In this report, we present a fluorescence-based approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers.

No abstract

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.

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.