Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations

Background

Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.

Results

We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).

Conclusion

Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.     

Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC) lines from Rett syndrome patients as in vitro disease model

Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5-6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.     

Spectrum of MECP2 mutations in Rett syndrome

Mutations in the MECP2 (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. We have collected the results of MECP2 analysis conducted in four laboratories in France. A total of 301 RTT alleles have been analyzed, demonstrating a total of 69 different mutations so far observed and accounting for 64% of MECP2 genes in RTT patients living in France. R168X (11.5%) is the most common of MECP2 mutations, followed by R255X (10.9%), R270X (10.5%), T158M (7.8%), and R306C (6.8%). Only 10 mutations had a relative frequency > 2%. A total of 59 mutations were found in a small number of RTT alleles (from 1 to 2). These data demonstrate the high allelic heterogeneity of RTT in France and provide information relevant to the development of strategies for molecular diagnosis and genetic counseling in RTT families.     

Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: Report of two novel mutations

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome.     

Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function

Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in?vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.     

MeCP2 R168X male and female mutant mice exhibit Rett‐like behavioral deficits

Rett syndrome (RTT) is a regressive developmental disorder characterized by motor and breathing abnormalities, anxiety, cognitive dysfunction and seizures. Approximately 95% of RTT cases are caused by more than 200 different mutations in the X‐linked gene encoding methyl‐CpG‐binding protein 2 (MeCP2). While numerous transgenic mice have been created modeling common mutations in MeCP2, the behavioral phenotype of many of these male and, especially, female mutant mice has not been well characterized. Thorough phenotyping of additional RTT mouse models will provide valuable insight into the effects of Mecp2 mutations on behavior and aid in the selection of appropriate models, ages, sexes and outcome measures for preclinical trials. In this study, we characterize the phenotype of male and female mice containing the early truncating MeCP2 R168X nonsense point mutation, one of the most common in RTT individuals, and compare the phenotypes to Mecp2 null mutants. Mecp2^R168X mutants mirror many clinical features of RTT. Mecp2^R168X/y males exhibit impaired motor and cognitive function and reduced anxiety. The behavioral phenotype is less severe and with later onset in Mecp2^R168X/+ females. Seizures were noted in 3.7% of Mecp2^R168X mutant females. The phenotype in Mecp2^R168X/y mutant males is remarkably similar to our previous characterizations of Mecp2 null males, whereas Mecp2^R168X/+ females exhibit a number of phenotypic differences from females heterozygous for a null Mecp2 mutation. This study describes a number of highly robust behavioral paradigms that can be used in preclinical drug trials and underscores the importance of including Mecp2 mutant females in preclinical studies .     

Reduced MeCP2 Expression is Frequent in Autism Frontal Cortex and Correlates with Aberrant MECP2 Promoter Methylation

Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are “pervasive developmental disorders” and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.     

Novel Mutation in the <Emphasis Type="Italic">MECP2</Emphasis> Gene Identified in a Group of Rett Syndrome Patients from Ukraine

Mutations in the MECP2 gene are known to cause Rett syndrome (RTT)—a neurodevelopmental disorder, one of the most common causes of intellectual disability in females, with an incidence of 1 in 10000–15000. We have investigated exons 3 and 4 of the MECP2 gene, that coding MBD and TRD domains of the MeCP2 protein, in 21 RTT patients from Ukraine by PCR-DGGE analysis followed by Sanger sequencing of PCR fragments with abnormal migration profiles. In 13 of 21 (61.9%) patients 7 different mutations were identified one nonsense mutation—c. NC_000023.11:g.154031326G>A (MECP2:c.502C>T) and 4 missense mutation NC_000023.11:g.154031409G>T (MECP2:c.419C>T), NC_000023.11:g.154031355G>A (MECP2:c.473C>T), NC_000023.11:g.154031354A>C (MECP2:c.472A>C), NC_000023.11:g.154031431G>A (MECP2:c.397C>T) located in exon 4, a rare RTT-causing splice site mutation NC_000023.10:g.153296903T>G (MECP2:c.378-2A>C) in intron 3 and deletion NC_000023.10:g.1532 96079_153296122del44 in exon 4. The novel mutation MECP2:c.472A>C identified in our study in patients withclassic RTT phenotype leds to T158P substitution. It is one more confirmation of crucial role that 158 codon in MECP2 protein function.     

Profiling beneficial and potential adverse effects of MeCP2 overexpression in a hypomorphic Rett syndrome mouse model

De novo loss-of-function mutations in methyl-CpG-binding protein 2 (MeCP2) lead to the neurodevelopmental disorder Rett syndrome (RTT). Despite promising results from strategies aimed at increasing MeCP2 levels, additional studies exploring how hypomorphic MeCP2 mutations impact the therapeutic window are needed. Here, we investigated the consequences of genetically introducing a wild-type MECP2 transgene in the Mecp2 R133C mouse model of RTT. The MECP2 transgene reversed the majority of RTT-like phenotypes exhibited by male and female Mecp2 R133C mice. However, three core symptom domains were adversely affected in female Mecp2^R133C/+ animals; these phenotypes resemble those observed in disease contexts of excess MeCP2. Parallel control experiments in Mecp2^Null/+ mice linked these adverse effects to the hypomorphic R133C mutation. Collectively, these data provide evidence regarding the safety and efficacy of genetically overexpressing functional MeCP2 in Mecp2 R133C mice and suggest that personalized approaches may warrant consideration for the clinical assessment of MeCP2-targeted therapies.     

Characterization of the MeCP2R168X Knockin Mouse Model for Rett Syndrome

Rett syndrome, one of the most common causes of mental retardation in females, is caused by mutations in the X chromosomal gene MECP2. Mice deficient for MeCP2 recapitulate some of the symptoms seen in patients with Rett syndrome. It has been shown that reactivation of silent MECP2 alleles can reverse some of the symptoms in these mice. We have generated a knockin mouse model for translational research that carries the most common nonsense mutation in Rett syndrome, R168X. In this article we describe the phenotype of this mouse model. In male MeCP2^R168X mice life span was reduced to 12–14 weeks and bodyweight was significantly lower than in wild type littermates. First symptoms including tremor, hind limb clasping and inactivity occurred at age 27 days. At age 6 weeks nest building, rotarod, open-field and elevated plus maze experiments showed impaired motor performance, reduced activity and decreased anxiety-like behavior. Plethysmography at the same time showed apneas and irregular breathing with reduced frequency. Female MeCP2^R168X mice showed no significant abnormalities except decreased performance on the rotarod at age 9 months. In conclusion we show that the male MeCP2^R168X mice have a phenotype similar to that seen in MECP2 knockout mouse models and are therefore well suited for translational research. The female mice, however, have a much milder and less constant phenotype making such research with this mouse model more challenging.     

Novel double deletions in the MECP2 gene in Tunisian Rett patient

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively girls. Rett patients present an apparently normal psychomotor development during the first 6-18 months of life. Thereafter, they show a short period of developmental stagnation followed by a rapid regression in language and motor development. RTT is currently considered as monogenic X-linked dominant disorder due to mutations in the MECP2 gene, encoding the methyl-CpG binding protein 2. The aim of this study was to perform a mutational analysis of the MECP2 gene in a classical Rett patient.The results showed the presence of a novel point mutation c.C1142T (p.P381L) and two deletions at the heterozygous state: a novel deletion c.1075delTTC (p.S359) and a known one c.1157del44 (p.L386Q fs X2) in the C-terminal region of MeCP2.