X chromosome inactivation does not necessarily determine the severity of the phenotype in Rett syndrome patients

Rett syndrome; X chromosome inactivation; MECP2 geneSíndrome de Rett; Inactivación del cromosoma X; Gen MECP2Síndrome de Rett; Inactivació del cromosoma X; Gen MECP2Rett syndrome (RTT) is a severe neurological disorder usually caused by mutations in the MECP2 gene. Since the MECP2 gene is located on the X chromosome, X chromosome inactivation (XCI) could play a role in the wide range of phenotypic variation of RTT patients; however, classical methylation-based protocols to evaluate XCI could not determine whether the preferentially inactivated X chromosome carried the mutant or the wild-type allele. Therefore, we developed an allele-specific methylation-based assay to evaluate methylation at the loci of several recurrent MECP2 mutations. We analyzed the XCI patterns in the blood of 174 RTT patients, but we did not find a clear correlation between XCI and the clinical presentation. We also compared XCI in blood and brain cortex samples of two patients and found differences between XCI patterns in these tissues. However, RTT mainly being a neurological disease complicates the establishment of a correlation between the XCI in blood and the clinical presentation of the patients. Furthermore, we analyzed MECP2 transcript levels and found differences from the expected levels according to XCI. Many factors other than XCI could affect the RTT phenotype, which in combination could influence the clinical presentation of RTT patients to a greater extent than slight variations in the XCI pattern.The work was supported by grants from the Spanish Ministry of Health (Instituto de Salud Carlos III/FEDER, PI15/01159); Crowdfunding program PRECIPITA, from the Spanish Ministry of Health (Fundacion Espanola para la Ciencia y la Tecnologia); the Catalan Association for Rett Syndrome; Fondobiorett and Mi Princesa Rett

Comprehensive analysis of GABAA-A1R developmental alterations in Rett Syndrome: setting the focus for therapeutic targets in the time frame of the disease

Rett syndrome, a serious neurodevelopmental disorder, has been associated with an altered expression of different synaptic-related proteins and aberrant glutamatergic and γ-aminobutyric acid (GABA)ergic neurotransmission. Despite its severity, it lacks a therapeutic option. Through this work we aimed to define the relationship between MeCP2 and GABAA.-A1 receptor expression, emphasizing the time dependence of such relationship. For this, we analyzed the expression of the ionotropic receptor subunit in different MeCP2 gene-dosage and developmental conditions, in cells lines, and in primary cultured neurons, as well as in different developmental stages of a Rett mouse model. Further, RNAseq and systems biology analysis was performed from post-mortem brain biopsies of Rett patients. We observed that the modulation of the MeCP2 expression in cellular models (both Neuro2a (N2A) cells and primary neuronal cultures) revealed a MeCP2 positive effect on the GABAA.-A1 receptor subunit expression, which did not occur in other proteins such as KCC2 (Potassium-chloride channel, member 5). In the Mecp2+/- mouse brain, both the KCC2 and GABA subunits expression were developmentally regulated, with a decreased expression during the pre-symptomatic stage, while the expression was variable in the adult symptomatic mice. Finally, the expression of the gamma-aminobutyric acid (GABA) receptor-related synaptic proteins from the postmortem brain biopsies of two Rett patients was evaluated, specifically revealing the GABA A1R subunit overexpression. The identification of the molecular changes along with the Rett syndrome prodromic stages strongly endorses the importance of time frame when addressing this disease, supporting the need for a neurotransmission-targeted early therapeutic intervention

Comprehensive analysis of diagnostic approaches and molecular landscape in Rett syndrome spectrum disorders

[eng] Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by a regression in acquired skills, such as purposeful hand use and language, after an apparently normal early development. RTT affects almost exclusively females and is mainly caused by mutations in the X-linked MECP2 gene, encoding methyl-CpGbinding protein 2 (MeCP2). MeCP2 is a global regulator of gene expression that operates through different mechanisms, including transcriptional regulation, chromatin architecture, splicing modulation, and miRNA processing. Nevertheless, the precise pathomechanisms by which MeCP2 deficiency leads to RTT remain elusive. MeCP2 plays a pivotal role in neuronal maturation and maintenance in the postnatal brain, and its deficiency causes severe defects in dendritic arborization and synaptogenesis. Currently, RTT has no cure or any effective pharmacological treatment, but the delineation of the downstream effects of MeCP2 deficiency could lead to the identification of biomarkers and potential therapeutic targets for RTT. The reversibility of RTT-like features in Mecp2-null mouse models upon Mecp2 reactivation strongly suggests that symptomatic patients could benefit from counteracting the effects of MeCP2 deficiency. This doctoral thesis aims to profile the molecular landscape of RTT in different ways to contribute to the understanding of the pathomechanisms behind this disorder. MECP2 being an X-linked gene, it has been long hypothesized that X chromosome inactivation (XCI) patterns may influence the phenotype of RTT patients. Therefore, this thesis has studied XCI patterns in blood and brain samples of RTT patients with different recurrent MECP2 mutations to investigate their potential correlation with the severity of the clinical phenotype. Although the main features of RTT are neurologic in nature, MeCP2 is a ubiquitously expressed protein. In this thesis, we have characterized gene expression levels in primary fibroblast cell cultures directly derived from RTT patients using an integrative multi-omics approach that combines transcriptomic and proteomic data to identify the most robust gene expression changes. We have identified an enrichment in cellular processes such as cytoskeletal activity, vesicular transport, energy metabolism and RNA processing, with important implications for neurological phenotypes despite having studied an extraneurological tissue. Moreover, we have investigated the effects of MeCP2 deficiency on the expression of GABAergic synapse proteins, and identified a developmental stage-dependent positive regulation of their expression by MeCP2, linking GABAergic neurotransmission defects with early events in RTT pathophysiology. With the advent of next-generation sequencing, many patients with a clinical diagnosis of RTT have been found to have mutations in genes other than MECP2. Understanding the relationships and interactions between these genes may help identifying common pathomechanisms leading to the overlapping phenotypes and pinpoint common therapeutic targets. In this thesis, we have used comprehensive multi-omics genomic testing to solve cases with no molecular diagnosis, and we have studied the molecular alterations in RTT-spectrum patients fibroblasts searching for common gene expression changes also found in RTT patients

Comprehensive analysis of diagnostic approaches and molecular landscape in Rett syndrome spectrum disorders

Tesi realitzada a l’Institut de Recerca Sant Joan de Déu / Programa de Doctorat en Genètica[eng] Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by a regression in acquired skills, such as purposeful hand use and language, after an apparently normal early development. RTT affects almost exclusively females and is mainly caused by mutations in the X-linked MECP2 gene, encoding methyl-CpGbinding protein 2 (MeCP2). MeCP2 is a global regulator of gene expression that operates through different mechanisms, including transcriptional regulation, chromatin architecture, splicing modulation, and miRNA processing. Nevertheless, the precise pathomechanisms by which MeCP2 deficiency leads to RTT remain elusive. MeCP2 plays a pivotal role in neuronal maturation and maintenance in the postnatal brain, and its deficiency causes severe defects in dendritic arborization and synaptogenesis. Currently, RTT has no cure or any effective pharmacological treatment, but the delineation of the downstream effects of MeCP2 deficiency could lead to the identification of biomarkers and potential therapeutic targets for RTT. The reversibility of RTT-like features in Mecp2-null mouse models upon Mecp2 reactivation strongly suggests that symptomatic patients could benefit from counteracting the effects of MeCP2 deficiency. This doctoral thesis aims to profile the molecular landscape of RTT in different ways to contribute to the understanding of the pathomechanisms behind this disorder. MECP2 being an X-linked gene, it has been long hypothesized that X chromosome inactivation (XCI) patterns may influence the phenotype of RTT patients. Therefore, this thesis has studied XCI patterns in blood and brain samples of RTT patients with different recurrent MECP2 mutations to investigate their potential correlation with the severity of the clinical phenotype. Although the main features of RTT are neurologic in nature, MeCP2 is a ubiquitously expressed protein. In this thesis, we have characterized gene expression levels in primary fibroblast cell cultures directly derived from RTT patients using an integrative multi-omics approach that combines transcriptomic and proteomic data to identify the most robust gene expression changes. We have identified an enrichment in cellular processes such as cytoskeletal activity, vesicular transport, energy metabolism and RNA processing, with important implications for neurological phenotypes despite having studied an extraneurological tissue. Moreover, we have investigated the effects of MeCP2 deficiency on the expression of GABAergic synapse proteins, and identified a developmental stage-dependent positive regulation of their expression by MeCP2, linking GABAergic neurotransmission defects with early events in RTT pathophysiology. With the advent of next-generation sequencing, many patients with a clinical diagnosis of RTT have been found to have mutations in genes other than MECP2. Understanding the relationships and interactions between these genes may help identifying common pathomechanisms leading to the overlapping phenotypes and pinpoint common therapeutic targets. In this thesis, we have used comprehensive multi-omics genomic testing to solve cases with no molecular diagnosis, and we have studied the molecular alterations in RTT-spectrum patients fibroblasts searching for common gene expression changes also found in RTT patients

Technological Improvements in the Genetic Diagnosis of Rett Syndrome Spectrum Disorders

Rett syndrome (RTT) is a severe neurodevelopmental disorder that constitutes the second most common cause of intellectual disability in females worldwide. In the past few years, the advancements in genetic diagnosis brought by next generation sequencing (NGS), have made it possible to identify more than 90 causative genes for RTT and significantly overlapping phenotypes (RTT spectrum disorders). Therefore, the clinical entity known as RTT is evolving towards a spectrum of overlapping phenotypes with great genetic heterogeneity. Hence, simultaneous multiple gene testing and thorough phenotypic characterization are mandatory to achieve a fast and accurate genetic diagnosis. In this review, we revise the evolution of the diagnostic process of RTT spectrum disorders in the past decades, and we discuss the effectiveness of state-of-the-art genetic testing options, such as clinical exome sequencing and whole exome sequencing. Moreover, we introduce recent technological advancements that will very soon contribute to the increase in diagnostic yield in patients with RTT spectrum disorders. Techniques such as whole genome sequencing, integration of data from several “omics”, and mosaicism assessment will provide the tools for the detection and interpretation of genomic variants that will not only increase the diagnostic yield but also widen knowledge about the pathophysiology of these disorders

Identification of molecular signatures and pathways involved in Rett syndrome using a multi-omics approach

Abstract Background Rett syndrome (RTT) is a neurodevelopmental disorder mainly caused by mutations in the methyl-CpG-binding protein 2 gene (MECP2). MeCP2 is a multi-functional protein involved in many cellular processes, but the mechanisms by which its dysfunction causes disease are not fully understood. The duplication of the MECP2 gene causes a distinct disorder called MECP2 duplication syndrome (MDS), highlighting the importance of tightly regulating its dosage for proper cellular function. Additionally, some patients with mutations in genes other than MECP2 exhibit phenotypic similarities with RTT, indicating that these genes may also play a role in similar cellular functions. The purpose of this study was to characterise the molecular alterations in patients with RTT in order to identify potential biomarkers or therapeutic targets for this disorder. Methods We used a combination of transcriptomics (RNAseq) and proteomics (TMT mass spectrometry) to characterise the expression patterns in fibroblast cell lines from 22 patients with RTT and detected mutation in MECP2, 15 patients with MDS, 12 patients with RTT-like phenotypes and 13 healthy controls. Transcriptomics and proteomics data were used to identify differentially expressed genes at both RNA and protein levels, which were further inspected via enrichment and upstream regulator analyses and compared to find shared features in patients with RTT. Results We identified molecular alterations in cellular functions and pathways that may contribute to the disease phenotype in patients with RTT, such as deregulated cytoskeletal components, vesicular transport elements, ribosomal subunits and mRNA processing machinery. We also compared RTT expression profiles with those of MDS seeking changes in opposite directions that could lead to the identification of MeCP2 direct targets. Some of the deregulated transcripts and proteins were consistently affected in patients with RTT-like phenotypes, revealing potentially relevant molecular processes in patients with overlapping traits and different genetic aetiology. Conclusions The integration of data in a multi-omics analysis has helped to interpret the molecular consequences of MECP2 dysfunction, contributing to the characterisation of the molecular landscape in patients with RTT. The comparison with MDS provides knowledge of MeCP2 direct targets, whilst the correlation with RTT-like phenotypes highlights processes potentially contributing to the pathomechanism leading these disorders

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs

Additional file 9 of Identification of molecular signatures and pathways involved in Rett syndrome using a multi-omics approach

Additional file 9: Table S6. Summary for the DEG and DEP from RTT-MECP2 vs MDS and RTT-MECP2 versus RTT-like

Additional file 4 of Identification of molecular signatures and pathways involved in Rett syndrome using a multi-omics approach

Additional file 4: Table S1. Detailed description of the study cohort