Altered expression of neuropeptides in FoxG1-null heterozygous mutant mice.

Foxg1 gene encodes for a transcription factor essential for telencephalon development in the embryonic mammalian forebrain. Its complete absence is embryonic lethal while Foxg1 heterozygous mice are viable but display microcephaly, altered hippocampal neurogenesis and behavioral and cognitive deficiencies. In order to evaluate the effects of Foxg1 alteration in adult brain, we performed expression profiling in total brains from Foxg1+/- heterozygous mutants and wild-type littermates. We identified statistically significant differences in expression levels for 466 transcripts (P<0.001), 29 of which showed a fold change ≥ 1.5. Among the differentially expressed genes was found a group of genes expressed in the basal ganglia and involved in the control of movements. A relevant (three to sevenfold changes) and statistically significant increase of expression, confirmed by qRT-PCR, was found in two highly correlated genes with expression restricted to the hypothalamus: Oxytocin (Oxt) and Arginine vasopressin (Avp). These neuropeptides have an important role in maternal and social behavior, and their alteration is associated with impaired social interaction and autistic behavior. In addition, Neuronatin (Nnat) levels appear significantly higher both in Foxg1+/- whole brain and in hippocampal neurons after silencing Foxg1, strongly suggesting that it is directly or indirectly repressed by Foxg1. During fetal and neonatal brain development, Nnat may regulate neuronal excitability, receptor trafficking and calcium-dependent signaling and, in the adult brain, it is predominantly expressed in parvalbumin-positive GABAergic interneurons. Overall, these results implicate the overexpression of a group of neuropeptides in the basal ganglia, hypothalamus, cortex and hippocampus in the pathogenesis FOXG1 behavioral impairments.

GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

Epigenetic and copy number variation analysis in retinoblastoma by MS-MLPA.

Retinoblastoma is the most common primary intraocular malignancy in children. Two step inactivation of RB1 (M1-M2) represents the key event in the pathogenesis of retinoblastoma but additional genetic and epigenetic events (M3-Mn) are required for tumor development. In the present study, we employed Methylation Specific Multiplex Ligation Probe Assay to investigate methylation status and copy number changes of 25 and 39 oncosuppressor genes, respectively. This technique was applied to analyse 12 retinoblastomas (5 bilateral and 7 unilateral) and results were compared to corresponding normal retina. We identified hypermethylation in seven new genes: MSH6 (50%), CD44 (42%), PAX5 (42%), GATA5 (25%), TP53 (8%), VHL (8%) and GSTP1 (8%) and we confirmed the previously reported hypermethylation of MGMT (58%), RB1 (17%) and CDKN2 (8%). These genes belong to key pathways including DNA repair, pRB and p53 signalling, transcriptional regulation, protein degradation, cell-cell interaction, cellular adhesion and migration. In the same group of retinoblastomas, a total of 29 copy number changes (19 duplications and 10 deletions) have been identified. Interestingly, we found deletions of the following oncosuppressor genes that might contribute to drive retinoblastoma tumorigenesis: TP53, CDH13, GATA5, CHFR, TP73 and IGSF4. The present data highlight the importance of epigenetic changes in retinoblastoma and indicate seven hypermethylated oncosuppressors never associated before to retinoblastoma pathogenesis. This study also confirms the presence of copy number variations in retinoblastoma, expecially in unilateral cases (mean 3 ± 1.3) where these changes were found more frequently respect to bilateral cases (mean 1.4 ± 1.1).

p53 Arg72Pro and MDM2 309 SNPs in hereditary retinoblastoma.

The tumor suppressor p53 and its negative regulator MDM2 have crucial roles in a variety of cellular functions such as the control of the cell cycle, senescence, genome stability and apoptosis, and are frequently deregulated in carcinogenesis. Previous studies have highlighted the contribution of the common functional polymorphisms p53 p.Arg72Pro and MDM2 309SNP to the risk of both common cancers and Li-Fraumeni syndrome. Their possible role in retinoblastoma has recently been addressed by Castéra et al, who however only studied the MDM2 309SNP. Here, for the first time, we analyzed both single nucleotide polymorphisms (SNPs) in a case-control study of 111 Italian hereditary retinoblastoma patients. We found a significant association of the p53 Pro/Pro genotype with the disease (odds ratio=3.58, P=0.002). The MDM2 309SNP showed a weak negative association of allele G that deserves further investigation. These findings further support the hypothesis that genetic variability of the p53 pathway contributes to the individual susceptibility to retinoblastoma, as shown for Li-Fraumeni syndrome and a variety of non-hereditary cancers.