We report on mutation analysis of five genes involved in the receptor tyrosine 
kinase (RET) or the endothelin-signalling pathways in 28 sporadic Japanese 
patients with Hirschsprung disease. Analysis of DNA obtained from peripheral 
blood cells revealed six mutations in the RET proto-oncogene, four of which were 
disease-causing mutations in exon 9 (D584G), the splice donor site of intron 10 
(+2T to A), exon 11 (A654T), and exon 12 (T706A). A heterozygous A to G 
transition was found in 47 bases upstream from the 5' end of exon 2 in two HD 
patients but was also seen in one control subject (2/28; 1/24). A silent 2307T 
to G transversion was observed in exon 13. Two disease-causing mutations were 
detected in the endothelin receptor (EDNRB) gene, in the non-coding region of 
exon 1 (-26 G to A) and in exon 4 (A301T); the latter mutation was a novel one. 
One silent mutation was observed in exon 4 (codon 277). One heterozygous T to C 
mutation was found in the glial cell line-derived neurotrophic factor gene in 25 
bases upstream of the coding region in exon 1. No nucleotide changes were 
detected in either the endothelin-3 or neurturin genes. Disease-causing mutation 
rates in the RET proto-oncogene and the EDNRB gene were estimated at 14.3% 
(4/28) and 10.7% (3/28), respectively. In addition to mutations in the RET and 
EDNRB genes, embryonic environmental factors and/or other genetic factors appear 
to be involved in the development of Hirschsprung disease. Further systematic 
studies of genetic variation in a large series of patients and controls are 
necessary for elucidating the pathogenesis of this disorder.
CONCLUSION: This study provides further gene alterations as disease-causing 
mutations in Japanese cases of sporadic Hirschsprung disease. However, the low 
mutation rate of the susceptibility genes may indicate that Hirschsprung disease 
arises from a combination of genetic and environmental factors.