HIRSCHSPRUNG'S DISEASE
Principal Investigators; A. Krantis, PhD. Wm. Staines, PhD.
Resident Research Associate: E. Donat, MD
Graduate Trainee: Amy Peaire (PhD)
Collaborators: Dr S. Rubin (CHEO), Drs P. Russo and M. Di Lorenzo ( Ste Justine Hosp. Montreal)
Hirschsprung's Disease (congenital megacolon) is the most common form of congenital bowel obstruction. Intestinal obstruction often occurs immediately after birth. The condition is characterized by an aganglionic non-functional segment of distal colon. There are three main theories for the aganglionosis. The first and most popular theory holds that there is a defect in the vagal neural crest resulting in disordered neuronal migration and population of the distal gut. The second theory holds that the enteric neuronal precursors colonize the gut normally, but subsequently, there is a region specific neuronal cell death as a result of some toxic event. The third theory suggests that the enteric microenvironment is abnormal and as such, unable to support or else locally excludes enteric precusors. Our studies show the presence of disrupted ganglia and surviving NO neurons in some Hirschsprung's disease (HSCR) aganglionic intestine samples. Little is known about NO in the pathological mammalian gut, in particular the human. Selective survival of NO synthesizing neurones occurs in CNS lesions such as Huntington's chorea and we believe that we have discovered the same phenomenon in the gut. It thus appears that in some forms of HSCR, there is selective sparing of NOS-containing neurons. Therefore characterising this disease as an aganglionosis is an oversimplification. Although there is a familial association for the disease, the genetics is not understood.The molecular genetics pertaining to HSCR clearly supports the notion that this disease can have origins other than failure of neural crest cell migration. It has been shown that a receptor tyrosine kinase (RET) proto-oncogene is associated with both sporadic and familial forms of autosomal dominant HSCR. In addition, mutations in the gene for endothelin or in the gene for one of its receptors, the endothelin B receptor gene (EDNRB) can give rise to the disease. To date some 21 mutations within these genes have been pinpointed as probable causes of this disorder. More importantly this data points to at least two mechanistic routes for the development of this disease. In an attempt to address questions on the mechanisms of this disorder we are examining whether we can correlate determination of a specific gene mutation with the neurochemistry of the surviving enteric neurones at the lesion boundary and patient histories for given HSCR cases. These studies include graduate and postgraduate training projects which are described below.
The RET protooncogene signalling pathway: Dr Wu (Pediatric Surgeon, Univ Beijing) joined our group in December 19 96 and is funded through a fellowship from the Dept. of Surgery and the Research Institute of the Children,s Hospital of Eastern Ontario to undertake research with the DDRG. His training in biochemical and molecular analysis of human tissue, including intestine of HSCR patients, compliments our expertise in this area. Dr Wu,s research efforts focus on the role of RET in the pathogenesis of HSCR. More specifically, he is undertaking mutation analysis using a combination of denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism (SSCP). The smooth muscle of the diseased colon itself is less favorable for neuronal development than the normally innervated region, with nerve cell diameter growth being consistently inhibited when neurons are added to smooth muscle cultures from the aganglionic region of HSCR patient. On this basis, it has been proposed that an intrinsic membrane-linked factor of the aganglionic region itself inhibits neuronal survival, as opposed to neuronal absence due to failure of neural crest migration. We postulate, that the microenvironment at the late stage of ENS development is responsible for the enteric neurone mutations in the RET loci. We believe that a treatment for HSCR may be developed based on targeting RET or the signalling pathway that is initiated when this receptor is activated. We are currently evaluating the putative ligand for the RET site, GDNF. This glial cell derived neurotrophic factor, is necessary for the survival of enteric neurones.
Endothelins and Hirschsprung's Disease: Studies have shown that the signal conveyed by endothelin-3 via the endothelin-B receptor is specifically required for the normal development of enteric ganglion neurons in mice and humans. In parallel with our study of RET, Amy Peaire (PhD trainee) is investigating endothelins and the endothelin B receptor involvement in HSCR. In addition, this project involves characterization of those cells which survive endothelin-induced, NO mediated neurotoxic events such as occur in Huntington's Chorea, and which we postulate to occur in some forms of Hirschsprung's disease. This research involves neurochemical characterization of cells surviving in the distal colon of HSCR, and correlation of cell survival with known gene mutations implicated in HSCR. A large number of HSCR cases are being screened to determine the prevalence of HSCR subpopulations. It is proposed that the NOS neuron-preserving subpopulation of HSCR patients may be correlated with a specific gene mutation. The recent identification of several previously unidentified HSCR mutations combined with our large number of HSCR specimens, permits characterization of the new mutations with regard to resulting length of aganglionic segment, heritability, and gene penetrance. Also of interest is the number of sporadic vs. familial HSCR cases investigated. We are examining the hypothesis that HSCR and Huntington's are due to a disruption in endothelin levels. Endothelins are known to be able to overstimulate neurons and cause neuronal death. Therefore, we are undertaking lesion experiments utilising primary cultures of enteric neurones to examine whether endothelins, which can effect neuronal lesion in the CNS, can induce enteric neuronal cell death.
Heat Shock proteins: Dr. E Donat MD (Senior Gastroenterology Resident) is undertaking research to study cell signalling, in particular the role of the haemoxygenase derived gas CO in the human enteric nerve layers. In addition, Dr Donat is investigating the colon of HSCR patients for evidence of ganglionic injury and hence examine the notion that neural disruption in the colon of HSCR patients can occur other than by failure of the development of the enteric neural networks in this region. We believe that there is enough evidence that at least some cases of HSCR are acquired through injury to the enteric ganglia. The focus of this research is to examine the expression of heat shock protein, heme oxygenase-1 (HO-1) which is induced in cells undergoing metabolic stress. We have determined that neurones in the transition zone between ganglionic and aganglionic region display dramatic increase in HO-1 expression and this likely represents neurones that are under stress but still viable.