Correlation between multiple RET mutations and severity of Hirschsprung’s disease

The enteric nervous system (ENS), comprising neurons and glial cells, organized as interconnected ganglia within the gut wall, controls peristalsis and the production of secretions. The RET receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development. Humans with mutations in the RET locus have Hirschsprung’s disease (HSCR), and mice lacking RET exhibit total intestinal aganglionosis. Although a number of mutations with the potential for causing HSCR have been reported, their precise correlation with phenotype and symptom severity in HSCR is not clearly understood. Our study investigates the correlation between mutations in the RET locus and symptom severity in HSCR.

We performed a comprehensive nucleotide analysis of the RET coding region in 18 HSCR patients and 87 controls, performed cellular biological analysis by Western blotting using the expression vector, and analyzed cell proliferation with anti-Ki67 antibody under immunofluorescence confocal microscopy (ICM).

We identified three novel mutations, D489N, L769L, and V778D in the RET coding region in our HSCR patients. In the allelic distribution of D489N and L769L, the difference between HSCR patients and controls reached statistical significance (p = 0.0373 and p = 0.0004, respectively), whereas no statistical difference was observed in the allelic distribution of V778D (p = 0.1073). One HSCR patient who died from total colonic aganglionosis had a combination of homozygous mutation of D489N, L769L, and heterozygous mutation of V778D. Western blotting of full mutant RET from this patient showed significantly increased 150kD-band, which corresponds to the immature form compared with wild-type and single mutant RET. ICM showed that overexpression of full mutant RET significantly reduced cellular proliferation in comparison with wild-type and single mutant RET.

A combination of mutations in the RET locus may correlate with symptom severity in HSCR as a consequence of reduced cellular proliferation secondary to altered maturation of RET.