Transcript Diapositiva 1

MEDICEL – Bologna
April 5th 2011
Environmental and Genetic Risk
Factors for Coeliac Disease
Luigi Greco,
European Laboratory for Food Induced Disease
Naples, Italy
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PREVENT CD (EUFP6)
“Nutritional risk factors in Coeliac Disease : possible induction
of tolerance to gluten in genetically predisposed infants
during breast feeding
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Israel
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PREVENT CD PARTNERS
PREVENT CELIAC DISEASE ???
• 1319 infants from families with a first degree
relative with CD were recruited.
• 905 of them, who were HLADQ2 and /or DQ8
positive, were prospectively followed-up for the
development of CD.
• Biopsies were performed if symptoms appeared
and/or if two or three consecutive samples were
positive for anti-tissue transglutaminase or antigliadin
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Intervention : 100 mg gluten at 4 mon.
Till December 2010, 787, 450, and 207
infants were older than 12, 24 and 36
months respectively.
48 biopsies were performed in 47 children
31 diagnosis of CD diagnosis were made
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Prevention or infection ?
Of the 207 children who reached the age of
36 mo, 14 were diagnosed with CD
between the 2nd and 3rd year (prevalence =
6,76%). Expected < 5%
Of the 243 children aged 12-24 mo 15 new
cases occurred (6,17%). Two more
infants were diagnosed before the age of
12 mo. Expected < 2,5%
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The great Swedish Epidemic
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Just a stimulation of the phenotype to unveal
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Breast feeding in cases and controls
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Delay from symptoms to diagnosis in months
Milk
N
Latency (mt)
Breast
26,0
6,3
Bottle
120,0
4,0
F
p
5,15
0,025
0,708
0,552
Age at gluten introduction (mt)
1°
7,0
6,0
2°
18,0
5,7
3°
79,0
4,1
4°
37,0
3,8
>5°
5,0
6,1
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Mean age at first symptoms
Milk
N
Mean (mt)
Breast
26,0
8,91
Bottle
120,0
6,30
F
p
6,64
0,01
0,604
0,60
Age at gluten introduction (mt)
1°
7,0
7,4
2°
18,0
6,0
3°
79,0
6,9
4°
37,0
6,9
>5°
5,0
4,2
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PROFILE OF CD CANDIDATE GENES
an alternative point of view
• COMMON TO OTHER AUTOIMMUNE DISEASES
• CELL STRUCTURE AND SHAPE
MOLECULAR RECEPTION OF VIRAL (?) PARTICLES
• NATURAL IMMUNITY AND INFLAMMATION
• THE NF-kB SYSTEM
• HLA SPECIFIC RECOGNITION AND PRESENTATION
• T-CELL STIMULATION AND ACTIVATION
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MOLECULAR RECEPTION OF VIRAL (?) PARTICLES
1. Molecular mimicry between Gluten fractions and Viral Antigen is
likely and may suggest the alternative use of the same pathway
2. Double stranded RNA viruses naturally link to Toll Like receptor
(TLR3) and then they can activate Transglutaminase.
3. Tissue Transglutaminase is activated by viral or bacterial
polysaccarides (poly-C) , through Toll Like Receptors
CD CANDIDATE GENES
 TLR7 and TLR8: Key players in the antiviral response
 TNFRSF14 also engages the herpes virus entry mediator.
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Viral (& Gluten?) Receptors
1. TNFRSF14 gene
Function: This receptor was identified as a cellular mediator of herpes simplex
virus (HSV) entry.
2. TNFSF14 gene
Function: Binding to the decoy receptor TNFRSF modulates its effects.
Activates NFKB, stimulates the proliferation of T-cells, and inhibits growth of
the adenocarcinoma HT-29. Acts as a receptor for Herpes simplex virus
3. TLR 7 gene & TLR 8 gene
Function : play a fundamental role in pathogen recognition and activation of
innate immunity. They recognize pathogen-associated molecular patterns
(PAMPs) GLUTEN??? and mediate the production of cytokines necessary for
the development of effective immunity. Acts via MYD88 and TRAF6, leading to
NF-kappa-B activation, cytokine secretion and the inflammatory response.
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TNFSF14 with its receptor TNFRSF14
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TLR7/TLR8 receptors
• Two of these orphan
receptors TLR7/TLR8 act
as sensors for single
stranded RNA.
• Activation of TLRs leads to
the generation of an
adaptive immune response
resulting in the eradication
of pathogens.
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An important contribution by
A. Fasano , Baltimora!
J Immunol. 2006 Feb 15;176(4):2512-21.
Gliadin stimulation of murine macrophage
inflammatory gene expression and intestinal
permeability are MyD88-dependent: role of the innate
immune response in Celiac disease.
Thomas KE, Sapone A, Fasano A, Vogel SN.
Department of Microbiology and Immunology, University of Maryland School of
Medicine, Baltimore, 21201, USA.
Unfortunately they are not able to identify the specific Toll
Like Rec. involved : they try with TLR2 and TLR4 and find that
they are not stimulated by gliadin
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TLR7 and TLR8: Key players in the
antiviral response
gluten
Nuclear
Activation
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Is Rotavirus Infection
a Risk Factor ?
 Rotavirus infection has been reported marginally
increased in CD cases vs controls (Stene & Norris 2007)
 In Celiac Disease, a Subset of Autoantibodies
against Transglutaminase Binds Toll-Like Receptor 4
and Induces Activation of Monocytes (Zanoni et al., XX)
 A subset of TGASeAb recognize the viral protein VP-7,
suggesting a possible involvement of rotavirus infection
through a mechanism of molecular mimicry.
 Rotavirus Infection was not increased in CD vs controls
(Rostami-Nejad M. et al , 2010)
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EPIDEMIOLOGY DOES NOT SUPPORT THIS !
Trend of infective Gastro Enteritis vs Celiac
Disease
7
6
5
4
Enteritis
3
Celiac
2
1
0
50
55
60
65
70
75
80
YEARS
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85
90
95
100
Evolutionary and Functional Analysis of Celiac Risk
Loci Reveals SH2B3 as a Protective Factor against
Bacterial Infection
The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.05.004
• The improved response to bacterial ligands, followed by
positive selection of different SH2B3 gene variants
suggest:
•
• a possible mechanism of how this polymorphism
contributes to the increased risk of developing immunerelated diseases
• that the cause of the signature of positive selection
should be sought in improved host defense against
infections.
( … We observed a dose-response
relationship of the
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risk-allele A with IL1b production (p = 0.034 for trend)…
PROFILE OF CD CANDIDATE GENES
an alternative point of view
• COMMON TO OTHER AUTOIMMUNE DISEASES
• CELL STRUCTURE AND SHAPE
• MOLECULAR RECEPTION OF VIRAL (?) PARTICLES
 NATURAL IMMUNITY AND INFLAMMATION
THE NF-kB SYSTEM
• HLA SPECIFIC RECOGNITION AND PRESENTATION
• T-CELL STIMULATION AND ACTIVATION
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TNFAIP3 gene - OR = 1,26
(Trynka G et al., GUT 2009)
 Inhibits NF-kappa B activation as well as TNF-mediated
apoptosis.
 Interacts with NAF1 and inhibits TNF-induced NF-kappa-Bdependent gene expression by interfering with an RIP- or
TRAF2-mediated transactivation signal.
 Has deubiquitinating activity that is directed towards
'Lys-48' or 'Lys-63'-linked polyubiquitin chains
 The A20 gene product is required for termination of the
NF-kB signal mediated by innate immune receptors via
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de-ubiquitination of several
NF-kB signalling factors.
Where TNFAIP3 works
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African-Derived Genetic Polymorphisms in TNFAIP3
Mediate Risk for Autoimmunity
James P. Lodolce,*,1 Lauren E. Kolodziej,*,1 Lesley Rhee,*
The Journal of Immunology, 2010, 184, 7001 -7009
 In African-American SLE patients a novel African-derived
risk haplotype (odds ratio = 1.6; p = 0.006) was identified
in TNFAIP3 gene
 In addition, a rare protective haplotype was defined by
A125V (odds ratio = 0.31, p = 0.027).
 Although A125V was associated with protection from
SLE, surprisingly the same allele was associated with
increased risk of inflammatory bowel disease.
 Functional activity: the A125V coding-change variant
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alters the DUB activity of the protein.
Structure of a NF-kB/DNA complex.
The NF-kB consists of two subunits: p50 (green) and p65
(red). NF-kB works only when two members form a dimer.
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We
pubblished
10 years
ago!
 NF-κB is the specific molecular mechanism by which
inflammation is activated in celiac mucosa
 NF-κB activation in mucosal tissue culture from healed
celiac patients exposed to gluten peaks very early at 6 hour
after exposition and fades after 24 hours
 NF-κB is costituvely activated in celiac mucosa
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NF-KB c-Rel Pathway
GLUTEN ?
NF_kB
Nuclear
Activation
Complex
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Candidate Gene in the NF-kB domain
REL gene
Function
 The REL gene encodes c-Rel, a transcription factor
that is a member of the Rel/NFKB family .
 Functionally, the gene participates in several
processes:
 positive regulation of I-kappaB kinase/NF-kappaB
cascade,
 cytokine production,
 positive regulation of interleukin-12 biosynthetic process,
 positive regulation of transcription, DNA-dependent,
 transcription from RNA polymerase II promoter).
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NF-kB include 5 genes .Three subunits, c-Rel, p65 o RelA, e RelB,
have a transactivation domain. These members of the
NF-kB family assemble homo- or heterodimers to produce
gene regulatory complexes with specific properties
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C-Rel is a main candidate
gene in CD
Family Study
Improving the recurrence risk in
sibs
180 FAMILIES WITH PROBANDS AND UNKNOWN PHENOTYPE OF SIBS
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Genotypic TDT based on three pseudo-controls
SNP
Gene
Model
Risk Allele
OR (95% CI)
p-value
rs1464510*
LPP
Additive
A
2.36 (1.64-3.41)
3∙10-6
rs2816316*
RGS1
Dominant
A
1.75 (1.07-2.86)
0.025
rs842647*
REL
Dominant
A
1.66 (1.04-2.65)
0.034
rs2327832
OLIG3
Additive
G
1.35 (0.90-2.03)
0.150
rs6441961
CCR1/CCR3
Additive
A
1.24 (0.89-1.72)
0.189
rs6822844
IL2/IL21
Additive
C
1.43 (0.82-2.49)
0.210
rs1738074
TAGAP
Dominant
A
1.31 (0.79-2.16)
0.293
rs3184504
SH2B3
Additive
A
1.19 (0.86-1.63)
0.294
rs17810546
IL12A
Additive
G
1.10 (0.80-1.51)
0.572
rs9811792
IL12A/SCHIP1
Dominant
G
1.10 (0.59-2.05)
0.753
*statistically significant results
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HLA only and HLA + 3 genes BS classifications
HLA groups from 1 to 3
VS
HLA groups 4-5
HLA-BS
Healthy
CD
Healthy
CD
Test +
105
31
55
24
Test -
111
2
161
9
Considered at risk
136 (55%)
69 (28%)
Sensitivity
(95% CI)
0.94 (0.86-0.95)
0.73 (0.58-0.88)
Specificity
(95% CI)
0.51 (0.44-0.58)
0.75 (0.69-0.80)
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Risk BY HLA HAPLOTYPE
0.25
0.20
0.15
0.10
0.05
0.00
DQ-
DQ8
DQ2
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DQ2T
DQ22
Risk Ranks for each DQ class
INCREASING RISK BY A ALLELES IN 3 GENES
0.25
0.20
0.15
RISK
DQ22
DQ2T
DQ2
DQ8
0.10
DQ-
0.05
0.00
AA Min
AAA 0,25
AAAA 0,5 AAAAA 0,75
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AAAAAA
Max
What we learn from these studies ?
EARLY NUTRITION : very difficult, it may
act on the individual case. Breast feeding
and gluten work on the phenotype.
• INFECTIONS : we do have anecdotical
experience that infections unveil the
disease, but … risk factor ?
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GENETIC PROFILE
• Certainly the most interesting and
promising domain
• Susceptibility to infections is important
• Genes regulating autoimmunity
• Possible population differences
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THEN : WHAT WE DO ?
• Start or continue a good collection of data
and files
• Whole Families are very important
• A center based BIOBANK is the next step
• Serum, DNA, Saliva : different methods to
reinforce your own collection
• Apply for research projects
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