Abstract

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses-often de novo-contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

Highlights

  • The enteric neurons and glia in the intestine form the enteric nervous system (ENS), which is derived from the neural crest

  • Hirschsprung disease (HSCR) patients were subdivided in three groups for the Copy Number Variation (CNV) detection study: patients with HSCR and additional anatomical malformations or neurological defects (HSCR-AAM), but without a REarranged during Transfection gene (RET) pathogenic variant, or other causal genetic defect, patients with HSCR and a known variant in RET or another causal gene, and patients with only HSCR, without a deleterious RET coding variant or other causal genetic defect

  • Large copy number losses reveal novel Hirschsprung disease candidate genes We find that these rare Copy Number (CN) losses contained more “ENS genes” that were a constrained coding regions (CCR)

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Summary

Introduction

The enteric neurons and glia in the intestine form the enteric nervous system (ENS), which is derived from the neural crest. Proliferation, differentiation or survival of these ENCCs [2] is known to result in Hirschsprung disease (HSCR). This congenital enteric neuropathy is characterized by an absence of the ENS, or called enteric ganglia, in the distal colon (called aganglionosis). The common haplotypes influence HSCR penetrance and include haplotypes near RET. They are known to increase disease risk substantially, especially if homozygous, in specific combinations (rs2506030, rs7069590, rs2435357) [10,11], together with other risk loci near the

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