PREVENIRE LA CELIACHIA ? CASERTA 19 MAGGIO 2012 luigi greco.

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1 PREVENIRE LA CELIACHIA ? CASERTA 19 MAGGIO 2012 luigi greco

2 Fattori Causali di una Patologia Varianza Totale di una Malattia Multifattoriale Genetica Ereditata dai Genitori Esperienza Ambientale Condivisa Esperienza non condivisa Genetica Ereditata dai Genitori Esperienza Ambientale Condivisa Esperienza non condivisa Genetica Ereditata dai Genitori Esperienza Ambientale Condivisa Esperienza non condivisa INFEZIONI MALATTIE GENETICHE

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4 Gut. 2006 Jun;55(6):803-8. Concordance, disease progression, and heritability of coeliac disease in Italian twins. Nisticò L. Fagnani C., Coto I, Percopo S, Cotichini R, Limongelli MG, Paparo F, D’Alfonso S, Giordano M, Sferlazzas C, Magazzu G, Momigliano-Richiardi P, Greco L, Stazi MA

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6 Geni + Esperienza Condivisa + Esperienza Individuale ? (peso % di ciascun elemento) CASI ‘CLINICI’ 1:1000 GENI57% Fattori comuni42% Fattori Individuali 1% INCIDENZA 1:100 GENI87% Fattori comuni12% Fattori Individuali 1%

7 La finestra della prevenzione ? Risk of celiac disease autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. JAMA. 2005 May 18;293(19):2343-51. Glutine a mesi CeliaciControlliOdds Ratio CI Estimate OR 1-3 mesi3 (6%)400,83-10,42,9 4-6 mesi12 (2%)57411 > 7 mesi36 (4%)8950,92-3,421,78 511509Solo 25 casi con biopsia

8 PREVENT-CD PROTOCOL ENROLLMENT Families with at least one member affected by CD BIRTH HLA analysis on cordon blood HLA DQ2/DQ8 positiveHLA DQ2/DQ8 negative Breastfeeding Intervention between 4°-6° month Gradual introduction of gluten after 6° month Clinical and serological evaluation every 3-6 months Persistent positivity of serological tests Clinical symptoms INTESTINAL BIOPSY Clinical follow up every year Daily intake of 100 mg of gluten AGA IgA, tTG IgA Basilio Malamisura Centro Diagnosi Celiachia - Cava de’ Tirreni

9 At 1 year 0.6% At 2 years 4.6% At 3 years 7.6% Incidence CD Basilio Malamisura Centro Diagnosi Celiachia - Cava de’ Tirreni Recruited: 1348 Randomised: 952

10 Infant at family risk of CD 4- 6 months 12 months AGA and TTG at 24, 36 and 60 months HLA-DQ2/DQ8 + tot IgA AGA and TTG at 15 months Gluten- containing diet at age 12 months Exclusive milk feeding from birth to 4-6 months Continue GFD Up to 12 months Gluten (wheat flour) introduced at 6 months Group A Group B Basilio Malamisura Centro Diagnosi Celiachia - Cava de’ Tirreni Age at gluten introduction and risk of celiac disease (CD)

11 Biopsy-proven CD 15m24m36mTot Gruppo A Gruppo B 8 17 5 30 (group size) (377) (329)(294) 1 6 10 17 (345) (325)(283) Basilio Malamisura Centro Diagnosi Celiachia - Cava de’ Tirreni

12 Journal oJ Pediatric Gastroenterology and Nutrition 7:395-399 @ 1988 Raven Press, Ltd., New York Case Control Study on Nutritional Risk Factors in Celiac Disease L. Greco, S. Auricchio, M. Mayer, and M. Grimaldi Department of Clinical Pediatrics, 2nd Faculty of Medicine, University of Naples, Naples, ltaly

13 L’allattamento al seno rinvia di circa 4 mesi la comparsa dei sintomi di celiachia L’introduzione del Glutine non ha effetto

14 Abbiamo lavorato già 20 anni fa ! Changing pattern of childhood coeliac disease in Finland Acta Paediatr Scand. 1988 May;77(3):408-12 Long breast-feeding seemed to postpone the symptoms but the introduction of gluten was of no significance We do not believe that coeliac disease has disappeared but that it will be found during the next decade Different features of coeliac disease in two neighbouring countries Arch Dis Child. 1993 Sep;69(3):375-80 It is concluded that the intake of infant cereal protein might influence when and how clinical coeliac disease appears

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16 HLA ASSOCIATED RISK

17 RISCHI DEI GRUPPI GENOTIPICI NELLA POPOLAZIONE RISCHI DEI GRUPPI GENOTIPICI NELLA POPOLAZIONEGruppo Genotipo DR Gruppo genotipico DQ Rischio % H1/H1DR3/DR3 G1 (Doppio DQ2) 21 % H1/H2DR3/DR7 H2/H3DR5/DR7G2 (DQ2 in trans) 17 % H1/H3DR3/DR5 G3 (DQ2 in cis) 6 % H1/H4DR3/DR4 H1/H5DR3/DRX* H2/H2DR7/DR7 G4 (1/2 DQ2 e/o DQ8) 5 % H2/H4DR7/DR4 H4/H4DR4/DR4 H5altriG5 0,6 % rischio per un individuo di sviluppare la malattia secondo il suo gruppo genotipo

18 La soglia della sensibilità al glutine dipende dallo status HLA e dalla quantità di peptidi della gliadina riconosciuti e presentati alle cellule T, fino a superare una soglia di ‘intollerabilità’ in soggetti con specifici profili genomici

19 Effetto dose del Glutine Il glutine è capace, in soggetti con peculiare profilo genetico, di stimolare la risposta adattativa dei CD8 T nella lamina propria, ma anche di espandere i Linfociti Intraepiteliali indipendentemente dalla presentazione HLA. Questo produce induzione di apoptosi, proteine da stress MIC-A/B, inibizione dell’endocitosi da Epidermal Growth Factor, stimolazione delle MAP kinasi e produzione eccessiva di IL-15 Un peptide del glutine lega la classe I dell’HLA-E, ligando delle cellule Natural Killer CD94 – NKG2A Un Toll-like receptor è coinvolto nella azione del glutine nell’immunità innata

20 Stima del rischio in base al genotipo dei genitori Rischio > 20% 15% < Rischio < 20% 10% < Rischio < 15% 1% < Rischio < 10% Rischio < 1% Rischio per un fratello di un probando in accordo al genotipo DQ dei genitori DQB1*02/02 DQA 05/05 DQA 07/03

21 PROBABILITA’ PER UN FRATELLO DI UN PROBANDO DI APPARTENERE A Gi E RISCHIO CORRISPONDENTE PROBABILITA’ PER UN FRATELLO DI UN PROBANDO DI APPARTENERE A Gi E RISCHIO CORRISPONDENTE 0.12 0.17 0.24 0.05 0.42 Risk < 1 % 1% < Risk < 10 % Risk > 20 % Doppio DQ2 Un solo DQ2 DQ8 o 1/DQ2

22 Che dobbiamo fare ? Scoraggiare un matrimonio ?

23 IT IS NOT ONLY HLA !!!

24 0,20 0,04

25 ChrSNPLocusA1A2 CD cases, Controls Ratios MAF CD MAF Controls χ2χ2 p- value OR (95% CI) 1rs2816316RGS1AC1092:182,1169:2530.140.186.100.01351.299 (1.055-1.598) 2rs917997IL18RAPGA965:309, 1092:3300.240.230.4080.52310.9438 (0.7901-1.127) 2rs842647RELAG967:307, 1080:3420.24 0.0010.97740.9974 (0.8357-1.190) 3rs6441961CCR3GA741:533, 906:5160.410.368.7070.00320.7918 (0.6780-0.9247) 3rs17810546SCHIP1AG1158:116, 1312:1100.090.071.6410.20020.8370 (0.6373-1.099) 3rs1464510LPPCA646:628, 844:5780.490.4020.326.5E-060.7045 (0.6048-0.8206) 4rs13119723KIAA1109AG1135:139, 1220:1960.110.145.2850.02151.312 (1.040-1.654) 4rs13151961KIAA1109AG1139:135, 1225:1950.100.146.1430.01321.343 (1.063-1.697) 6rs2327832OLIG3AG1008:248,1162:2560.190.231.2460.26430.8955 (0.7376-1.087) 6rs1738074TAGAPGA681:593, 816:6040.460.424.3760.03650.8500 (0.7299-0.9899) 19rs3760746TNFSF14TC318:276, 461:3750.420.410.3620.54730.9372 (0.7588-1.158) Chr: chromosome; A2: minor allele; MAF: minor allele frequency; OR: odds ratio; CI: confidence interval; χ 2 : χ-square test; allelic p-value was calculated by 2x2 two-sided χ-square test; bold indicates p-value < 0.05 372 CD-Cases vs 451 Controls Regulator of G-protein signalling Case-control association analyses at the single SNP level Chemokine receptor Structural role in maintaining cell shape and motility; it may be involved in signal trasduction and activation of gene transcription Endopeptidase Rho GTPase activatin protein Subunit of NF-kB

26 Pathway of c-Rel in NF-KB GLUTINE ? COMPLESSO DI ATTIVAZIONE NUCLEARE NF_kB

27 Integration of immunological pathways and celiac disease (CD)-associated genes into a model of CD pathogenesis. The figure is subdivided into three distinct anatomical regions in which T cell differentiation (thymus), T cell polarization (inductive site), and effector immune response (effector site) take place. Genes associated with CD by genome-wide association studies are listed in red according to their potential implication in distinct immunological pathways. THEMIS and RUNX3 are involved in the thymic differentiation of CD4 and CD8 T cells, respectively. Dendritic cells located in the lamina propria acquire a proinflammatory phenotype upon viral recognition (TLR7/8 and IRF4) and migrate to the mesenteric lymph nodes (inductive site). There, they present gluten peptides (HLA-DQA1, HLA-DQB1, and CIITA) to naive CD4 T cells and promote T cell activation (e.g., CD28, CD80, CTLA4, CD247, PTPN2, SH2B3, TAGAP, IL2, and FASLG) and differentiation into inflammatory effector T cells (IL12A, IL18R1, IL18RAP, IL1RL1, and IL1RL2). In addition, transglutaminase 2 (TG2) and gluten-specific B cells (that have internalized gluten-TG2 complexes) receive help from gluten-specific T cells, become activated, and differentiate into immunoglobulin (Ig)A- and IgG-producing plasma cells (ICOS, ICOSLG, IL21, and RGS1). Other genes regulate activation and migration of cytotoxic intraepithelial lymphocytes (IELs) (MAP3K7, IL-21, CCR9, and RGS1). Finally, some genes are involved in cell migration [e.g., genes coding for chemokine receptors (CCRs) and ITGA4], and others regulate tumor necrosis factor (TNF)-dependent pathways (TNFAIP3, TNFSF4, TNFSF18, TNFRSF9, and TNFRSF14). Even though their genes have not been identified by genetic studies, interleukin (IL)-15 and interferon (IFN)-α play a critical role in orchestrating the immune responses that lead to CD pathogenesis. IL-15 upregulates activating natural killer cell (NK) receptors and licenses IELs to kill epithelial cells, whereas IFN-α promotes the differentiation of proinflammatory dendritic cells. Abbreviations: HLA, human leukocyte antigen; TGF, transforming growth factor; Th, T helper cell. RGS1 : controls the homing of IEL

28 Lpp -/- mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Vervenne et al., 2009 Lpp -/- mouse model

29 LPPc-RELRGS1 DQ22DQ2TDQ2DQ8DQ-- CCAG+GGAC+CC 0,210,170,060,050,01 CCAAAC+CC 0,240,200,060,070,01 CCAG+GGAA 0,230,190,080,060,01 CCAA 0,250,210,070,080,01 ACAG+GGAC+CC 0,260,220,090,080,01 ACAAAC+CC 0,280,230,10 0,02 ACAG+GGAA 0,270,230,120,090,02 ACAA 0,290,240,11 0,02 AAAG+GGAC+CC 0,290,240,130,120,03 AA AC+CC 0,300,250,150,130,04 AAAG+GGAA 0,300,250,150,120,03 AA 0,300,260,160,140,05 Increase of the a priori HLA Risk by adding LPP, c-REL, RGS1

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31 No Dq2 or DQ8 Double DQB1*02 DQ8

32 Coeliac Sibs

33 ROC - Performance of the predicting model

34 Università degli Studi di Napoli “Federico II” But the GENOMA is a written book : Life is the EXPRESSION of Genes

35 EXPRESSION Biopsie intestinali - 1 Sperandeo et al, 2011 4q27

36 Discriminant Analysis between Coeliacs and Controls by the Expression of Candidate Genes in Intestinal Mucosa Wilks' Lambda df1 Exact F Sig. 1TNFAIP3,404 159,002,000 2IL21,300 245,521,000 3REL,261 335,809,000 4RGS1,235 430,143,000 5 LPP,222 525,272,000 Wilk’s Lambda shows the ability to discriminate between Coeliac and Controls 1--------------------------------0 Wilks’ Lambda

37 Classification results Predicted Group Membership Total STATUSControlCeliac CountControl19120 Celiac22022 %Control955100 Celiac1090100 92,9% of original grouped cases correctly classified.

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39 EXPRESSION Monociti da Sangue Periferico  Endopeptidasi  Si suppone che nei mammiferi abbia un ruolo nella regolazione della crescita delle cellule epiteliali e nella differenziazione e sviluppo tumorale (Kuo et al., 2006)  Subunità del complesso NF-kB  Può innescare ma anche terminare (feedback - ) i meccanismi di azione pro-infiammatoria di NF-kB (Bonizzi et al, 2004, Lawrence et al, 2005).  Conosciuto anche come Herpesvirus Entry Mediator (HVEM). Interessamento infiammatorio a livello periferico

40 Classification based on the expression of 4 gene in blood leucocytes

41 Diseases in symptomatic cases RegionsGI SymptomsAnaemiaOsteopeniaAbnormal Liver Short Stature Children North214.069100.73875.55425.18510.047 N.East18.5578.7336.5502.183927 M.East123.44758.09343.57014.5238.849 East52.90324.89618.6726.2244.535 South252.846118.98689.24029.74721.125 MED tot657.119309.232231.92477.30841.609 Possiamo prevenirne almeno una parte ????