Single-nucleus transcriptomics reveals multicellular remodeling beyond the aganglionic segment in Hirschsprung disease

Hirschsprung disease (HSCR) is defined by the absence of enteric ganglion cells in the distal bowel, yet postoperative bowel dysfunction and Hirschsprung-associated enterocolitis (HAEC) can persist after resection of the aganglionic segment. Here, we generated a segment-resolved single-nucleus RNA sequencing atlas (snRNA-seq) of ganglionic, transition and aganglionic colonic segments from three children with short-segment HSCR and transverse-colon tissues from three age-matched control infants. After quality control, 93,256 nuclei were retained and annotated as 14 major colonic cell types. Enteric neurons from HSCR ganglionic segments showed reduced representation of molecular programs associated with synaptic organization, neuronal projection development, neurotransmission, and cholinergic synapses. Enteric glial cells showed compositional remodeling, while canonical enteric glia exhibited inflammation-associated transcriptional changes. Stromal analyses indicated segment-specific remodeling, with relative enrichment of inflammation-associated S4 fibroblasts in ganglionic and transition segments and more prominent myofibroblasts and collagen deposition in the aganglionic segment. Epithelial cells in the ganglionic segment showed transcriptional alterations in programs related to barrier homeostasis. CellChat analysis further suggested remodeling of candidate stromal–epithelial and neuron–glia communication networks. These findings provide a human single-nucleus resource and generate testable hypotheses regarding segment-specific multicellular remodeling in HSCR-affected bowel.