NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE: COPY NUMBER VARIATION, METHYLATION AND COREGULATION IN NFkB-RELATED GENES Nora Fernandez Jimenez eman ta zabal zazu Universidad del País Vasco Euskal Herriko Unibertsitatea 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 • Clinical Features and Diagnosis Intestinal features: diarrhea, abdominal distensión, vomiting… Symptoms Atypical symptoms: neurological symptoms, infertility, osteoporosis… 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 Introduction GLOBAL PUBLIC HEALTH PROBLEM Introduction HLA genotype explains around 40% of heritability HLA is necessary but not sufficient to develop CD Abadie et al, 2011 Introduction Genome-Wide Association Studies 39 non-HLA loci associated 63 genes PROPOSED >24,000 samples UK Italy Netherlands Spain Poland India From Trynka et al, 2011 Introduction … all together, these variants explain ≈ 50% of heritability in CD. Where can we look for the missing heritability? Introduction Whole genome expression analysis in CD ITGB5 TIMP2 ExtraCellular Matrix PLOD2 LAMB3 P4HB LAMB2 adherens ACVR1B IQGAP1 junction LAMA1 laminin CD36 Collagen receptor cell adhesion LAMA1/B2/B3 TGF CD36 integrin CD36 GZMB ACVR1B CASP10 GRB2 CFLAR Smads CASP3 GFD p53 RANTES BIRC3 MAPK1 BIRC3 ERBB2 P JAK2/3 PI3K STAT1 Cell death catenin CAPN5 GST2/4 connexins PPP2R3A Cdc2 EPB41L3 MAPK1 GRB2 STAT1 IL15R JAK3 DIAGNOSIS JAK2 CAPN5 DIAGNOSIS GFD E2F5 SOCS1 JAK2 [Ca++] TMEM37 ** ACTB ACTB IQGAP1 MAPK1 INSR ERBB2 PTPRF PTPRF PTPRF SSX2IP SSX2IP SSX2IP ACVR1B PLCB3 IFNG CCND2 CCND2 PIK3R3 CASP8 PPP3C junction MAPK signalingSTAT1 pathway STAT1 CASP8 Survival SSX2IP ** tight STAT1 PIK3R3 BCL12L14 MAPK1 IFNGR2 ACTB MYH14 IFNAR1 Actin cytoeskeleton GRB2 STAT1 cadherin Apoptosis MAPK signaling pathway E2F5 IL15RA INFR1 PPP3C HMMR DIAGNOSIS IFNG P BMPR1A CASP3 GZMB CASP8 MAGI1 PPP2R3A EPB41L3 JAM1 PPP2R3A GNAI1 ACTB ACTB MYH14 ZAK ID3 CASP10 P CD47 PI3K gap junction MAGI1 BMP2 BMPR1A perforin CD47 JAM1 BMP8B INFR2 PRF1 CD47 FasL INF BMP2/8B PTPRF IFN BMP2 ITGA9 PIK3R3 ITGA9/B5 CD36 CDC2 PLCB3 MAPK1 GRB2 GNAI1 EDG2 EDG2 RAF1 PRKG2 MAP3K2 ADCY9 ITGB5 MMP3/12/28 E2F3 E2F3 STAT1 GFD PTTG1 DIAGNOSIS GFD CDC7 CDC14B CDC25C BUB1 PTTG CCNB1 CDC27 P UBE1 IkB IKK complex APC4/7 Cell cycle APC ubiquitin P NFkB CDC25C G0 S CCND2 CCND2 PSMB2 PSMA5 PSMD14 MMP3 PSMA1 PSMA1 PSMA1/4/5/6 SUMO1 PSMA4 PSMD13 TAP1/2 SOCS1 RANTES ID3 ATR E2F3 CDC2/7 BUB3 ATR E2F5 CDC14 CCDN2 DIAGNOSIS GFD PSMB1/2 PSMA1/4/5/6 PSME1/2 activation T cell activation and Th2 mediated inflammation PSMA1 UBE1 CDC2 E2F5 PSMA6 UBD CDC27 G1 CDC25B CCND2 PSMB1 SUMO1 APC4/7 G2 CCNB1 CDC25B APC4 Apoptosis CFLAR NDFIP1 CDC27 activation PSME1/2 CASP8 + regulation of NFkB cascade NFkB UBD ubiquitin UBE1 BUB3 APC7 RAB6IP2 Cell RAB6IP2 cycle APC BUB1 Mitosis CDC2 IkB IKK complex proteasome immunoproteasome proteasome PSMB1/2 PSMD/13/14 degradation CALR TAP1/2 TAP1/2 transport protelytic degradation protelytic TAP2 CALR CALR CALR CALR ER transport HSP70/90 PSMA1 PSMA1 PSMD/13/14 immunoproteasome TAP1 MMP3 TAP1/2 TGM2 iNOS HSP70 ER HSP70/90 HSP70 HSP90 Antigenpresentation presentation Antigen transport transport vesiclevesicle HSP90 HSP90 DIAGNOSIS Castellanos-Rubio et al, 2010 GFD Ubiquitin–proteasome system: Selection of NFkB pathway for further analyses Introduction Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics Non coding RNAs Cis and trans gene expression regulators Da Sacco et al, 2012 Introduction Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics DNA methylation and histone modifications Sun et al, 2013 Introduction Other putative sources of genetic variability affecting susceptibility in complex diseases Epigenetics DNA methylation Sun et al, 2013 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 NFkB coregulation and modulation in CD First specific aim: 2. To look into the constitutive activation of the NFkB pathway in celiac disease, through an extensive expression analysis of 93 NFkB-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 NFkB signaling route by MALT1 inhibition. Expression and modulation study 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) 4h- in vitro experiment Duodenal biopsy pieces from each patient: Basal Ø Gliadin Gliadin + Z Z-VRPR-FMK (Z) gliadin 93 gene expression analysis in Taqman Low Density Array (TLDA) format T test for group comparisons and Pearson’s correlation matrixes for coexpression analyses Expression and modulation study •Basal biopsy experiment Downregulation Confirmation of the constitutive overexpression of the route Upregulation Expression and modulation study •Basal biopsy experiment Active vs. Control Active and GFD vs. Control Active vs. 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). Expression and modulation study •Basal biopsy experiment Coexpression matrixes no coexpression p<0.05 P<0.01 P<0.001 NFkB-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 Methylation study - 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. Implication in celiac disease NFkB-related gene selection criteria: -CpG-enriched promoters or first exons -Predesigned available methylation assays Methylation commercially -Regulatory and central functions in the NFkB biological route Gene symbol Assay Number MALT1 PM00185143 MAP3K7 PM00122850 MAP3K7IP1 PM00199521 MAP3K14 PM00177569 NFKBIA PM00056287 RELA PM00048895 TNFAIP3 PM00122129 TRADD PM00061369 Conventional pyrosequencing used for methylation level assessment T test for group comparisons and Pearson’s correlation for co-methylation analyses Implication in celiac disease •Methylation study in celiac disease Methylation level differences among groups (%) Active CD Treated CD Controls 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 Conclusions Most of the studied genes that are constitutively upregulated in celiac disease belong to the core of the NFkB 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 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, GFDtreated 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.
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