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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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 anti-gliadin

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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 NLatency (mt)

F p

Breast 26,0 6,3    

Bottle 120,0 4,0    

      5,15 0,025

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    

      0,708 0,552

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Mean age at first symptoms

Milk N Mean (mt) F p

Breast 26,0 8,91    

Bottle 120,0 6,30    

      6,64 0,01

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    

      0,604 0,60

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PROFILE OF CD CANDIDATE GENESan alternative point of view

• COMMON TO OTHER AUTOIMMUNE DISEASES• CELL STRUCTURE AND SHAPEMOLECULAR 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 geneFunction: This receptor was identified as a cellular mediator of herpes simplex virus (HSV) entry.

2. TNFSF14 geneFunction: 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 geneFunction : 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

Nuclear Activation

gluten

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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

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Evolutionary and Functional Analysis of Celiac Risk Loci Reveals SH2B3 as a Protective Factor against

Bacterial InfectionThe 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 immune-related 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

risk-allele A with IL1b production (p = 0.034 for trend)…

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PROFILE OF CD CANDIDATE GENESan alternative point of view

• COMMON TO OTHER AUTOIMMUNE DISEASES• CELL STRUCTURE AND SHAPE• MOLECULAR RECEPTION OF VIRAL (?) PARTICLESNATURAL 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-B-dependent 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 de-ubiquitination of several NF-kB signalling factors.ELFID-UNINA

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

alters the DUB activity of the protein.ELFID-UNINA

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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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

We pubblished

10 years ago!

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NF-KB c-Rel PathwayGLUTEN ?

NF_kB Nuclear

Activation Complex

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Candidate Gene in the NF-kB domain 

REL geneFunction

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

C-Rel is a main candidate gene in CDELFID-UNINA

Family Study

Improving the recurrence risk in sibs

180 FAMILIES WITH PROBANDS AND UNKNOWN PHENOTYPE OF SIBS

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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

Genotypic TDT based on three pseudo-controls

*statistically significant results ELFID-UNINA

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HLA groups from 1 to 3VS

HLA groups 4-5HLA-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)

HLA only and HLA + 3 genes BS classifications

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Risk Ranks for each DQ class

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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 differencesELFID-UNINA

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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