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Hirschsprung disease (HSCR) is a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis. HSCR demonstrates a complex pattern of inheritance, with the RET proto-oncogene acting as a major gene and with several additional susceptibility loci related to the Ret-signaling pathway or to other developmental programs of neural crest cells. To test how the HSCR phenotype may be affected by the presence of genetic variants, we investigated the role of a single-nucleotide polymorphism (SNP), 2508C-->T (S836S), in exon 14 of the RET gene, characterized by low frequency among patients with HSCR and overrepresentation in individuals affected by sporadic medullary thyroid carcinoma. Typing of several different markers across the RET gene demonstrated that a whole conserved haplotype displayed anomalous distribution and nonrandom segregation in families with HSCR. We provide genetic evidence about a protective role of this low-penetrant haplotype in the pathogenesis of HSCR and demonstrate a possible functional effect linked to RET messenger RNA expression.

Hirschsprung disease (HSCR), the most common hereditary cause of intestinal obstruction, shows considerable variation and complex inheritance. Coding sequence mutations in RET, GDNF, EDNRB, EDN3 and SOX10 lead to long-segment (L-HSCR) and syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). We conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11 and 19q12 that seem to be necessary and sufficient to explain recurrence risk and population incidence. The gene at 10q11 is probably RET, supporting its crucial role in all forms of HSCR; however, coding sequence mutations are present in only 40% of linked families, suggesting the importance of noncoding variation. Here we show oligogenic inheritance of S-HSCR, the 3p21 and 19q12 loci as RET-dependent modifiers, and a parent-of-origin effect at RET. This study demonstrates by a complete genetic dissection why the inheritance pattern of S-HSCR is nonmendelian.

The MEN2A oncogene encodes for a constitutive active member of the RET receptor tyrosine kinase family. Here, we report that MEN2A-RET activates Signal Transducer and Activator of Transcription 3 (STAT3) via two YxxV/Q STAT3 docking sites, Tyr752 and Tyr928. MEN2A-RET induces both Tyr705 and Ser727 phosphorylation of STAT3, and STAT3 serine phosphorylation is required for its maximal transcriptional activity. Stable NIH3T3 cell lines expressing both MEN2A-RET and STAT3alpha but not STAT3beta, are characterized by enhanced proliferation and cyclin-D1 promoter activity, and enhanced growth in soft agar. These data indicate that malignant cell growth induced by MEN2A-RET involves its activation of STAT3.

Hirschsprung disease (HSCR) is an inherited disorder characterised by absence of intrinsic ganglion cells in the distal gastrointestinal tract. Different susceptibility genes, involved in either the Ret-tyrosine kinase or the endothelin signalling pathways, contribute to HSCR phenotype. Interestingly, alterations of these genes are detected in only 30-50% of all HSCR patients, suggesting the involvement of modifier genes and/or additional genetic or environmental risk factors. In complex disorders common polymorphic variants can be associated with the disease phenotype, thus modifying the risk of recurrence. To investigate whether sequence variants of the RET proto-oncogene may be associated with the development of the HSCR phenotype, we analysed 92 Italian patients for the 2508C > T synonymous substitution in exon 14 (S836S) finding that the T allele is clearly less frequent than in control individuals (Fisher exact test P = 0.0002). On the other hand, this RET variant allele is overrepresented in patients affected with medullary thyroid carcinoma. Assuming a direct effect of this single-nucleotide polymorphism in predisposing to RET associated pathologies, we have performed functional tests which excluded any possible involvement of the C and T alleles in DNA-protein binding, transcript stability and RNA splicing and editing.

Background: RET mutations have been reported variously to affect 7% to 41% of Hirschsprung's disease (HSCR) patients depending on familial or sporadic occurrence of the disease, length of aganglionosis and possible association with other disease phenotypes. The authors report a study of the incidence of RET mutations in unselected HSCR patients from two regional centers and correlate their genotypes and phenotypes., Methods: The records of HSCR patients treated in 2 regional centers with a combined population of 5 million were reviewed, and blood samples were obtained from 57 patients. During the same period, 39 patients with similar demographic data refused or provided insufficient blood for study. DNA was extracted, and the 21 exons of the RET protooncogene were screened for mutations using denaturing gradient gel electrophoresis (DGGE)., Results: Of 57 patients, 48 were sporadic, and 9 were familial. Lengths of aganglionosis were total colonic, 4; long, 11; short, 39; ultrashort, 1; unclassified, 2. Associated anomalies were present in 20. Causative mutations were identified in 4 (7%): missense or "silent" in 3 (exons 5, 11, 13) and deletion in 1. The silent mutation of exon 11 recently has been shown to have effects on correct RET mRNA splicing. One mutation occurred in total colonic aganglionosis (25%), 1 in long segment dysganglionosis (9%), and 2 in short segment aganglionosis (5%). Surprisingly, all these mutations occurred in sporadic cases (10%). Five patients (9%) had rare polymorphic alleles at exon 14 (n = 1) and exon 18 (n = 4). Fifty patients (88%) showed common polymorphic alleles (sequence variants) in 1 or more exons (> 4, n = 5)., Conclusions: RET mutation as a primary cause for Hirschsprung's disease in the general surgical population is less frequent than previously thought. This observation is compatible with the hypothesis that HSCR could be a polygenic disease caused by additive subclinical effects of more than one gene, including RET.

Using a PCR strategy based on an initial set of 15 couples of primers designed from the known cDNA sequence, we identified 18 introns in the human RET proto-oncogene and sequenced the corresponding 5' and 3' exon-intron junctions. This approach was successful in locating all the introns contained in fragments short enough to be amplified by PCR. Thus 19 exons were identified which, together with the previously reported exon subjected to alternative splicing, brings the total number of RET exons to 20. This information is relevant for the screening of recently reported missense mutations of RET which cause Multiple Endocrine Neoplasia 2A (MEN2A) and for the search of additional point mutations of the same gene which might cause two other neural crest disorders, MEN2B and Hirschsprung disease, mapping in the same region as MEN2A.