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.

De novo variants in FBXO11 cause a syndromic form of intellectual disability with behavioral problems and dysmorphisms.

Determining pathogenicity of genomic variation identified by next-generation sequencing techniques can be supported by recurrent disruptive variants in the same gene in phenotypically similar individuals. However, interpretation of novel variants in a specific gene in individuals with mild-moderate intellectual disability (ID) without recognizable syndromic features can be challenging and reverse phenotyping is often required. We describe 24 individuals with a de novo disease-causing variant in, or partial deletion of, the F-box only protein 11 gene (FBXO11, also known as VIT1 and PRMT9). FBXO11 is part of the SCF (SKP1-cullin-F-box) complex, a multi-protein E3 ubiquitin-ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. Twenty-two variants were identified by next-generation sequencing, comprising 2 in-frame deletions, 11 missense variants, 1 canonical splice site variant, and 8 nonsense or frameshift variants leading to a truncated protein or degraded transcript. The remaining two variants were identified by array-comparative genomic hybridization and consisted of a partial deletion of FBXO11. All individuals had borderline to severe ID and behavioral problems (autism spectrum disorder, attention-deficit/hyperactivity disorder, anxiety, aggression) were observed in most of them. The most relevant common facial features included a thin upper lip and a broad prominent space between the paramedian peaks of the upper lip. Other features were hypotonia and hyperlaxity of the joints. We show that de novo variants in FBXO11 cause a syndromic form of ID. The current series show the power of reverse phenotyping in the interpretation of novel genetic variances in individuals who initially did not appear to have a clear recognizable phenotype.

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.

Diagnostic exome sequencing in 100 consecutive patients with both epilepsy and intellectual disability.

OBJECTIVE: Epilepsy is highly prevalent among patients with intellectual disability (ID), and seizure control is often difficult. Identification of the underlying etiology in this patient group is important for daily clinical care. We assessed the diagnostic yield of whole exome sequencing (WES). In addition, we evaluated which clinical characteristics influence the likelihood of identifying a genetic cause and we assessed the potential impact of the genetic diagnosis on (antiepileptic) treatment strategy. METHODS: One hundred patients with both unexplained epilepsy and (borderline) ID (intelligence quotient ≤ 85) were included. All patients were evaluated by a clinical geneticist, a (pediatric) neurologist, and/or a specialist ID physician. WES analysis was performed in two steps. In step 1, analysis was restricted to the latest versions of ID and/or epilepsy gene panels. In step 2, exome analysis was extended to all genes (so-called full exome analysis). The results were classified according to the American College of Medical Genetics and Genomics guidelines. RESULTS: In 58 patients, the diagnostic WES analysis reported one or more variant(s). In 25 of the 100 patients, these were classified as (likely) pathogenic, in 24 patients as variants of uncertain significance, and in the remaining patients the variant was most likely not related to the phenotype. In 10 of 25 patients (40%) with a (likely) pathogenic variant, the genetic diagnosis might have an impact on the treatment strategy in the future. SIGNIFICANCE: This study illustrates the clinical diagnostic relevance of WES for patients with both epilepsy and ID. It also demonstrates that implementing WES diagnostics might have impact on the (antiepileptic) treatment strategy in this population. Confirmation of variants of uncertain significance in (candidate) genes may further increase the yield.

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.

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.

PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature.

BACKGROUND: De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia. OBJECTIVES: To delineate the clinical spectrum of PURA syndrome and study genotype-phenotype correlations. METHODS: Diagnostic or research-based exome or Sanger sequencing was performed in individuals with ID. We systematically collected clinical and mutation data on newly ascertained PURA syndrome individuals, evaluated data of previously reported individuals and performed a computational analysis of photographs. We classified mutations based on predicted effect using 3D in silico models of crystal structures of Drosophila-derived Pur-alpha homologues. Finally, we explored genotype-phenotype correlations by analysis of both recurrent mutations as well as mutation classes. RESULTS: We report mutations in PURA (purine-rich element binding protein A) in 32 individuals, the largest cohort described so far. Evaluation of clinical data, including 22 previously published cases, revealed that all have moderate to severe ID and neonatal-onset symptoms, including hypotonia (96%), respiratory problems (57%), feeding difficulties (77%), exaggerated startle response (44%), hypersomnolence (66%) and hypothermia (35%). Epilepsy (54%) and gastrointestinal (69%), ophthalmological (51%) and endocrine problems (42%) were observed frequently. Computational analysis of facial photographs showed subtle facial dysmorphism. No strong genotype-phenotype correlation was identified by subgrouping mutations into functional classes. CONCLUSION: We delineate the clinical spectrum of PURA syndrome with the identification of 32 additional individuals. The identification of one individual through targeted Sanger sequencing points towards the clinical recognisability of the syndrome. Genotype-phenotype analysis showed no significant correlation between mutation classes and disease severity.

Rett syndrome - biological pathways leading from MECP2 to disorder phenotypes.

Rett syndrome (RTT) is a rare disease but still one of the most abundant causes for intellectual disability in females. Typical symptoms are onset at month 6-18 after normal pre- and postnatal development, loss of acquired skills and severe intellectual disability. The type and severity of symptoms are individually highly different. A single mutation in one gene, coding for methyl-CpG-binding protein 2 (MECP2), is responsible for the disease. The most important action of MECP2 is regulating epigenetic imprinting and chromatin condensation, but MECP2 influences many different biological pathways on multiple levels although the molecular pathways from gene to phenotype are currently not fully understood. In this review the known changes in metabolite levels, gene expression and biological pathways in RTT are summarized, discussed how they are leading to some characteristic RTT phenotypes and therefore the gaps of knowledge are identified. Namely, which phenotypes have currently no mechanistic explanation leading back to MECP2 related pathways? As a result of this review the visualization of the biologic pathways showing MECP2 up- and downstream regulation was developed and published on WikiPathways which will serve as template for future omics data driven research. This pathway driven approach may serve as a use case for other rare diseases, too.

New insights in Rett syndrome using pathway analysis for transcriptomics data.

The analysis of transcriptomics data is able to give an overview of cellular processes, but requires sophisticated bioinformatics tools and methods to identify the changes. Pathway analysis software, like PathVisio, captures the information about biological pathways from databases and brings this together with the experimental data to enable visualization and understanding of the underlying processes. Rett syndrome is a rare disease, but still one of the most abundant causes of intellectual disability in females. Cause of this neurological disorder is mutation of one single gene, the methyl-CpG-binding protein 2 (MECP2) gene. This gene is responsible for many steps in neuronal development and function. Although the genetic mutation and the clinical phenotype are well described, the molecular pathways linking them are not yet fully elucidated. In this study we demonstrate a workflow for the analysis of transcriptomics data to identify biological pathways and processes which are changed in a Mecp2 (-/y) mouse model.

Neurophysiology versus clinical genetics in Rett syndrome: A multicenter study.

Many studies have attempted to establish the genotype-phenotype correlation in Rett syndrome (RTT). Cardiorespiratory measurements provide robust objective data, to correlate with each of the different clinical phenotypes. It has important implications for the management and treatment of this syndrome. The aim of this study was to correlate the genotype with the quantitative cardiorespiratory data obtained by neurophysiological measurement combined with a clinical severity score. This international multicenter study was conducted in four European countries from 1999 to 2012. The study cohort consisted of a group of 132 well-defined RTT females aged between 2 and 43 years with extended clinical, molecular, and neurophysiological assessments. Diagnosis of RTT was based on the consensus criteria for RTT and molecular confirmation. Genotype-phenotype analyses of clinical features and cardiorespiratory data were performed after grouping mutations by the same type and localization or having the same putative biological effect on the MeCP2 protein, and subsequently on eight single recurrent mutations. A less severe phenotype was seen in females with CTS, p.R133C, and p.R294X mutations. Autonomic disturbances were present in all females, and not restricted to nor influenced by one specific group or any single recurrent mutation. The objective information from non-invasive neurophysiological evaluation of the disturbed central autonomic control is of great importance in helping to organize the lifelong care for females with RTT. Further research is needed to provide insights into the pathogenesis of autonomic dysfunction, and to develop evidence-based management in RTT. © 2016 Wiley Periodicals, Inc.

Eye Gaze Technology as a Form of Augmentative and Alternative Communication for Individuals with Rett Syndrome: Experiences of Families in The Netherlands.

This paper provides a brief report on families' experiences of eye gaze technology as one form of augmentative and alternative communication (AAC) for individuals with Rett syndrome (RTT), and the advice, training and support they receive in relation to this. An online survey exploring communication and AAC was circulated to 190 Dutch families; of the 67 questionnaires that were returned, 63 had answered questions relating to eye gaze technology. These 63 were analysed according to parameters including: experiences during trial periods and longer-term use; expert knowledge, advice and support; funding; communicative progress; and family satisfaction. 20 respondents were using or had previous experience of using an eye gaze system at the time of the survey, 28 of those with no prior experience wanted to try a system in the future. Following a trial period, 11 systems had been funded through health insurance for long-term use and two families had decided a system was not appropriate for them. Levels of support during trials and following long-term provision varied. Despite frustrations with the technology, satisfaction with the systems was higher than satisfaction with the support. The majority of families reported progress in their child's skills with longer term use. These findings suggest that although eye gaze technologies offer potential to individuals with RTT and their families, greater input from suppliers and knowledgeable AAC professionals is essential for individuals and families to benefit maximally. Higher levels of training and support should be part of the 'package' when an eye gaze system is provided.

De novo loss-of-function mutations in WAC cause a recognizable intellectual disability syndrome and learning deficits in Drosophila.

Recently WAC was reported as a candidate gene for intellectual disability (ID) based on the identification of a de novo mutation in an individual with severe ID. WAC regulates transcription-coupled histone H2B ubiquitination and has previously been implicated in the 10p12p11 contiguous gene deletion syndrome. In this study, we report on 10 individuals with de novo WAC mutations which we identified through routine (diagnostic) exome sequencing and targeted resequencing of WAC in 2326 individuals with unexplained ID. All but one mutation was expected to lead to a loss-of-function of WAC. Clinical evaluation of all individuals revealed phenotypic overlap for mild ID, hypotonia, behavioral problems and distinctive facial dysmorphisms, including a square-shaped face, deep set eyes, long palpebral fissures, and a broad mouth and chin. These clinical features were also previously reported in individuals with 10p12p11 microdeletion syndrome. To investigate the role of WAC in ID, we studied the importance of the Drosophila WAC orthologue (CG8949) in habituation, a non-associative learning paradigm. Neuronal knockdown of Drosophila CG8949 resulted in impaired learning, suggesting that WAC is required in neurons for normal cognitive performance. In conclusion, we defined a clinically recognizable ID syndrome, caused by de novo loss-of-function mutations in WAC. Independent functional evidence in Drosophila further supported the role of WAC in ID. On the basis of our data WAC can be added to the list of ID genes with a role in transcription regulation through histone modification.

Mutations in DDX3X Are a Common Cause of Unexplained Intellectual Disability with Gender-Specific Effects on Wnt Signaling.

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

Further delineation of the KBG syndrome phenotype caused by ANKRD11 aberrations.

Loss-of-function variants in ANKRD11 were identified as the cause of KBG syndrome, an autosomal dominant syndrome with specific dental, neurobehavioural, craniofacial and skeletal anomalies. We present the largest cohort of KBG syndrome cases confirmed by ANKRD11 variants reported so far, consisting of 20 patients from 13 families. Sixteen patients were molecularly diagnosed by Sanger sequencing of ANKRD11, one familial case and three sporadic patients were diagnosed through whole-exome sequencing and one patient was identified through genomewide array analysis. All patients were evaluated by a clinical geneticist. Detailed orofacial phenotyping, including orthodontic evaluation, intra-oral photographs and orthopantomograms, was performed in 10 patients and revealed besides the hallmark feature of macrodontia of central upper incisors, several additional dental anomalies as oligodontia, talon cusps and macrodontia of other teeth. Three-dimensional (3D) stereophotogrammetry was performed in 14 patients and 3D analysis of patients compared with controls showed consistent facial dysmorphisms comprising a bulbous nasal tip, upturned nose with a broad base and a round or triangular face. Many patients exhibited neurobehavioural problems, such as autism spectrum disorder or hyperactivity. One-third of patients presented with (conductive) hearing loss. Congenital heart defects, velopharyngeal insufficiency and hip anomalies were less frequent. On the basis of our observations, we recommend cardiac assessment in children and regular hearing tests in all individuals with a molecular diagnosis of KBG syndrome. As ANKRD11 is a relatively common gene in which sequence variants have been identified in individuals with neurodevelopmental disorders, it seems an important contributor to the aetiology of both sporadic and familial cases.

De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum.

Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

A recurrent deletion syndrome at chromosome bands 2p11.2-2p12 flanked by segmental duplications at the breakpoints and including REEP1.

We identified an identical and recurrent 9.4-Mbp deletion at chromosome bands 2p11.2-2p12, which occurred de novo in two unrelated patients. It is flanked at the distal and proximal breakpoints by two homologous segmental duplications consisting of low copy repeat (LCR) blocks in direct orientation, which have >99% sequence identity. Despite the fact that the deletion was almost 10 Mbp in size, the patients showed a relatively mild clinical phenotype, that is, mild-to-moderate intellectual disability, a happy disposition, speech delay and delayed motor development. Their phenotype matches with that of previously described patients. The 2p11.2-2p12 deletion includes the REEP1 gene that is associated with spastic paraplegia and phenotypic features related to this are apparent in most 2p11.2-2p12 deletion patients, but not in all. Other hemizygous genes that may contribute to the clinical phenotype include LRRTM1 and CTNNA2. We propose a recurrent but rare 2p11.2-2p12 deletion syndrome based on (1) the identical, non-random localisation of the de novo deletion breakpoints in two unrelated patients and a patient from literature, (2) the patients' phenotypic similarity and their phenotypic overlap with other 2p deletions and (3) the presence of highly identical LCR blocks flanking both breakpoints, consistent with a non-allelic homologous recombination (NAHR)-mediated rearrangement.

[Rett syndrome]. / Het Rett-syndroom.

BACKGROUND: Rett syndrome is caused by mutations in the X-linked MECP2 gene, encoding MeCP2 protein. This protein is essential for the transcription and repression of other genes and is important for the development and plasticity of the central nervous system. Children with Rett syndrome initially develop normally but after a few months their development deteriorates. CASE DESCRIPTION: The case describes a girl aged 3 years 7 months whose development had initially been normal but then stagnated and was followed by a phase of regression. Her speech was lost and she developed severe dyspraxia with stereotypic hand movements characteristic of the condition. The clinical diagnosis of Rett syndrome was confirmed through genetic testing. Later on she developed epileptic seizures and a severe scoliosis for which surgical correction and stabilisation was carried out. CONCLUSION: Rett syndrome is a severe neurological developmental disorder that occurs almost exclusively in females and for which there is still no causal treatment. The treatment is multidisciplinary and based on clinical experience.