PRDM9 Deficiency Drives Mosaic Promoter Deletions in Sporadic Hirschsprung Disease and Supports Blood-Based Molecular...

Hirschsprung disease (HSCR) is a congenital enteric neuropathy with distal aganglionosis and dysmotility. Germline mutations explain many familial cases, but most sporadic cases lack a molecular explanation. We sought noncanonical mechanisms and blood-accessible biomarkers.

We performed RNA-sequencing (RNA-seq) on paired aganglionic and ganglionic colon biopsy specimens from 103 sporadic HSCR patients, with control colon samples (n = 22). Blood whole-genome sequencing (WGS) was available for 41 patients with RNA-seq profiles. We assessed PR/SET domain 9 (PRDM9) in patient tissues, perturbed prdm9 in zebrafish and mice, and performed mechanistic profiling in PRDM9-knockout human-induced pluripotent stem cell-derived enteric neural crest cells. Mosaic promoter deletions (MPDs) were called from colon WGS (n = 30; matched blood, n = 27) and summarized as a blood MPD score in blood WGS cohorts (discovery: 89 HSCR and 43 controls; validation: 165 HSCR and 42 controls), alone and combined with the polygenic risk score.

A predominant subgroup (79.6% [82 of 103]) showed coordinated repression of neurogenesis programs. Promoter motif enrichment implicated PRDM9, which we localized to normal enteric nervous system but found down-regulated in aganglionic tissue with promoter hypermethylation. Prdm9 perturbation reduced HuC/D-positive differentiated enteric neurons and impaired motility in zebrafish and mice. In enteric neural crest cells, PRDM9 loss redistributed DNA double-strand breaks toward promoters and enhancers, generated MPDs at neurogenesis loci, and correlated with transcriptional repression and impaired neuronal differentiation. Blood MPD score discriminated HSCR from controls in a discovery cohort (area under the receiver operating characteristic curve [AUROC], 0.78) and an independent validation cohort (AUROC, 0.82), and improved with polygenic risk score (AUROC, 0.89 and 0.91, respectively).

PRDM9 deficiency links ectopic DNA breaks to MPDs and impaired enteric neuronal differentiation in a predominant molecular subgroup within sporadic HSCR and enables complementary, noninvasive molecular stratification.