Hirschsprung disease (HSCR) is a common cause of functional colonic obstruction in children. The currently available genetic testing is often inadequate as it mainly focuses on RET and several other genes, accounting for only 15-20% of cases. To identify novel, potentially pathogenic variants, we isolated a panel of genes from a whole-exome sequencing study and from the published mouse aganglionosis phenotypes, enteric nervous system development, and a literature review. The coding exons of 172 genes were analyzed in 83 sporadic patients using next-generation sequencing. Rare stop-gain, splice-site variants, frameshift and in-frame insertions/deletions and non-synonymous variants (conserved and predicted to be deleterious) were prioritized as the most promising variants to have an effect on HSCR and subjected to burden analysis. GeneMANIA interaction database was used to identify protein-protein interaction-based networks. In addition, 6 genes (PTPN13, PHKB, AGL, ZFHX3, LAMA1, and AP3B2) were prioritized for follow-up studies: both their time-space expression patterns in mouse and human colon showed that they are good candidates for predicting pathogenicity. The results of this study broaden the mutational spectrum of HSCR candidate genes, and they provide an insight into the relative contributions of individual genes to this highly heterogeneous disorder. © 2017 The Author(s).