Rett syndrome (RTT) is an early-onset neurodevelopmental disorder that primarily affects females, resulting in severe cognitive and physical disabilities, and is one of the most prevalent causes of intellectual disability in females. More than fifty years after the first publication on Rett syndrome, and almost two decades since the first report linking RTT to the MECP2 gene, the research community’s effort is focused on obtaining a better understanding of the genetics and the complex biology of RTT and Rett-like phenotypes without MECP2 mutations. Herein, we review the current molecular genetic studies, which investigate the genetic causes of RTT or Rett-like phenotypes which overlap with other genetic disorders and document the swift evolution of the techniques and methodologies employed. This review also underlines the clinical and genetic heterogeneity of the Rett syndrome spectrum and provides an overview of the RTT-related genes described to date, many of which are involved in epigenetic gene regulation, neurotransmitter action or RNA transcription/translation. Finally, it discusses the importance of including both phenotypic and genetic diagnosis to provide proper genetic counselling from a patient’s perspective and the appropriate treatment.

MECP2 duplication syndrome (MDS) is an X‐linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho‐motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation‐dependent probe amplification (MLPA). Using, a custom in‐house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype‐phenotype correlations, and thus more personalized genetic counselling.

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Rett 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.

Methyl CpG binding protein 2 (MECP2) is located at Xq28 and is a multifunctional gene with ubiquitous expression. Loss-of-function mutations in MECP2 are associated with Rett syndrome (RTT), which is a well-characterized disorder that affects mainly females. In boys, however, mutations in MECP2 can generate a wide spectrum of clinical presentations that range from mild intellectual impairment to severe neonatal encephalopathy and premature death. Thus, males can be more difficult to classify and diagnose than classical RTT females. In addition, there are some variants of unknown significance in MECP2, which further complicate the diagnosis of these children. Conversely, the entire duplication of the MECP2 gene is related to MECP2 duplication syndrome (MDS). Unlike in RTT, in MDS, males are predominantly affected. Usually, the duplication is inherited from an apparently asymptomatic carrier mother. Both syndromes share some characteristics, but also differ in some aspects regarding the clinical picture and evolution. In the following review, we present a thorough description of the different types of MECP2 variants and alterations that can be found in males, and explore several genotype–phenotype correlations, although there is still a lot to understand.

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.

Noncoding RNAs play regulatory roles in physiopathology, but their involvement in neurodevelopmental diseases is poorly understood. Rett syndrome is a severe, progressive neurodevelopmental disorder linked to loss-of-function mutations of the MeCP2 gene for which no cure is yet available. Analysis of the noncoding RNA profile corresponding to the brain-abundant circular RNA (circRNA) and transcribed-ultraconserved region (T-UCR) populations in a mouse model of the disease reveals widespread dysregulation and enrichment in glutamatergic excitatory signaling and microtubule cytoskeleton pathways of the corresponding host genes. Proteomic analysis of hippocampal samples from affected individuals confirms abnormal levels of several cytoskeleton-related proteins together with key alterations in neurotransmission. Importantly, the glutamate receptor GRIA3 gene displays altered biogenesis in affected individuals and in vitro human cells and is influenced by expression of two ultraconserved RNAs. We also describe post-transcriptional regulation of SIRT2 by circRNAs, which modulates acetylation and total protein levels of GluR-1. As a consequence, both regulatory mechanisms converge on the biogenesis of AMPA receptors, with an effect on neuronal differentiation. In both cases, the noncoding RNAs antagonize MeCP2-directed regulation. Our findings indicate that noncoding transcripts may contribute to key alterations in Rett syndrome and are not only useful tools for revealing dysregulated processes but also molecules of biomarker value.

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.