Background Hirschsprung disease (HSCR) is a rare neurodevelopmental disorder caused by disrupted migration and proliferation of enteric neural crest cells during enteric nervous system development. Genetic studies suggest a complex etiology involving both rare and common variants, but the contribution of ultra-rare pathogenic variants (PAs) remains poorly understood.

Methods We perform whole-exome sequencing (WES) on 301 HSCR probands and 109 family trios, employing advanced statistical methods and gene prioritization strategies to identify genes carrying de novo and ultra-rare coding pathogenic variants. Multiple study designs, including case-control, de novo mutation analysis and joint test, are used to detect associated genes. Candidate genes are further prioritized based on their biological and functional relevance to disease associated tissues and onset period (i.e., human embryonic colon).

Results We identify 19 risk genes enriched with ultra-rare coding pathogenic variants in HSCR probands, including four known genes (RET, EDNRB, ZEB2, SOX10) and 15 novel candidates (e.g., COLQ, NES, FAT3) functioning in neural proliferation and neuromuscular synaptic development. These genes account for 17.5% of the population-attributable risk (PAR), with novel candidates contributing 6.5%. Notably, a positive correlation between pathogenic mutational burden and disease severity is observed. Female cases exhibit at least 42% higher ultra-rare pathogenic variant burden than males (P = 0.05).

Conclusions This first-ever genome-wide screen of ultra-rare variants in a large, phenotypically diverse HSCR cohort highlights the substantial contribution of ultra-rare pathogenic variants to the disease risk and phenotypic variability. These findings enhance our understanding of the genetic architecture of HSCR and provide potential targets for genetic screening and personalized interventions.

The authors have declared no competing interest.

This study was funded by NIH grants DK135089 to AC and SC, HD028088 to AC and HD116004 to SC. The funders had no role in design of the study or data interpretation.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics committee/IRB of New York University Grossman School of Medicine gave ethical approval for this work with IRB number i17-01813

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WES data of the HSCR case cohort: The data can be available upon reasonable request from the corresponding author, A.C. Due to the inclusion of individual genetic information, the data is not publicly available to protect the privacy of research participants.

WES data of the UKB controls: The data is available by applying for access through UK Biobank website.

ASD control trio data: The data is available from Iossifov et al., 2014 (Supplementary Table 2). Single cell RNA sequencing data of human embryonic gut: The data can be downloaded with the link https://cellgeni.cog.sanger.ac.uk/gutcellatlas/final_fetal_object_cellxgene.h5ad