Involvement of extracellular vesicle microRNA clusters in developing healthy and Rett syndrome brain organoids.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression.

Burden of illness in Rett syndrome: initial evaluation of a disorder-specific caregiver survey.

BACKGROUND: Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder associated with multiple neurologic impairments. Previous studies have shown challenges to the quality of life of individuals with RTT and their caregivers. However, instruments applied to quantify disease burden have not adequately captured the impact of these impairments on affected individuals and their families. Consequently, an international collaboration of stakeholders aimed at evaluating Burden of Illness (BOI) in RTT was organized. METHODS: Based on literature reviews and qualitative interviews with parents of children and adults with RTT, a caregiver questionnaire was constructed to evaluate 22 problems (inclusive of core characteristics, functional impairments, and comorbidities) often experienced with RTT, rated mainly with a 5-level Likert scale. The questionnaire was administered anonymously online to an international sample of 756 caregivers (predominantly parents) of girls and women with RTT. Descriptive statistics were used to identify problems of high frequency and impact on affected individuals and caregivers. Chi-square tests characterized the relationship between problem severity and impact responses, while nonparametric ANOVAs of raw and z-score adjusted scores identified agreement between severity and impact on individual and caregiver. Secondary inferential tests were used to determine the roles of age, clinical type, and country of residence on BOI in RTT. RESULTS: There was variability in reported frequency of problems, with the most prevalent, severe and impactful being those related to the core features of RTT (i.e., communication and fine and gross motor impairments). Chi-square analyses demonstrated interdependence between severity and impact responses, while ANOVAs showed that many problems had disproportionately greater impact than severity, either on affected individuals (e.g., hand stereotypies) or their caregivers (e.g., sleep difficulties, seizures, pain, and behavioral abnormalities). With certain exceptions (e.g., breath-holding, seizures), age, clinical type, or country of residence did not influence these BOI profiles. CONCLUSIONS: Our data demonstrate that core features and related impairments are particularly impactful in RTT. However, problems with mild severity can also have disproportionate impact on affected individuals and, particularly, on their caregivers. Future analyses will examine the role of factors such as treatment outcomes, healthcare services, and healthcare provider's perspectives, in these BOI profiles.

A novel insight into neurological disorders through HDAC6 protein-protein interactions.

Due to its involvement in physiological and pathological processes, histone deacetylase 6 (HDAC6) is considered a promising pharmaceutical target for several neurological manifestations. However, the exact regulatory role of HDAC6 in the central nervous system (CNS) is still not fully understood. Hence, using a semi-automated literature screening technique, we systematically collected HDAC6-protein interactions that are experimentally validated and reported in the CNS. The resulting HDAC6 network encompassed 115 HDAC6-protein interactions divided over five subnetworks: (de)acetylation, phosphorylation, protein complexes, regulatory, and aggresome-autophagy subnetworks. In addition, 132 indirect interactions identified through HDAC6 inhibition were collected and categorized. Finally, to display the application of our HDAC6 network, we mapped transcriptomics data of Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis on the network and highlighted that in the case of Alzheimer's disease, alterations predominantly affect the HDAC6 phosphorylation subnetwork, whereas differential expression within the deacetylation subnetwork is observed across all three neurological disorders. In conclusion, the HDAC6 network created in the present study is a novel and valuable resource for the understanding of the HDAC6 regulatory mechanisms, thereby providing a framework for the integration and interpretation of omics data from neurological disorders and pharmacodynamic assessments.

Microglial phagolysosome dysfunction and altered neural communication amplify phenotypic severity in Prader-Willi Syndrome with larger deletion.

Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of cases. Two distinct genetic subtypes of PWS deletions are characterized, where type I (PWS T1) carries four extra haploinsufficient genes compared to type II (PWS T2). PWS T1 individuals display more pronounced physiological and cognitive abnormalities than PWS T2, yet the exact neuropathological mechanisms behind these differences remain unclear. Our study employed postmortem hypothalamic tissues from PWS T1 and T2 individuals, conducting transcriptomic analyses and cell-specific protein profiling in white matter, neurons, and glial cells to unravel the cellular and molecular basis of phenotypic severity in PWS sub-genotypes. In PWS T1, key pathways for cell structure, integrity, and neuronal communication are notably diminished, while glymphatic system activity is heightened compared to PWS T2. The microglial defect in PWS T1 appears to stem from gene haploinsufficiency, as global and myeloid-specific Cyfip1 haploinsufficiency in murine models demonstrated. Our findings emphasize microglial phagolysosome dysfunction and altered neural communication as crucial contributors to the severity of PWS T1's phenotype.

Food-Related Brain Activation Measured by fMRI in Adults with Prader-Willi Syndrome.

(1) Background: Prader-Willi syndrome (PWS) is characterized by hyperphagia, resulting in morbid obesity if not controlled. The primary aim of this study was to investigate whether PWS patients show altered activation of brain areas involved in hunger. As a secondary objective, we assessed whether there is an association between these brain areas and several endocrine and metabolic factors in the fasting state. (2) Methods: 12 PWS adults and 14 healthy controls (siblings) performed a food-related experimental task after an overnight fast while brain activation in regions of interest was measured by functional MRI. (3) Results: In controls, significantly more activation was found in the left insula (p = 0.004) and the bilateral fusiform gyrus (p = 0.003 and 0.013) when the individuals were watching food as compared to non-food pictures, which was absent in PWS patients. Moreover, in PWS adults watching food versus non-food pictures a significant negative correlation for glucose and right amygdala activation (p_fwe = 0.007) as well as a positive correlation for leptin and right anterior hippocampus/amygdala activation (p_fwe = 0.028) was demonstrated. No significant associations for the other hormonal and metabolic factors were found. (4) Conclusions: PWS individuals show aberrant food-related brain activation in the fasting state. Leptin is associated with activation within the neural motivation/reward circuitry, while the opposite is true for glucose.

The role of Extracellular Vesicles during CNS development.

With a diverse set of neuronal and glial cell populations, Central Nervous System (CNS) has one of the most complex structures in the body. Intercellular communication is therefore highly important to coordinate cell-to-cell interactions. Besides electrical and chemical messengers, CNS cells also benefit from another communication route, what is known as extracellular vesicles, to harmonize their interactions. Extracellular Vesicles (EVs) and their subtype exosomes are membranous particles secreted by cells and contain information packaged in the form of biomolecules such as small fragments of DNA, lipids, miRNAs, mRNAs, and proteins. They are able to efficiently drive changes upon their arrival to recipient cells. EVs actively participate in all stages of CNS development by stimulating neural cell proliferation, differentiation, synaptic formation, and mediating reciprocal interactions between neurons and oligodendrocyte for myelination process. The aim of the present review is to enlighten the presence and contribution of EVs at each CNS developmental milestone.

A resource to explore the discovery of rare diseases and their causative genes.

Here, we describe a dataset with information about monogenic, rare diseases with a known genetic background, supplemented with manually extracted provenance for the disease itself and the discovery of the underlying genetic cause. We assembled a collection of 4166 rare monogenic diseases and linked them to 3163 causative genes, annotated with OMIM and Ensembl identifiers and HGNC symbols. The PubMed identifiers of the scientific publications, which for the first time described the rare diseases, and the publications, which found the genes causing the diseases were added using information from OMIM, PubMed, Wikipedia, whonamedit.com, and Google Scholar. The data are available under CC0 license as spreadsheet and as RDF in a semantic model modified from DisGeNET, and was added to Wikidata. This dataset relies on publicly available data and publications with a PubMed identifier, but by our effort to make the data interoperable and linked, we can now analyse this data. Our analysis revealed the timeline of rare disease and causative gene discovery and links them to developments in methods.

A catalogue of 863 Rett-syndrome-causing MECP2 mutations and lessons learned from data integration.

Rett syndrome (RTT) is a rare neurological disorder mostly caused by a genetic variation in MECP2. Making new MECP2 variants and the related phenotypes available provides data for better understanding of disease mechanisms and faster identification of variants for diagnosis. This is, however, currently hampered by the lack of interoperability between genotype-phenotype databases. Here, we demonstrate on the example of MECP2 in RTT that by making the genotype-phenotype data more Findable, Accessible, Interoperable, and Reusable (FAIR), we can facilitate prioritization and analysis of variants. In total, 10,968 MECP2 variants were successfully integrated. Among these variants 863 unique confirmed RTT causing and 209 unique confirmed benign variants were found. This dataset was used for comparison of pathogenicity predicting tools, protein consequences, and identification of ambiguous variants. Prediction tools generally recognised the RTT causing and benign variants, however, there was a broad range of overlap Nineteen variants were identified that were annotated as both disease-causing and benign, suggesting that there are additional factors in these cases contributing to disease development.

Extracellular Vesicles in CNS Developmental Disorders.

The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics.

Development of consensus-based guidelines for managing communication of individuals with Rett syndrome.

Difficulties with communication have a profound impact on the lives of individuals with Rett syndrome and their caregivers. Globally, many families report difficulty accessing appropriate and timely information and services from professionals with expertise in augmentative and alternative communication (AAC) as it pertains to Rett syndrome. To address this need, international consensus-based guidelines for managing the communication of individuals with Rett syndrome were developed by combining available evidence and lived experience with expert opinion. A two-phase Delphi survey was built on statements and recommendations extracted from a review of over 300 pieces of literature combined with survey responses from communication professionals and caregivers. All statements that reached a pre-determined threshold of ≥70% agreement were incorporated into guidelines that consist of 268 statements and recommendations relating to (a) rights of the individual; (b) beliefs and attitudes of communication partners; (c) professional knowledge and team work; (d) strategies to optimize engagement; (e) assessment; and (f) intervention (targets and goals, techniques), including the use of AAC. To date, this project is the largest of its kind, with 650 participants from 43 countries contributing to development of consensus-based guidelines for Rett syndrome.

[Formula: see text] Measuring visual matching and short-term recognition memory with the CANTAB® Delayed Matching to Sample task in schoolchildren: Effects of demographic influences, multiple outcome measures and regression-based normative data.

The study aims to establish demographically corrected, pediatric norms for the computerized Delayed Matching to Sample (DMS) test, a measure of "visual matching ability and short-term visual recognition memory, for non-verbalisable problems". The DMS was administered to n = 184 children aged 5.10 to 14.5 years old. The DMS is a 4-choice recognition task of non-verbal, abstract patterns. The child has "to select, among four different choice patterns, the one that matches a complex visual pattern presented," i.e., (the target stimulus). The DMS consists of two conditions: a) the overt condition in which the target stimulus and four choice patterns are shown simultaneously and b) the covert condition, in which the choice patterns are shown after the target pattern is covered. The DMS test provides three outcome measures: the accuracy score (i.e., the number of correct patterns selected), latency (i.e., the response speed) and the probability of making an error after an incorrect response. These outcome measures were calculated for both conditions and for both conditions combined. Results showed that demographic variables, such as age, sex, and/or level of parental education (LPE) affected scores on these outcome measures. Based on these data, demographically corrected norms were established for all outcome measures, per condition and for both conditions combined.

Integrated analysis of human transcriptome data for Rett syndrome finds a network of involved genes.

OBJECTIVES: Rett syndrome (RTT) is a rare disorder causing severe intellectual and physical disability. The cause is a mutation in the gene coding for the methyl-CpG binding protein 2 (MECP2), a multifunctional regulator protein. Purpose of the study was integration and investigation of multiple gene expression profiles in human cells with impaired MECP2 gene to obtain a robust, data-driven insight in molecular disease mechanisms. METHODS: Information about changed gene expression was extracted from five previously published studies, integrated and the resulting differentially expressed genes were analysed using overrepresentation analysis of biological pathways and gene ontology, and network analysis. RESULTS: We identified a set of genes, which are significantly changed not in all but several transcriptomics datasets and were not mentioned in the context of RTT before. We found that these genes are involved in several processes and molecular pathways known to be affected in RTT. Integrating transcription factors we identified a possible link how MECP2 regulates cytoskeleton organisation via MEF2C and CAPG. CONCLUSIONS: Integrative analysis of omics data and prior knowledge databases is a powerful approach to identify links between mutation and phenotype especially in rare disease research where little data is available.

Beyond Pathway Analysis: Identification of Active Subnetworks in Rett Syndrome.

Pathway and network approaches are valuable tools in analysis and interpretation of large complex omics data. Even in the field of rare diseases, like Rett syndrome, omics data are available, and the maximum use of such data requires sophisticated tools for comprehensive analysis and visualization of the results. Pathway analysis with differential gene expression data has proven to be extremely successful in identifying affected processes in disease conditions. In this type of analysis, pathways from different databases like WikiPathways and Reactome are used as separate, independent entities. Here, we show for the first time how these pathway models can be used and integrated into one large network using the WikiPathways RDF containing all human WikiPathways and Reactome pathways, to perform network analysis on transcriptomics data. This network was imported into the network analysis tool Cytoscape to perform active submodule analysis. Using a publicly available Rett syndrome gene expression dataset from frontal and temporal cortex, classical enrichment analysis, including pathway and Gene Ontology analysis, revealed mainly immune response, neuron specific and extracellular matrix processes. Our active module analysis provided a valuable extension of the analysis prominently showing the regulatory mechanism of MECP2, especially on DNA maintenance, cell cycle, transcription, and translation. In conclusion, using pathway models for classical enrichment and more advanced network analysis enables a more comprehensive analysis of gene expression data and provides novel results.

Challenges in evaluating the oculomotor function in individuals with Rett syndrome using electronystagmography.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder characterized by a broad spectrum of symptoms. Communication is a major area of difficulty. Use of eye tracking technology offers a potentially effective method of communication when underpinned by intact oculomotor function. In this study, oculomotor function was assessed using electronystagmography (ENG). However, challenges were encountered when examining individuals with RTT. PURPOSE: To improve oculomotor examination in individuals with RTT by evaluating the challenges encountered during ENG examination. MATERIAL AND METHODS: Oculomotor function was examined in 17 girls and young women with RTT and 16 typically developing (TD) individuals using ENG. Observational analysis of both performance and results indicated that challenges in examination were mainly related to quality of attention and quality of signals. Subsequently these outcome values were explored quantitatively according to percentage looking time for attention and drift for signal quality. RESULTS: A significantly reduced level of attention and suboptimal electrode signals were evident in the RTT group when compared with the TD group for all tests except torsion swing. CONCLUSION: The challenges in testing confirm that regular oculomotor examination should be adjusted to meet the needs of individuals with RTT. It is hypothesized that the RTT group's higher quality of attention on the torsion swing can be explained by the more forceful vestibular rather than visual-ocular stimulus operating in this test. Suggested adaptations include reducing the number of electrodes, changing the picture stimuli and bringing them closer, performing observational assessments rather than ENG, and using virtual reality goggles.

Prader-Willi syndrome and Angelman syndrome: Visualisation of the molecular pathways for two chromosomal disorders.

Objectives: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two syndromes that are caused by the same chromosomal deletion on 15q11.2-q13. Due to methylation patterns, different genes are responsible for the two distinct phenotypes resulting in the disorders. Patients of both disorders exhibit hypotonia in neonatal stage, delay in development and hypopigmentation. Typical features for PWS include hyperphagia, which leads to obesity, the major cause of mortality, and hypogonadism. In AS, patients suffer from a more severe developmental delay, they have a distinctive behaviour that is often described as unnaturally happy, and a tendency for epileptic seizures. For both syndromes, we identified and visualised molecular downstream pathways of the deleted genes that could give insight on the development of the clinical features.Methods: This was done by consulting literature, genome browsers and pathway databases to identify molecular interactions and to construct downstream pathways.Results: A pathway visualisation was created and uploaded to the open pathway database WikiPathways covering all molecular pathways that were found.Conclusions: The visualisation of the downstream pathways of PWS- and AS-deleted genes shows that some of the typical symptoms are caused by multiple genes and reveals critical gaps in the current knowledge.

Review and gap analysis: molecular pathways leading to fetal alcohol spectrum disorders.

Alcohol exposure during pregnancy affects the development of the fetus in various ways and may lead to Fetal Alcohol Spectrum Disorders (FASD). FASD is one of the leading preventable forms of neurodevelopmental disorders. In the light of prevention and early intervention, knowledge on how ethanol exposure induces fetal damage is urgently needed. Besides direct ethanol and acetaldehyde toxicity, alcohol increases oxidative stress, and subsequent general effects (e.g., epigenetic imprinting, gene expression, and metabolite levels). The current review provides an overview of the existing knowledge about specific downstream pathways for FASD that affects e.g., the SHH pathway, cholesterol homeostasis, neurotransmitter signaling, and effects on the cytoskeleton. Available human data vary greatly, while animal studies with controlled ethanol exposition are only to a certain limit transferable to humans. The main deficits in knowledge about FASD are the lack of pathophysiological understanding and dose-response relationships, together with the lack of reliable biomarkers for either FASD detection or estimation of susceptibility. In addition to single outcome experiments, omics data should be generated to overcome this problem. Therefore, for future studies we recommend holistic data driven analysis, which allows integrative analyses over multiple levels of genetic variation, transcriptomics and metabolomics data to investigate the whole image of FASD development and to provide insight in potential drug targets for intervention.

Oculomotor Function in Individuals With Rett Syndrome.

BACKGROUND: Individuals with Rett syndrome (RTT) are notoriously reliant on the use of eye gaze as a primary means of communication. Underlying an ability to communicate successfully via eye gaze is a complex matrix of requirements, with an intact oculomotor system being just one element. To date, the underlying neural and motor pathways associated with eye gaze are relatively under-researched in RTT. PURPOSE: This study was undertaken to plug this gap in knowledge and to further the understanding of RTT in one specific area of development and function, namely oculomotor function. MATERIAL AND METHODS: The eye movements of 18 girls and young women with RTT were assessed by electronystagmography (ENG). This tested their horizontal saccadic and smooth pursuit eye movements as well as optokinetic nystagmus and vestibulo-ocular reflex. Their results were compared with normative data collected from 16 typically developing children and teenagers. RESULTS: Overall, the individuals with RTT demonstrated a range of eye movements on a par with their typically developing peers. However, there were a number of difficulties in executing the ENG testing with the RTT cohort which made quantitative analysis tricky, such as reduced motivation and attention to test materials and low-quality electrode signals. CONCLUSIONS: This study suggests that individuals with RTT have an intact oculomotor system. However, modifications should be made to the ENG assessment procedure to combat problems in testing and add strength to the results. Further investigation into these testing difficulties is warranted in order to inform such modifications.

MECP2 variation in Rett syndrome-An overview of current coverage of genetic and phenotype data within existing databases.

Rett syndrome (RTT) is a monogenic rare disorder that causes severe neurological problems. In most cases, it results from a loss-of-function mutation in the gene encoding methyl-CPG-binding protein 2 (MECP2). Currently, about 900 unique MECP2 variations (benign and pathogenic) have been identified and it is suspected that the different mutations contribute to different levels of disease severity. For researchers and clinicians, it is important that genotype-phenotype information is available to identify disease-causing mutations for diagnosis, to aid in clinical management of the disorder, and to provide counseling for parents. In this study, 13 genotype-phenotype databases were surveyed for their general functionality and availability of RTT-specific MECP2 variation data. For each database, we investigated findability and interoperability alongside practical user functionality, and type and amount of genetic and phenotype data. The main conclusions are that, as well as being challenging to find these databases and specific MECP2 variants held within, interoperability is as yet poorly developed and requires effort to search across databases. Nevertheless, we found several thousand online database entries for MECP2 variations and their associated phenotypes, diagnosis, or predicted variant effects, which is a good starting point for researchers and clinicians who want to provide, annotate, and use the data.

Abnormal Foot Position and Standing and Walking Ability in Rett Syndrome: an Exploratory Study.

This study aimed to determine whether there is a relationship between abnormal foot position and standing and walking ability in individuals with Rett syndrome (RTT), a rare neurological condition primarily affecting females, often accompanied by impaired gross motor function and musculoskeletal deformities. Through means of an online survey, physiotherapists were asked to share information about their work and experience with individuals with RTT. They were asked about their clients' scores on the Rett Syndrome Gross Motor Scale and measures of their foot deformity, passive range of motion of dorsiflexion of the foot, use of supportive footwear, pressure load on the foot, and symmetry in weight bearing. 45 physiotherapists gave answers relating to 67 individuals with RTT who ranged in age from 2 to over 50 years. Almost 80% had an abnormal foot position which required support of special shoes or orthoses. Approximately 55% experienced abnormal pressure load on the foot and 65% demonstrated asymmetrical weight-bearing; 22% could sit independently and 17% were able to stand and walk independently. Of all the variables investigated, only abnormal distribution of pressure on the foot and asymmetry in weight bearing through the legs were found to be (negatively) correlated with standing and walking ability. Physiotherapists can use this information to give advice on othopedic support for the feet of individuals with RTT.