Hirschsprung's disease is a genetic disorder of neural crest development affecting 1 in 5,000 births. It is characterized by the absence of intramural ganglion cells in the hindgut, which often results in partial to complete intestinal obstruction during the first years of life. An autosomal dominant gene causing this disease was recently mapped to chromosome 10q11.2 (refs 1,2), using an interstitial deletion of this region isolated in a cell hybrid. It was subsequently localized to a 250-kilobase interval which contains the RET proto-oncogene. Using flanking intronic sequences as primers to amplify 12 of the 20 exons of RET from genomic DNA of 27 Hirschsprung's disease patients, we have now identified four mutations (one frameshift and three missense) that totally disrupt or partially change the structure of the tyrosine kinase domain of the RET protein (Ret). Mutations in the extracellular cysteine-rich domain of Ret have been identified previously in patients with multiple endocrine neoplasia type 2A, and a targeted mutation in the tyrosine kinase domain of the same gene produces intestinal aganglionosis and kidney agenesis in homozygous transgenic mice. Our results support the hypothesis that RET, in addition to its potential role in tumorigenesis, plays a critical role in the embryogenesis of the mammalian enteric nervous system.
To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, we have studied the clinical features of HoltOram syndrome caused by 10 different TBX5 mutations. Defects predicted to create null alleles caused substantial abnormalities both in limb and heart. In contrast, missense mutations produced distinct phenotypes: Gly80Arg caused significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp caused extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences.
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