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NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE: COPY NUMBER VARIATION, METHYLATION AND COREGULATION IN NF k B-RELATED GENES Nora Fern a ndez Jimenez. Doctoral thesis Leioa, 2/28/2014. - PowerPoint PPT Presentation
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NOVEL ASPECTS IN THE GENETICS OF CELIAC DISEASE:COPY NUMBER VARIATION, METHYLATION AND
COREGULATION IN NFkB-RELATED GENESNora 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
MAPK1PLCB3
GRB2GNAI1EDG2EDG2RAF1
PRKG2MAP3K2ADCY9
MAGI1PPP2R3AEPB41L3
JAM1PPP2R3A
GNAI1ACTBACTB
MYH14ZAK
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
PPP2R3AEPB41L3
**
MitosisG1
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 E2F3 CDC2/7
E2F5
IKK complex
RAB6IP2
IkB
UBD
P
proteasome
ubiquitin
PSMA1/4/5/6
PSMB1/2
PSMD/13/14PSME1/2
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
IFNAR1IFNGR2
GRB2STAT1STAT1STAT1STAT1STAT1IL15RJAK3IFNG
CCND2CCND2PIK3R3JAK2
SOCS1JAK2
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/13/14PSME1/2
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
MMP3 RANTESTAP1/2
SOCS1
CCDN2
E2F5ID3
Ubiquitin–proteasome system: Selection of NFkB pathway for further analyses
Castellanos-Rubio et al, 2010
Whole genome expression analysis in CD
IntroductionOther 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. ControlActive and GFD vs. ControlActive vs. GFD vs. ControlColocalizationsPhysical interactions
Expression and modulation study
Expression and modulation study •Basal biopsy experiment
Coexpression matrixes
GENE B
no coexpressionGENE A
p<0.05P<0.01P<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 assessmentT 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.