Free Powerpoint Templates 1 La nuova celiachia ? Diagnosi Serologica, Istologica o… molecolare ! Discutiamone con luigi greco L’Anno del Signore 2013 European.

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1 Free Powerpoint Templates 1 La nuova celiachia ? Diagnosi Serologica, Istologica o… molecolare ! Discutiamone con luigi greco L’Anno del Signore 2013 European Laboratory for Food Induced Diseases Univ. Federico II

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4 4 Come primo test si raccomanda la ricerca di IgA anti-TG2 Gli anticorpi anti-endomisio sono di conferma per la diagnosi I kit rapidi non sono destinati a sostituire le prove di laboratorio o a fornire una diagnosi definitiva. Devono sempre essere confermati. I test per la rilevazione di anticorpi IgG o IgA antigliadina non devono essere utilizzati per la diagnosi di malattia celiaca. I test che misurano gli anticorpi sierici contro peptidi deamidati della gliadina non aggiungono alcuna precisione meglio degli Anti-TGASI Anticorpi della celiachia: raccomandazioni

5 Free Powerpoint Templates 5 Correlazione tra sierologia e istologia La severità della lesione istologica correla con i valori di IgA anti-tTG La specificità degli anticorpi IgA anti-tTG per la diagnosi della celiachia è del 93% (92-95); ed è più alta nel caso degli EMA (96%; 94-98). In queste circostanze la biopsia è ancora necessaria?

6 Free Powerpoint Templates 6 Il Network MEDICEL – Mediterranean Celiac

7 Free Powerpoint Templates 7 Tutto si basa sulla relazione tra livello di TTGASI e Danno Istologico (Marsh stage) - Italia

8 Free Powerpoint Templates 8 Correlation between tTG-log and Marsh stage CountrySpearman Rhop value Spain-0.090.5 France0.0620.6 Naples0.530.0001 Sicily0.370.008 Malta0.180.9 Slovenia0.130.33 Croatia-0.070.6 Bosnia0.090.58 Albania0.420.1 Montenegro0.250.079 Greece0.0070.9 Turkey0.2010.16 Egypt0.170.3 Tunisia0.1940.17 Algeria-0.1410.32 Morocco0.330.018 Ma questa base di sicurezza non c’è dovunque !

9 Free Powerpoint Templates 9 DQ2-8 DR3-7 Chrom 5q31-33 CTLA4 Chrom 19p13.1 MIC-A-B / TNF Chrom 11, 9, 15 HLA DR Extended Carmela è ….. …ggeniale !! E centinaia? di altri geni ad effetto minore

10 Free Powerpoint Templates 10 Quali geni spiegano la fortissima predisposizione genetica ?

11 Free Powerpoint Templates 11 Manca almeno metà della ereditarietà : Chi spiega il resto ?? Nature Genetics, May 2012, 483 MalattiaEreditarietà% Varianza spiegata N. di Polimorfismi SNPs Celiachia0,5-0,8744% (40-47)2550 Diabete0,2-0,6949% (46-53)2919 Artrite Reumatoide 0,53-0,6818% (15-20)2231 Infarto0,3-0,6348% (43-54)1766 Polimorfismi in geni molto comuni con rischio bassissimo : da 1,001 a 1,1 : cioè quasi indistinguibile dal non malato, ma speculabile con enormi 10.000 campioni !

12 Free Powerpoint Templates 12 Siete pronti ??? Fisiologia ??? European Laboratory for Food Induced Diseases Dobbiamo sceglierne qualcuno !!

13 Free Powerpoint Templates 13 Ci sarà un 1% di differenza anche tra di loro !!!!

14 Free Powerpoint Templates 14 COSA CI DICONO I GENI ? Non vi sono geni ‘deficitari’ : tutto funziona come nei normali Vi sono geni di vantaggio per le difese da virus e batteri : I nostri ‘antibiotici’ naturali I geni della ‘celiachia’ sono quelli comunemente usati nella complessa reazione immunitaria Condividiamo molti geni con altre patologie immunitarie European Laboratory for Food Induced Research Federico II

15 Free Powerpoint Templates 15 cREL AND TNFAIP3…REGULATORS OF NFkB – Maiuri & Greco 2993 TNFAIP3 cREL Just 6 hours after gluten exposure the NF-kB complex is fully activated. cREL is one of the subunit of the complex TNFAIP3 acts in a negative feedback loop to control NF- kB-dependent gene expression

16 Free Powerpoint Templates 16 IL-21: A CRUCIAL ROLE IN THE ACTIVATION OF THE GLUTEN-INDUCED CD4 CELLS IL-21

17 Free Powerpoint Templates 17 RGS1: REGULATES THE HOMING OF I.E.L RGS1 Elevated RGS1 levels profoundly reduce T cell migration to lymphoid-homing chemokines, whereas RGS-1 depletion selectively enhances such hemotaxis in gut T cells. Its capacity to limit egress of inflammatory and/or autoimmune cells could clearly promote immunopathology.[

18 Free Powerpoint Templates 18 LPP: AN INTEGRAL COMPONENT OF CELL MIGRATION. LPP gene has an activity as cell adhesion and is an integral component of cell migration. Regulation of LPP expression results in a increase or decrease cell migration. Over-expression of LPP increased Epidermal Growth Factor-stimulated migration of vascular SMCs induced by TGF-β1, confirming the role of LPP in cell motility.

19 Free Powerpoint Templates 19 KIAA1109: TH17 CELLS DIFFERENTIATION The KIAA1109 gene is in the region of /Tenr/IL2/IL21 in chromosome 4q27, many times replicated in association studies OF CD. This region is involved in differentiation of naïve human CD4+ T cells into Th17 cells, which produce a variety of cytokines including IL-17A, IL- 17F, IL-21 and IL-22. Genetic alterations in the 4q27 locus could results in non- functional IL-21 and hence lack of IL-17A or vice versa. KIAA1109/Tenr/IL2/ IL21

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21 Free Powerpoint Templates 21 Rischio a posteriori per classe HLA sommando gli alleli di rischio “A” DQ2 omozigote: 21%  30% DQ2 in trans: 17%  26% DQ2 eterozigote: 6%  17% DQ8: 5%  13% DQ negativi: 0.6%  5% Percentuali di rischio in base all’HLA rispetto all’aggiunta degli alleli di rischio per i tre SNP associati. DQ2 omozigote: 21%  30% DQ2 in trans: 17%  26% DQ2 eterozigote: 6%  17% DQ8: 5%  13% DQ negativi: 0.6%  5% Percentuali di rischio in base all’HLA rispetto all’aggiunta degli alleli di rischio per i tre SNP associati.

22 Free Powerpoint Templates 22 No Dq2 or DQ8 Double DQB1*02 DQ8 N° di alleli di rischio “A” Aumento del rischio a priori per classe HLA sommando gli alleli di rischio “A” per i 3 SNP analizzati

23 Free Powerpoint Templates 23 Risultati dell’applicazione del modello predittivo Soggetti in esame Rischio HLA Rischio NON-HLA Combinazione rischio HLA + NON-HLA 101 fratelli BASSO Classe G5 20 INTERMEDIO Classi G3-G4 48 ALTO Classe G1-G2 33 ALTO Score bayes ≥ mediana 26 ALTO 33+26= 59 BASSO 20+22= 42 BASSO Score bayes ≤ mediana 22

24 Free Powerpoint Templates 24 ROC - Performance of the predicting model

25 Free Powerpoint Templates 25 STUDI DI ESPRESSIONE GENICA I Step: Analisi dell’espressione genica dei geni candidati in pazienti celiaci e controlli sani in diversi tessuti umani: Mucosa intestinale Linee cellulari di linfociti T Monociti isolati da sangue periferico I Step: Analisi dell’espressione genica dei geni candidati in pazienti celiaci e controlli sani in diversi tessuti umani: Mucosa intestinale Linee cellulari di linfociti T Monociti isolati da sangue periferico La genomica da sola è in grado di spiegare solo una quota della componente genetica della celiachia, la rimanente percentuale è attribuibile a meccanismi di epigenetica: Regolazione dell’espressione genica Metilazione del DNA Azione di micro-RNA La genomica da sola è in grado di spiegare solo una quota della componente genetica della celiachia, la rimanente percentuale è attribuibile a meccanismi di epigenetica: Regolazione dell’espressione genica Metilazione del DNA Azione di micro-RNA

26 Free Powerpoint Templates 26 DUODENAL BIOPSIY SH2B3 expression was higher in CD mucosa than in controls TAGAP was significantly up-regolated in CD mucosa TNFSF14 levels were higher in CD patients and in CD-GFD patients TNFRSF14.TNFAIP3, and RGS1mRNA levels show a modest up- regulation in CD patients versus controls also if this difference is not statistically significant SH2B3 expression was higher in CD mucosa than in controls TAGAP was significantly up-regolated in CD mucosa TNFSF14 levels were higher in CD patients and in CD-GFD patients TNFRSF14.TNFAIP3, and RGS1mRNA levels show a modest up- regulation in CD patients versus controls also if this difference is not statistically significant

27 Free Powerpoint Templates 27 DUODENAL BIOPSY IL-21 are more elevated in CD than in control IL-2, KIAA1109 mRNA do not show significantly variations among the two groups but they seem to have an up-regulating trend in CD cREL expression was lower in CD monocytes compared with controls IL-21 are more elevated in CD than in control IL-2, KIAA1109 mRNA do not show significantly variations among the two groups but they seem to have an up-regulating trend in CD cREL expression was lower in CD monocytes compared with controls

28 Free Powerpoint Templates 28 DISCRIMINANT ANALYSIS IN DUODENAL BIOPSY The aim of the discriminant analysis is to weigh the discriminating capacity of each single gene to obtain a single new composite variable. 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 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 1-------------------------------0 Wilks’ Lambda Wilk’s Lambda shows the ability to discriminate between Coeliac and Controls

29 Free Powerpoint Templates 29 CLASSIFICATION RESULTS 92,9% OF ORIGINAL GROUPED CASES CORRECTLY CLASSIFIED.

30 Free Powerpoint Templates 30 DIFFERENT EXPRESSION PROFILES IN CASES AND CONTROLS

31 Free Powerpoint Templates 31 PERIPHERAL BLOOD MONOCYTES KIAA1109 gene was over-expressed in CD, Crohn and CD-GFD vs controls c-REL was lower in CD monocytes SH2B3 was lower in CD monocytes LPP expression was lower in CD monocytes TNFAI3 mRNA showed a modest diminution in CD patient among control and CD-GFD RGS1 was lower in CD monocytes KIAA1109 gene was over-expressed in CD, Crohn and CD-GFD vs controls c-REL was lower in CD monocytes SH2B3 was lower in CD monocytes LPP expression was lower in CD monocytes TNFAI3 mRNA showed a modest diminution in CD patient among control and CD-GFD RGS1 was lower in CD monocytes

32 Free Powerpoint Templates 32 DISCRIMINANT ANALYSIS IN MONOCYTES we developed a similar linear discriminant analysis for the gene expression in peripheral blood monocytes. By stepwise statistic entered the expression of four candidate genes were selected by multivariate analysis of CD, similar to the results obtained in the duodenal tissue. By computing the discriminant score and the relative membership probability, 95.5% of patients (91% controls and 100% celiac patients) were correctly classified.

33 Free Powerpoint Templates 33 HOW MUCH GENE EXPRESSION IN MONOCYTES SEPARATE CELIACS FROM CONTROLS ?

34 Free Powerpoint Templates 34 DISCRIMINANT ANALYSIS IN PERIPHERAL BLOOD MONOCYTES The D-Score for active celiac patients was negative in all cases, while it was positive for all the other groups on differentiated clusters.

35 Free Powerpoint Templates 35 OUR BIOLOGICAL HYPOTHESIS! RGS1 KIAA110 9 RGS1 IL-21 RGS1 LPP ??? LPP ??? RGS1 IL-21 cREL/TNFAIP 3

36 Free Powerpoint Templates 36 CONCLUSIONS We obtained a stimulating suggestion of the possible relationships among the expression of candidate genes in the target mucosa, and in the peripheral blood monocytes. The analysis of the expression of each single genes does not permit to identified an overview of pathogenic mechanism of CD, whereas with a discriminant analisys approach we identified different functional pathways may be involved in the complex gluten induced abnormal responses! We now suggest that the expression of a small set of candidate genes identified CD patients without considering clinical data, HLA and anti-tTG antibodies.

37 Free Powerpoint Templates 37 Per quanto ancora ? FAREMO UNA BIOPSIA ‘VIRTUALE PER ESPLORARE LO STATO FUNZIONALE DELLA MUCOSA INTESTINALE ?