Phelan-McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan-McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan-McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan-McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region.
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 dys-regulating 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.Arg306C) and 2 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 delta 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 down-regulated in both MECP2 and CDKL5-mutated iPS cells and up-regulated 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.
Rett Syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan Glutamate receptor Delta-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses towards the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1+/− patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1+/− mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-α1) were increased, while the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1, PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1+/− patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses.
Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.
Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.
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