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

Abstract

[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.