Transcript Novel aspects in the genetics of celiac disease

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
INFR1
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
INFR2
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.