Whole exome sequencing of Rett syndrome-like patients reveals the mutational diversity of the clinical phenotype.

Classical Rett syndrome (RTT) is a neurodevelopmental disorder where most of cases carry MECP2 mutations. Atypical RTT variants involve mutations in CDKL5 and FOXG1. However, a subset of RTT patients remains that do not carry any mutation in the described genes. Whole exome sequencing was carried out in a cohort of 21 female probands with clinical features overlapping with those of RTT, but without mutations in the customarily studied genes. Candidates were functionally validated by assessing the appearance of a neurological phenotype in Caenorhabditis elegans upon disruption of the corresponding ortholog gene. We detected pathogenic variants that accounted for the RTT-like phenotype in 14 (66.6 %) patients. Five patients were carriers of mutations in genes already known to be associated with other syndromic neurodevelopmental disorders. We determined that the other patients harbored mutations in genes that have not previously been linked to RTT or other neurodevelopmental syndromes, such as the ankyrin repeat containing protein ANKRD31 or the neuronal acetylcholine receptor subunit alpha-5 (CHRNA5). Furthermore, worm assays demonstrated that mutations in the studied candidate genes caused locomotion defects. Our findings indicate that mutations in a variety of genes contribute to the development of RTT-like phenotypes.

Mutations in JMJD1C are involved in Rett syndrome and intellectual disability.

PURPOSE: Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders. METHODS: We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect. RESULTS: We found seven JMJD1C variants that were not present in any control sample (~ 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity. CONCLUSIONS: Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability.Genet Med 18 1, 378-385.

An increase in MECP2 dosage impairs neural tube formation.

Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.

Dysregulation of the long non-coding RNA transcriptome in a Rett syndrome mouse model.

Mecp2 is a transcriptional repressor protein that is mutated in Rett syndrome, a neurodevelopmental disorder that is the second most common cause of mental retardation in women. It has been shown that the loss of the Mecp2 protein in Rett syndrome cells alters the transcriptional silencing of coding genes and microRNAs. Herein, we have studied the impact of Mecp2 impairment in a Rett syndrome mouse model on the global transcriptional patterns of long non-coding RNAs (lncRNAs). Using a microarray platform that assesses 41,232 unique lncRNA transcripts, we have identified the aberrant lncRNA transcriptome that is present in the brain of Rett syndrome mice. The study of the most relevant lncRNAs altered in the assay highlighted the upregulation of the AK081227 and AK087060 transcripts in Mecp2-null mice brains. Chromatin immunoprecipitation demonstrated the Mecp2 occupancy in the 5'-end genomic loci of the described lncRNAs and its absence in Rett syndrome mice. Most importantly, we were able to show that the overexpression of AK081227 mediated by the Mecp2 loss was associated with the downregulation of its host coding protein gene, the gamma-aminobutyric acid receptor subunit Rho 2 (Gabrr2). Overall, our findings indicate that the transcriptional dysregulation of lncRNAs upon Mecp2 loss contributes to the neurological phenotype of Rett syndrome and highlights the complex interaction between ncRNAs and coding-RNAs.

Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum.

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in S. cerevisiae, Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null ddp1(Δ) mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic ddp1(15.1) mutation. Similar results were obtained in the fission yeast Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly.

Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice.

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763.

Drosophila DDP1, a multi-KH-domain protein, contributes to centromeric silencing and chromosome segregation.

BACKGROUND: The Drosophila melanogaster DDP1 protein is a highly evolutionarily conserved protein that is characterised by the presence of 15 tandemly organized KH domains, known for mediating high-affinity binding to single-stranded nucleic acids (RNA and ssDNA). Consistent with its molecular organization, DDP1 binds single-stranded nucleic acids with high affinity, in vitro. It was shown earlier that, in polytene chromosomes, DDP1 is found in association with chromocenter heterochromatin, suggesting a contribution to heterochromatin formation and/or maintenance. RESULTS: In this paper, the actual contribution of DDP1 to the structural and functional properties of heterochromatin was determined through the analysis of the phenotypes associated with the hypomorphic ddp1(15.1) mutation that was generated through the mobilization of a P element inserted in the second intron of ddp1. ddp1(15.1) behaves as a dominant suppressor of PEV in the variegated rearrangement In(1)w(m4) as well as in several transgenic lines showing variegated expression of a hsp70-white(+) transgene. In polytene chromosomes from homozygous ddp1(15.1) larvae, histone H3-K9 methylation and HP1 deposition at chromocentre heterochromatin are strongly reduced. Our results also show that, when the maternal contribution of DDP1 is reduced, chromosome condensation and segregation are compromised. Moreover, in a ddp1(15.1) mutant background, transmission of the nonessential Dp1187 minichromosome is reduced. CONCLUSIONS: We conclude that DDP1 contributes to the structural and functional properties of heterochromatin. These results are discussed in the context of current models for the formation and maintenance of heterochromatin; in these models, HP1 deposition depends on H3-K9 methylation that, in turn, requires the contribution of the RNAi pathway.

Circadian cycle-dependent MeCP2 and brain chromatin changes.

Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.

Improvement of the Rett syndrome phenotype in a MeCP2 mouse model upon treatment with levodopa and a dopa-decarboxylase inhibitor.

Rett Syndrome is a neurodevelopmental autism spectrum disorder caused by mutations in the gene coding for methyl CpG-binding protein (MeCP2). The disease is characterized by abnormal motor, respiratory, cognitive impairment, and autistic-like behaviors. No effective treatment of the disorder is available. Mecp2 knockout mice have a range of physiological and neurological abnormalities that resemble the human syndrome and can be used as a model to interrogate new therapies. Herein, we show that the combined administration of Levodopa and a Dopa-decarboxylase inhibitor in RTT mouse models is well tolerated, diminishes RTT-associated symptoms, and increases life span. The amelioration of RTT symptomatology is particularly significant in those features controlled by the dopaminergic pathway in the nigrostratium, such as mobility, tremor, and breathing. Most important, the improvement of the RTT phenotype upon use of the combined treatment is reflected at the cellular level by the development of neuronal dendritic growth. However, much work is required to extend the duration of the benefit of the described preclinical treatment.

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues.

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.

Whole-genome bisulfite DNA sequencing of a DNMT3B mutant patient.

The immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is associated to mutations of the DNA methyl-transferase DNMT3B, resulting in a reduction of enzyme activity. Aberrant expression of immune system genes and hypomethylation of pericentromeric regions accompanied by chromosomal instability were determined as alterations driving the disease phenotype. However, so far only technologies capable to analyze single loci were applied to determine epigenetic alterations in ICF patients. In the current study, we performed whole-genome bisulphite sequencing to assess alteration in DNA methylation at base pair resolution. Genome-wide we detected a decrease of methylation level of 42%, with the most profound changes occurring in inactive heterochromatic regions, satellite repeats and transposons. Interestingly, transcriptional active loci and ribosomal RNA repeats escaped global hypomethylation. Despite a genome-wide loss of DNA methylation the epigenetic landscape and crucial regulatory structures were conserved. Remarkably, we revealed a mislocated activity of mutant DNMT3B to H3K4me1 loci resulting in hypermethylation of active promoters. Functionally, we could associate alterations in promoter methylation with the ICF syndrome immunodeficient phenotype by detecting changes in genes related to the B-cell receptor mediated maturation pathway.

Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer.

PURPOSE: Epithelial ovarian cancer (EOC) is the fifth leading cause of death in women diagnosed with gynecologic malignancies. The low survival rate is because of its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. So, the development of effective innovative therapeutic strategies to overcome cisplatin resistance remains a high priority. EXPERIMENTAL DESIGN: To investigate new treatments in in vivo models reproducing EOCs tumor growth, we generated a preclinical model of ovarian cancer after orthotopic implantation of a primary serous tumor in nude mice. Further, matched model of acquired cisplatin-resistant tumor version was successfully derived in mice. Effectiveness of lurbinectedin (PM01183) treatment, a novel marine-derived DNA minor groove covalent binder, was assessed in both preclinical models as a single and a combined-cisplatin agent. RESULTS: Orthotopically perpetuated tumor grafts mimic the histopathological characteristics of primary patients' tumors and they also recapitulate in mice characteristic features of tumor response to cisplatin treatments. We showed that single lurbinectedin or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. CONCLUSIONS: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance.

Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies.

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.

Disrupted microRNA expression caused by Mecp2 loss in a mouse model of Rett syndrome.

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate post-transcriptional gene expression. They influence a wide range of physiological functions, including neuronal processes, and are regulated by various mechanisms, such as DNA methylation. This epigenetic mark is recognized by transcriptional regulators such as the methyl CpG binding protein Mecp2. Rett syndrome is a complex neurological disorder that has been associated with mutations in the gene coding for Mecp2. Thus, we examined the possible miRNA misregulation caused by Mecp2 absence in a mouse model of Rett syndrome. Using miRNA expression microarrays, we observed that the brain of Rett syndrome mice undergoes a disruption of the expression profiles of miRNAs. Among the significantly altered miRNAs (26%, 65 of 245), overall downregulation of these transcripts was the most common feature (71%), whilst the remaining 30% were upregulated. Further validation by quantitative RT-PCR demonstrated that the most commonly disrupted miRNAs were miR-146a, miR-146b, miR-130, miR-122a, miR-342 and miR-409 (downregulated), and miR-29b, miR329, miR-199b, miR-382, miR-296, miR-221 and miR-92 (upregulated). Most importantly, transfection of miR-146a in a neuroblastoma cell line caused the downregulation of IL-1 receptor-associated kinase 1 (Irak1) levels, suggesting that the identified defect of miR-146a in Rett syndrome mice brains might be responsible for the observed upregulation of Irak1 in this model of the human disease. Overall, we provide another level of molecular deregulation occurring in Rett syndrome that might be useful for understanding the disease and for designing targeted therapies.

Chromatin dynamics coupled to DNA repair.

In order to protect and preserve the integrity of the genome, eukaryotic cells have developed accurate DNA repair pathways involving a coordinated network of DNA repair and epigenetic factors. The DNA damage response has to proceed in the context of chromatin, a packaged and compact structure that is flexible enough to regulate the accession of the DNA repair machinery to DNA-damaged sites. Chromatin modifications and ATP-remodeling activities are both necessary to ensure efficient DNA repair. Here we review the current progress of research into the importance of chromatin modifications and the ATP-remodeling complex to the DNA damage response, with respect to the sensing and signaling of DNA lesions, DNA repair and the processes that restore chromatin structure.

The multi-KH domain protein of Saccharomyces cerevisiae Scp160p contributes to the regulation of telomeric silencing.

Multi-KH domain proteins are highly evolutionarily conserved proteins that associate to polyribosomes and participate in RNA metabolism. Recent evidence indicates that multi-KH domain proteins also contribute to the structural organization of heterochromatin both in mammals and Drosophila. Here, we show that the multi-KH domain protein of Saccharomyces cerevisiae, Scp160p, contributes to silencing at telomeres and at the mating-type locus, but not to ribosomal silencing. The contribution of Scp160p to silencing is independent of its binding to the ribosome as deletion of the last two KH domains, which mediate ribosomal binding, has no effect on silencing. Disruption of SCP160 increases cell ploidy but this effect is also independent of the contribution of Scp160p to telomeric silencing as strong relief of silencing is observed in Deltascp160 cells with normal ploidy and, vice versa, Deltascp160 cells with highly increased ploidy show no significant silencing defects. The TPE phenotype of Deltascp160 cells associates to a decreased Sir3p deposition at telomeres and, in good agreement, silencing is rescued by SIR3 overexpression and in a Deltarif1Deltarif2 mutant. Scp160p shows a distinct perinuclear localization that is independent of its ability to bind ribosomes. Moreover, telomere clustering at the nuclear envelope is perturbed in Deltascp160 cells and disruption of the histone deacetylase RPD3, which is known to improve telomere clustering, rescues telomeric silencing in Deltascp160 cells. These results are discussed in the context of a model in which Scp160p contributes to silencing by helping telomere clustering.

Analysing the contribution of nucleic acids to the structure and properties of centric heterochromatin.

A class of repetitive DNA sequences frequently found at centromeric regions are R/Y-satellites showing an asymmetric distribution of residues resulting in one strand being rich in purines (R-strand) while the complementary strand is pyrimidine-rich (Y-strand). The dodeca-satellite of Drosophila belongs to this class of centromeric satellites. In vitro, the dodeca-satellite forms altered DNA structures in which the R-strand forms very stable intramolecular fold-backs that are stabilised by the formation of tandem G x A mismatches. A single-stranded nucleic acids binding protein, DDP1, binds the unstructured dodeca-satellite Y-strand with high affinity. In polytene chromosomes, DDP1 associates with the heterochromatic chromocenter and, at the euchromatic chromosome arms, co-localises with HP1. DDP1 is a vigilin. Vigilins are highly conserved multi-KH-domain proteins. Scp160p, the vigilin from S. cerevisiae, is involved in the control of ploidy. DDP1 complements a deltascp160 deletion.