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Background and Aims An expanding number of monogenic defects have been identified as causative of severe forms of very early-onset inflammatory bowel diseases [VEO-IBD]. The present study aimed at defining how next-generation sequencing [NGS] methods can be used to improve identification of known molecular diagnosis and to adapt treatment. Methods A total of 207 children were recruited in 45 paediatric centres through an international collaborative network [ESPGHAN GENIUS working group] with a clinical presentation of severe VEO-IBD [n = 185] or an anamnesis suggestive of a monogenic disorder [n = 22]. Patients were divided at inclusion into three phenotypic subsets: predominantly small bowel inflammation, colitis with perianal lesions, and colitis only. Methods to obtain molecular diagnosis included functional tests followed by specific Sanger sequencing, custom-made targeted NGS, and in selected cases whole exome sequencing [WES] of parents-child trios. Genetic findings were validated clinically and/or functionally. Results Molecular diagnosis was achieved in 66/207 children [32%]: 61% with small bowel inflammation, 39% with colitis and perianal lesions, and 18% with colitis only. Targeted NGS pinpointed gene mutations causative of atypical presentations, and identified large exonic copy number variations previously missed by WES. Conclusions Our results lead us to propose an optimised diagnostic strategy to identify known monogenic causes of severe IBD.

Early-onset inflammatory bowel diseases can result from a wide spectrum of rare mendelian disorders. Early molecular diagnosis is crucial in defining treatment and in improving life expectancy. Herein we aimed at defining the mechanism of an immunodeficiency-polyendrocrinopathy and enteropathy-X-linked (IPEX)-like disease combined with a severe immunodeficiency in 2 siblings born from distantly related parents.Whole exome sequencing was performed on blood-extracted genomic DNA from the 2 affected children and their parents on the genomic platform of Institut IMAGINE. Candidate gene mutation was identified using the in-house software PolyWeb and confirmed by Sanger sequencing. Protein expression was determined by western blot. Flow cytometry was used to assess consequences of the mutation on lymphocyte phenotype and nuclear factor-kappa B (NF-κB) activation at diagnosis and after treatment by hematopoietic stem cell transplantation.We identified a homozygous missense mutation in mucosa-associated lymphoid tissue lymphoma translocation 1 gene (MALT1), which precluded protein expression. In keeping with the known function of MALT1, NF-κB-dependent lymphocyte activation was severely impaired. Moreover, there was a drastic reduction in Forkhead box P3 (FOXP3) regulatory T cells accounting for the IPEX-like phenotype. Following identification of the mutation, both children received hematopoietic stem cell transplantation, which permitted full clinical recovery. Immunological workup at 6 and 12 months after transplantation showed normal NF-κB activation and correction of regulatory T cells frequency.Along with FOXP3, interleukin 2 receptor alpha chain (IL2RA), and cytotoxic T-lymphocyte protein 4 precursor (CTLA-4) mutations, MALT1 deficiency should now be considered as a possible cause of IPEX-like syndrome associated with immunodeficiency that can be cured by hematopoietic stem cell transplantation.

Background: Lamina propria T lymphocytes (LPL) of the intestinal mucosa are chronically activated in Crohn’s disease (CD). Defective apoptosis of activated LPL was proposed as a key pathogenic mechanism. In fact, increased expression of antiapoptotic molecules was observed in CD LPL. In the present work, we aimed to analyse the effects and underlying molecular mechanisms of 5-amino salicylic acid (5-ASA) and derivatives on apoptosis of LPL and peripheral blood lymphocytes (PBL) in patients with CD compared with ulcerative colitis (UC) and in non-inflammatory controls. Methods: PBL and LPL were isolated by Ficoll-Hypopaque gradient centrifugation and the EGTA-collagenase method, respectively. PBL/LPL were stimulated with FasL, 5-ASA, sulphapyridine, and sulphasalazine for 24/48 hours and apoptosis was quantified by flow cytometry (annexin V- propidium iodide method) and immunofluorescence. The molecular mechanisms of drug induced apoptosis were analysed in wild-type and FADD−/− Jurkat T cells using western blots and caspase assays. Results: While PBL displayed a normal apoptosis pattern after Fas stimulation in patients with active CD, LPL from inflammatory areas were highly resistant. Comparable resistance to apoptosis was observed in LPL of UC patients. In contrast with 5-ASA, which did not induce apoptosis in lymphocytes, sulphasalazine proved to be a potent proapoptotic agent. Sulphasalazine induced T lymphocyte apoptosis was independent of the Fas pathway but associated with marked downregulation of antiapoptotic bcl-xl and bcl2, activation of the mitochondrial apoptosis signalling pathway, and subsequent activation of caspase-9 and caspase-3. Conclusion: The beneficial effect of sulphasalazine in treating inflammatory bowel disease is at least in part attributable to its proapoptotic effects on LPL which allows potent downregulation of lymphocyte activation.

Early-onset inflammatory bowel disease starting within the first months of life could be due to a particular genetic defect. We set up the GENetically determined ImmUne-mediated enteropathieS (GENIUS) network and collected infants with a proven defect of the IL10 axis for accurate phenotyping of disease presentation and evolution.Ten patients with early-onset inflammatory bowel disease with confirmed mutations in IL10, IL10RA, or IL10RB genes were characterized on clinical, endoscopic-histological, immunobiological, and radiological findings. Functional assays to confirm defective responses to IL10 were performed on peripheral blood mononuclear cells.A functional defect in IL10 signaling was confirmed in all IL10R patients tested. Disease started with severe diarrhea within the first 12 weeks in all patients. All infants showed Crohn's disease-like ulcerations limited to the colon with marked perianal inflammation (fissures, abscess, and fistula); disease progression to the small bowel occurred in only 1 patient. Four of the 10 patients had granulomata on histology, and all patients showed Crohn's disease-like mesenteric infiltration on imaging. Disease pattern was indistinguishable between IL10R alpha or beta chain or IL10 defects; autoimmunity was not observed. Mutations in IL10 were more frequently associated with bacterial and viral infections. Patients responded partially to treatment with steroids or anti-tumor necrosis factor drugs, whereas hematopoietic stem cell transplantation proved efficacious.The importance of the IL10 pathway within the colonic mucosa is highlighted by the development of severe colitis within a few weeks in infants with mutations in IL10, IL10RA, or IL10RB. Immunosuppression failed to correct the defect in this pathway, which seems to be a key to controlling inflammation in the colon.

Background: Interleukin (IL)-23, IL-17A, IL-17F, and interferon-gamma (IFN-γ) are important mediators of inflammatory colitis and are potential therapeutic targets in inflammatory bowel disease (IBD). Their expression profile in the different parts of normal noninflammatory intestine is unclear and their changes during pathology have not yet been addressed in pediatric IBD patients. Methods: We quantified the transcriptional expression of IL-23, IL-12, IL-17A, IL-17F, IL-6, and IL-10 in healthy, noninflammatory duodenum, ileum, and colon and in inflamed and noninflamed biopsies of pediatric patients with Crohn's disease (CD) and ulcerative colitis (UC). Results: In healthy tissue, expression of IL-17A is highest in the ileum, with IFN-γ expression lowest in the colon. Compared to healthy sections, CD patients displayed increased IL-12p35 and IFN-γ levels in noninflamed ileum and colon, respectively. Modifications of cytokine expression between noninflamed and inflamed tissues was characterized by increased IL-17A in UC colon, IFN-γ in CD colon, and IL-17A, IFN-γ and IL-6 in CD ileum. Elevated IL-17A levels were positively correlated with IFN-γ in both inflammatory CD and UC but IL-17A and IFN-γ were correlated with IL-23p19 in CD ileum only. Conclusions: The expression of Th1 and Th17 cytokines varies along the intestine, indicating local specific regulation mechanisms. However, the cytokine expression patterns in the same tissue depends on the pathology, with a Th1 or a Th17 profile in the colon of CD and UC patients, respectively, and a Th1/Th17 profile in the ileum of CD patients. This indicates overlapping but distinct immune mechanisms driving intestinal inflammation in these two pathologies. (Inflamm Bowel Dis 2011)

Objectives Early onset inflammatory bowel diseases (EO-IBD) developing during the first year of life are likely to reflect inherited defects in key mechanism(s) controlling intestinal homeostasis, as recently suggested for interleukin 10 (IL10). Thus, we aimed to further elaborate the hypothesis of defective anti-inflammatory responses in patients with IBD. Methods The capacities of transforming growth factor β (TGFβ) and IL10 to inhibit proinflammatory cytokine production by monocyte-derived dendritic cells (MoDC) or peripheral blood cells (PBMC) was analyzed in 75 children with IBD, including 13 infants with EO-IBD (in whom autoimmune diseases or classical immunodeficiencies were ruled out). IL10 receptor-A/-B expression, STAT3 activation in response to IL6, IL10, IL21, IL22 were analyzed by FACS and western blotting. IL10RA and B genes were sequenced. The response to IL22 was tested in ileal/colonic tissue cultures. Tissue gene expression was analyzed by Taqman real-time polymerase chain reaction. Results Production of IL10 in response to bacterial motifs was normal in all IBD patients. In contrast to our original hypothesis, no defect of the anti-inflammatory potential of TGFβ and IL10 was observed in children with IBD or EO-IBD except two infants who presented with granuloma-positive colitis at 3 months of life: no response to IL10 was observed secondary to mutations in the α (p.R262C) or β (p.E141X) chain of IL10R, respectively, although a fully functional Jak-STAT3 pathway was present in both patients. When analyzing the regulation of intestinal bacterial clearance, we detected a defect in the patient with absent IL10 RB to upregulate protective transcripts in response to IL22, whereas all other EO-IBD patients, including the patient with an abnormal α chain, responded normally. Conclusions Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.