The ten "hard" questions in pediatric functional constipation.

Functional constipation is a common problem in childhood and has a great impact on social, physical, and emotional functioning of affected children and their caregivers. No organic cause of the constipation can be found in approximately 95% of children, defining the "so-called" chronic functional constipation. Its prevalence has been reported to range from 0.7 to 29.6%, with a median of 12%. The diagnosis of functional constipation is exclusively clinical based on the pediatric diagnostic Rome criteria for functional gastrointestinal disorders and does not routinely require laboratory and/or radiological investigations. In case of alarm signs and symptoms that may suggest organic diseases, further investigations can be required. The therapeutic management is based on non-pharmacological and pharmacological approaches. Education, demystification of constipation and reward-based toilet training represent the cornerstones of nonpharmacological management. Disimpaction, maintenance treatment and weaning of medication are all elements of pharmacological treatment. Osmotic laxatives, mainly polyethylene glycol (PEG), are considered the first-choice laxative for both disimpaction and maintenance treatment. The aim of this review is to provide pediatric gastroenterologists with a practical tool to support the clinical and therapeutic management of children and adolescents affected by chronic functional constipation.

Diagnostic Delay of Celiac Disease in Childhood.

Importance: The extent and factors associated with risk of diagnostic delay in pediatric celiac disease (CD) are poorly understood. Objectives: To investigate the diagnostic delay of CD in childhood, and to assess factors associated with this delay. Design, Setting, and Participants: Multicenter, retrospective, cross-sectional study (2010-2019) of pediatric (aged 0-18 years) patients with CD from 13 pediatric tertiary referral centers in Italy. Data were analyzed from January to June 2023. Main Outcomes and Measures: The overall diagnostic delay (ie, the time lapse occurring from the first symptoms or clinical data indicative of CD and the definitive diagnosis), further split into preconsultation and postconsultation diagnostic delay, were described. Univariable and multivariable linear regression models for factors associated with diagnostic delay were fitted. Factors associated with extreme diagnostic delay (ie, 1.5 × 75th percentile) and misdiagnosis were assessed. Results: A total of 3171 patients with CD were included. The mean (SD) age was 6.2 (3.9) years; 2010 patients (63.4%) were female; and 10 patients (0.3%) were Asian, 41 (1.3%) were Northern African, and 3115 (98.3%) were White. The median (IQR) overall diagnostic delay was 5 (2-11) months, and preconsultation and postconsultation diagnostic delay were 2 (0-6) months and 1 (0-3) month, respectively. The median (IQR) extreme overall diagnostic delay (586 cases [18.5%]) was 11 (5-131) months, and the preconsultation and postconsultation delays were 6 (2-120) and 3 (1-131) months, respectively. Patients who had a first diagnosis when aged less than 3 years (650 patients [20.5%]) showed a shorter diagnostic delay, both overall (median [IQR], 4 [1-7] months for patients aged less than 3 years vs 5 [2-12] months for others) and postconsultation (median [IQR], 1 [0-2] month for patients aged less than 3 years vs 2 [0-4] months for others). A shorter delay was registered in male patients, both overall (median [IQR], 4 [1-10] months for male patients vs 5 [2-12] months for female patients) and preconsultation (median [IQR], 1 [0-6] month for male patients vs 2 [0-6] months for female patients). Family history of CD was associated with lower preconsultation delay (odds ratio [OR], 0.59; 95% CI, 0.47-0.74) and lower overall extreme diagnostic delay (OR, 0.75; 95% CI, 0.56-0.99). Neurological symptoms (78 patients [21.5%]; OR, 1.35; 95% CI, 1.03-1.78), gastroesophageal reflux (9 patients [28.1%]; OR, 1.87; 95% CI, 1.02-3.42), and failure to thrive (215 patients [22.6%]; OR, 1.62; 95% CI, 1.31-2.00) showed a more frequent extreme diagnostic delay. A previous misdiagnosis (124 patients [4.0%]) was more frequently associated with gastroesophageal reflux disease, diarrhea, bloating, abdominal pain, constipation, fatigue, osteopenia, and villous atrophy (Marsh 3 classification). Conclusions and Relevance: In this cross-sectional study of pediatric CD, the diagnostic delay was rather short. Some factors associated with risk for longer diagnostic delay and misdiagnosis emerged, and these should be addressed in future studies.

Diagnosis and management of anemia in pediatric inflammatory bowel diseases: Clinical practice guidelines on behalf of the SIGENP IBD Working group.

Anemia is one of the most frequent extra-intestinal manifestations of inflammatory bowel disease. Insidious onset, variability of symptoms and lack of standardized screening practices may increase the risk of underestimating its burden in children with IBD. Despite its relevance and peculiarity in everyday clinical practice, this topic is only dealt with in a few documents specifically for the pediatric field. The aim of the current guidelines is therefore to provide pediatric gastroenterologists with a practical update to support the clinical and therapeutic management of children with IBD and anemia. A panel of 19 pediatric gastroenterologists and 1 pediatric hematologist with experience in the field of pediatric IBD was agreed by IBD Working group of the Italian Society of Gastroenterology, Hepatology and Nutrition (SIGENP) to produce the present article outlining practical clinical approaches to the pediatric patient with IBD and anemia. The levels of evidence and recommendations have been defined for each part of the statement according to the GRADE system.

Mobile Health technology in pediatric esophagogastroduodenoscopy quality indicators assessment: results from a national program of the Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition.

BACKGROUND AND AIMS: Upper endoscopy procedures (UEP, esophagogastroduodenoscopy [EGDS] and retrograde endoscopic retrograde cholangiography [ERCP]) are an established standard of care in pediatric gastroenterology. The Pediatric endoscopy quality improvement network (PEnQuIN) recently published its pediatric-specific endoscopy quality guidelines. This study, initiated by the Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition (SIGENP), aims to evaluate the adherence of Italian Pediatric Endoscopy Centers to these established quality standards. METHODS: Conducted between April 2019 and March 2021, this nationwide study utilized a smartphone app-based approach. Data encompassing pediatric endoscopy facilities, patient profiles, endoscopy indications, 17 procedure-related PEnQuIN indicators, and a GHAA-9m patient satisfaction questionnaire were systematically collected. RESULTS: A comprehensive analysis of 3582 procedures from 24 PECs revealed that 2654 (76%) were UEP. The majority of centers (75%) involved more than one operator, with 9 PEC incorporating adult endoscopists, responsible for 5% of UEPs. Overall, adherence to quality standards was good; however, areas of improvement include sub-optimal reporting of sedation details, adherence to disease-specific guidelines, and patient satisfaction questionnaire completeness (56%). The complication rate aligned with literature standards (1%), and patient satisfaction was generally high. A noteworthy observation was a 30% decrease monthly reporting rate and a shift in disease-specific patterns following the COVID-19 outbreak. CONCLUSIONS: Pediatric UEP practices in Italy adhere well to established quality standards. Emphasizing the adoption of disease-specific guidelines is crucial for optimizing resources, enhancing diagnostic accuracy, and minimizing unnecessary procedures. Prioritizing patient satisfaction is important for immediate enhancements in practice as well as for future research endeavors.

Italian guidelines for the management of irritable bowel syndrome in children and adolescents : Joint Consensus from the Italian Societies of: Gastroenterology, Hepatology and Pediatric Nutrition (SIGENP), Pediatrics (SIP), Gastroenterology and Endoscopy (SIGE) and Neurogastroenterology and Motility (SINGEM).

The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder (FGID), whose prevalence has widely increased in pediatric population during the past two decades. The exact pathophysiological mechanism underlying IBS is still uncertain, thus resulting in challenging diagnosis and management. Experts from 4 Italian Societies participated in a Delphi consensus, searching medical literature and voting process on 22 statements on both diagnosis and management of IBS in children. Recommendations and levels of evidence were evaluated according to the grading of recommendations, assessment, development, and evaluation (GRADE) criteria. Consensus was reached for all statements. These guidelines suggest a positive diagnostic strategy within a symptom-based approach, comprehensive of psychological comorbidities assessment, alarm signs and symptoms' exclusion, testing for celiac disease and, under specific circumstances, fecal calprotectin and C-reactive protein. Consensus also suggests to rule out constipation in case of therapeutic failure. Conversely, routine stool testing for enteric pathogens, testing for food allergy/intolerance or small intestinal bacterial overgrowth are not recommended. Colonoscopy is recommended only in patients with alarm features. Regarding treatment, the consensus strongly suggests a dietary approach, psychologically directed therapies and, in specific conditions, gut-brain neuromodulators, under specialist supervision. Conditional recommendation was provided for both probiotics and specific fibers supplementation. Polyethylene glycol achieved consensus recommendation for specific subtypes of IBS. Secretagogues and 5-HT4 agonists are not recommended in children with IBS-C. Certain complementary alternative therapies, antispasmodics and, in specific IBS subtypes, loperamide and rifaximin could be considered.

Precision nutrition in pediatric IBD: A position paper from the ESPGHAN special interest group for basic science and translational research, the IBD Porto group, and allied health professionals.

Stratified and precision nutrition refers to disease management or prevention of disease onset, based on dietary interventions tailored to a person's characteristics, biology, gut microbiome, and environmental exposures. Such treatment models may lead to more effective management of inflammatory bowel disease (IBD) and reduce risk of disease development. This societal position paper aimed to report advances made in stratified and precision nutritional therapy in IBD. Following a structured literature search, limited to human studies, we identified four relevant themes: (a) nutritional epidemiology for risk prediction of IBD development, (b) food-based dietary interventions in IBD, (c) exclusive enteral nutrition (EEN) for Crohn's disease (CD) management, and (d) pre- and probiotics for IBD management. There is scarce literature upon which we can make recommendations for precision or stratified dietary therapy for IBD, both for risk of disease development and disease management. Certain single-nucleotide polymorphisms related to polyunsaturated fatty acid (PUFA) metabolism may modify the effect dietary PUFA have in increasing the risk of IBD development. Non-colonic CD, mild-to-moderate CD, and high microbiota richness may predict success of EEN and may be used both for prediction of treatment continuation, but also for early cessation in nonresponders. There is currently insufficient evidence to make recommendations for precision or stratified dietary therapy for patients with established IBD. Despite the great interest in stratified and precision nutrition, we currently lack data to support conclusive recommendations. Replication of early findings by independent research groups and within structured clinical interventions is required.

Gastrointestinal bleeding in children: diagnostic approach.

Different conditions may underlie gastrointestinal bleeding (GIB) in children. The estimated prevalence of GIB in children is 6.4%, with spontaneous resolution in approximately 80% of cases. Nonetheless, the initial approach plays a pivotal role in determining the prognosis. The priority is the stabilization of hemodynamic status, followed by a systematic diagnostic approach. GIB can originate from either upper or lower gastrointestinal tract, leading to a broad differential diagnosis in infants and children. This includes benign and self-limiting disorders, alongside serious conditions necessitating immediate treatment. We performed a nonsystematic review of the literature, in order to describe the variety of conditions responsible for GIB in pediatric patients and to outline diagnostic pathways according to patients' age, suspected site of bleeding and type of bleeding which can help pediatricians in clinical practice. Diagnostic modalities may include esophagogastroduodenoscopy and colonoscopy, abdominal ultrasonography or computed tomography and, when necessary, magnetic resonance imaging. In this review, we critically assess these procedures, emphasizing their respective advantages and limitations concerning specific clinical scenarios.

Nutraceuticals and biotics in pediatric gastrointestinal disorders.

In recent years there has been growing interest in the use of nutraceuticals and biotics in both pediatric and adult clinical practice. The overlapping and often ambiguous symptoms of both functional and organic gastrointestinal disorders have led to a search for alternative therapeutic approaches that avoid the use of synthetic or chemical treatments. However, while nutraceuticals and natural supplements are widely used, their health benefits are often not supported by adequate scientific evidence, and an unregulated use of nutraceuticals can be potentially harmful. The correct use of nutraceuticals, prebiotics, and probiotics can optimize the results of drug therapy in some cases and reduce the risk of side effects. This review aims to provide clinicians with guidance on the use of complementary therapies for pediatric gastrointestinal symptoms and disorders, highlighting the scarcity of studies on the kinetics and dynamics of nutraceuticals and biotics. While it is generally difficult to associate their intakes with adverse events due to the often-coexisting pharmacological treatments, it is essential to avoid the abandonment of traditional drugs with proven efficacy in the treatment of single diseases. Overall, the use of nutraceuticals, prebiotics, and probiotics in pediatric gastroenterological practice requires caution and medical supervision. Further research is needed to determine the effects of alternative therapies on pediatric gastrointestinal symptoms and disorders, and to ensure their safe and effective use in the clinical practice.

Disease course of ulcerative proctitis in children: A population-based study on behalf of the SIGENP IBD Group.

BACKGROUND: The natural history of ulcerative proctitis (UP) has been poorly investigated in children. AIMS: We aimed to compare the disease course of children with UP at diagnosis to the other locations and to identify extension predictors. METHODS: This was a multicenter, observational study carried out from data prospectively entered in the SIGENP-IBD-Registry. Children with ulcerative colitis (UC) diagnosis and at least 1-year follow-up were included. On the basis of Paris classification UP patients were identified and compared with the other locations. RESULTS: 872 children were enrolled (median age at diagnosis: 11.2 years; M/F: 426/446), of whom 78 (9%) with UP. Kaplan-Meier analysis demonstrated increased cumulative probabilities of disease extension in the E1 group [1 year: 20.3%; 5 years: 52.7%; 10 years: 72.4%] compared to E3 group [1 year: 8.5%; 5 years: 24.9% and 10 years: 60.1%, p=0.001]. No differences were observed comparing E1 and E2 groups [p=0.4]. Cumulative probabilities of surgery at 1, 5 and 10 years were 1.3, 2.8 and 2.8% in the E1 group and 2.5, 8 and 12.8% in the E2-E3-E4 group, respectively (p=0.1). Cox regression analysis demonstrated that PUCAI>35 at diagnosis was associated with endoscopic extension (HR=4.9; CI 95% 1.5-15.2, p=0.006). CONCLUSIONS: UP is associated with similar short and long-term outcomes compared to other locations.

Infliximab therapy decreases the expression of serum and faecal miR-126 and miR-20a in paediatric Crohn's disease: A pilot study.

AIM: We aimed to evaluate the serum and faecal expression of miR-126 and miR-20a in children with Crohn's disease (CD) during infliximab (IFX) therapy. METHODS: In this prospective observational study, serum and faeces from CD patients were collected before IFX therapy (T0), after induction (T1) and after 6 months from IFX (T2). IFX levels were determined by Enzyme-linked immunosorbent assay at T1 and T2. miRNAs were profiled through Real-Time RT-PCR. The activity of disease was evaluated through the Paediatric Crohn's disease activity index (PCDAI), serum C-reactive protein (CRP) and faecal calprotectin. RESULTS: Nine CD children were enrolled. Serum and faecal miR-126 and miR-20a levels were higher at T0 and showed a time-dependent decrease, being significantly down-regulated after IFX treatment at T2. Specifically, IFX levels recorded at T1 and T2 negatively correlated with the serum and faecal expression of miR-126 and miR-20a. Serum and faecal changes of miR-126 and miR20-a were positively associated with the decrease of the inflammatory marker CRP and PDCAI at all time points. CONCLUSION: In children with CD, IFX therapy decreases the expression of serum and faecal miR-126 and miR-20a, suggesting an involvement of these two miRNAs in the action of the drug.

Prevalence of functional gastrointestinal disorders in Italian children living in different regions: analysis of the difference and the role of diet.

BACKGROUND: Functional Gastrointestinal Disorders (FGIDs) are common in pediatric age. AIMS: To estimate the prevalence of FGIDs in Italian children and evaluate the impact of diet. METHODS: Healthy children aged 4-18 years were recruited in a multicenter cross-sectional study. We evaluated their eating habits and the presence of FGIDs, using Rome IV criteria, 3-day food diaries and Mediterranean Diet Quality Index (KIDMED) questionnaires. RESULTS: Seven hundred forty subjects were enrolled:369 children aged 4-9 years (Group A), and 371 adolescents 10-18 years old (Group B). The overall prevalence of FGIDs was 26.4% in Group A and 26.2% in Group B, with a significant higher prevalence in females in both groups. The most frequent disorders were functional constipation, functional dyspepsia, and abdominal migraine. No significant difference in FGIDs prevalence was found between Northern and Southern Italy, despite significant variation in diet. In Group A there was a significant difference in KIDMED between North and South (5.3 ± 1 vs 6 ± 1.2, respectively; p = 0.001). A significant association between FGIDs and KIDMED was found in Group A (OR=0.83, p = 0.034), but not in Group B (OR=0.89, p = 0.166). CONCLUSIONS: FGIDs are common in Italian children, with a higher prevalence in females. Despite significant differences in dietary habits between North and South, FGIDs prevalence does not vary significantly.

The Role of Gluten in Gastrointestinal Disorders: A Review.

Gluten is only partially digested by intestinal enzymes and can generate peptides that can alter intestinal permeability, facilitating bacterial translocation, thus affecting the immune system. Few studies addressed the role of diet with gluten in the development of intestinal inflammation and in other gastrointestinal disorders. The aim of this narrative review was to analyse the role of gluten in several gastrointestinal diseases so as to give a useful overview of its effectiveness in the prevention and management of these disorders.

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

BACKGROUND: In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. METHODS: CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). RESULTS: We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. CONCLUSIONS: The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. TRIAL REGISTRATION: ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . IMPACT: Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

Pediatric Gastroenterology and Hepatology in Italy before and after the COVID-19: Lessons learned and management changes by SIGENP.

Around the world, the 2019 Coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised serious public health problems and major medical challenges. The Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition (SIGENP) published several papers on the impact of COVID-19 on the current management, diagnosis, and treatment of acute and chronic gastrointestinal, hepatic, immune-mediated, and functional disorders. The present article summarizes the most relevant SIGENP reports and consensus during and after the peak of the COVID-19 outbreak, including the diagnosis and treatment of inflammatory bowel disease (IBD), indications and timing of digestive endoscopy, and insights into the novel hepatitis.

The use of calgranulin-C (S100A12) and fecal zonulin as possible non-invasive markers in children with inflammatory bowel disease: a clinical study.

Calgranulin-C (S100A12) and zonulin are considered markers of intestinal inflammation. Our aim was to evaluate fecal S100A12 (f-S100A12) and fecal zonulin (f-zonulin) in children with inflammatory bowel disease (IBD), compared to fecal calprotectin (FC) and serum inflammatory markers. We enrolled children with a previous diagnosis of Crohn's disease (CD) and ulcerative colitis (UC). F-S100A12, f-zonulin, and FC were determined by enzyme-linked immunosorbent assay (ELISA). Endoscopic examination was considered in the patients who underwent ileocolonoscopy within 2 weeks from the enrollment. One hundred seventeen children, 39.3% with CD and 60.7% with UC were enrolled. In both CD and UC, there was a significant direct correlation between FC and f-S100A12 levels. In children with CD and UC, both FC and f-S100A12 correlated with markers of serum inflammation. We found difference in FC and f-S100A12 levels between patients in clinical relapse and remission (FC: mean 1027 ± 818 mcg/ml vs 580 ± 695 mcg/ml respectively, p = 0.028; f-S100A12: mean 66.4 ± 48.2 mcg/ml vs 42.7 ± 40 mcg/ml, respectively p = 0.02). Moreover, we found difference in FC between children with endoscopic inflammation and remission (mean 825 ± 779 mcg/ml vs 473.3 ± 492 mcg/ml, respectively p = 0.048), as well as for f-S100A12 (53 ± 43 mcg/ml vs mean 31 ± 33 mcg/ml vs, respectively p = 0.019). No significant results were found for f-zonulin. CONCLUSION: Our data suggest that f-S100A12 and FC are both useful non-invasive biomarkers in the management of pediatric IBD in follow up and in monitoring endoscopic and clinical relapse. WHAT IS KNOWN: • Fecal calprotectin (FC), fecal S100A12 (f- S100A12), and fecal zonulin represent potential noninvasive markers of gut inflammation. • Since S100A12 is predominantly expressed by granulocytes, high levels of f-S100A12 should be more specific for inflammation than FC. WHAT IS NEW: • FC and f-S100A12 were correlated to each other and despite the lack of correlation with disease location, they were associated with endoscopic inflammation and clinical relapse in children with IBD. • No significant correlations were found between f-zonulin and the inflammatory parameters.

Role of inflammation in pediatric irritable bowel syndrome.

BACKGROUND: IBS affects a large number of children throughout the world and is thought to be the result of disturbed neuroimmune function along with the brain-gut axis. Although the underlying pathophysiologic mechanisms are not clear, the role of low-grade inflammation and mucosal immune activation in IBS symptom generation has become evident also in subsets of pediatric patients. Animal models provided meaningful insight in the causal relationship between abnormal mucosal immune activation and changes in gastrointestinal (GI) sensory-motor function. Likewise, the development of long-standing GI symptoms fulfilling the current criteria for functional GI disorders after infection gastroenteritis and in patients with IBD or celiac disease in remission further supports this hypothesis. Immune activation, its impact on gut sensory-motor function, and potential implications for symptom generation emerged in both children and adults with IBS. PURPOSE: The aim of this review is to summarize the main evidence on the presence of low-grade inflammation and immune activation in children with IBS, its possible role in symptom generation, and its potential implication for new therapeutic strategies.

Increased expression of CB2 receptor in the intestinal biopsies of children with inflammatory bowel disease.

OBJECTIVES: The Cannabinoid Receptor type 2 (CB2) is involved in inflammation and immune cell modulation. In previous studies, we demonstrated the association between the CNR2 rs35761398 polymorphism and the risk for pediatric inflammatory bowel disease (IBD). In this study, we analyzed the intestinal biopsies from Crohn disease (CD) and ulcerative colitis (UC) pediatric patients at the diagnosis to evaluate the expression of CB2 and several factors associated with IBD inflammatory pathways. METHODS: We enrolled five patients with CD, five with UC, and five controls (CTR). We analyzed ileum and rectum biopsies from patients of each group evaluating the expression of CB2, Toll-like receptor 4, interleukin-6, and interleukin-1ß by western blot and immunofluorescence. RESULTS: Western blot analysis showed a significant increase of CB2 in the CD ileum and in the UC rectum biopsies and an increase of TLR4 in the UC rectum. We also observed a significant over-expression of the IL-6 in UC rectum. The immunofluorescence analysis confirmed western blot data, showing also a T-lymphocytes infiltration colocalized with CB2 expression in the CD ileum and UC rectum. CONCLUSIONS: Our results show an upregulation of CB2 in pediatric IBD, which might have implications for drug discovery. IMPACT: The Cannabinoid Receptor type 2 (CB2) is involved in the inflammation and modulation of the immune response in pediatric inflammatory bowel disease (IBD). CB2 receptor is more expressed in the inflamed intestine of pediatric IBD patients. CB2 could be used as a potential therapeutic target to reduce IBD-related inflammatory state in childhood.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.