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The molecular genetic basis of HSCR. Search for HSCR modifier loci in humans

We have extensively studied Hirschsprung disease (HSCR) as a model complex genetic disorder. In particular,
(1) genetic analysis at the RET locus has been carried out to identify variants conferring a modified risk of HSCR development.

We have confirmed the view that RET variants defining common haplotypes act as low penetrant alleles in the development of the disease, even in the absence of clear causative mutations, by SNPs genotyping, haplotype reconstruction and search for RET functional/hypomorphic variants. A high LD in the region, defining two distinct blocks, and a clear association of the 5 region of the gene with HSCR disease have been reported. In addition, our two groups participated to an International HSCR Consortium aiming to genotype a number of SNPs across the RET locus and using the transmission disequilibrium test (TDT) to determine transmission from heterozygous parents to affected offspring. In this study the relative contributions of CDS mutations and of an enhancer mutation at risk of HSCR already described have been examined with respect to various HSCR-associated features in a total of 1,000 patients (as trios) providing a consistent explanation of the genetic features of a classical multifactorial disease. In addition, we have obtained results suggesting that the HSCR predisposing haplotype is associated with low RET gene expression, a finding in agreement with the overrepresentation and non random transmission of this haplotype to HSCR patients.

We have also investigated a HSCR protective RET haplotype finding association with two nucleotide substitutions located nearby the third and fourth polyadenylation sites. Data obtained in vitro, using proper constructs containing the 3 terminal sequences of the RET gene, clearly demonstrate that one of these variant alleles interferes with normal RET mRNA half-life thus allowing to conclude that a more stable RET mRNA can lead to an increase of endogenous transcript, and probably of the amount of total protein produced, and providing a consistent explanation of the protective effect previously described.

(2) molecular characterization of the genetic defect underlying a HSCR associated phenotype: the Congenital Central Hypoventilation Syndrome (CCHS)
Between our two groups, we have collected so far a total of more than 180 CCHS patients, 28% of whom showing association with HSCR. Mutation screening of the PHOX2B gene has allowed to detect heterozygous frameshift mutations and in-frame changes leading to 5 to 13 polyalanine expansions of the PHOX2B gene, vastly occurred de novo, in over 85 % CCHS patients studied, thus concluding that PHOX2B is the major gene in CCHS pathogenesis, with autosomal dominant inheritance and reduced penetrance. In addition, we have shown i) a particular susceptibility to neuroblastoma in CCHS patients with truncating mutation of the PHOX2B gene as opposed to those with polyalanine expansion mutations, ii) the possibility of germline PHOX2B gene mutation predisposing to neuroblastoma independent of the CCHS phenotype and that iii) the two sets of PHOX2B mutations found in CCHS patients play different roles in the transcriptional regulation of different target genes. In particular, we have shown a correlation between the length of polyalanine expansions and the severity of reduced transcriptional activity caused by retention of the mutated protein in the cytoplasm or in the nuclear aggregates. On the other hand, frameshift mutations did not impair the PHOX2B nuclear income, suggesting a different mechanism through which they would exert the observed effects on target promoters

(3) functional reconstruction of the gene network underlying correct development of Autonomic Nervous System as an approach to identify novel candidates for ENS dysfuntions and/or HSCR modifier genes

We aimed to isolate factors regulating expression and targets of two homeobox genes, Tlx2 and Tlx3, involved in the development of branches of autonomic nervous system, and to identify genes cooperating in the RET transcriptional regulation. In particular, i) transient transfections, electrophoretic mobility shift and chromatin immunoprecipitation assays have all indicated that PHOX2B is able to bind sequences of a cell-specific element in the 5-flanking region of the human TLX2 gene, determining its transactivation in neuroblastoma cells, and confirming a physiological role of the PHOX2B-TLX2 promoter interaction in the transcription factors cascade underlying the differentiation of autonomic neuronal lineages during human embryogenesis, ii) after identifying the transcription initiation points, we have demonstrated that the 5-untranslated region of the TLX3 gene is necessary for the basal promoter activity in cell lines from different origin. Moreover, two tandem CCAAT boxes have been localized as critical elements, also showing their interaction with the nuclear factor Y (NFY).

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