Kiminori Hosoda

Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice

Endothelins act on two subtypes of G protein-coupled receptors, termed endothelin-A and endothelin-B receptors. We report a targeted disruption of the mouse endothelin-B receptor (EDNRB) gene that results in aganglionic megacolon associated with coat color spotting, resembling a hereditary syndrome of mice, humans, and other mammalian species. Piebald-lethal (sl) mice exhibit a recessive phenotype identical to that of the EDNRB knockout mice. In crossbreeding studies, the two mutations show no complementation. Southern blotting revealed a deletion encompassing the entire EDNRB gene in the sl chromosome. A milder allele, piebald (s), which produces coat color spotting only, expresses low levels of structurally intact EDNRB mRNA and protein. These findings indicate an essential role for EDNRB in the development of two neural crest-derived cell lineages, myenteric ganglion neurons and epidermal melanocytes. We postulate that defects in the human EDNRB gene cause a hereditary form of Hirschsprungs disease that has recently been mapped to human chromosome 13, in which EDNRB is located.

Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.

Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting (ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprungs disease.

A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease

Hirschsprungs disease (HSCR) is characterized by an absence of enteric ganglia in the distal colon and a failure of innervation in the gastrointestinal tract. We recently mapped a recessive susceptibility locus (HSCR2) to human chromosome 13q22, which we now demonstrate to be the endothelin-B receptor gene (EDNRB). We identified in HSCR patients a G-->T missense mutation in EDNRB exon 4 that substitutes the highly conserved Trp-276 residue in the fifth transmembrane helix of the G protein-coupled receptor with a Cys residue (W276C). The mutant W276C receptor exhibited a partial impairment of ligand-induced Ca2+ transient levels in transfected cells. The mutation is dosage sensitive, in that W276C homozygotes and heterozygotes have a 74% and a 21% risk, respectively, of developing HSCR. Genotype analysis of patients in a Mennonite pedigree shows HSCR to be a multigenic disorder.