Size matters: large copy number losses reveal novel Hirschsprung disease genes

Abstract Background Hirschsprung disease (HSCR) is characterized by absence of ganglia in the intestine. Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). HSCR is a complex genetic disease in which the loss of enteric ganglia stems from a combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Pinpointing the responsible culprits within a large CNV is challenging as often many genes are affected. We investigated if we could find deleterious CNVs and if we could identify the genes responsible for the aganglionosis. Results Deleterious CNVs were detected in three groups of patients: HSCR-AAM, HSCR patients with a confirmed causal genetic variant and HSCR-isolated patients without a known causal variant and 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 HSCR-AAM patients had larger copy number losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes in Copy Number 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 in vivo . Conclusion Rare large Copy Number losses - often de novo - contribute to the disease in HSCR-AAM patients specifically. We proved the involvement of five genes in enteric nervous system development and Hirschsprung disease.