The Role of and Ultra-Rare Variants in Hirschsprung Disease (HSCR): Extended Gene Discovery for Risk Profiling of Patients.

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 and ultra-rare coding pathogenic variants. Multiple study designs, including case-control, 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 ( , , , ) and 15 novel candidates (e.g., , , ) 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.