APOBEC mutagenesis is a common process in normal human small intestine.

APOBEC mutational signatures SBS2 and SBS13 are common in many human cancer types. However, there is an incomplete understanding of its stimulus, when it occurs in the progression from normal to cancer cell and the APOBEC enzymes responsible. Here we whole-genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals and found that SBS2/SBS13 mutations were present in 17% of crypts, more frequent than most other normal tissues. Crypts with SBS2/SBS13 often had immediate crypt neighbors without SBS2/SBS13, suggesting that the underlying cause of SBS2/SBS13 is cell-intrinsic. APOBEC mutagenesis occurred in an episodic manner throughout the human lifespan, including in young children. APOBEC1 mRNA levels were very high in the small intestine epithelium, but low in the large intestine epithelium and other tissues. The results suggest that the high levels of SBS2/SBS13 in the small intestine are collateral damage from APOBEC1 fulfilling its physiological function of editing APOB mRNA.

Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.

BACKGROUND & AIMS: Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function. METHODS: Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function. RESULTS: Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation. CONCLUSIONS: Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.

Disease Prognostic Biomarkers in Inflammatory Bowel Diseases-A Reality Check.

Inflammatory bowel diseases [IBD] such as Crohn's disease [CD] and ulcerative colitis [UC] are complex conditions presenting with a wide range of phenotypes. Given major variation in disease severity and outcomes as well as response to existing therapies, a personalised treatment approach stands the chance of improving the overall disease outcome as well as minimising potentially harmful side effects. However, disease activity or distribution at the point of diagnosis are poor predictors of future disease outcome. Hence, the urgent need to develop biomarkers that could either predict the overall disease course [i.e., disease prognostic biomarkers] or the response to individual therapies [i.e., disease predictive biomarkers]. Despite the widely accepted need for such biomarkers to improve the management of IBD patients, their development has proven to be challenging for a number of reasons. Based on our own experience in this field, we perform a reality check on existing evidence, discuss main challenges, and outline future perspectives.

Sclerosing Cholangitis in Pediatric Inflammatory Bowel Disease: Early Diagnosis and Management Affect Clinical Outcome.

OBJECTIVE: To describe the characteristics and clinical course of children and young persons with inflammatory bowel disease (IBD) and sclerosing cholangitis (SC). STUDY DESIGN: Retrospective analysis of clinical characteristics, management, and outcome of two separate cohorts of children and young persons with IBD-SC managed in a tertiary pediatric gastroenterology center and in a tertiary pediatric hepatology center in the UK. RESULTS: Eighty-two pediatric patients (31% female) with IBD-SC and a mean age at diagnosis of 11.9 ± 2.8 years were followed up for a mean of 6.8 ± 3.3 years. The most common type of IBD was ulcerative colitis (55%), followed by unclassified IBD (30%) and Crohn's disease (15%). Autoimmune SC (ASC) was diagnosed in 72%, and small duct SC was diagnosed in 28%. Complication-free and native liver survival were 96% and 100%, respectively, at 5 years after diagnosis and 75% and 88%, respectively, at 10 years after diagnosis. Patients in the gastroenterology center, who were diagnosed with liver disease sooner after diagnosis of IBD compared with the hepatology center cohort (mean, 2.7 ± 6.1 months vs 9.3 ± 19.4 months; P = .03), did not develop liver-related complications during follow-up. CONCLUSIONS: Our data suggest that children with IBD-SC have better clinical outcomes than have been reported previously, particularly if diagnosed early. We recommend prompt assessment for SC, including liver biopsy and biliary imaging, when liver function abnormalities are detected in a children diagnosed with IBD.

Guidance on the interpretation of faecal calprotectin levels in children.

BACKGROUND: Faecal calprotectin (FCP) is a powerful tool to predict inflammatory bowel disease (IBD) in patients with gastrointestinal symptoms. In the paediatric patient population, the reference value of < 50 µg/g and the influence of age on FCP levels result in a high number of redundant investigations and specialist referrals. We assessed paediatric FCP levels, their diagnostic value and corresponding referral pathways from primary and secondary care. METHODS: We analysed two cohorts from a precisely defined catchment area: one consisted of all FCPs measured in this area (n = 2788). The second cohort-a subset of the first cohort-consisted of FCP values and corresponding clinical data from children who were referred for possible IBD to our department (n = 373). RESULTS: In the first cohort, 47% of FCP levels were > 50 µg/g, 15% were ≥ 250 µg/g. Children < 1y had significantly (p < 0.001) higher FCP than older children. In the second cohort, 6.7% of children with an FCP of < 250 µg/g (or 8.6% with an FCP of < 600 µg/g) had IBD-all featured symptoms suggestive of IBD (e.g. bloody diarrhoea, nocturnal abdominal pain, weight loss) or abnormal blood tests. 76% of patients in whom raised FCP (> 50 µg/g) was the sole reason for being referred for suspected IBD did not have IBD. CONCLUSION: Children with an FCP < 600 µg/g and without matching symptoms suggestive of IBD are unlikely to have IBD. A higher FCP reference value may provide cost-effective improvement that could avoid redundant investigations and specialist referrals. A guideline for specialist referrals is proposed.

Transcription and DNA Methylation Patterns of Blood-Derived CD8+ T Cells Are Associated With Age and Inflammatory Bowel Disease But Do Not Predict Prognosis.

BACKGROUND & AIMS: Gene expression patterns of CD8+ T cells have been reported to correlate with clinical outcomes of adults with inflammatory bowel diseases (IBD). We aimed to validate these findings in independent patient cohorts. METHODS: We obtained peripheral blood samples from 112 children with a new diagnosis of IBD (71 with Crohn's disease and 41 with ulcerative colitis) and 19 children without IBD (controls) and recorded medical information on disease activity and outcomes. CD8+ T cells were isolated from blood samples by magnetic bead sorting at the point of diagnosis and during the course of disease. Genome-wide transcription (n = 192) and DNA methylation (n = 66) profiles were generated using Affymetrix and Illumina arrays, respectively. Publicly available transcriptomes and DNA methylomes of CD8+ T cells from 3 adult patient cohorts with and without IBD were included in data analyses. RESULTS: Previously reported CD8+ T-cell prognostic expression and exhaustion signatures were only found in the original adult IBD patient cohort. These signatures could not be detected in either a pediatric or a second adult IBD cohort. In contrast, an association between CD8+ T-cell gene expression with age and sex was detected across all 3 cohorts. CD8+ gene transcription was clearly associated with IBD in the 2 cohorts that included non-IBD controls. Lastly, DNA methylation profiles of CD8+ T cells from children with Crohn's disease correlated with age but not with disease outcome. CONCLUSIONS: We were unable to validate previously reported findings of an association between CD8+ T-cell gene transcription and disease outcome in IBD. Our findings reveal the challenges of developing prognostic biomarkers for patients with IBD and the importance of their validation in large, independent cohorts before clinical application.

The growing gap between demand and availability of clinical psychology in Paediatric Gastroenterology: a retrospective analysis of clinical routine care.

Clinical psychology intervention in paediatric gastroenterology is vital given the biopsychosocial aetiology of paediatric functional gastrointestinal disorders, and the psychological impact of chronic conditions. The aim was to assess the availability and benefit of clinical psychology in paediatric gastroenterology across the UK and Germany. A retrospective assessment of referrals (n = 936 referrals) to clinical psychology was performed at our tertiary paediatric gastroenterology centre between 2010 and 2018. The availability of clinical psychologists and outcome of psychology intervention for children with functional abdominal pain were also assessed. Access to clinical psychology across the UK and Germany was assessed using an online questionnaire. We observed a substantial rise in the number of clinical psychology referrals between 2010 and 2018. Increasing demand was not matched by sufficient increase in availability of clinical psychology, leading to longer waiting times. A major benefit of clinical psychology intervention was highlighted with 95% of patients (n = 20) reporting a significant reduction in symptoms. Of the 12 centres who responded, 11 centres have direct access to clinical psychology with a mean of 13% of patients requiring psychology referrals annually.Conclusion: Despite evidence of its benefit and increasing demand, there is insufficient access to clinical psychological services, highlighting the urgent need to address this important issue. What is known: • The biopsychosocial pathophysiology of functional gastrointestinal disorders involves a disordered brain-gut interaction, which emphasizes the close link between psychological factors and altered gut function. • Psychological intervention, as an adjunct to medical treatment, improves outcomes in paediatric patients with gastrointestinal (GI) disease such as functional gastrointestinal disorders and inflammatory bowel diseases What is new: • There is a rising number of referrals from paediatric gastroenterology to clinical psychology in our centre which is not met by a sufficient increase in the availability of clinical psychologists. Similarly, access to clinical psychological services is lacking in several paediatric gastroenterology centres in the UK and Germany. • Strategic action is required to address this important gap in the care of children suffering from GI diseases.

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease.

Somatic mosaicism and common genetic variation contribute to the risk of very-early-onset inflammatory bowel disease.

Very-early-onset inflammatory bowel disease (VEO-IBD) is a heterogeneous phenotype associated with a spectrum of rare Mendelian disorders. Here, we perform whole-exome-sequencing and genome-wide genotyping in 145 patients (median age-at-diagnosis of 3.5 years), in whom no Mendelian disorders were clinically suspected. In five patients we detect a primary immunodeficiency or enteropathy, with clinical consequences (XIAP, CYBA, SH2D1A, PCSK1). We also present a case study of a VEO-IBD patient with a mosaic de novo, pathogenic allele in CYBB. The mutation is present in ~70% of phagocytes and sufficient to result in defective bacterial handling but not life-threatening infections. Finally, we show that VEO-IBD patients have, on average, higher IBD polygenic risk scores than population controls (99 patients and 18,780 controls; P < 4 × 10-10), and replicate this finding in an independent cohort of VEO-IBD cases and controls (117 patients and 2,603 controls; P < 5 × 10-10). This discovery indicates that a polygenic component operates in VEO-IBD pathogenesis.

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.

OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. RESULTS: We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. CONCLUSIONS: Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.

Early Treatment Response Predicts Outcome in Paediatric Ulcerative Colitis: GASTROENTEROLOGY: INFLAMMATORY BOWEL DISEASE.

The disease course of children with ulcerative colitis (UC) varies substantially. Published data on predictors of disease outcomes in children remain scarce. We validate clinical predictors of outcomes in 93 children with UC in a single centre (age range: 2-18 years, minimum follow-up: 18 months). We stratified children into 3 groups according to their disease course, that is, 1 = mild (38/93, 40.9%), 2 = moderate (38/93, 40.9%), 3 = severe (17, 18.2%). Comparison of clinical and biochemical parameters was performed between groups using Chi-square, Mann-Whitney, and log-rank tests. Predictors of a severe disease course included pancolitis (P 0.01), low albumin (P 0.005), low haemoglobin at diagnosis (P 0.04), paediatric ulcerative colitis activity index (PUCAI) at 3 months, and nonresponse to steroids at 3 months (P 0.0001). In our cohort, failure to achieve remission at 3 months implied an 80% likelihood to require biologics or major surgery within 18 months. A specific 3-month review point is recommended to guide future management.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome.

BACKGROUND & AIMS: We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis. METHODS: We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn's disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed. RESULTS: We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents. CONCLUSIONS: We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.

Clinical course and outcomes of diagnosing Inflammatory Bowel Disease in children 10 years and under: retrospective cohort study from two tertiary centres in the United Kingdom and in Italy.

BACKGROUND: Most children with Inflammatory Bowel Disease (IBD) are diagnosed between 11 and 16 years of age, commonly presenting with features of typical IBD. Children with onset of gut inflammation under 5 years of age often have a different underlying pathophysiology, one that is genetically and phenotypically distinct from other children with IBD. We therefore set out to assess whether children diagnosed after the age of 5 years, but before the age of 11, have a different clinical presentation and outcome when compared to those presenting later. METHODS: Retrospective cohort study conducted at two European Paediatric Gastroenterology Units. Two cohorts of children with IBD (total number = 160) were compared: 80 children diagnosed between 5 and 10 years (Group A), versus 80 children diagnosed between 11 and 16 (Group B). Statistical analysis included multiple logistic regression. RESULTS: Group A presented with a greater disease activity (p = 0.05 for Crohn's disease (CD), p = 0.03 for Ulcerative Colitis (UC); Odds Ratio 1.09, 95 % Confidence Interval: 1.02-1.1), and disease extent (L2 location more frequent amongst Group A children with CD (p = 0.05)). No significant differences were observed between age groups in terms of gastro-intestinal and extra-intestinal signs and symptoms at disease presentation, nor was there a difference in the number of hospitalisations due to relapsing IBD during follow-up. However, children in Group A were treated earlier with immunosuppressants and had more frequent endoscopic assessments. CONCLUSION: While clinicians feel children between 5 and 10 years of age have a more severe disease course than adolescents, our analysis also suggests a greater disease burden in this age group. Nevertheless, randomized trials to document longer-term clinical outcomes are urgently needed, in order to address the question whether a younger age at disease onset should prompt per se a more "aggressive" treatment. We speculate that non-clinical factors (e.g. genetics, epigenetics) may have more potential to predict longer term outcome than simple clinical measures such as age at diagnosis.

Paediatric gastrointestinal endoscopy: a qualitative study.

OBJECTIVE: Gastrointestinal endoscopy is an invasive procedure used to diagnose and/or treat diseases of the gut. As with any invasive procedure, there is a small risk for complications, and it is therefore important that due consideration be taken when reviewing the indications for endoscopy, particularly in children. Despite this, there remains a wide variation nationally in clinical practice among paediatric gastroenterologists. In the absence of a standard, we critically reviewed current endoscopy practice at Addenbrooke's Hospital with the aim of creating robust measures that could be used to achieve and maintain an efficient, high-quality paediatric endoscopy service. Specifically, we assessed the proportion of referred patients undergoing endoscopy and the clinical outcomes of these procedures. DESIGN: A retrospective list of new clinic patients who had undergone endoscopy within 3 months of their appointment was obtained, dating from 1 January 2011 to 31 December 2011. Individual electronic medical records (eMR) records were reviewed for the type of endoscopy, indications, interval to procedure, histology findings, diagnoses, and impact on management. RESULTS: Out of the 674 children seen at clinic over the year, 17% (n=114) went on to have an endoscopic procedure; half of these patients underwent both upper endoscopy (i.e., oesophagogastroduodenoscopy) and colonoscopy. Out of the children who underwent colonoscopy, 43% (n=25) were confirmed to have inflammatory bowel disease. CONCLUSION: Our results provide a useful initial reference point against which other tertiary units can assess their own practice, with all striving to provide appropriate, efficient and cost-effective endoscopy services for children.

Epigenetics in Paediatric Gastroenterology, Hepatology, and Nutrition: Present Trends and Future Perspectives.

Epigenetics can be defined as stable, potentially heritable changes in the cellular phenotype caused by mechanisms other than alterations to the underlying DNA sequence. As such, any observed phenotypic changes including organ development, aging, and the occurrence of disease could be driven by epigenetic mechanisms in the presence of stable cellular DNA sequences. Indeed, with the exception of rare mutations, the human genome-sequence has remained remarkably stable over the past centuries. In contrast, substantial changes to our environment as part of our modern life style have not only led to a significant reduction of certain infectious diseases but also seen the exponential increase in complex traits including obesity and multifactorial diseases such as autoimmune disorders. It is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment, and a large body of existing evidence supports the importance of environmental factors such as diet and nutrition, infections, and exposure to toxins on human health. This seems to be particularly the case during vulnerable periods of human development such as pregnancy and early life. Importantly, as the first point of contact for many of such environmental factors including nutrition, the digestive system is being increasingly linked to a number of "modern" pathologies. In this review article, we aim to give a brief introduction to the basic molecular principals of epigenetics and provide a concise summary of the existing evidence for the role of epigenetic mechanisms in gastrointestinal health and disease, hepatology, and nutrition.

Differential expression of mucosal trefoil factors and mucins in pediatric inflammatory bowel diseases.

In the intestinal mucosa trefoil factors (TFF) and mucins (Muc) - primarily produced by goblet cells - are thought to play a major role in providing barrier function during infection and inflammation. To investigate their role in pediatric Crohn's disease (CD) and ulcerative colitis (UC) we obtained mucosal biopsies of children with CD, UC and healthy controls and analyzed genetic expression. Levels of TFF2 mRNA were lower in inflamed mucosal samples (terminal ileum (TI) and duodenum) of children with CD, but higher in non-inflamed mucosal samples when compared to healthy controls (p < 0.05). Similarly, TFF2 levels in the TI were significantly lower in inflamed UC tissue. Adjustment for goblet cell density revealed slightly less marked, yet significantly different gene expression in IBD and controls. Furthermore, TI expression of TFF2 and Muc2 was inversely correlated with interleukin-8 expression in CD (p = 0.027). In Summary, our data demonstrate significant changes in Muc and TFF mRNA expression in pediatric patients with IBD suggesting a role in mucosal healing. Further studies are needed to elucidate a potential use as biomarkers for disease progression.

Coeliac disease in children with type 1 diabetes: are current guidelines proving difficult to implement in practice?

Updated European guidelines for the diagnosis of coeliac disease (CD) in the paediatric population (the European Society for Gastroenterology, Hepatology, and Nutrition, January 2012) outlined distinct diagnostic algorithms for patients with type 1 diabetes mellitus (T1DM). In this short report we demonstrate a period prevalence of CD in the T1DM population of 5.8% at a large tertiary centre. In addition to this, using a questionnaire circulated to paediatricians, we assessed present practice in the diagnosis of CD in T1DM 16 months following the European Society for Gastroenterology, Hepatology, and Nutrition guideline publication. Our results indicate that present practice and adherence to guidelines varies substantially. Further dissemination and perhaps simplification of guidelines may be required.