Background/purpose: Mucin glycoproteins (mucins) recently have been shown to be deficient in the colonic mucosa of patients with Hirschsprung's disease (HD). The authors performed a detailed histo- and immunohistochemical analysis of mucins in the colonic mucosa and studied the expression of mucin genes to characterize histologically mucin quality and gene expression in HD compared with controls.
Methods: Paraffin-embedded 4-microm thick sections from patients with HD (n = 11 ganglionic, 10 aganglionic) and controls (n = 19) were taken. Slides were stained with mild periodic acid Schiff with and without saponification with KOH (reacts with O-actylated mucins), high iron diamine/alcian blue (differentiates sulphated v nonsulphated mucins), the monoclonal antimucin antibodies, PR3A5 (against di- and tri-O-acetylated sialic acids) and 91.9H (against sulphated mucins). O-acetylation and sulphation both confer an increased resistance of mucins to bacterial degradation and are thought to be important in the defensive function of the colonic mucus gel layer. In situ hybridization was used to study expression of the mucin genes MUC 1, 2, 3, 4, 5AC, 5B, 6, 7, and 8. [35S]-sulphate-labelled antisense oligonucleotide 48mer probes designed to the known tandem repeat domains of MUC genes were used. After hybridization and washing the slides were opposed to Hyperfilm MP for 7 days. The autoradiographs were scored by three independent observers for differences in expression and by image analysis. Those with positive findings were dipped in photographic emulsion, developed, and counterstained for photomicrographs.
Results: There were different patterns of staining dependent on the region of the colon and especially the age of the patient with three reagents. No significant differences in the histological staining pattern was detected between HD patients and controls. The colonic mucins in HD were found to be primarily O-acetylated and sulphated. The MUC gene expression was similar in patients and controls. MUC2 and 4 were strongly expressed, MUC1, 3, and 5B had moderate to weak expression, and MUC 5AB, 6, 7, and 8 had baseline expression.
Conclusions: The mucin glycoproteins in children with HD, although quantitatively deficient, show no qualitative differences on histo- and immunohistochemical staining from normal controls. The expression of all the known mucin genes, the genetic control of mucin secretion, and the quality of mucins, is similar to normal controls.