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

COREGULATION IN NFkB-RELATED GENES

Nora Fernandez Jimenez

Doctoral thesisLeioa,

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 casesGood 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 samplesUK

ItalyNetherlands

SpainPolandIndia

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

Introduction

ITGA9/B5

integrin

laminin

LAMA1/B2/B3

MMP3/12/28TIMP2

CD36

Collagen receptorcell adhesion

DIAGNOSIS GFD

HMMR

CD47

CD47

ITGA9

CD36

CD36

CD36

LAMA1

LAMB2

LAMB3

ITGB5

ITGB5

CD47

ExtraCellular Matrix

P4HBPLOD2

FasL

CASP10 CASP8

CASP3

CFLARPI3K

Cell deathSurvival

BIRC3

p53

BCL12L14

PIK3R3

GST2/4

[Ca++]

PPP3C

CAPN5

GZMB

GZMB

PRF1

perforin

TMEM37DIAGNOSIS GFD

CAPN5

CASP8

CASP8

PIK3R3

PPP3C

BIRC3

CASP3

CASP10

**

CDC2

MAPK1

PLCB3

GRB2

GNAI1

EDG2

EDG2

RAF1

PRKG2

MAP3K2

ADCY9

MAGI1

PPP2R3A

EPB41L3

JAM1

PPP2R3A

GNAI1

ACTB

ACTB

MYH14

ZAK

ACTBACTB

IQGAP1MAPK1INSR

ERBB2PTPRFPTPRFPTPRFSSX2IPSSX2IPSSX2IPACVR1B

DIAGNOSIS GFD

MAPK signaling pathway

PTPRF

catenin

IQGAP1

cadherin

adherens junction

Actin cytoeskeleton

JAM1

connexins

MAPK1

Cdc2

gap junctionMAGI1

tight junction

PLCB3

MYH14

SSX2IP

ACTB

ERBB2

GRB2

PPP2R3A

EPB41L3

**

Mitosis

G1

G0S

G2

DIAGNOSIS GFD

CDC14

ATR

BUB3

CCND2

CCND2

CDC25C

CDC25B

CDC2

CCNB1

BUB1

PTTG

CDC7

E2F3

E2F3

E2F5

CDC25C

CDC2

CCNB1

BUB3

CDC25B

CCND2

CDC14BBUB1

PTTG1

ATR E2F3CDC2/7

E2F5

IKK complex

RAB6IP2

IkB

UBD

P

proteasome

ubiquitin

PSMA1/4/5/6

PSMB1/2

PSMD/13/14

PSME1/2

NFkB

SUMO1

immunoproteasome

protelytic degradation

TAP1/2

transport vesicle

transportER

Antigen presentation

DIAGNOSIS GFD

APC7

APC4

UBE1

CDC27

PSMB1

PSMA6

PSMB2

PSMA5

PSMD14

PSMA1

PSMA1

PSMA1

PSMA4

TAP1

PSMA1

PSMA1

TAP2

CALR

CALR

CALR

HSP70

HSP70

HSP90

HSP90

HSP90

HSP70/90

CALR

PSMD13

APCCell

cycle

APC4/7

CDC27

UBE1

activation

P P

ACVR1B

TGF BMP2/8B

BMPR1A

MAPK1

INF

INFR2

IFN

JAK2/3

STAT1

Smads

RANTES P PI3KApoptosis

MAPK signaling pathway

ACVR1B

BMP8B

BMP2

BMP2

BMPR1A

IFNG

E2F5

MAPK1

ID3

IFNAR1

IFNGR2

GRB2

STAT1

STAT1

STAT1

STAT1

STAT1

IL15R

JAK3

IFNG

CCND2

CCND2

PIK3R3

JAK2

SOCS1

JAK2

STAT1

IL15RA

GRB2

INFR1

DIAGNOSIS GFD

CFLAR

NDFIP1

CASP8regulation of

NFkB cascade+ Apoptosis

T cell activation and Th2 mediated

inflammation

IKK complex

RAB6IP2

IkB

UBD

P

proteasome

ubiquitin

PSMA1/4/5/6

PSMB1/2

PSMD/13/14

PSME1/2

NFkB

SUMO1

immunoproteasome

protelytic degradation

TAP1/2

transport vesicle

transportER

Antigen presentation

HSP70/90

CALR

iNOSMMP3 TAP1/2

TGM2

APCCell

cycle

APC4/7

CDC27

UBE1

activation

MMP3RANTES

TAP1/2

SOCS1

CCDN2

E2F5ID3

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

NFkB 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.

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

93 gene expression analysis in Taqman Low Density Array (TLDA)

format

Modulation experiment in vitro challenge with pepsin-trypsin digested gliadin (PTG) and the NFkB modulator (Z)

T test for group comparisons and Pearson’s correlation matrixes for

coexpression analyses

gliadin

Z-VRPR-FMK (Z)

4h- in vitro experimentDuodenal biopsy pieces from

each patient: Basal Ø Gliadin Gliadin + Z

Expression and modulation study

•Basal biopsy experiment

Confirmation of the constitutive overexpression of the route

Downregulation

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. ControlActive vs. GFD vs. ControlColocalizations

Physical interactions

Expression and modulation study

Expression and modulation study

•Basal biopsy experiment

Coexpression matrixes

GENE

B

no coexpressionGENE

A

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

- 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.

Methylation study

Implication in celiac disease

NFkB-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 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

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

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, 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.