Modeling antisense oligonucleotide therapy in MECP2 duplication syndrome human iPSC-derived neurons reveals gene expression programs responsive to MeCP2 levels.

Genomic copy-number variations (CNVs) that can cause neurodevelopmental disorders often encompass many genes, which complicates our understanding of how individual genes within a CNV contribute to pathology. MECP2 duplication syndrome (MDS or MRXSL in OMIM; OMIM#300260) is one such CNV disorder caused by duplications spanning methyl CpG-binding protein 2 (MECP2) and other genes on Xq28. Using an antisense oligonucleotide (ASO) to normalize MECP2 dosage is sufficient to rescue abnormal neurological phenotypes in mouse models overexpressing MECP2 alone, implicating the importance of increased MECP2 dosage within CNVs of Xq28. However, because MDS CNVs span MECP2 and additional genes, we generated human neurons from multiple MDS patient-derived induced pluripotent cells (iPSCs) to evaluate the benefit of using an ASO against MECP2 in a MDS human neuronal context. Importantly, we identified a signature of genes that is partially and qualitatively modulated upon ASO treatment, pinpointed genes sensitive to MeCP2 function, and altered in a model of Rett syndrome, a neurological disorder caused by loss of MeCP2 function. Furthermore, the signature contained genes that are aberrantly altered in unaffected control human neurons upon MeCP2 depletion, revealing gene expression programs qualitatively sensitive to MeCP2 levels in human neurons. Lastly, ASO treatment led to a partial rescue of abnormal neuronal morphology in MDS neurons. All together, these data demonstrate that ASOs targeting MECP2 benefit human MDS neurons. Moreover, our study establishes a paradigm by which to evaluate the contribution of individual genes within a CNV to pathogenesis and to assess their potential as a therapeutic target.

Deletions in the CDKL5 5' untranslated region lead to CDKL5 deficiency disorder.

Pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene are associated with CDKL5 deficiency disorder (CDD), a severe X-linked developmental and epileptic encephalopathy. Deletions affecting the 5' untranslated region (UTR) of CDKL5, which involve the noncoding exon 1 and/or alternatively spliced first exons (exons 1a-e), are uncommonly reported. We describe genetic and phenotypic characteristics for 15 individuals with CDKL5 partial gene deletions affecting the 5' UTR. All individuals presented characteristic features of CDD, including medically refractory infantile-onset epilepsy, global developmental delay, and visual impairment. We performed RNA sequencing on fibroblast samples from three individuals with small deletions involving exons 1 and/or 1a/1b only. Results demonstrated reduced CDKL5 mRNA expression with no evidence of expression from alternatively spliced first exons. Our study broadens the genotypic spectrum for CDD by adding to existing evidence that deletions affecting the 5' UTR of the CDKL5 gene are associated with the disorder. We propose that smaller 5' UTR deletions may require additional molecular testing approaches such as RNA sequencing to determine pathogenicity.

Psychometric evaluation of clinician- and caregiver-reported clinical severity assessments for individuals with CDKL5 deficiency disorder.

OBJECTIVE: The CDKL5 Clinical Severity Assessment (CCSA) is a comprehensive, content-validated measurement tool capturing the diverse challenges of cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD), a genetically caused developmental epileptic encephalopathy (DEE). The CCSA is divided into clinician-reported (CCSA-Clinician) and caregiver-reported (CCSA-Caregiver) assessments. The aim of this study was to evaluate the factor structure of these measures through confirmatory factor analysis (CFA) and evaluate their validity and reliability. METHODS: Participants were recruited from the International CDKL5 Clinical Research Network to take part in an in-clinic CCSA-Clinician evaluation (n = 148) and/or complete the CCSA-Caregiver questionnaire (n = 198). CFA was used to determine domains, and factor loadings and validity were assessed. For the CCSA-Clinician, inter-rater reliability was assessed by nine CDD experienced clinicians via 14 pre-recorded evaluations. Eight clinicians re-viewed and re-scored the videos after 4 weeks to evaluate intra-rater reliability. The CCSA-Caregiver was completed on a second occasion by 34 caregivers after 2-4 weeks to assess test-retest reliability. RESULTS: CFA resulted in three domains for the CCSA-Clinician (motor and movement, communication, vision) and four domains for the CCSA-Caregiver (seizures, behavior, alertness, feeding), with good item loadings across both measures. Structural statistics, internal consistency, discriminant validity, and reliability were satisfactory for both measures, and scores were consistent between known groups. SIGNIFICANCE: This study provides strong evidence that the CCSA measures are suitable to assess the clinical severity of individuals with CDD, supporting their use in clinical trials. Further evaluation of responsiveness to change in a longitudinal assessment is planned. Use may also be appropriate in similar DEEs but would require validation in those populations.

Epileptic spasms in CDKL5 deficiency disorder: Delayed treatment and poor response to first-line therapies.

OBJECTIVE: We aimed to assess the treatment response of infantile-onset epileptic spasms (ES) in CDKL5 deficiency disorder (CDD) vs other etiologies. METHODS: We evaluated patients with ES from the CDKL5 Centers of Excellence and the National Infantile Spasms Consortium (NISC), with onset from 2 months to 2 years, treated with adrenocorticotropic hormone (ACTH), oral corticosteroids, vigabatrin, and/or the ketogenic diet. We excluded children with tuberous sclerosis complex, trisomy 21, or unknown etiology with normal development because of known differential treatment responses. We compared the two cohorts for time to treatment and ES remission at 14 days and 3 months. RESULTS: We evaluated 59 individuals with CDD (79% female, median ES onset 6 months) and 232 individuals from the NISC database (46% female, median onset 7 months). In the CDD cohort, seizures prior to ES were common (88%), and hypsarrhythmia and its variants were present at ES onset in 34%. Initial treatment with ACTH, oral corticosteroids, or vigabatrin started within 1 month of ES onset in 27 of 59 (46%) of the CDD cohort and 182 of 232 (78%) of the NISC cohort (p < .0001). Fourteen-day clinical remission of ES was lower for the CDD group (26%, 7/27) than for the NISC cohort (58%, 106/182, p = .0002). Sustained ES remission at 3 months occurred in 1 of 27 (4%) of CDD patients vs 96 of 182 (53%) of the NISC cohort (p < .0001). Comparable results were observed with longer lead time (≥1 month) or prior treatment. Ketogenic diet, used within 3 months of ES onset, resulted in ES remission at 1 month, sustained at 3 months, in at least 2 of 13 (15%) individuals with CDD. SIGNIFICANCE: Compared to the broad group of infants with ES, children with ES in the setting of CDD often experience longer lead time to treatment and respond poorly to standard treatments. Development of alternative treatments for ES in CDD is needed.

DoMY-Seq: A yeast two-hybrid-based technique for precision mapping of protein-protein interaction motifs.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and coimmunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast two-hybrid and next-generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast two-hybrid reporter system. Next-generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.

Gastrointestinal Health Questionnaire for Rett Syndrome: Tool Development.

OBJECTIVES: We report the development and validation of a tool to assess gastrointestinal health in Rett syndrome (RTT). We hypothesized that the Gastrointestinal Health Questionnaire (GHQ) is a valid clinical outcomes measure of gastrointestinal health in RTT. PATIENTS AND METHODS: We used parent interviews, surveys, and literature review to generate a questionnaire related to gastrointestinal health and function, mood and behaviors, and parental concerns for individuals with RTT. Parents of affected and unaffected individuals provided responses to the GHQ, assessed the relevance and importance of statements, and completed 5 surveys related to gastrointestinal health, child-related mood and behaviors, and parent concerns. We used multivariate item analysis, 2-sample t tests, and correlations to assess the validity of the GHQ. RESULTS: We documented acceptable internal consistency of statements related to gastrointestinal health and function (Cronbach-α = 0.91), RTT-related mood and behaviors (Cronbach-α = 0.89), and parent concerns (Cronbach-α = 0.95) in the GHQ. We documented favorable external validity based on differences in response scores between parents of affected and unaffected individuals (P < 0.001) and correlations in parental response scores between the GHQ and 5 validated questionnaires addressing similar issues (P < 0.001). CONCLUSION: The GHQ is a valid tool for the assessment of gastrointestinal health in RTT and offers the opportunity to field test the safety and efficacy of novel drug therapies in clinical trials for individuals affected with this disorder.

QTY code enables design of detergent-free chemokine receptors that retain ligand-binding activities.

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5QTY, CXCR4QTY, and CXCR7QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.

Characterizing the phenotypic effect of Xq28 duplication size in MECP2 duplication syndrome.

Individuals with methyl CpG binding protein 2 (MECP2) duplication syndrome (MDS) have varying degrees of severity in their mobility, hand use, developmental skills, and susceptibility to infections. In the present study, we examine the relationship between duplication size, gene content, and overall phenotype in MDS using a clinical severity scale. Other genes typically duplicated within Xq28 (eg, GDI1, RAB39B, FLNA) are associated with distinct clinical features independent of MECP2. We additionally compare the phenotype of this cohort (n = 48) to other reported cohorts with MDS. Utilizing existing indices of clinical severity in Rett syndrome, we found that larger duplication size correlates with higher severity in total clinical severity scores (r = 0.36; P = 0.02), and in total motor behavioral assessment inventory scores (r = 0.31; P = 0.05). Greater severity was associated with having the RAB39B gene duplicated, although most of these participants also had large duplications. Results suggest that developmental delays in the first 6 months of life, hypotonia, vasomotor disturbances, constipation, drooling, and bruxism are common in MDS. This is the first study to show that duplication size is related to clinical severity. Future studies should examine whether large duplications which do not encompass RAB39B also contribute to clinical severity. Results also suggest the need for creating an MDS specific severity scale.

The array of clinical phenotypes of males with mutations in Methyl-CpG binding protein 2.

Mutations in the X-linked gene MECP2 are associated with a severe neurodevelopmental disorder, Rett syndrome (RTT), primarily in girls. It had been suspected that mutations in Methyl-CpG-binding protein 2 (MECP2) led to embryonic lethality in males, however such males have been reported. To enhance understanding of the phenotypic spectrum present in these individuals, we identified 30 males with MECP2 mutations in the RTT Natural History Study databases. A wide phenotypic spectrum was observed, ranging from severe neonatal encephalopathy to cognitive impairment. Two males with a somatic mutation in MECP2 had classic RTT. Of the remaining 28 subjects, 16 had RTT-causing MECP2 mutations, 9 with mutations that are not seen in females with RTT but are likely pathogenic, and 3 with uncertain variants. Two subjects with RTT-causing mutations were previously diagnosed as having atypical RTT; however, careful review of the clinical history determined that an additional 12/28 subjects met criteria for atypical RTT, but with more severe clinical presentation and course, and less distinctive RTT features, than females with RTT, leading to the designation of a new diagnostic entity, male RTT encephalopathy. Increased awareness of the clinical spectrum and widespread comprehensive genomic testing in boys with neurodevelopmental problems will lead to improved identification.

The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.

Glc7, the yeast protein phosphatase 1, is a component of the cleavage and polyadenylation factor (CPF). Here we show that downregulation of Glc7, or its dissociation from CPF in the absence of CPF subunits Ref2 or Swd2, results in similar snoRNA termination defects. Overexpressing a C-terminal fragment of Sen1, a superfamily I helicase required for snoRNA termination, suppresses the growth and termination defects associated with loss of Swd2 or Ref2, but not Glc7. Suppression by Sen1 requires nuclear localization and direct interaction with Glc7, which can dephosphorylate Sen1 in vitro. The suppressing fragment, and in a similar manner full-length Sen1, copurifies with the snoRNA termination factors Nrd1 and Nab3, suggesting loss of Glc7 from CPF can be compensated by recruiting Glc7 to Nrd1-Nab3 through Sen1. Swd2 is also a subunit of the Set1c histone H3K4 methyltransferase complex and is required for its stability and optimal methyltransferase activity.

Development and application of a DNA microarray-based yeast two-hybrid system.

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein-protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.

Dendritic arborization and spine dynamics are abnormal in the mouse model of MECP2 duplication syndrome.

MECP2 duplication syndrome is a childhood neurological disorder characterized by intellectual disability, autism, motor abnormalities, and epilepsy. The disorder is caused by duplications spanning the gene encoding methyl-CpG-binding protein-2 (MeCP2), a protein involved in the modulation of chromatin and gene expression. MeCP2 is thought to play a role in maintaining the structural integrity of neuronal circuits. Loss of MeCP2 function causes Rett syndrome and results in abnormal dendritic spine morphology and decreased pyramidal dendritic arbor complexity and spine density. The consequences of MeCP2 overexpression on dendritic pathophysiology remain unclear. We used in vivo two-photon microscopy to characterize layer 5 pyramidal neuron spine turnover and dendritic arborization as a function of age in transgenic mice expressing the human MECP2 gene at twice the normal levels of MeCP2 (Tg1; Collins et al., 2004). We found that spine density in terminal dendritic branches is initially higher in young Tg1 mice but falls below control levels after postnatal week 12, approximately correlating with the onset of behavioral symptoms. Spontaneous spine turnover rates remain high in older Tg1 animals compared with controls, reflecting the persistence of an immature state. Both spine gain and loss rates are higher, with a net bias in favor of spine elimination. Apical dendritic arbors in both simple- and complex-tufted layer 5 Tg1 pyramidal neurons have more branches of higher order, indicating that MeCP2 overexpression induces dendritic overgrowth. P70S6K was hyperphosphorylated in Tg1 somatosensory cortex, suggesting that elevated mTOR signaling may underlie the observed increase in spine turnover and dendritic growth.

Development and validation of parent-reported gastrointestinal health scale in MECP2 duplication syndrome.

BACKGROUND/AIMS: We aimed to develop a validated patient-reported Gastrointestinal Health Scale (GHS) specific to MECP2 Duplication Syndrome (MDS) to be used in clinical trials. METHODS: MDS parents completed a Gastrointestinal Health Questionnaire (GHQ) to investigate the most relevant and important items associated with gastrointestinal problems in MECP2-related disorders. Item reduction was executed according to EORTC guidelines. We performed reliability and validity studies for the finalized scale. RESULTS: A total of 106 surveys were eligible for item reduction and validation processes. The initial 55 items were reduced to 38 items based on parent responses, expert opinion, and initial confirmatory factor analysis (CFA). The final MDS-specific GHS included 38 items and 7 factors that underwent further reliability and validity assessments. The power of the study was at least 0.982. The Cronbach's alphas of the instruments were General Health: 0.799, Eating-Chewing-Swallowing: 0.809, Reflux: 0.794, Motility: 0.762, Mood: 0.906, Medication: 0.595, Parenting: 0.942 and all items together: 0.928. The correlation coefficient between total and individual item scores ranged from 0.215 to 0.730. Because of the ordinal nature of the variables, the diagonal weighted least squares estimation (DWLS) method was used to execute the CFA and Structural Equation Modeling. The GHS had excellent model fit with the acceptable range of fit indices values. CONCLUSIONS: We developed a parent-reported, reliable, and valid MDS-specific GHS. This scale can be utilized in clinical settings or as an outcome measure in translational and clinical research.

Sensory experiences questionnaire unravels differences in sensory profiles between MECP2-related disorders.

The methyl CpG-binding protein-2 (MECP2) gene is located on the Xq28 region. Loss of function mutations or increased copies of MECP2 result in Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), respectively. Individuals with both disorders exhibit overlapping autism symptoms, yet few studies have dissected the differences between these gene dosage sensitive disorders. Further, research examining sensory processing patterns in persons with RTT and MDS is largely absent. Thus, the goal of this study was to analyze and compare sensory processing patterns in persons with RTT and MDS. Towards this goal, caregivers of 50 female individuals with RTT and 122 male individuals with MDS, between 1 and 46 years of age, completed a standardized measure of sensory processing, the Sensory Experiences Questionnaire. Patterns detected in both disorders were compared against each other and against normative values. We found sensory processing abnormalities for both hyper- and hypo-sensitivity in both groups. Interestingly, abnormalities in MDS were more pronounced compared with in RTT, particularly with items concerning hypersensitivity and sensory seeking, but not hyposensitivity. Individuals with MDS also exhibited greater sensory symptoms compared with RTT in the areas of tactile and vestibular sensory processing and for both social and nonsocial stimuli. This study provides a first description of sensory symptoms in individuals with RTT and individuals with MDS. Similar to other neurodevelopmental disorders, a variety of sensory processing abnormalities was found. These findings reveal a first insight into sensory processing abnormalities caused by a dosage sensitive gene and may ultimately help guide therapeutic approaches for these disorders.

Generation of five induced pluripotent stem cell lines from patients with MECP2 Duplication Syndrome.

MECP2 Duplication Syndrome (MDS) is a rare, severe neurodevelopmental disorder arising from duplications in the Xq28 region containing the MECP2 gene that predominantly affects males. We generated five human induced pluripotent stem cell (iPSC) lines from the fibroblasts of individuals carrying between 0.355 and 11.2 Mb size duplications in the chromosomal locus containing MECP2. All lines underwent extensive testing to confirm MECP2 duplication and iPSC-related features such as morphology, pluripotency markers, and trilineage differentiation potential. These lines are a valuable resource for molecular and functional studies of MDS as well as screening for a variety of therapeutic approaches.

Modification of a parent-report sleep scale for individuals with CDKL5 deficiency disorder: a psychometric study.

STUDY OBJECTIVES: Sleep difficulties are common in CDKL5 deficiency disorder (CDD), a developmental and epileptic encephalopathy (DEE). This study evaluated the factor structure of the Disorders of Initiating and Maintaining Sleep (DIMS), Disorders of Excessive Daytime Somnolence (DOES) and Sleep Breathing Disorders (SBD) domains of the Sleep Disturbance Scale for Children (SDSC) for CDD. METHODS: A cross-sectional psychometric study design was used. Data were collected for 125 individuals aged 3 years or older who attended a US Centers of Excellence clinic or registered with the International CDKL5 Disorder Database. RESULTS: The median age was 10.3 years (range 3.2 - 40.7 years) and 105 (84%) were female. Two of the three SBD items related were not observed by most respondents and analysis was restricted to the DIMS and DOES domains. Using all items in the initial confirmatory factor analysis, two items in the DIMS domain and one item in the DOES domain loaded poorly. After deleting these items and repeating the analysis, item loading (0.524-0.814) and internal consistency (DIMS: 0.78, DOES: 0.76) statistics were good. The square of the inter-domain correlation coefficient was 0.17, less than Average Variance Extracted values for both domains and indicating good discriminant validity. The Tucker-Lewis and Comparative Fit indices were slightly lower than the threshold of >0.9 for establishing goodness of fit. CONCLUSIONS: The modified DIMS and DOES domains from the SDSC could be suitable clinical outcome assessments of insomnia and related impairments in CDD and potentially other DEE conditions.

Clinical Features and Disease Progression in Older Individuals with Rett Syndrome.

Although long-term survival in Rett syndrome (RTT) has been observed, limited information on older people with RTT exists. We hypothesized that increased longevity in RTT would be associated with genetic variants in MECP2 associated with milder severity, and that clinical features would not be static in older individuals. To address these hypotheses, we compared the distribution of MECP2 variants and clinical severity between younger individuals with Classic RTT (under 30 years old) and older individuals (over 30 years old). Contrary to expectation, enrichment of a severe MECP2 variant (R106W) was observed in the older cohort. Overall severity was not different between the cohorts, but specific clinical features varied between the cohorts. Overall severity from first to last visit increased in the younger cohort but not in the older cohort. While some specific clinical features in the older cohort were stable from the first to the last visit, others showed improvement or worsening. These data do not support the hypothesis that mild MECP2 variants or less overall severity leads to increased longevity in RTT but demonstrate that clinical features change with increasing age in adults with RTT. Additional work is needed to understand disease progression in adults with RTT.

The Cross-and-Capture system: a versatile tool in yeast proteomics.

High-throughput technologies such as affinity purification and yeast two-hybrid (Y2H) screening have been applied to explore protein-protein interactions (PPIs) in the model organism Saccharomyces cerevisiae. The "Cross-and-Capture" system is an alternative approach for an assessment of PPIs using two arrayed collections of differentially tagged yeast strains. In its current implementation, Cross-and-Capture encompasses ∼500 endogenously tagged yeast proteins, predominantly with roles in DNA metabolism and maintenance. The tagged arrays can also serve other purposes in yeast proteomics, such as monitoring of protein expression and the detection of posttranslational protein modifications. In this article, I summarize the development of this tool and describe its application to detect protein complexes and to screen for novel PPIs.