Upper gastrointestinal disorders of gut-brain interaction (DGBI) present from infancy through adolescence. The Rome V Criteria have expanded to include DGBI of the esophagus, disorders of air-transit, and feeding disorders, as well as rumination syndrome, cyclic vomiting, chronic nausea syndrome, and functional dyspepsia. This expansion provides a diagnostic framework for patients presenting with chest and throat pain, feeding difficulties, belching, pain with eating, nausea, and vomiting. Given the advances in impedance technology and high-resolution manometry, testing plays a greater role in many of these diagnostic criteria than they have in past Rome iterations. This harmony between symptoms and testing results in more precision in therapeutic approaches that are critically multidisciplinary. The ability to assign new, positive diagnoses across the upper gastrointestinal tract offers new opportunities for pediatric-focused therapeutic trials.

Esophageal reconstruction is one of the most challenging procedures in gastrointestinal surgery. While conventional therapeutic approaches, such as gastric pull-up and intestinal interposition, can restore continuity, they often fail to replicate native physiology. This limitation frequently leads to long-term complications, including dysphagia, stricture, and reflux, which can significantly affect the patients' quality of life. Tissue engineering approaches offer promising alternatives aimed at developing esophageal constructs that restore both structure and function, addressing the shortcomings of current treatment methods. This review highlights recent progress in esophageal tissue engineering (ETE), focusing on the requirements for ideal ETE scaffolds and examining available biomaterials, including natural, synthetic, and hybrid. We discuss advances in fabrication techniques and various cell-based approaches, such as primary cells, stem cells, and organoids. Furthermore, we also review the steps necessary to transition ETE constructs from the laboratory to clinical settings (ongoing human trials), including preclinical studies conducted on rodent, rabbit, canine, and porcine models with the expected functional outcomes and regeneration capabilities. Early translational efforts in ETE are addressed, along with the regulatory and ethical considerations regarding good manufacturing practice (GMP) compliance, traceability, and long-term surveillance. While significant advancements in ETE have been made in preclinical models, the review also discusses the challenges of moving to clinical studies. Potential strategies to address these challenges, such as 4-dimensional printing, smart materials, artificial intelligence-driven scaffold optimization, and organoid-based models, are introduced to help bridge the gap from preclinical research to successful clinical trials. In summary, ETE is transitioning from an experimental advancement to a translational reality by integrating significant achievements in biomaterials, fabrication technologies, and cell biology while following health regulatory standards. These efforts aim to provide regenerative solutions that overcome the limitations of current therapeutic approaches in clinical settings, ultimately facilitating healing and improving the patients' quality of life.

The development of the esophagus and trachea following the septation of the anterior foregut is a highly regulated process involving bidirectional communication between the endoderm and mesoderm. Signaling pathways such as the Bone Morphogenetic Protein family, Wnt/β-catenin, Sonic Hedgehog, and Fibroblast Growth Factor family mediate this complex crosstalk to induce the dorsal-ventral patterning of the anterior foregut as well as lineage specification. Even though the mechanisms are not fully understood, dysregulation of signaling pathways may lead to congenital malformations such as tracheomalacia, laryngeal-tracheal clefts and multiple types of esophageal atresia with/without tracheoesophageal fistula (EA/TEF). Human induced pluripotent stem cells (iPSCs) provide a robust in vitro platform to monitor the normal and abnormal development of esophagus and trachea and to understand the roles of the endoderm and mesoderm during anterior foregut development. Recent studies have demonstrated that direct differentiation of iPSCs into epithelial and mesenchymal lineages can recapitulate the key stages of foregut development. In this regard, in the current paper, we review the signaling pathways involved in the development of organs deriving from the anterior foregut as well as the roles of the endoderm and mesoderm revealed by previous studies. Furthermore, we discuss the use of iPSCs as a valuable model for investigating the bidirectional communications between the endoderm and mesoderm, which can broaden our knowledge and understanding of the critical mechanisms leading to normal and abnormal development of the esophagus and trachea.

Microfluidic systems are increasingly utilized in preclinical research, however, developing platforms that are compatible with large-scale manufacturing remains a critical challenge. In this regard, we show that thermoplastic elastomer (TPE)-based microfluidic chips could be a viable alternative to traditional poly(dimethylsiloxane) (PDMS) devices for drug testing applications. Microfluidic concentration gradient generators are essential components in these systems, enabling local spatial and temporal control of biochemical stimuli for applications such as cellular response studies and drug screening. TPEs offer key advantages, especially, optical transparency, flexibility, and compatibility with high-throughput fabrication processes compared to PDMS, thereby enhancing commercialization potential. This study demonstrates the design and fabrication of TPE-based microfluidic devices incorporating concentration gradient generators that allow precise control over solute distributions. Gradient formation was validated through numerical modeling and co-flow experiments using fluorescent dyes. The platform supported the co-culture of human lung fibroblast cells and human colon adenocarcinoma cells (HT-29), which exhibited coordinated responses to gradients of fetal bovine serum (FBS) and sepantronimum bromide (YM-155). Performance benchmarks against PDMS chips demonstrated comparable functionality, underscoring the potential of TPE-based devices for future scalable and cost-effective drug testing and cellular assays.

BACKGROUND Disorders of gut-brain interaction (DGBI) are defined as a variable combination of chronic or recurrent gastrointestinal symptoms. Early-life stressors have been implicated as possible contributing factors. AIM To determine if prematurity and neonatal factors influence the development of DGBI in adults. METHODS A case-control study was carried out at a tertiary referral center from July 2019 to July 2021. Cases (adults born with extremely premature < 29 weeks of gestation) were recruited from the Health of Adults Born Preterm Investigation cohort. Control subjects were recruited from the general population. All participants completed the Rome IV diagnostic questionnaire online. Cases completed anxiety and depression questionnaires (Patient-Reported Outcomes Measurement Information System-29 items, Generalized Anxiety Disorder-7 items, Patient Health Questionnaire-9 items). Neonatal data and sociodemographic status were collected. RESULTS A total of 79 cases and 124 controls were enrolled in the study. The group of adults born preterm exhibited a significantly higher prevalence of functional bowel disorders (P = 0.01) and a trend suggesting a higher prevalence of functional gastroduodenal disorders (P = 0.06). Among women born prematurely, the prevalence of functional gastroduodenal disorders, functional bowel disorders, and functional constipation was significantly higher compared to the female control group (P = 0.02 for all). The identified risk factors are categorized as directly linked to prematurity (e.g. , chorioamnionitis), indirectly related to prematurity (e.g. , anxiety, depression, and social skills as consequences of prematurity), or independent of prematurity (e.g. , female sex). CONCLUSION This is the first case-control study reporting the prevalence of DGBI in a cohort of well-characterized adults born prematurely. We confirm that prematurity is a risk factor for developing a DGBI.

Hirschsprung disease (HSCR) is a deadly congenital disorder where the enteric nervous system (ENS) is absent from the distal bowel. Current surgical treatment is generally life-saving but is often accompanied by long-term bowel problems and comorbidities. As alternative, we are developing a regenerative therapy based on rectal administration of the neurotrophic factor GDNF. We previously showed that administering GDNF enemas to HSCR mice soon after birth suffice to permanently induce a new ENS from tissue-resident neural progenitors. Here, we clarify the underlying mechanism using single-cell transcriptomics, signal transduction inhibitors and cell lineage tracing tools. Notably, we found that the neurogenic effect of GDNF is mediated by NCAM1, rather than by its canonical signaling receptor RET. We also unveiled the existence of multiple differentiation pathways that involve a larger than expected repertoire of tissue-resident neural progenitors, including a surprising one not derived from the usual neural crest. ### Competing Interest Statement RS and NP are co-founders of the biotech company Neurenati Therapeutics, which had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The remaining authors declare no competing interests. Canadian Institutes of Health Research, https://ror.org/01gavpb45, PJT-180290

Objectives:Pediatric intestinal pseudo-obstruction (PIPO) is a rare, heterogeneous, and severe gut motility disorder. In 2014, Chetaille et al. described chronic atrial and intestinal dysrhythmia (CAID) syndrome associated with a recessive SGO1 mutation (p.Lys23Glu) linking it to both intestinal pseudo-obstruction and cardiac dysrhythmia. This study aimed to describe the clinical and nutritional features and outcomes of pediatric patients with the homozygous SGO1 (p.Lys23Glu) mutation. Methods:We retrospectively enrolled children under 18 years with PIPO and homozygous (p.Lys23Glu) SGO1 mutation. Results:Eight patients were included (five girls), two were first-degree relatives. All exhibited a typical PIPO clinical presentation, but with a later onset than usually seen in primary PIPO (median age: 6.3 years). Contrast studies revealed massively distended small bowel and colon in all patients. Antroduodenal and/or colonic manometry, performed in six patients, revealed a neuropathic pattern. A full-thickness intestinal biopsy showed variable fibrosis of the smooth muscle internal layer associated with an abnormal presence of Cajal cells within the muscular layers. All patients required parenteral nutrition (PN) at a median age of 11.8 years. Five patients needed an ileostomy. At 18 years, one patient was off PN. Three patients developed sinus dysfunction: one at the time of PIPO diagnosis and two later. All required pacemakers. One patient died in her twenties secondary to cardiac complications. Conclusions:Recessive SGO1 mutation is a form of late onset PIPO associated with sinus dysfunction. Long-term outcome is unclear. Cardiac, intestinal, and neurologic follow-up is recommended as cerebral small vessel disease has been described in adults.

The International Network on Esophageal Atresia (INoEA) stands as a beacon of collaboration in addressing the complexities of this congenital condition on a global scale. The eleven board members, from various countries (USA, Canada, France, Australia, Italy, Sweden, Germany, and The Netherlands) and backgrounds (pediatric gastroenterology, pediatric surgery, pediatric pulmonology, nursing, and parents) met in a face-to-face symposium in Lille in November 2023, to identify challenges and solutions for improving global collaboration of the network.

BACKGROUND:To review the outcomes of premature patients with type C esophageal atresia (EA). METHODS:In this retrospective cohort study, charts of patients of type C EA patients were reviewed from 1992 to 2022. Outcomes of premature patients were compared to term patients. Preterm patients were analyzed to compare outcomes of infants with very low birth weights (VLBW) to patients >1,500 g as well as primary versus delayed anastomosis. RESULTS:Among 192 type C EA, 67 were premature. Median and interquartile range (IQR) gestational age and birth weight of preterm patients were 34 [33-36] weeks and 1965 [1740-2290] g. Delayed anastomosis was performed in 12 (18%) preterm vs. 3 (2%) term patients (p = 0.0003). Short-term postoperative outcomes were similar between preterm and term patients, except for recurrent fistula (16% vs. 6%, p = 0.01). Prematurity was associated with an increased need for long-term enteral tube feeding (56% vs. 10%, p = 0.0001) and parenteral nutrition (10 days vs. 0 days, p = 0.0004). The length of stay was 3 times longer when patients were premature (50 days vs. 17 days, p = 0.002). Delayed surgery in preterm patients was associated with post-operative leaks, strictures, recurrent fistula, prolonged enteral tube feeding, and gastrostomy insertion. Patients with very low birth weight (VLBW) were compared to other preterm patients and showed no difference in terms of rate of delayed surgery, and post-operative outcomes. CONCLUSION:Despite increased prematurity-related comorbidities and low birth weight, premature infants with type C EA/TEF have similar post-operative outcomes to term patients though recurrent fistula was more frequent with prematurity. TYPE OF STUDY:Retrospective cohort study. LEVEL OF EVIDENCE:III.

BACKGROUND. Glucosensing elements are widely distributed throughout the body and relay information about circulating glucose levels to the brain via the vagus nerve. However, while anatomical wiring has been established, little is known about the physiological role of the vagus nerve in glucosensing. The contribution of the vagus nerve to inflammation in the fetus is poorly understood. Increased glucose levels and inflammation act synergistically when causing organ injury, but their interplay remains incompletely understood. We hypothesized that vagotomy (Vx) will trigger a rise in systemic glucose levels and this will be enhanced during systemic and organ-specific inflammation. Efferent vagus nerve stimulation (VNS) should reverse this phenotype. METHODS. Near-term fetal sheep (n = 57) were surgically prepared using vascular catheters and ECG electrodes as the control and treatment groups (lipopolysaccharide (LPS), Vx + LPS, Vx + LPS + selective efferent VNS). The experiment was started 72 h postoperatively to allow for post-surgical recovery. Inflammation was induced with LPS bolus intravenously (LPS group, 400 ng/fetus/day for 2 days; n = 23). For the Vx + LPS group (n = 11), a bilateral cervical vagotomy was performed during surgery; of these n = 5 received double the LPS dose, LPS800. The Vx + LPS + efferent VNS group (n = 8) received cervical VNS probes bilaterally distal from Vx in eight animals. Efferent VNS was administered for 20 min on days 1 and 2 +/10 min around the LPS bolus. Fetal arterial blood samples were drawn on each postoperative day of recovery (-72 h, -48 h, and -24 h) as well as at the baseline and seven selected time points (3–54 h) to profile inflammation (ELISA IL-6, pg/mL), insulin (ELISA), blood gas, and metabolism (glucose). At 54 h post-LPS, a necropsy was performed, and the terminal ileum macrophages’ CD11c (M1 phenotype) immunofluorescence was quantified to detect inflammation. The results are reported for p < 0.05 and for Spearman R2 > 0.1. The results are presented as the median (IQR). RESULTS. Across the treatment groups, blood gas and cardiovascular changes indicated mild septicemia. At 3 h in the LPS group, IL-6 peaked. That peak was decreased in the Vx + LPS400 group and doubled in the Vx + LPS800 group. The efferent VNS sped up the reduction in the inflammatory response profile over 54 h. The M1 macrophage activity was increased in the LPS and Vx + LPS800 groups only. The glucose and insulin concentrations in the Vx + LPS group were, respectively, 1.3-fold (throughout the experiment) and 2.3-fold higher vs. control (at 3 h). The efferent VNS normalized the glucose concentrations. CONCLUSIONS. The complete withdrawal of vagal innervation resulted in a 72-h delayed onset of a sustained increase in glucose for at least 54 h and intermittent hyperinsulinemia. Under the conditions of moderate fetal inflammation, this was related to higher levels of gut inflammation. The efferent VNS reduced the systemic inflammatory response as well as restored both the concentrations of glucose and the degree of terminal ileum inflammation, but not the insulin concentrations. Supporting our hypothesis, these findings revealed a novel regulatory, hormetic, role of the vagus nerve in the immunometabolic response to endotoxin in near-term fetuses.

Oesophageal atresia–tracheoesophageal fistula (EA-TEF) is a common congenital digestive disease. Patients with EA-TEF face gastrointestinal, surgical, respiratory, otolaryngological, nutritional, psychological and quality of life issues in childhood, adolescence and adulthood. Although consensus guidelines exist for the management of gastrointestinal, nutritional, surgical and respiratory problems in childhood, a systematic approach to the care of these patients in adolescence, during transition to adulthood and in adulthood is currently lacking. The Transition Working Group of the International Network on Oesophageal Atresia (INoEA) was charged with the task of developing uniform evidence-based guidelines for the management of complications through the transition from adolescence into adulthood. Forty-two questions addressing the diagnosis, treatment and prognosis of gastrointestinal, surgical, respiratory, otolaryngological, nutritional, psychological and quality of life complications that patients with EA-TEF face during adolescence and after the transition to adulthood were formulated. A systematic literature search was performed based on which recommendations were made. All recommendations were discussed and finalized during consensus meetings, and the group members voted on each recommendation. Expert opinion was used when no randomized controlled trials were available to support the recommendation. The list of the 42 statements, all based on expert opinion, was voted on and agreed upon. Currently, systematic care for adults and adolescents with oesophageal atresia–tracheoesophageal fistula (EA-TEF) as they transition into adulthood is lacking. A multidisciplinary group of experts structured this Consensus Statement offering guidelines for the care of adolescents and adults with EA-TEF during transition.

OBJECTIVES:Aerodigestive disorders encompass various pathological conditions affecting the lungs, upper airway, and gastrointestinal tract in children. While advanced care has primarily occurred in specialty centers, many children first present to general pediatric gastroenterologists with aerodigestive symptoms necessitating awareness of these conditions. At the 2021 Annual North American Society for Pediatric Gastroenterology, Hepatology and Nutrition meeting, the aerodigestive Special Interest Group held a full-day symposium entitled, Pediatric Aerodigestive Medicine: Advancing Collaborative Care of Children with Aerodigestive Disorders. The symposium aimed to underline the significance of a multidisciplinary approach to achieve better outcomes for these complex patients.METHODS:The symposium brought together leading experts to highlight the growing aerodigestive field, promote new scientific and therapeutic strategies, share the structure and benefits of a multidisciplinary approach in diagnosing common and rare aerodigestive disorders, and foster multidisciplinary discussion of complex cases while highlighting the range of therapeutic and diagnostic options. In this article, we showcase the diagnostic and therapeutic approach to oropharyngeal dysphagia (OPD), one of the most common aerodigestive conditions, emphasizing the role of a collaborative model.CONCLUSIONS:The aerodigestive field has made significant progress and continues to grow due to a unique multidisciplinary, collaborative model of care for these conditions. Despite diagnostic and therapeutic challenges, the multidisciplinary approach has enabled and greatly improved efficient, high-quality, and evidence-based care for patients, including those with OPD.

Background: The aim of this study was to establish the feasibility and safety of the use of indocyanine green technology during pediatric intestinal resections. While indocyanine green fluorescence angiography (ICG-FA) has been advocated as an imaging technique to assess bowel perfusion in adults, few studies have evaluated this technology in a pediatric context.Methods: A prospective clinical trial was conducted. Patients 16 years old or younger undergoing a surgery potentially requiring an intestinal resection were eligible. Patients received a standardized intravenous injection of indocyanine green and intestinal perfusion was evaluated. The study endpoints included safety, impact on bowel resection and feasibility and acceptance of ICG-FA in this population. Results: From May 2020 to March 2021, 30 consecutive patients were included in this trial. Final analysis was done on 28 patients with a median age of 15.00 [6.36,85.00] weeks and weight of 5.58 [3.64,11.70] kg at surgery. Adequate fluorescence was achieved in less than one minute for all cases with an average dose of 0.14 mg/kg. No adverse event related to indocyanine green occurred. ICG-FA versus standard assessment of potential resection sites differed in 62% (95% IC 0.41-0.82) of our cases. Qualitative analysis demonstrated that 95% of the surgical team agreed that ICG-FA was safe.Conclusions: The use of ICG-FA is feasible and safe for pediatric intestinal resections. Introduction of ICGFA was simple and acceptance rates were high within the surgical team. This fluorescence imaging may be a valuable imaging technology for intestinal resections in pediatric surgery. (c) 2022 Elsevier Inc. All rights reserved.

Background and Aim To assess the outcomes of children born with esophageal atresia/tracheoesophageal fistula (EA-TEF) with concomitant SARS-CoV-2 infection. Methods An international survey was circulated to the International Network of Esophageal Atresia ( INoEA) members from April 2020 to May 2022. Information on demography, type of EA-TEF, co-morbidities, complications, hospitalization, and therapies administered for SARS-CoV-2 infection was collected for all patients. Results Forty-two patients from April 2020-May 2022, with a mean age of 6.8 years were reported from Argentina, Switzerland, Netherlands, Canada, France, Italy, Australia and Turkey. 34 patients (81%) had a type C, EA-TEF. 30 had respiratory comorbidities, 14 had associated cardiac malformations and 14 had a history of recurrent anastomotic stricture. Reported medications included proton-pump inhibitors (n=14), inhaled bronchodilators (n=3) and inhaled corticosteroids (n=4). Six patients (14%) were hospitalised. Three required respiratory support and one required extra-corporal membranous oxygenation. There were no deaths. Respiratory, cardiac and gastrointestinal comorbidities were not associated with increased risk of hospitalization. Concomitant medication at time of infection was associated with increased risk for hospitalization with SARS-CoV-2 infection (p=0.0035), however PPI alone was not significantly associated with increased risk for hospitalization (p=0.16). Conclusion Rates of hospitalization with SARS-CoV-2 are higher for patients with EA-TEF than the general pediatric population, with increased risk in those on medication in patients. 67% of those admitted required respiratory support. Infection likely does not represent a risk for severe respiratory complications or severe outcome.

Background: Vagus nerve stimulation (VNS) reduced inflammation induced by lipopolysaccharide (LPS) in an adult rat sepsis model. A multi-dimensional heart rate variability (HRV) index reliably tracked the inflammatory profile in near-term sheep fetuses. The effects of VNS on neonates are not known. First, in a neonatal piglet model of sepsis, we present an approach to evaluate the effect of VNS on systemic inflammatory response induced by a high dose of LPS to mimic late-onset neonatal sepsis. Second, we present an analytical pipeline to validate our fetal-sheep-derived HRV inflammatory index in neonatal piglets to test its performance in different species, older developmental stages, and more robust septic responses. Methods: Three neonatal piglets of 7–14 days of age with 2.4–4 kg in body weight were used in this proof-of-principle study. Following anesthesia, electrodes were attached bilaterally to the cervical portion of the vagus nerve to allow for stimulation of the left vagus (VNS) and bilateral vagus electroneurogram (VENG). Electrocardiogram (ECG), blood pressure (BP), and VENG were recorded for the duration of the experiment. After baseline recording, the piglets were administered LPS at 2 mg/kg IV bolus. In the VNS-treated piglet, the vagus nerve was stimulated for 10 min prior to and 10 min after the injection of LPS. In both groups, each 15 min post LPS, an arterial blood sample was drawn for blood gas, lactate, and glucose as well as the inflammatory cytokines measured by a quantitative ELISA multiplex panel. At the end of the experiment, the piglets were euthanized. BP and ECG-derived HRV were calculated and VENG was analyzed. Results: The piglets developed a potent inflammatory response to the LPS injection with TNF-α, IL-1β, IL-6 and IL-8 peaking between 45 and 90-min post-injection. VNS diminished the LPS-induced systemic inflammatory response, with a decrease in measured cytokines levels ranging from two to ten-fold. We present a low-cost, easy-to-implement design of a VNS/VENG probe and a framework to analyze VENG in response to LPS. Furthermore, the HRV index accurately tracked cytokine temporal profiles’ which was reflected in the power-spectral and complex properties of VENG when applying the machine learning model derived from HRV. Discussion: We present a novel method to model, manipulate, and track neonatal sepsis using VNS/VENG. Our supportive findings suggest that (1) the HRV index of the systemic inflammatory response applies across species pre- and postnatally, (2) the HRV index performs well at different degrees of sepsis (i.e., nanogram and milligram doses of LPS), and (3) the present VNS paradigm effectively suppresses LPS-induced inflammation, even with high doses of LPS; an effect that is reflected by changes in the shared mathematical properties of both VENG and HRV. These findings suggest that the HRV inflammatory index reflects underlying changes in the VENG activity. The presented method lays a foundation for larger studies to investigate the mechanisms and therapeutic potential of early postnatal VNS intervention to counteract sepsis progression. Moreover, the presented experimental and analytical frameworks highlight the potential for HRV monitoring to serve as an early biomarker for tracking the systemic inflammatory response.

PurposeAnorexia Nervosa (AN) is a serious and potentially lethal mental disorder characterised by a deliberate quest to reduce one's weight. It can have multiple physical and psychological consequences. The clinical presentation of AN can include gastrointestinal symptoms, however, the pathophysiology of these symptoms in the context of AN remains uncertain. It is hypothesised that patients with AN may have an increase in intestinal permeability, which could lead to an increase in faecal calprotectin (fCP), a marker of intestinal inflammation. No relation between AN and elevation of fCP has been previously described in literature. MethodsEight patients hospitalised for AN have a dosage of fCP. ResultsCalprotectine was found to be elevated in 50% of cases, with or without any underlying comorbid gastrointestinal disease. Only the duration of illness tended to be associated with the increase in fCP suggesting a greater alteration as a function related to the time of denutrition. ConclusionAlthough these findings provide insights in the potential pathophysiology of gastrointestinal symptoms in AN, further studies that evaluate the factors associated with elevated fCP in patients with AN are needed.

Christophe Faure, Nikhil Thapar, Carlo Di Lorenzo (eds.), Pediatric Neurogastroenterology, https://doi.org/ 978-3-031-15228-3

Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify novel genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein altering de novo coding variants in complex cases (p=3.3e-4), especially in genes that are intolerant of loss of function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. This research may have implications for the mechanisms of other rare congenital anomalies.