ヒルシュスプルング病（全結腸型又は小腸型）

Hirschsprung disease (HSCR) is a congenital enteric neuropathy caused by disrupted development of enteric neural crest-derived cells (ENCDCs). Although pathogenic coding variants in RET account for many cases, the largest genetic contribution to HSCR risk arises from a common noncoding variant (rs2435357) within a SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. However, the ENS cell types affected by this enhancer and the mechanisms by which these transcriptional changes lead to HSCR remain unknown. Here, we investigated the role of this enhancer by generating mice carrying a deletion of the orthologous Ret mcs+9.7 enhancer (Δmcs+9.7). Single-cell RNA sequencing of E14.5 embryonic gut demonstrated that enhancer deletion reduced Ret expression by 8% without altering ENS cell composition. However, reduced Ret expression was restricted to differentiating neurons and inhibitory motor neuron lineages, revealing cell type-specific enhancer activity. To determine the functional consequences of further reducing Ret dosage, we generated compound heterozygous mice carrying both the enhancer deletion and a Ret coding null allele (+/Δmcs+9.7;+/CFP). These mice exhibited additive reductions in Ret expression, altered Sox10 expression, dysregulation of cell-cycle and neuronal differentiation programs, and selective depletion of developing inhibitory motor neuron lineages. These findings establish a cell type-specific role for the mcs+9.7 enhancer in modulating Ret dosage and reveal how subtle enhancer perturbations alter neural subtype specification without overt hypoganglionosis, suggesting that HSCR arises from a cascade of cellular defects triggered by >50% loss of Ret function.

BACKGROUND: Current surgical treatment of Hirschsprung disease (HSCR), a rare congenital intestinal disorder, is frequently complicated by defecation dysfunction. The role of glycogen synthase kinase 3 beta (GSK3β), aberrantly highly expressed in HSCR tissues, in the HSCR pathogenesis remains unclear. METHODS: Publicly available scRNA-seq data of HSCR were analyzed. Histopathological characteristics of clinical samples were observed from HSCR children using Hematoxylin-Eosin staining. Regulation of the CXCL9:SPP1 (CS) polarization of glial cells by the GSK3β-NOTCH axis was assessed in both clinical samples and Schwann cells through immunofluorescence (IF), qRT-PCR, and Western blot. Regulation of neuronal development by the CS polarization was assessed in a co-culture system of Schwann cells and mouse dorsal root ganglion neurons using TUNEL staining, ELISA, IF, and patch-clamp techniques. In an Ednrb knockout mouse model, we verified in vivo that GSK3β regulated CS polarization of enteric glial cells via the NOTCH signaling pathway, which exacerbated developmental abnormalities of the enteric nervous system (ENS) and accelerated the progression of HSCR in vivo. RESULTS: Enteric glial cells were significantly increased in HSCR mouse samples, mainly as an SPP1+ subpopulation where GSK3β was highly expressed and the NOTCH signaling pathway was significantly enriched. GSK3β knockdown significantly attenuated CS polarization and blocked the NOTCH pathway. Mechanistically, GSK3β promoted enteric glial cell CS polarization by activating the NOTCH signaling pathway and aggravated the developmental abnormalities of the ENS, ultimately exacerbating postnatal ENS dysfunction and thereby potentially worsening HSCR-associated complications. CONCLUSION: GSK3β upregulation promotes NOTCH signaling pathway activation, which in turn regulates CS polarization and potentially exacerbates postnatal ENS dysfunction and aggravates HSCR-associated complications.

PURPOSE: This study evaluates the role of intraoperative frozen-section circumferential biopsies ("donuts") in preventing transition zone (TZ) pull-through (PT) in patients with Hirschsprung disease (HSCR). METHODS: We performed a single-institution retrospective review of intraoperative and final pathology reports for all PTs between 2020-2026 in which a frozen-section donut was obtained from planned anastomotic site based on previously confirmed normal full-thickness biopsy. If the initial donut was abnormal, additional proximal donuts were obtained. Donuts were considered normal when ganglion cells were present circumferentially, and fewer than two hypertrophic submucosal nerves were observed per high-power field. RESULTS: Frozen-section donuts were sent for intraoperative consultation in 134 PTs. Fourteen cases required two donuts, and two cases required three; among these 16 cases, 14 were due to abnormal pathology, and two for technical reasons. Final pathology was concordant with frozen-section findings in 130 patients (97.0%). Additional donuts prevented TZ PT in 13 patients (9.6%). Two patients (1.5%) had circumferential ganglion cells with hypertrophic nerves on final pathology despite normal frozen-section results. Non-circumferential ganglion cells were identified in two patients: one was accepted after two abnormal donuts and discussion with family; the other resulted from inaccurate donut processing. CONCLUSION: In this series, frozen-section circumferential biopsies reduced the incidence of TZ in PT, with only 3.0% of patients demonstrating TZ on final pathology and an additional 9.6% prevented intraoperatively. Routine use of this technique, alongside ongoing improvements in pathologic processing, enhances intraoperative decision-making and may contribute to safer, more effective PT procedures for HSCR. LEVEL OF EVIDENCE: IV.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of inherited cornification disorders caused by defects in epidermal barrier formation. Mutations in CYP4F22 are an uncommon cause of ARCI and are associated with variable phenotypes including lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE). This is a report of a genetically confirmed case of CYP4F22-related ARCI in a child born to consanguineous parents who presented with collodion membrane at birth followed by persistent ichthyosiform scaling and palmoplantar keratoderma. Genetic analysis identified a homozygous pathogenic CYP4F22 variant, c.1303C>T p.(His435Tyr). In addition to cutaneous findings, the patient had Hirschsprung disease managed surgically during infancy and was followed by pediatric nephrology for Bartter-like manifestations associated with hypokalemia and bilateral renal stones. This case highlights the importance of recognizing phenotypic heterogeneity of CYP4F22-associated ARCI and describes unusual extracutaneous manifestations in association with this rare genodermatosis.

BACKGROUND: Accurate identification of ganglionated bowel is essential during laparoscopic pull-through for Hirschsprung Disease (HD), yet intraoperative biopsy interpretation is time-sensitive and operator-dependent. Fluorescence confocal microscopy (FCM) provides rapid imaging of fresh tissue, and deep-learning (DL) has the potential to extract diagnostic patterns from these images automatically. However, DL development is limited by HD rarity and images scarcity. Generative-AI may address this gap by synthesizing realistic images to expand underrepresented data, improving model robustness. This study aims to evaluate whether conditional generative adversarial network (cGAN)-synthetic FCM images can improve DL performance for discriminating ganglionic cells. METHODS: Between November 2024 and November 2025, intraoperative FCM was routinely performed during laparoscopic pull-through for HD. High-resolution images were annotated to identify ganglionic and aganglionic regions, subdivided into 256×256-pixel tiles (resolution 0.5x0.5μm/pixel), and grayscale (128 levels) converted. Three convolutional neural networks (CNN), having the same architecture, were trained on different datasets: CNN-1 considered only real tiles (461 ganglionic and 1374 aganglionic), CNN-2 considered the same real tiles (461 ganglionic and 1374 aganglionic) and 913 conditional-GAN simulated ganglionic tiles, and CNN-3 considered the same real tiles (461 ganglionic and 1374 aganglionic) and 913 data augmented ganglionic tiles. CNNs were validated in the same independent testing dataset composed of real 590 aganglionic and 198 ganglionic tiles. RESULTS: CNN-1, CNN-2 and CNN-3 identified ganglionic tiles with 75%, 84% and 80% accuracy, 78%, 91% and 79% sensitivity, 73%, 78% and 75% specificity, respectively. CNN-2 achieved a significantly higher ROC-AUC than CNN-1 (0.94 vs 0.84 and 0.80; P<0.01). CONCLUSION: Synthetic augmentation with cGAN-generated ganglionic tiles significantly improved CNN discrimination of ganglionic bowel on real FCM images, supporting generative-AI as a scalable strategy to strengthen DL for discriminating ganglionic bowel in HD.