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

Unraveling Molecular Pathways Altered in MeCP2-Related Syndromes, in the Search for New Potential Avenues for Therapy

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked epigenetic modulator whose dosage is critical for neural development and function. Loss-of-function mutations in MECP2 cause Rett Syndrome (RTT, OMIM #312750) while duplications in the Xq28 locus containing MECP2 and Interleukin-1 receptor-associated kinase 1 (IRAK1) cause MECP2 duplication syndrome (MDS, OMIM #300260). Both are rare neurodevelopmental disorders that share clinical symptoms, including intellectual disability, loss of speech, hand stereotypies, vasomotor deficits and seizures. The main objective of this exploratory study is to identify novel signaling pathways and potential quantitative biomarkers that could aid early diagnosis and/or the monitoring of disease progression in clinical trials. We analyzed by RT-PCR gene expression in whole blood and microRNA (miRNA) expression in plasma, in a cohort of 20 females with Rett syndrome, 2 males with MECP2 duplication syndrome and 28 healthy controls, and correlated RNA expression with disease and clinical parameters. We have identified a set of potential biomarker panels for RTT diagnostic and disease stratification of patients with microcephaly and vasomotor deficits. Our study sets the basis for larger studies leading to the identification of specific miRNA signatures for early RTT detection, stratification, disease progression and segregation from other neurodevelopmental disorders. Nevertheless, these data will require verification and validation in further studies with larger sample size including a whole range of ages

Study of expression levels in MECP2 related disorders using transcriptomics and proteomics: characterizing Rett syndrome and MECP2 duplication syndrome

[eng] MECP2 is a multifunctional gene involved in multiple processes such as transcription regulation, chromatin remodelling, splicing and miRNA regulation. Malfunction of MECP2 due to loss of function mutations leads to Rett syndrome (RTT) whereas its overexpression triggers MECP2 duplication syndrome (MDS). Besides, variants in MECP2 can cause a wide spectrum of phenotypes, from severe congenital encephalopathy with early death to mild intellectual disability (ID). RTT and MDS are two well characterized rare diseases with a partly overlapping phenotype consisting of neurodevelopmental delay, ID, impaired muscle tone, lack or unstable ambulation, little or absent speech, gastrointestinal problems, autism like behaviour and hand stereotypies. With next generation sequencing derived methodologies, gigantic breakthroughs have been done in diagnostics and research. These new omic strategies, such as transcriptomic or proteomic, can be applied to patient-derived samples to obtain answers to some of the still unknown aspects of the molecular effect of MECP2 in RTT and MDS. For the present thesis project, patients with alterations in MECP2 were gathered and three cohorts were created and thoroughly studied and characterized: a classic RTT girls group with large deletions within MECP2, a group of patients with MDS together with their duplication carrier mothers, and a group of boys with ID and neurodevelopmental delay with variants in MECP2. Genotype-phenotype correlations were also attempted for these cohorts. In order to further study patients with classic RTT and MDS we decided to use a multi-omic (transcriptomic and proteomic) approach. For that, 22 classic RTT, 17 MDS, 10 MECP2 duplication carriers and 13 healthy controls were gathered and primary cultured cell lines were established from their skin biopsies. DNA, RNA and proteins were extracted from them all and RNA sequencing and tandem mass tag-mass spectrometry (TMT-MS) experiments were performed. The obtained data was analysed in a case-control approach. The multi-omic analysis revealed shared and distinct altered biological processes for each cohort studied. The gene causing RTT and MDS is the same, but its downstream molecular effects might be opposite. Being able to obtain RNA and protein profiles from these patient cohorts seems to be a promising way to better understand MECP2’s role in the underlying pathomechanism triggering RTT and MDS. Differentially expressed genes and proteins involved in cytoskeleton, vesicular activity or immune system were found, and some of them are highlighted as potential biomarker and therapeutic target candidates. Altogether, we aimed to fill the gap by exploring the patients’ genetics, transcriptomics and proteomics in order to get closer to identifying therapeutic targets and biomarkers that could be used in future clinical trials

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

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

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

Additional file 7: Table S4. Enrichment analysis results for the RTT-MECP2 versus healthy controls, RTT versus MDS, RTT versus RTT-like, transcriptomic and proteomic data

Additional file 6 of Identification of molecular signatures and pathways involved in Rett syndrome using a multi-omics approach

Additional file 6: Table S3. RNAseq and proteomics differential expression results for RTT - MECP2 versus healthy controls