Molecular and Pathological characterization of a non-aganglionic congenital megacolon in the rabbit

Congenital megacolon (CM) is a severe colonic dysfunction usually associated with an underlying aganglionosis of the enteric nervous system (ENS). CM unrelated to aganglionosis may also occur with a largely unknown pathogenesis. Aim: to characterize a new, non-rodent natural model of non-aganglionic congenital megacolon. The CM phenotype is related to an incomplete dominant allele at the English spotting locus (En) and appears only in homozygous En/En animals. Methods: An F1 population of 80 animals was created crossing En/en rabbits. En/En rabbits (almost completely with a white fur due to the absence of melanocytes in the skin) and littermate controls (en/en) (normally coloured) have been monitored since birth up to severe deterioration of En/En animals with the CM phenotype. Cecum and ascending colon of controls (n=4) and CM (n=6) were processed for quantitative double label immunohistochemistry (using antibodies for structural and neurochemical markers of the ENS: Hu, substance P [SP] and neural nitric oxide synthase [nNOS]) and electron microscopy analysis. DNA was extracted from blood samples collected from all F1 animals and used for candidate gene analysis. Results: Compared to controls (en/en), En/En rabbits were subvital showing feeding abnormalities, reduced body weight and a massive colonic distension predominant in the cecum and ascending colon. Genetic tests confirmed the effects and segregation of the En alleles in the F1 population. Sequencing and genotyping of identified single nucleotide polymorphisms (SNP) in a few candidate genes showed complete co-segregation of a SNP in the KIT gene with the coat colour effects of the English spotting locus (LOD = 37.93;  = 0.00). Quantitative gene expression in colon and cecum specimens showed that the level of Kit gene in En/En rabbits was only 5-10% vs that of en/en rabbits. Morphometric data on whole mounts of cecum and ascending colon showed a decreased number of Hu- and SP-immunoreactive (IR) neurons in En/En vs. en/en rabbits (950110 vs 1440120 and 7614 vs 16024, respectively; P<0.05). Although not statistically significant, nNOS-IR neurons were less abundant in En/En vs en/en. Compared to en/en, electron microscopy analysis of En/En tissues showed neuronal (rough endoplasmic reticulum with dilated cisternae, chaotically arranged cytoskeleton and nerve endings with empty synaptic vesicles) and interstitial cells of Cajal (ICC) (few cells with immature or altered features) abnormalities, particularly in the ascending colon. Conclusions: Combined neuronal and ICC network alterations underlie this non-aganglionic model of CM. Kit mutations may account for ICC abnormalities. The present findings can help understanding neuro-muscular changes occurring in human non-aganglionic CM