gliadin Gliadin + Z

16. Expression and modulation study • Basal biopsyexperiment Upregulation Downregulation Confirmationoftheconstitutiveoverexpressionoftheroute

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

18. Expression and modulation study • Basal biopsyexperiment Coexpression matrixes GENE A GENE B no coexpression p<0.05 P<0.01 P<0.001 NFkB-related gene coexpression is a feature of health

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

20. Methylationstudy - To determine whether changes in methylation in promoters and first exons of several NFkB-related genes occur in the celiac intestinal mucosa. • - To check how methylation levels of several NFkB-related genes vary in the different stages of celiac disease.

21. Implication in celiac disease NFkB-related gene selectioncriteria: -CpG-enriched promoters or first exons -Predesigned methylation assays commercially available -Regulatory and central functions in the NFkB biological route Conventionalpyrosequencingusedformethylationlevelassessment T test forgroupcomparisonsandPearson’scorrelationforco-methylationanalyses

22. Implication in celiac disease • Methylationstudy in celiacdisease Methylationleveldifferencesamonggroups(%) Active CD Treated CD Controls

23. Implication in celiac disease • Methylationstudy in celiacdisease Comethylation in celiacpatientspartiallydisruptscoexpression

24. Implication in celiac disease Expressionandmodulationstudy + Methylationanalysis

25. Conclusions

26. Conclusions Most of the studied genes that are constitutively upregulated in celiac disease belong to the core of the NFkBroute 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 upregulatedonly 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 gliadinin both expression levels and coexpressionpatterns, proposing MALT1 inhibition as a putative therapeutic target for acute symptoms in celiac disease.

27. Conclusions Several NFkB-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.