MeCP2 was initially identified as an abundant protein in the brain, with an affinity for methylated DNA in vitro. Interestingly, both deficiency and excess of the protein leads to severe neurological problems, such as Rett syndrome, which is the result of mutations in the MECP2 gene. Subsequent transfection experiments showed that MeCP2 can recruit corepressor complexes and inhibit gene expression in vivo. MeCP2 was therefore thought to repress specific gene targets and the aetiology of Rett syndrome was proposed to result from aberrant gene expression in the MeCP2-deficient brain. Although gene expression is perturbed in the Mecp2-null mouse brain, few specific targets have been verified and alternative hypotheses for MeCP2 function have been put forward. Previous binding studies have also failed to clearly identify MeCP2 targets. To shed light on these matters, a novel technique was generated to isolate neuronal and glial nuclei and established that the amount of MeCP2 is unexpectedly high in neurons, with an abundance approaching that of the histone octamer. Chromatin immunoprecipitation experiments on mature mouse brain showed widespread binding of MeCP2, consistent with its high abundance, tracking the methyl-CpG density of the genome. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. The mutant brain also shows elevated transcription of repetitive elements, which are distributed throughout the mouse genome. Based on this data, we propose that MeCP2 binds genome wide and suppresses spurious transcription through binding in a DNA methylation dependent manner.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016. / A Deficiência Intelectual (DI), é um grande problema de saúde pública, visto que sua prevalência é de aproximadamente 2%-3%. Sua maior prevalência em homens se deve ao grande número de formas de DI Ligada ao cromossomo X (DILX). Essa podem ser, classificadas como sindrômicas (S-DILX) ou não-sindrômicas (NS-DILX). Uma das causas mais comuns de DILX em meninos é a duplicação do gene MECP2. Estudos anteriores relatam frequências de 1 a 15% de portadores de CNVs incluindo o gene MECP2 em coortes de meninos com DI. Este trabalho buscou rastrear a presença de microduplicações do gene MECP2 em uma população de 265 pacientes do sexo masculino, sendo 138 analisados pela técnica de qPCR e 127 pacientes provenientes do banco de dados de análise cromossômica por microarray do laboratório de Genética da UnB. Não foram identificados CNVs do gene MECP2 nos 265 pacientes estudados. A frequência de CNVs de MECP2 nesse estudo foi menor que a reportada anteriormente na literatura e sua baixa prevalência não justifica a triagem individual de alterações de número de cópias de MECP2 em meninos com DI. / The Intellectual Disability (ID) is a big problem of public health, since its prevalence is approximately 1%. Its higher prevalence in men is due to the large number of forms of ID linked to the X chromosome (DILX). It can be classified as syndromic (S-DILX) or non-syndromic (NS-DILX). One of the most common causes of DILX in boys is the duplication of the MECP2 gene. Earlier studies reported frequencies from 1 to 15% of CNVs carriers including MECP2 in cohort of boys with ID. This study aimed to trace the presence of microduplications of the MECP2 gene in a population of 265 male patients, 138 analysed by qPCR technique and 127 patients from the chromosomal analysis database by microarray from the Genetics Laboratory at UnB. We have not identified none of the MECP2 gene in 265 patients studied. The frequency of CNVs from MECP2 in this study was lower than reported previously in the literature and its low prevalence does not justify the individual triage of the alteration of MECP2 number of copies in boys with ID.

Rett syndrome (RTT) is an X-linked neurological disorder primarily caused by mutations in the MECP2 gene. The majority of RTT mutations disrupt the interaction of MeCP2 with DNA or TBL1X/TBL1XR1, which forms the scaffold of NCoR/SMRT co-repressor complex. Patients with RTT show no signs of neuronal death, although they have abnormal neuronal morphology, indicating that it is a neurodevelopmental rather than a neurodegenerative disease. It has been shown that reactivation of silenced MeCP2 in mice rescues the RTT phenotype, which implies that the disease is treatable. The RTT mutations in MeCP2 cluster to two regions - the methyl-CpG-binding domain (MBD) and NCoR/SMRT Interaction Domain (NID). While the interaction between MBD and DNA has been biochemically and structurally characterised, there are no structural data about the interaction between MeCP2 NID and TBL1XR1. The aim of this work was to understand how mutations in the NID cause RTT by characterising the interaction between MeCP2 and TBL1XR1. I have solved the structure of MeCP2 NID bound to TBL1XR1 WD40 domain. I show that a small region of the MeCP2 NID makes extensive contacts with TBL1XR1, and that these contacts are mediated primarily by MeCP2 residues known to be mutated in RTT. I also measured the affinities between TBL1XR1 and MeCP2-derived peptides using fluorescence anisotropy and surface plasmon resonance assays. I determined the affinity between MeCP2 NID peptide and TBL1XR1 to be around 10- 20 μM, and show that mutations in either MeCP2 or TBL1XR1 can abolish this interaction. Taken together, these data strongly suggest that the abolition of the interaction between MeCP2 NID and TBL1XR1 WD40 domain is sufficient to cause RTT. This knowledge can help with the rational design of small drug-like molecules that might be able to mediate the interaction between mutated MeCP2 and TBL1XR1, potentially helping to treat the disease.

I denna pilotstudie utvärderas KomRett, en kommunikationskurs för närstående till personer med Rett syndrom som genomfördes för första gången hösten 2009. Målsättningarna med KomRett var att öka kursdeltagarnas kunskap om kommunikation och strategier för att stimulera kommunikation samt grafisk AKK med eller utan ljud. Kursen utformades med utgångspunkt från innehållet i föräldrakurserna i projektet AKKTIV vid DART Center i Göteborg (Alternativ och Kompletterande Kommunikation Tidig InterVention till föräldrar som har barn med kommunikationssvårigheter). Kommunikationspartners till fyra personer med Rett syndrom deltog i studien. I utvärderingen användes analys av olika skattningsformulär samt videoanalys av filmer från två kursdeltagare. Resultaten visar att tre av sex kursdeltagarna började använda bilder i sin kommunikation med personerna med Rett syndrom efter kursen. I videoanalysen framkom att en av två kursdeltagare oftare använde en responsiv kommunikationsstil i vald aktivitet efter kursen. Höga poäng på utvärderingsformuläret tydde på att kursdeltagarna uppfattade kursen positivt och flera av kursdeltagarna uppgav att de var mer uppmärksamma på kommunikationen efter kursen.

Intractable epilepsy remains one of the top issues affecting the quality of living in Rett children. While several MeCP2-deficient mouse models of Rett Syndrome have been established, minimal information exists on how the loss of MeCP2 affects brain network activity. To address this issue, in vivo recordings of the hippocampus and somatosensory cortex of MeCP2-deficient mice were taken during exploration, immobility, and sleep. The frequency of hippocampal theta oscillations was significantly attenuated in MeCP2-deficient mice during exploration. A subset of MeCP2-heterozygotes displayed spontaneous, cortical epileptiform-like discharges in the immobile-awake state. Similar epileptiform-like discharges were observed in one of the four Mecp2-null mice recorded. Aside from these EEG abnormalities, basal network activity was preserved. Further, convulsive seizures were not seen. Collectively, these findings indicate that a deficiency of MeCP2 in mice leads to only subtle alterations in brain wave activity, contrasting the severely abnormal EEG observed in Rett girls.

Intractable epilepsy remains one of the top issues affecting the quality of living in Rett children. While several MeCP2-deficient mouse models of Rett Syndrome have been established, minimal information exists on how the loss of MeCP2 affects brain network activity. To address this issue, in vivo recordings of the hippocampus and somatosensory cortex of MeCP2-deficient mice were taken during exploration, immobility, and sleep. The frequency of hippocampal theta oscillations was significantly attenuated in MeCP2-deficient mice during exploration. A subset of MeCP2-heterozygotes displayed spontaneous, cortical epileptiform-like discharges in the immobile-awake state. Similar epileptiform-like discharges were observed in one of the four Mecp2-null mice recorded. Aside from these EEG abnormalities, basal network activity was preserved. Further, convulsive seizures were not seen. Collectively, these findings indicate that a deficiency of MeCP2 in mice leads to only subtle alterations in brain wave activity, contrasting the severely abnormal EEG observed in Rett girls.

Several years ago a ten year child with a disability died from "severe malnutrition" according to a Coroners Service inquest jury. The inquest evidence shows that approximately one week prior to this child's death three health care providers conducted individual assessments of the child. Using institutional ethnography as a theoretical and methodological framework, the author conducts a textual analysis of the health care providers' documents generated during their provision of service to this child. Obtained as public documents from the Coroners Service, this data includes: the hospital form, the hospice society records and home care nursing records. / Graduate / 0566

abstract: Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression with highest levels of expression in mature neurons. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). At the cellular level, MECP2 mutations cause both synaptic and dendritic defects. Despite identification of MECP2 as a cause for RTT nearly 16 years ago, little progress has been made in identifying effective treatments. Investigating major cellular and molecular targets of MECP2 in model systems can help elucidate how mutation of this single gene leads to nervous system and behavioral defects, which can ultimately lead to novel therapeutic strategies for RTT and MDS. In the work presented here, I use the fruit fly, Drosophila melanogaster, as a model system to study specific cellular and molecular functions of MECP2 in neurons. First, I show that targeted expression of human MECP2 in Drosophila flight motoneurons causes impaired dendritic growth and flight behavioral performance. These effects are not caused by a general toxic effect of MECP2 overexpression in Drosophila neurons, but are critically dependent on the methyl-binding domain of MECP2. This study shows for the first time cellular consequences of MECP2 gain-of-function in Drosophila neurons. Second, I use RNA-Seq to identify KIBRA, a gene associated with learning and memory in humans, as a novel target of MECP2 involved in the dendritic growth phenotype. I confirm bidirectional regulation of Kibra by Mecp2 in mouse, highlighting the translational utility of the Drosophila model. Finally, I use this system to identify a novel role for the C-terminus in regulating the function of MECP in apoptosis and verify this finding in mammalian cell culture. In summary, this work has established Drosophila as a translational model to study the cellular effects of MECP2 gain-of-function in neurons, and provides insight into the function of MECP2 in dendritic growth and apoptosis. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2015

Rett syndrome is a debilitating autistic spectrum disorder affecting one in ten thousand girls. Patients develop normally for up to eighteen months before a period of regression involving stagnation in head growth, loss of speech, hand use and mobility. It is almost exclusively caused by mutation in Methyl CpG binding Protein 2 (MeCP2). MeCP2 has traditionally been thought of as a transcriptional repressor, although its exact function remains unknown and it has recently been shown that the protein can also bind to hydroxymethylation and non-CpG methylation, which occurs predominantly at CAC sites in the mature nervous system. Genotype-phenotype studies of the most common Rett-causing mutations in affected patients revealed that a missense mutation, R133C results in a milder form of Rett syndrome. The reasons for this are unclear, as the mutation lies right in the heart of the methylated DNA binding domain. Previous in vitro studies of R133C showed a severe deficit in binding to methylated cytosine. A subsequent study found that R133C binding to hydroxymethylated cytosine was specifically impaired, whereas binding to methylated cytosine was indistinguishable from wildtype. Defining the DNA binding impairment of MeCP2R133C would yield important insights into Rett disease pathophysiology and provide an explanation for the phenotypic spectrum seen in patients. To shed light on these matters, a novel mouse model of the R133C mutation was created. The R133C mouse had a phenotype that was less severe than other missense mutant mice, in terms of survival, growth, Rett-like phenotypic score and some behavioural paradigms thus recapitulating the patient data. At the molecular level in adult mouse brain, MeCP2R133C protein abundance was reduced. Immunohistochemistry showed that MeCP2R133C had an abnormal pattern of localisation in the nucleus of neurons. In vitro electrophoretic mobility shift assays suggested that MeCP2R133C binding to (hydroxy)methyl-CAC may be reduced to a greater extent than binding to mCpG. Chromatin immunoprecipitation experiments confirmed the deficit in binding to methylated sites and supported a disproportionate reduction in binding to methylation in a CAC sequence context. Analysis of adult mouse cerebellar gene expression revealed a subtle upregulation of long genes and downregulation of short genes. Based on these data, it is proposed that Rett syndrome caused by the R133C mutation results from a combination of protein instability and defective binding to methylated DNA. Methyl-CAC binding is potentially abolished. The downstream biological consequence of this is a length-dependent deregulation of gene expression in the brain.

Orientador : Iscia T. Lopes-Cendes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-01T18:03:09Z (GMT). No. of bitstreams: 1 Facchin_Daniela_M.pdf: 13970036 bytes, checksum: ad97f904ffe393b72f0a4ec57b941290 (MD5) Previous issue date: 2002 / Resumo: A síndrome de Rett (RS) é considerada um transtorno invasivo do desenvolvimento, primeiramente descrita em 1966 pelo austríaco Andreas Rett. Clinicamente é caracterizada por desenvolvimento neuropsicomotor aparentemente nonnal entre os seis e 18 meses de idade. Após este período o desenvolvimento fica estagnado, atinge um platô e posteriormente ocorre regressão motora progressiva. A prevalência da RS é ao redor de 1:1O000 a 1:22 000, no sexo feminino. Até outubro de 1999, o diagnóstico era feito somente através da aplicação de critérios clínicos, pois não havia um marcador bioquímico, citogenético ou molecular conhecido, associado à patologia. Diferentes modelos de herança foram propostos para RS a saber: mutações no DNA mitocondrial, dissomia uniparental, inativação preferencial do cromossomo X, mutações dinâmicas (repetição de tripletos) e aumento na freqüência de sítios frágeis. Recentemente, mutações no gene MECP2, localizado na região Xq28, foram identificadas em 33% a 80% de pacientes com quadro clínico clássico da RS e em pacientes outros fenótipos. o objetivo principal deste projeto foi investigar a presença de ligação genética entre marcadores de DNA localizados no cr X, incluindo a região Xq28. Para o cumprimento dos objetivos acima, contou-se com um total de 86 amostras, pertencentes a 23 famílias, das quais 25 pacientes com quadro clínico clássico de RS. Os estudos de ligação foram realizados através da genotipagem de marcadores anônimos de DNA do tipo microssatélite e a análise estatística da probabilidade de ligação calculada por métodos paramétricos, "lod score", através da utilização do programa de computador LINKAGE. Além disso, analisou-se, cuidadosamente, a segregação dos marcadores genotipados em em todas as famílias nas quais era possível estabelecer haplótipos. Obteve-se "lod scores" negativos significativos para todos os marcadores analisados, quando as famílias eram avaliadas em conjunto, sendo que extensas porções do cromossomo X foram excluídas a saber: DXS 1073 exclusão de 12 cM (compreendendo a região onde localiza-se o gene MECP2), DXS 8103 exclusão de 2 cM, DXS 8091 exclusão de 4 cM, DXS 8043 exclusão de 22 cM, DXS 1227 exclusão de 4 cM, e DXS 1192 / Abstract: Rett syndrome (RS) is a pervasive development disorder first described by Andreas Rett in 1966. It is eharaeterized by normal development during the first 6 to 18 months of age followed by developmental regression. Thereafter development sueeessively stagnates, plateaus and regresses. The estimated prevalenee is 1 in 10 000 to 22 000 females. Before oetober 1999, the diagnosis was based only on elinieal eriteria, sineethere were no bioquimie, citogenetie or moleeular marker for RS. Different models of inheritanee had been proposed for RS including uniparental disomy, skewed X inaetivation, mutations in the mitoehondrial DNA, and triplet repeat expansions. Recently, mutations in the MECP2 gene on ehromosome Xq28 have been found in 33 to 80% of patients with classieal RS and in patients with other phenotypes. The aim of the present study was to performed linkage analysis in 86 samples fiom 23 families including 25 girls with classieal RS using six mierosatellites markers on X ehromosome. Linkage studies were performed by genotyping marker using PCR and two point lod seore ealeulation using the program MLINK of the LINKAGE paekage. Lod score added for all 23 families. We have excluded a region of 12 eM around marker DXS 1073 (where the MECP2 gene is loeated), 2 eM around marker DXS 8103,4 eM around marker DXS 8091, 22 eM around marker DXS 8043, 4 eM around marker DXS 1227 and 10 eM around marker DXS 1192.No isolated families gave signifieant lod seore. However haplotype analysis of family 6 eomposed by two sisters with elassieal RS cleary show inherited of different haplotypes for five marker genotiped (Xq26 and Xqter). In eonclusion our results strengths the evidenee for genetie heterogeneity in RS, thus adding information whieh is likely to help the seareh for an alternative loeus for RS / Mestrado / Genetica Medica / Mestre em Ciências Médicas