Fig 1

(A) For this study we included 326 controls (group 4) and 58 HSCR patients. We determined the RET and / or known disease gene coding mutations of HSCR patients (n = 58). The HSCR patients with coding mutations were included in group 2 (n = 15). We determined the presence of associated anomalies in the rest of the HSCR patients, including them in either the group containing associated anomalies (HSCR-AAM; group 1, n = 23) or in the group without associated anomalies (group 3, n = 30). (B) For all subgroups of HSCR patients and the controls Copy Number profiles were determined. To select candidate genes, we ranked CNVs according to their frequency in the unaffected controls from the Deciphering Developmental Disorders project (for illustrative purposes a screenshot of their UCSC genome browser track; https://genome-euro.ucsc.edu/ is depicted). We determined if genes included in the rare CNVs were “ENS genes”: genes with increased expression in isolated ENS (with and without the addition of GDNF) cells compared to the whole intestine (for illustrative purposes a screenshot of a heatmap of differential gene expression of known disease genes derived from brb-array tools (https://brb.nci.nih.gov/BRB-ArrayTools/) is depicted. Additionally, we determined if a gene was known as a Constrained Coding Region (CCR)(For illustrative purposes a screenshot of gnomAD browser website where such scores can be found; https://gnomad.broadinstitute.org/). Next, we determined whether disruption of the main candidate genes resulted in a reduction of enteric neurons in zebrafish. (C) In parallel, we evaluated the contribution of predisposing haplotypes across groups. Combined, we suggest that the RSnc (predisposing haplotypes), RSrcv (deleterious rare variant burden) and RScnv (deleterious Copy Number Variation) result in the genetic risk for Hirschsprung disease. Abbreviations: CNV = Copy Number Variation, ENS = Enteric Nervous System, RSnc = Risk Score non-coding variants, RSrcv = Risk Score rare coding variants, RScnv = Risk Score CNV, CCR = Constrained Coding Region.