Silvia Borghini

Nuclear Factor Y Drives Basal Transcription of the Human TLX3, a Gene Overexpressed in T-Cell Acute Lymphocytic Leukemia

Based on a knocked-out mouse model and a few expression studies, TLX3 is regarded as a homeobox gene crucial for the development of the autonomic nervous system. This gene can undergo rearrangements or deregulation, giving rise to T-cell acute lymphocytic leukemia. The present report is focused on the identification of elements and factors playing a role in the TLX3 physiologic expression regulation and therefore likely to be involved in cancer development. In particular, after identifying the transcription start points, we have made use of in vitro transfection assays to show that the 5′-untranslated region of the gene is necessary for the basal promoter activity in cell lines from different origin. By site-directed mutagenesis, two tandem CCAAT boxes have been localized as critical elements of this region. In vivo chromatin immunoprecipitation and electrophoretic mobility shift assays have indicated that nuclear factor Y (NFY) recognizes these sites in all the analyzed cell lines. The physiologic role of such an interaction has been confirmed by a dominant-negative version of the NFY transcription factor that has turned out to decrease both in vitro TLX3 promoter activity and endogenous amount of mRNA. Finally, a consistent in vivo TLX3 expression impairment was also achieved after NFY mRNA knockdown. The full characterization of the TLX3 transcription regulation will ultimately provide crucial elements to define the involvement of this gene in T-cell acute lymphocytic leukemia development. (Mol Cancer Res 2006;4(9):635–43)

Search for pathogenetic variants of the SPRY2 gene in intestinal innervation defects

SPRY2 is an inducible inhibitor of signalling mediated by tyrosine kinases receptors, whose targeting causes intestinal hyperganglionosis in mice. In this light, we have undertaken a mutational analysis of the SPRY2 gene in patients affected with intestinal neuronal dysplasia (IND), without detecting nucleotide changes in any of the 26 DNA samples analysed, with the exception of two already known polymorphic variants. A role of the SPRY2 gene in IND pathogenesis can be thus excluded.

Candidate Genes in Patients with Autoinflammatory Syndrome Resembling Tumor Necrosis Factor Receptor-associated Periodic Syndrome Without Mutations in the TNFRSF1A Gene

Objective. Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autosomal-dominant multisystemic autoinflammatory condition. Patients display different mutations of the TNF receptor superfamily 1A gene (TNFRSF1A), coding for a nearly ubiquitous TNF receptor (TNFR1). No TNFRSF1A mutation has been identified in a proportion of patients with TRAPS-like phenotype. Methods. We investigated mechanisms downregulating the TNF-induced inflammatory response such as (1) receptor shedding, producing a secreted form acting as a TNF inhibitor; (2) receptor internalization with subsequent induction of apoptosis; and (3) negative regulation of nuclear factor-κB (NF-κB) transcription. We analyzed the sequence of genes known to play a pivotal role in these pathways, in 5 patients with TRAPS symptoms and showing shedding and/or apoptosis defects, but without mutations of the TNFRSF1A gene. Results. Sequence analysis of 3 genes involved in TNFR1 shedding (ERAP1, NUCB2, RBMX) and 3 genes involved in negative regulation of NF-κB signaling (TNFAIP3, CARP-2) or NF-κB transcription (ZFP36) revealed only a few unreported variants, apparently neutral. Conclusion. Our study rules out any involvement in the pathogenesis of TRAPS of some of the genes known to regulate TNFR1 shedding and TNF-induced NF-κB signaling and transcription. Gene(s) responsible for TRAPS-like syndrome remain to be investigated among currently unidentified genes likely involved in these pathways, or by applying the genome-wide function-free sequencing approach.

Hirschsprung associated GDNF mutations do not prevent RET activation

Hirschsprung disease (HSCR) is a complex disorder characterised by aganglia of distal gastrointestinal tracts. The highest proportion of both familial and sporadic cases is due to mutations of the RET proto-oncogene. Five germline mutations in the glial cell-line-derived neurotrophic factor (GDNF) gene, one of the RET ligands, have been detected in HSCR patients. Pedigrees analysis and the observed association between these GDNF alterations and RET variants in the same patients raised the question of whether the GDNF gene plays any causative/predisposing role in HSCR pathogenesis. In the present work, we have studied the ability of GDNF proteins, each bearing one of the reported mutations, to activate RET by performing a functional test in cultured neuroblastoma cells. Consistently with the lack of genotype/phenotype correlation in human subjects, our results indicate absence of detectable alterations of mutant GDNF induced RET activation.

Transcriptional regulation of TLX2 and impaired intestinal innervation: possible role of the PHOX2A and PHOX2B genes.

TLX2 (also known as HOX11L1, Ncx and Enx) is a transcription factor playing a crucial role in the development of the enteric nervous system, as confirmed by mice models exhibiting intestinal hyperganglionosis and pseudo-obstruction. However, congenital defects of TLX2 have been excluded as a major cause of intestinal motility disorders in patients affected with intestinal neuronal dysplasia (IND) or pseudo-obstruction. After demonstrating the direct regulation of TLX2 expression by the homeoprotein PHOX2B, in the present work, we have focused on its paralogue PHOX2A. By co-transfections, electrophoretic mobility shift assays and chromatin immunoprecipitation, we have demonstrated that PHOX2A, like PHOX2B, is involved in the cascade leading to TLX2 transactivation and presumably in the intestinal neuronal differentiation. Based on the hypothesis that missed activation of the TLX2 gene induces the development of enteric nervous system defects, PHOX2A and PHOX2B have been regarded as novel candidate genes involved in IND and pseudo-obstruction and consequently analyzed for mutations in a specific set of 26 patients. We have identified one still unreported PHOX2A variant; however, absence of any functional effect on TLX2 transactivation suggests that regulators or effectors other than the PHOX2 genes must act in the same pathway, likely playing a non redundant and direct role in the pathogenesis of such enteric disorders.

Cryopyrin-associated periodic syndromes in Italian Patients: Evaluation of the rate of somatic NLRP3 mosaicism and phenotypic characterization

Objective. To evaluate the rate of somatic NLRP3 mosaicism in an Italian cohort of mutation-negative patients with cryopyrin-associated periodic syndrome (CAPS). Methods. The study enrolled 14 patients with a clinical phenotype consistent with CAPS in whom Sanger sequencing of the NLRP3 gene yielded negative results. Patients’ DNA were subjected to amplicon-based NLRP3 deep sequencing. Results. Low-level somatic NLRP3 mosaicism has been detected in 4 patients, 3 affected with chronic infantile neurological cutaneous and articular syndrome and 1 with Muckle-Wells syndrome. Identified nucleotide substitutions encode for 4 different amino acid exchanges, with 2 of them being novel (p.Y563C and p.G564S). In vitro functional studies confirmed the deleterious behavior of the 4 somatic NLRP3 mutations. Among the different neurological manifestations detected, 1 patient displayed mild loss of white matter volume on brain magnetic resonance imaging. Conclusion. The allele frequency of somatic NLRP3 mutations occurs generally under 15%, considered the threshold of detectability using the Sanger method of DNA sequencing. Consequently, routine genetic diagnostic of CAPS should be currently performed by next-generation techniques ensuring high coverage to identify also low-level mosaicism, whose actual frequency is yet unknown and probably underestimated.

Somatic mosaicism of CIAS1/NLRP3 gene in two patients with chronic infantile neurologic, cutaneous, articular syndrome

CINCA syndrome (chronic, infantile, neurological , cutaneous, articular syndrome) also known as neonatal-onset multisystem inflammatory disease (NOMID) represents the most severe form of cryopyrin-associated periodic syndrome ( CAPS). This condition was found to be associated with missense mutations in the CIAS1/NALP3/PYPAF gene, which encodes cryopyrin. Cryopyrin is a member of the cytoplasmatic protein family CATERPILLER, which is involved in inflammasome molecular platform assembly, leading to inflammatory immune response and apoptosis regulation. It has been hypothesized that mutant cryopirin spontaneously oligomerizes and induces the inflammasome activation with elevated IL-1β production and autoinflammatory phenotype. Causative CIAS1 mutations have been detected in only half of the patients with CINCA and the genetic cause of the disease in patients who tested negative remains unclear.