GENETICA ED EPIGENETICA DEI GENI CANDIDATI NELLA MALATTIA CELIACA: ANALISI MOLECOLARE DEI MECCANISMI DI REGOLAZIONE

Per quanto concerne la malattia celiaca non è stata identificata una forte associazione con l’alterazione in un singolo gene, nonostante siano stati numerosissimi i lavori condotti in questo senso. Oltre all’implicazione di meccanismi di regolazione genica di altro tipo (epigenetica, microRNA ecc.), una possibile spiegazione del fallimento di questo tipo di approccio è che la patologia non sia determinata dal “default” di un singolo meccanismo, ma bensì sia il risultato da una serie di alterazioni a carico di processi fisiologici che nell’ insieme determinano l’insorgere del “fenotipo celiaco”. Lo scopo del nostro studio è stato quello di valutare le eventuali differenze nei livelli di espressione di alcuni geni candidati nella malattia celiaca in diversi distretti cellulari tra pazienti celiaci e controlli. In primo luogo ci siamo soffermati sull’ analisi dei livelli di espressione in cellule mononucleate di sangue periferico(PBMC) di soggetti predisposti geneticamente e con una accertata familiarità per la malattia celiaca. La coorte da noi analizzata consiste in bambini predisposti geneticamente (HLA DQ 2 e/o 8 positivi) con almeno un familiare di primo grado affetto da MC, arruolati e seguiti in follow-up dalla nascita al compimento di 6 anni di età. Grazie alle particolari caratteristiche di questi soggetti, che presentano un rischio molto elevato di sviluppare la celiachia, è stato possibile condurre un’ analisi longitudinale, avendo a disposizione campioni raccolti sia prima dello sviluppo della malattia, sia al momento della diagnosi, sia in remissione clinica. L’unicità della nostra popolazione ci ha permesso di indagare l’esistenza di eventuali alterazioni molecolari presintomatiche e di valutarne poi l’evoluzione al momento della diagnosi della patologia e dopo un anno a dieta senza glutine. In aggiunta alla valutazione dei livelli di espressione in questa è stata effettuata anche una analisi genotipo dei geni candidati, in quanto essendo soggetti appartenenti a famiglie a rischio era importante una corretta stratificazione dei fattori di rischio genetico di ciascun componente. Tutti gli studi che nella MC si sono concentrati sulla mucosa intestinale, hanno analizzato il tessuto senza fare distinzione tra i due compartimenti che lo compongono e che presentano caratteristiche molto diverse: l'epitelio e lamina propria. Ad oggi , non è stato mostrato chiaramente in che modo ciascuna componente della mucosa intestinale sia coinvolta nel processo patogenetico della celiachia. Quindi partendo da queste premesse nella seconda parte dello studio sono stati analizzati gli stessi geni candidati studiati nella prime parte della ricerca,la presenza di alterazioni sia geniche che epigenetiche(in particolare la metilazione) in cellule intestinali selezionate sia di natura epiteliale che appartenenti alla lamina propria. Lo scopo finale di questo lavoro di tesi è stato,dunque, la valutazione globale delle alterazioni di espressione genica in geni candidati, in modo da tentare di identificare a livello di singole e distinte popolazioni cellulari, eventuali alterazioni costanti e coerenti con il quadro patogenetico della MC, e in seconda battuta di definire i meccanismi di regolazione epigenetica che sottendono tali alterazioni

Respiratory infections and the risk of celiac disease

BACKGROUND AND OBJECTIVES: The increasing incidence of celiac disease (CD) suggests that common infections before the onset of autoimmune diseases could be an important factor in switching the immune response. We aimed to explore the relationship between early clinical events and the development of CD in genetically predisposed infants. METHODS: In this study, 373 newborns from families with at least 1 relative with CD were recruited, and human leukocyte antigen DQ2- or DQ8-positive infants were followed up with clinical and serological evaluations. Cross tabulation and odds ratios were used to explore the risk associated with single variables, and logistic regression analysis was performed to determine the variables that contributed to the risk of developing CD. Stepwise discriminant analysis was used to determine which variables could distinguish case patients from controls before diagnosis. RESULTS: The cumulative incidence of CD in this cohort was 6% at 3 years and 13.5% at 5 years of age, and l34 children (14%) developed CD before the sixth year of life. An analysis of adverse events showed a higher frequency of respiratory tract infections among CD patients during the first 24 months of life. In a stepwise discriminant analysis, which included sex and human leukocyte antigen risk class, only respiratory infections in the second and first years of life significantly contributed to discrimination of case patients versus controls. CONCLUSIONS: A multivariate model of discriminant analysis showed that the frequency of respiratory infections in the first 2 years of life could distinguish children who developed CD from those who did not

Gluten consumption and inflammation affect the development of celiac disease in at-risk children

: Gene expression, lipidomic and growth impairment findings suggest that the natural history of celiac disease (CD) starts before the gluten-induced immune response. Gluten intake in the first years of life is a controversial risk factor. We aimed to estimate the risk of developing CD associated with the amount of gluten intake and the serum inflammatory profile in genetically predisposed infants. From an Italian cohort of children at risk for CD, we enrolled 27 children who developed CD (cases) and 56 controls matched by sex and age. A dietary interview at 9, 12, 18, 24 and 36 months was performed. Serum cytokines (INFγ, IL1β, IL2, IL4, IL6, IL10 IL12p70, IL17, and TNFα) were analysed at 4 and 36 months. Infants who developed CD by 6 years showed an increase in serum cytokines (INFγ, IL1β, IL2, IL6, IL10, IL12p70 and TNFα) at 4 months of age before gluten introduction. CD cases ate significantly more gluten in the second year of life than controls, and gluten intake in the second year of life was strongly correlated with serum cytokines (INFγ, IL2, IL4, IL12p70, IL17) at 36 months only in CD cases. The dietary pattern of infants who developed CD was characterized by high consumption of biscuits and fruit juices and low intake of milk products, legumes, vegetables and fruits. Genetically predisposed infants who developed CD showed a unique serum cytokine profile at 4 months before gluten consumption. The amount of gluten was strongly correlated with an inflammatory profile in serum cytokines at 36 months only in infants who developed CD

Pre-symptomatic Diagnosis of Celiac Disease in Predisposed Children: the Role of Gene Expression Profile

The prevalence of Celiac Disease (CD) has increased significantly in recent years, and risk prediction and early diagnosis has become imperative especially in at risk families. In a previous study, we identified individuals with CD based on the expression profile of a set of candidate genes in peripheral blood monocytes. Here we evaluated the expression of a panel of CD candidate genes in peripheral blood mononuclear cells (PBMCs) from at risk infants long time before any symptom or production of antibodies

In Celiac Disease Patients the In Vivo Challenge with the Diploid Triticum monococcum Elicits a Reduced Immune Response Compared to Hexaploid Wheat

Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. We analysed gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1β, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFβ, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies

Gene Expression Profile of Peripheral Blood Monocytes: A Step towards the Molecular Diagnosis of Celiac Disease?

<div><p>Aim</p><p>Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls.</p> <p>Participants</p><p>Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn’s disease patients) were enrolled.</p> <p>Results</p><p>Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%.</p> <p>Conclusions</p><p>The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.</p> </div

mRNA expression of candidate genes in peripheral blood monocytes.

<div><p>A) <i>KIAA1109</i>expression was higher in CD (<i>p</i>=0.05) and in CD-GFD patients (<i>p</i>=0.05) than in controls, but also in Crohn peripheral monocytes (<i>p</i>=0.02); B) <i>c-REL</i> expression was lower in CD peripheral monocytes than in controls (<i>p</i><0.01), but become higher than controls (<i>p</i><0.01) and CD (<i>p</i><0.01) after one year of GFD; the same profile was observed in Crohn peripheral monocytes; C) <i>SH2B3</i> expression was lower in CD versus controls (<i>p</i>=0.04) whereas it was significantly higher in Crohn and CD-GFD patients versus controls (<i>p</i><0.01) and CD (<i>p</i><0.01). D) <i>LPP</i> expression was lower in CD peripheral monocytes than in controls (<i>p</i>=0.04); E-F) <i>TNFAIP3</i> and <i>RGS1</i> genes expression were lower in CD peripheral monocytes versus controls (<i>p</i><0.01) and higher in Crohn patients versus controls (<i>p</i><0.01) and CD patients (<i>p</i><0.01). Both genes expression levels normalized after one year of GFD;.</p> <p>RQ: relative quantification; Ctr: controls; CD: celiac disease; CD-GFD: celiac patients on a gluten-free diet; * <i>p</i><0.01, **<i>p</i><0.05.</p></div

Analysis of the levels of mRNA expression of associated genes in duodenal tissue.

<div><p>A) IL-21 is over-expressed in CD compared to controls (<i>p</i><0.01), B) IL-2 shows a very small trend of increase in CD compared to controls; C) and D) Expression of the KIAA1109 and cREL genes: the patterns are very similar and did not show any variations among the three groups.</p> <p>RQ: relative quantification; Ctr: controls; CD: celiac disease; CD-GFD: celiac patients on a gluten-free diet; * <i>p</i><0.01, **<i>p</i><0.05.</p></div

Distribution of the Discriminant Score of CD, Controls, Crohn and CD patients on gluten free diet.

<p>The D-score clearly separated the four groups of subjects evaluated. Only CD patients had a negative D-score. The D-score of CD patients on gluten free diet was intermediate between the scores of controls and Crohn patients.</p