NOX5 is Expressed Aberrantly but Not a Critical Pathogenetic Gene in Hirschsprung Disease

Background: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells in the distal gastrointestinal tract (GI), which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. Recent studies have suggested NADPH oxidase 5 (NOX5) as a candidate risk gene for HSCR. In this study, we examined the function of NOX5 to verify its role in the development of enteric nervous system (ENS). Methods: HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control specimens (n = 10) were obtained at the time of colostomy closure in patients. The NOX5 expression in aganglionic and ganglionic segments of HSCR colon and normal colon were analyzed by immunohistochemistry (IHC), western blot and RT-qPCR. The gene expression levels and spatiotemporal expression spectrum of NOX5 in different development stages of zebrafish embryo were investigated using real-time quantitative PCR (RT-qPCR) and in-situ hybridization (ISH). The enteric nervous system in NOX5 Morpholino (MO) knockdown and wild type (WT) zebrafish embryo was analyzed by whole-mount immunofluorescence (IF). Intestinal transit assay was performed to analyze the gastrointestinal motility in NOX5 knockdown and control larvae. Results: NOX5 is strongly expressed in the ganglion cells in the proximal segment of HSCR colons and all segments of normal colons. Moreover, the expression of NOX5 is markedly decreased in the aganglionic segment of HSCR colon compared to the ganglionic segment. In zebrafish, NOX5 mRNA level is the highest in one cell stage embryos and it is decreased by the development of the embryos. Interestingly, the expression of NOX5 appears to be enriched in the nervous system. However, the number of neurons in the GI tract and the GI motility were not affected upon NOX5 knockdown. Conclusions: NOX5 may play a role in the early development of zebrafish embryo, but not required for the ENS development and loss of NOX5 didn’t affect the GI motility in zebrafish. Nevertheless, we demonstrated that NOX5 specifically expressed in the ganglionic cell in colon tissue and markedly decreased in the aganglionic segment.