Use of anoctamin 1 (ANO1) to evaluate interstitial cells of Cajal in Hirschsprung's disease

Interstitial cells of Cajal (ICCs) are pacemaker cells involved in facilitating neurotransmission and the generation of slow electrical waves necessary for colonic peristalsis. Their distribution has been found to be abnormal in the aganglionic and ganglionic colon in Hirschsprung's disease (HSCR) using c-kit-labelling. Anoctamin-1 (ANO1) is a Ca2+-activated Cl- channel thought to be specifically expressed on ICCs. Unlike c-kit, it plays a key role in ICC pacemaker activity. We aimed to investigate the utility of ANO1 in evaluating the colonic ICC network in HSCR. We collected full-length pull-through specimens from children with HSCR (n = 10). Control colon specimens were collected at colostomy closure in children with anorectal malformation (n = 6). The distribution of ANO1 and c-kit expression was evaluated using immunofluorescence and confocal microscopy. ANO1 expression was quantified using Western blot analysis. ANO1 was not expressed on 23 % of c-kit immuno-positive cells in the circular muscle; however, 100 % of ANO1-positive ICCs were c-kit positive. The distribution of ANO1-positive ICCs was sparse in aganglionic colon, with a modest reduction in ICCs seen in the ganglionic colon in HSCR compared to controls (p = 0.044). ANO1 protein expression was reduced in aganglionic colon but similar in ganglionic colon relative to controls. ANO1 is preferential to c-kit in evaluating the ICC network in HSCR due to its specificity and functional importance. Abnormal distribution of ANO1-positive ICCs in the ganglionic colon in HSCR may contribute to persistent bowel symptoms in some patients after pull-through surgery.