Mutations in Smad-interacting protein 1 gene are responsible for absence of its expression in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a common congenital malformation of the enteric nervous system. The pathophysiological basis remains unclear. Recently, the SIP1 gene has been recognized as being involved in the pathogenesis of symptomatic HSCR patients with 2q22 chromosomal rearrangement. In this study, mutations in SIP1 were analyzed to explore the relationship between SIP1 and HSCR. All exons of SIP1 were amplified and then analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. SIP1 expression was determined by immunohistochemistry, Western blot and real-time quantitative PCR. By PCR-RFLP, three different electrophoretic bands of 536, 428 and 257 bp representing different genotypes were demonstrated accordingly. DNA sequencing revealed a heterozygous absence of codon 157 GTG → GTA exchange at exon 7. Simultaneously, exchanges of GCC → ACC at codon 351 and ACC → GCC at codon 395 were also observed in exon 8. All the exchanges caused a missense mutation. By immunohistochemistry, SIP1 was ectopically expressed in the aganglionic segment of HSCR without mutation. For comparison, in HSCR with mutation either in exon 7 or exon 8, SIP1 immunoreactivity disappeared in all structures. The protein and mRNA levels determined by Western blot and real-time quantitative PCR were consistent with that of immunohistochemistry. In summary, mutations of the SIP1 gene were detected in HSCR. These mutations in SIP1 were responsible for the absence of its expression in HSCR and contributed to the pathogenesis of this disease.