The ability of the brain to change structurally and functionally with experience is called brain plasticity. High levels of pro-inflammatory cytokines impair normal memory formation and consolidation. To better understand the role of pro-inflammatory cytokines in learning, the contribution of the cytokine interleukin-6 (IL-6) to a motor skill learning task investigated. The Fmr1 Knockout (KO) mouse, an animal model of Fragile X Syndrome, has demonstrated impaired neural plasticity and learning. Fmr1 KO and control wild-type (WT) mice were trained on the dowel and flat beam runways to study motor skill learning and motor activity respectively. The cerebellum from the animals was examined for IL-6 protein using ELISA. No significant differences in the levels of IL-6 in the cerebellum of the Fmr1 KO and WT normal mice were found. The expression of IL-6 was not altered by the behavioural training. These results suggest lack of association between IL-6, and FMRP and motor skill learning. / October 2016

Fragile X syndrome (FXS) is the leading single gene cause of intellectual disability and Autism Spectrum Disorder (ASD). It is caused by epigenetic silencing of the fragile X mental retardation gene (FMR1), causing a loss of Fragile-X Mental Retardation Protein (FMRP). Over the last 2 decades, much has been learned about the pathophysiology related to the loss of FMRP from mouse models of FXS. The recent generation of a rat model of FXS opens the door to: validate phenotypes across mammalian species, address cognitive dysfunction using paradigms that are more difficult to address in mice and explore candidate therapeutics more accurately. This thesis explored the validity of a new rat model for FXS (Fmr1 KO rat). I showed that Fmr1 KO rats exhibit normal spatial navigation memory, social interactions and anxiety levels. On the contrary, when subjects were tested in a battery of spontaneous exploration tasks: object recognition (OR), object-context (OC), object-place (OP), and object-place-context (OPC) recognition, which assess associative memory, Fmr1 KO rats showed a severe deficit in remembering the most complex (episodic-like) associations. Following these results, I sought to explore the development of associative memory from postnatal day 25 (P25) to adulthood (P71). Subjects were tested in the four spontaneous exploration tasks, previously mentioned, 8 times between P25 and P71 to assess the development of their ability to discriminate novel from familiar associations between objects, contexts and places. Fmr1 KO rats’ ability to discriminate novel from familiar object-place (spatial) and object-place-context (episodic-like) associations was significantly impaired (OP was delayed, and OPC ability did not develop). In the last part of this thesis I examined whether early therapeutic intervention with lovastatin can restore the cognitive deficits I observed. Subjects were fed either a diet containing lovastatin (“lovachow”) or an identically looking control diet, between P29 and P64, and tested in the four spontaneous exploration tasks, previously mentioned. Fmr1 KO rats demonstrated a developmental profile of associative memory indistinguishable from that of WT animals. At P64, lovachow was replaced with standard laboratory chow and the animals were tested 1 and 3 months later. Surprisingly, lovastatin treated Fmr1 KO animals maintained the ability to perform the OPC task even at 3 months after the end of treatment, whereas Fmr1 KO animals on control chow showed no improvement with age. The findings of this work indicate that transgenic rats can complement existing mouse models of FXS, providing valuable insights into the effects of FMRP loss on cognitive function. Furthermore, the results from the treatment study show that not only can lovastatin treatment prevent the emergence of cognitive deficits associated with Fragile X Syndrome but also that lovastatin (and perhaps pharmaceutical interventions more generally) may prevent the developmental deficits in neuronal circuit formation which can be maintained into adulthood.

La dyslexie est le plus fréquent des troubles neurodéveloppementaux de l’enfant. A ce jour, 9 loci de susceptibilité ont été identifiés parmi lesquels DYX 9 en Xq27. Nous avons réalisé la première étude de liaison française suivie de l’analyse de gènes candidats sur l’ensemble du génome de 58 sujets issus de 12 familles multiplex pour la dyslexie, en utilisant un phénotype catégoriel. Des résultats significatifs sont apparus pour la région Xq27.3, au sein de DYX9. Le Lod score multipoint maximal obtenu était 3.884 entre rs12558359 et rs454992. Dans cette région, les séquences codantes de 7 gènes candidats ayant un rôle dans le développement cérébral (CXORF1, CXORF51, SLITRK2, FMR1, FMR2, ASFMR1, FMR1NB), ont été étudiées à la recherche de mutations. Nous avons également étudié les séquences 5’ non codantes des gènes FMR1 et FMR2. Aucune mutation n’a été retrouvée. Ces résultats de liaison de la région Xq27.3, au locus XFRA, dans une population française confirment la région DYX9 comme région d’intérêt dans la dyslexie de développement. / Dyslexia is a frequent neurodevelopmental learning disorder. To date, 9 susceptibility loci have been determined among which DYX9 located in Xq27. We performed the first French SNP linkage study followed by candidate gene investigation in dyslexia by studying 12 multiplex families (58 subjects) with at least two children affected, according to categorical restrictive criteria for phenotype definition. Significant results emerged on Xq27.3 within DYX9. The maximum multipoint LOD score reached 3,884 between rs12558359 and rs454992. Within this region, 7 candidate genes were investigated for mutations in exonic sequences (CXORF1, CXORF51, SLITRK2, FMR1, FMR2, ASFMR1, FMR1NB), all having a role during brain development. We further looked for 5’UTR trinucleotide repeats in FMR1 and FMR2 genes. No mutation or polymorphism co-segregating with dyslexia was found. This finding in French families with Dyslexia showed significant linkage on Xq27.3 enclosing FRAXA, and consequently confirmed the DYX9 region as a robust susceptibility locus. We reduced the previously described interval from 6.8 Mb (DXS1227- DXS8091) to 4Mb also disclosing a higher Lod score.

The regulation of neural precursor cells (NPCs), which encompass neural progenitor and neural stem cells (NSCs), is fundamental for proper brain development and function. These cells are regulated by orchestrated signalling within their local environment. Aberrant aspects of cell proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome (FXS)—a disorder characterized by intellectual and social changes due to the silencing of the gene encoding FMRP. The biology of hippocampal NPCs in FXS during early postnatal development has not been studied, despite high FMRP expression levels in the hippocampus at the end of the first postnatal week. In this thesis, the Fmr1-knockout (KO) mouse model was used to study hippocampal cell biology during early postnatal development. A tissue culture assay, used to study the effect of astrocyte-secreted factors on the proliferation of NSCs, indicated that astrocyte secreted factors from Fmr1-KO brains enhanced the proliferation of wild type, but not Fmr1-KO NSCs (Chapter 3). Next, the proliferation and cell cycle profiles of NPCs in vitro and in vivo studied with immunocytochemistry, Western blotting, and flow cytometry revealed decreased proliferation of NPCs in the Fmr1-KO hippocampus (Chapter 4). Finally, cells isolated from the P7 dentate gyrus and characterized by flow cytometry, showed a reduced proportion of NSCs and an increased proportion of neuroblasts—neuronal committed progenitors—in Fmr1-KO mice. Together, these results indicate that hippocampal NPCs show aberrant proliferation and neurogenesis during early postnatal development. This could indicate stem-cell depletion, increased quiescence, or a developmental delay in relation to lack of FMRP and uncovers a new role for FMRP in the early postnatal hippocampus. In turn, elucidating the mechanisms that underlie FXS will aid in the development of targeted treatments. / Thesis / Doctor of Philosophy (PhD) / Fragile X syndrome is the leading inherited cause of intellectual impairment and autism spectrum disorder. The syndrome is caused by a defect in one gene. This gene has been suggested to play a role in regulating the birth of new brain cells termed neural precursor cells. The importance of neural precursor cells stems from their ability to generate neurons and glia, the main cells in the brain. In this thesis, I focus on studying neural precursor cells from the hippocampus, a brain region important for learning and memory. A mouse model was used to compare neural precursor cells from healthy and Fragile X mice during early postnatal development. I found that neural precursor cells do not divide as much as they should in the Fragile X mouse hippocampus. The results help to determine the causes for learning and memory deficits in Fragile X and potentially open avenues for intervention.

A proteína do retardo mental do X frágil (FMRP), codificada pelo gene do Retardo Mental do X Frágil (do inglês, Fragile Mental Retardation 1, FMR1) tem expressão significativa no encéfalo, gônadas e células proliferativas. A FMRP é uma proteína ligante de RNA, repressora traducional, que transita entre o núcleo celular, grânulos citoplasmáticos e polissomos. Sua associação a RNA pode se dar pelos domínios Tudor N-terminais, dois domínios centrais, com homologia à heteronucleoproteína K (KH) ou motivos RGG, ricos em arginina (R) e glicina (G), C-terminais. A abolição da expressão da FMRP por mutações no gene FMR1 é a causa mais frequente de deficiência intelectual hereditária entre homens. Transcritos desse gene sofrem splicing alternativo de quatro éxons, podendo gerar até 20 isoformas não redundantes da FMRP. A tradução de RNAm do FMR1 contendo o éxon 12 causa uma extensão em fase, em 21 aminoácidos na alça variável do segundo domínio KH (KH-2) da FMRP, cujos padrão de expressão e função ainda são desconhecidos. Embora a FMRP tenha alta similaridade com duas proteínas parálogas, proteínas relacionadas à FMRP, FRX1P e FXR2P, ela apresenta algumas características de expressão e função que lhes são próprias. A longa alça variável do domínio KH-2, por exemplo, não é observada nas parálogas e é característica somente de ortólogas da FMRP em mamíferos. Assim, é possível que o estudo deste segmento da proteína traga informações funcionais específicas para o encéfalo de mamífero. Demonstramos anteriormente, por qRT-PCR, que, em transcritos do Fmr1 de rato, a expressão da sequência do éxon 12 é regulada ao longo do desenvolvimento pós-natal precoce, de forma diferencialmente positiva no córtex cerebral frontal e cerebelo, em relação ao hipocampo. No presente trabalho, aprofundamos esses estudos, tendo como objetivo a análise cuidadosa da expressão desse éxon em isoformas da FMRP (FMRP+12ISO), pelo uso de um anticorpo dirigido ao segmento codificado por ele, em encéfalos do décimo segundo dia pós-natal (P12, controle positivo) ou embrionários. Para tal, foram realizadas análises por imunoistoquímica e, em P12, ensaios de cromatografia de exclusão molecular de partículas ribonucleoproteicas. Análises de níveis de RNAm e proteicos em fase embrionária (E12 a E20) do encéfalo do rato foram também conduzidas in vivo e in vitro, em cultivo primário de neuroesferas em suspensão, a partir da dissociação de vesículas telencefálicas de ratos em E14. Os dados de imunoistoquímica de encéfalo de ratos em P12 indicaram que (i) as camadas granular externa e a camada piramidal externa do córtex cerebral e as células de Purkinje no cerebelo são mais ricas em FMRP+12ISO; (ii) o giro denteado e CA3 foram fontes de FMRP+12ISO no hipocampo, porém em mais baixa intensidade; e (iii) o conjunto das isoformas da FMRP, incluindo as FMRP+12ISO, foram expressas em região periventricular dos ventrículos laterais em período pós-natal, sugestivo de células-tronco neurais do adulto ou recém diferenciadas. No córtex cerebral, as FMRP+12ISO foram expressas em áreas motora (segmento rostrodorsal), sensorial (segmentos dorsolaterais a laterais), auditiva (segmentos dorsolaterais), olfatória (córtex piriforme) e visual (segmentos ventrolaterais), além da área do cingulado (segmentos mediais) de ambos os hemisférios cerebrais. Os dados também confirmaram que, em P12, as FMRP+12ISO têm expressão mais pronunciada no córtex cerebral e cerebelo do que no hipocampo. À cromatografia, as FMRP+12ISO tiveram o mesmo padrão de distribuição que o conjunto das isoformas da FMRP, fracionando em complexos ribonucleoproteicos maiores que 600 kDa. De modo geral, a expressão das FMRP+12ISO foi baixa em E12 e E14. Houve concordância entre as análises por qRT-PCR, Western blotting e imunoistoquímica, corroborando a baixa expressão do éxon 12 do FMR1 na vesículas telencefálicas em E14. Observamos por imunoistoquímica poucas células sugestivas de progenitoras, na base do neuroepitélio, que expressassem FMRP+12ISO ou outras isoformas da FMRP. O córtex cerebral em E20 foi raramente positivo para FMRP+12ISO ou o conjunto das isoformas da FMRP. Por outro lado, células da camada mais superficial da placa cortical, indicativa de ser a camada I, mostraram expressão de isoformas da FMRP sem o segmento codificado pelo éxon 12 do Fmr1, em E18 e da FMRP, incluindo as FMRP+12ISO, em E20, de forma contínua em várias regiões corticais. Em neuroesferas em suspensão, a expressão das FMRP+12ISO foi muito baixa enquanto isoformas da FMRP, supostamente com a alça variável de KH- 2 em sua conformação curta, tiveram alta expressão nessas células. Desde as primeiras 24 horas sob condições de diferenciação neuronal in vitro, células de neuroesferas aumentaram a expressão das FMRP+12ISO, que se mantiveram alta no período analisado (12 dias in vitro), colocalizando-se com outras isoformas da FMRP. Ensaios preliminares in vitro pela interferência do RNA, em células imortalizadas C6, indicaram um RNA em fita dupla, entre dois testados, com capacidade de inibição de mensagens do Fmr1 que especificamente contenham o éxon 12. A expressão do éxon 12 do FMR1 no córtex cerebral frontal, humano, em envelhecimento foi baixa pela análise de RNAm, enquanto o total de transcritos deste gene apresentou-se em níveis significativos. Nossos dados sugerem que a expressão do éxon 12 do Fmr1 é mais significativa para FMRP+12ISO em células neuronais, durante um período crítico de sinaptogênese, no primeiro mês pós-natal do rato. O tecido telencefálico, embrionário não se mostrou uma fonte rica dessas isoformas, principalmente em células indiferenciadas, que foram francamente negativas / Fragile X Mental Retardation Protein (FMRP), codified by Fragile Mental Retardation 1 (FMR1) gene, is significantly expressed in the brain, gonads and proliferative cells. FMRP is an RNA-binding protein and acts as a translation repressor, which transits between cell nucleus, cytoplasmic granules and polysomes. Its association with RNA occurs via several domains, namely: Nterminal Tudor domains; two central domains with K-heteronucleoprotein (KH) homology; and C-terminal RGG motifs, that are rich in arginine (R) and glicine (G). The absence of FMRP expression triggered by mutations in the FMR1 gene is the most frequent cause of hereditary intellectual disability in human men. Four FMR1 exons may undergo alternative splicing, generating up to 20 non-redundant FMRP isoforms. The translation of the FMR1 mRNA containing exon 12 leads to an in-phase extension of 21 amino acids in the variable loop on FMRP the second KH domain (KH-2). The pattern of expression and function of this isoform are unknown. Although FMRP is highly similar to two paralogs proteins, FMRP-related proteins FRX1P and FRX2P, it presents some unique expression and function characteristics. The long variable loop of the KH-2 domain, for example, is not observed in these paralogs and is a hallmark of FMRP mammal orthologs. Therefore, the study of this protein segment can potentially bring information about its function specifically in the mammalian brain. Using qRT-PCR and Western blotting, we previously demonstrated that, for rat Fmr1 transcripts, the expression of sequences containing exon 12 is regulated during early postnatal development. In this period, expression of these segments in the frontal cerebral cortex and in the cerebellum is higher when compared to hippocampus expression. In the present work, we deepened these studies with the objective of carefully analysing the expression of FMRP isoforms containing FMRP exon 12 (FMRP+12ISO). For this purpose, we employed rat postnatal brains at the twelfth postnatal day (P12, used as a positive control) and rat embryo brain in immunohistochemistry assays with antibodies detecting peptides codified by exon 12. In this strategy, we also carried out molecular exclusion chromatography of ribonucleoproteins particles with lysates from rat P12 encephalon. Analysis of mRNA and protein levels in rat brain in the embryonic period [embryonic days 12 to 20 (E12 to E20)] were conducted in vivo and in vitro, in neurosphere suspension primary cultures, obtained by dissociation of telencephalic vesicles from E14 rats. Immunohistochemical data from P12 rat brain indicated that (i) the granular external layers and pyramidal external layer in the brain cortex and Purkinje cells in the cerebellum are richer in FMRP+12ISO; (ii) in the hippocampus, FMRP+12ISO can be found in dentate gyrus and CA3, although in lesser intensities; and (iii) FMRP isoforms, including FMRP+12ISO, are expressed in the periventricular regions from the lateral ventricles, suggesting their expression in adult neural stem cells or in differentiating cells. In the cerebral cortex, FMRP+12ISO are expressed in motor areas (rostrodorsal segment) and in sensorial areas (dorsolateral and lateral segments), specifically in auditory (dorsolateral segments), olfactory (piriform cortex) and visual (ventrolateral segments) areas, besides expression in the cingulate área (medial segment) in both hemispheres. Our data also confirmed that, in P12 brain, cerebral cortex and cerebellum have higher FMRP+12ISO expression when compared to the hippocampus. Chromatography data indicated that FMRP+12ISO have the same pattern of distribution than the FMRP isoform group, fractionating in ribonucleoproteics complexes heavier than 600kDa. Altogether, FMRP+12ISO expression is low in E12 and E14 rat brain. qRT-PCR, Western blotting and immunohistochemistry data were concordant, corroborating the low expression of exon 12 in E14 telencephalic vesicles. In immunohistochemistry images, we observed few cells with progenitor phenotype, at the basal neuroepithelium, expressing FMRP+12ISO or other FMRP isoforms. In E20, cerebral cortex was rarely positive for FMRP+12ISO or other FMRP isoforms. Still, cells in the superficial layer of the cortical plate, possibly layer 1, were positive for FMRP isoforms that do not contain the segment codified by exon 12 in E18, brains and positive for the ensemble of FMRP, isoforms, FMRP+12ISO included, in E20 brains, in continuous portions of several cortical regions. In suspension neurosphere cultures, FMRP+12ISO expression was very low, while the expression of FMRP isoforms, supposedly with the variable loop of KH-2 in its short conformation, was high in. After 24 hours under neuron differentiation conditions in vitro, neurosphere cells showed increasing expression of FMRP+12ISO, which remained high during the period analyzed (12 days in vitro), co-localizing with other FMRP isoforms. Preliminary assays using RNA interference in vitro in an immortalized glioma cell lineage (C6), disclosed a double-stranded RNA, among two tested samples, with the ability to suppress exon 12 containig Fmr1 mRNA. The expression of FMR1 transcripts containing exon 12 in aging human brain frontal cortex was low, while total FMR1 transcripts levels were significant. Our data suggest that the expression of exon 12 from Fmr1 is more significant in rat neuronal cells during a critical period of synaptogenesis in the first postnatal month. The embryonic telencephalic tissue is not rich in FMRP+12ISO, which were notably absent from undifferentiated cells

Made available in DSpace on 2016-08-10T10:38:39Z (GMT). No. of bitstreams: 1 ANDREIA PIRES AMANCIO.pdf: 1468482 bytes, checksum: cfd035e6b0a287fb6f6c412088f7aa20 (MD5) Previous issue date: 2013-01-31 / The Intellectual Disability (DM) is defined as an incomplete level of intellectual development, and its manifestation usually occurs before 18 years of age. It is one of the most common neuropsychiatric disorders, with a prevalence of 5% in our population. The Fragile X syndrome (FXS) is the most common cause of inherited mental retardation worldwide, and the second most common genetic cause of mental retardation. The phenotype of FXS is associated with mutations in FMR1 (Fragile X Mental Retardation-linked type 1), which is caused by the expansion of repetition of a trinucleotide sequence (CGG) in the regulatory region of the FMR1 gene, located on chromosome X (Xq27 .3). The importance of clinical recognition and specific diagnosis of FXS comes from the fact that theoretically all cases are hereditary and familial. In this context, this study aimed to validate the molecular genetic diagnosis simplified and cost, for patients suspected of FXS in the Laboratory of Cytogenetics and Molecular Genetics (LaGene) of the Department of Health of the State of Goiás in Goiânia using the PCR technique. We selected 35 patients referred by medical service of public health to LaGene with clinical indication for diagnosis of FXS. The patients' DNA was extracted and subjected to two PCR methods, here called PCR Screening (PCR-T) and PCR for Pre-mutation (PCR-P). The new methodology, PCRT, 88% produced conclusive results against 100% conclusiveness of the standard technique (PCR-P) and getting a p> 0.11. The alleles amplified by PCR-P allowed the diagnosis of pre-mutation in a sample. From these observations we propose a strategy for the diagnosis of the syndrome using PCR-P research in pre-mutation individuals in parents with inconclusive results. Given the successful results and improved the PCR, including the new and easy diagnosis of pre-mutation samples not completed by the technique of drawing, we suggest the implementation of both PCR genetics laboratory in the State of Goiás (LaGene ) for the diagnosis of FXS. / A Deficiência Mental (DM) é definida como sendo um nível incompleto do desenvolvimento intelectual, e sua manifestação ocorre normalmente antes dos 18 anos de idade. É um dos transtornos neuropsiquiátricos mais comuns, com uma prevalência de 5% na população brasileira. A síndrome do X-Frágil (SXF) é a causa mais comum de retardo mental hereditário em todo o mundo, e a segunda causa genética mais frequente de deficiência mental. O fenótipo da SXF está associado a mutações no gene FMR1 (Fragile X-linked Mental Retardation type 1), que é causada pela expansão da repetição de uma sequência de trinucleotídeos (CGG) na região reguladora do gene FMR1, localizado no cromossomo X (Xq27.3). A importância do reconhecimento clínico e diagnóstico específico da SXF vem do fato de que teoricamente todos os casos são hereditários e familiais. Nesse contexto, este estudo teve como objetivo validar o diagnóstico genético-molecular simplificado e de baixo custo, para pacientes com suspeita da SXF no Laboratório de Citogenética e Genética Molecular (LaGene) da Secretaria Estadual de Saúde do Estado de Goiás, na cidade de Goiânia, utilizando a técnica de PCR. Foram selecionados 35 pacientes encaminhados pelo serviço médico da rede pública de saúde ao LaGene com indicação clínica de diagnóstico da SXF. O DNA desses pacientes foi extraído e submetido a dois métodos de PCR, aqui denominados PCR de Triagem (PCR-T) e PCR para Pré-mutação (PCR-P). A nova metodologia, PCRT, produziu 88% de resultados conclusivos contra 100% de conclusividade pela técnica padrão (PCR-P) e obtendo um p>0,11. Os alelos amplificados pela PCR-P possibilitou o diagnóstico de pré-mutação em uma amostra. A partir destas observações propõe-se uma estratégia para o diagnóstico da síndrome utilizando a PCR-P na pesquisa de pré-mutação em pais de indivíduos com resultados inconclusivos. Considerando os resultados bem sucedidos e aprimorados da técnica de PCR, incluindo o novo e fácill diagnóstico da pré-mutação de amostras não concluídas pela técnica de triagem, sugere-se a implementação de ambas as PCR no laboratório de genética do Estado de Goiás (LaGene) para o diagnóstico da SXF.

Das Fehlen des FMR1-Genprodukts FMRP ist ursächlich für die Entstehung des Phänotyps bei Patienten mit Fragilem-X-Syndrom. Es kommt hierbei auf Grund einer Hypermethylierung der DNA im Bereich der FMR1-Promotorregion zu einer Hemmung der Transkription. Im Rahmen dieser Arbeit wurde untersucht, ob eine Behandlung mit Methotrexat, welches eine Hypomethylierung der DNA verursacht, eine Therapieoption für Patienten mit Fragilem-X-Syndrom darstellt. Es wurden Fibroblasten von Patienten mit Fragilem-X-Syndrom kultiviert und währenddessen mit Methotrexat behandelt. Hierbei trat eine Reaktivierung der FMR1-Transkription auf, FMRP konnte allerdings nicht reproduzierbar detektiert werden. Auch in der Untersuchung des Methylierungsstatus der FMR1-Promotorregion zeigte sich nach Behandlung keine Reduktion der Methylierung. Der fehlende Nachweis von FMRP in den behandelten Patientenzellen macht es unwahrscheinlich, dass MTX im Rahmen dieser Erkrankung klinische Anwendung finden wird.

O retardo mental (RM) representa um problema de saúde pública mundial ainda negligenciado no Brasil e, em especial nas regiões mais pobres como o Nordeste. A síndrome do X frágil (SXF) é uma das formas mais estudadas de RM hereditário em seres humanos. Esta doença monogênica, de herança ligada ao X dominante, é decorrente de uma mutação no exon 1 do gene FMR1, localizado na região Xq27.3. A mutação no FMR1 se caracteriza pelo aumento de repetições de trinucleotídios CGG em tandem na região 5 UTR desse gene, sendo a expansão dessas trincas o principal evento mutacional responsável pela SXF. De maneira geral, os fenótipos cognitivos de indivíduos do sexo masculino com a síndrome incluem deficiência intelectual de moderada à grave. No presente trabalho, realizamos um estudo transversal da SXF em indivíduos portadores de retardo mental de causa desconhecida, engajados em Programas de Educação Especial e em instituições psiquiátricas de São Luís-MA, rastreando amplificações de sequências trinucleotídicas no gene FMR1. A amostra foi composta por 238 indivíduos do sexo masculino, não aparentados, na faixa etária de 4 a 60 anos (média = 21 9 anos). O DNA dos participantes foi obtido a partir de 5 mL de sangue coletados em tubos com anti-coagulante EDTA e a análise molecular da região gênica de interesse foi realizada através da reação em cadeia da polimerase, utilizando-se três primers. Dentre os indivíduos triados quanto à presença de mutações no gene FMR1, apenas um apresentou um resultado inconclusivo e 2 (0,84%) foram positivos para a SXF, sendo que um deles (3503) apresentou mais de 200 repetições CGG no locus FRAXA e o outro indivíduo (3660) apresentou uma deleção de ~197 pb envolvendo parte das repetições CGG e uma região proximal às repetições CGG. Ambos possuíam história familiar de RM ligado ao X. No indivíduo 3503 observamos as seguintes características clínicas: temperamento dócil, orelhas grandes, mandíbula proeminente e flacidez ligamentar. O indivíduo 3660 apresentava hiperatividade, contato pobre com os olhos, orelhas grandes, mandíbula proeminente, pectus excavatum, macroorquidismo e pouca comunicação. O esclarecimento sobre a doença oferecido às famílias de ambos contribuiu sobremaneira para o entendimento da condição, do prognóstico e dos riscos de recorrência. A prevalência da SXF em nossa amostra, 0,84%, embora relativamente baixa, encontra-se na faixa de incidência de casos diagnosticados em outras populações que, em sua maioria, relatam incidências variando de 0 a 3%. Em parte, atribuímos o percentual encontrado aos critérios de inclusão utilizados em nosso estudo. Concluímos que o protocolo de triagem molecular utilizado em nosso estudo se mostrou eficiente e adequado para a realidade do Maranhão, podendo constituir uma ferramenta auxiliar a ser aplicada na avaliação de rotina dos portadores de RM, com grandes benefícios para o Estado. / Mental retardation (MR) is considered a global public health problem in Brazil and it is still ignored mainly in poor regions like Northeast Brazil. The fragile X syndrome (FXS) is one of the most common heritable disease in humans. it is a monogenic disease with X-linked dominant inheritance due to a mutation in exon 1 of the FMR1 gene, located at Xq27.3 region. The mutation in FMR1 is characterized by the increase in number of CGG repeats in the 5 'UTR of the gene. This expansion of CGG triplets in the first exon of the FMR1 gene is the main mutational event responsible for FXS. In general, the cognitive phenotypes of males with this syndrome include intellectual disabilities from moderate to severe. In this work, we conducted a cross-sectional study of FXS in individuals with MR of unknown cause, in Especial Education Programs and Psyquiatric Instituitions in São Luís-MA, by screening for amplifications of trinucleotide sequences within the FMR1 gene. The sample consisted of 238 unrelated males, which ages were from 4 to 60 years (mean = 21 9 years). The DNA of all individuals was obtained from 5 mL of peripheral blood which was colected in EDTA-anticoagulated tubes. The molecular analysis of the genetic region of interest was performed by polimerase chain reaction using three primers. Of the individuals screened for the presence of the mutation in the FMR1 gene, only one was inconclusive and two (0.84%) were positive for FXS. One (3503) presented more than 200 CGG repeats in FRAXA locus, and the other (3660) presented with a ~ 197 bp deletion involving part of CGG repeats and a proximal region to the CGG repeats. Both of these individuals have family history of X-linked Mental Retardation. The individual 3503 has the following clinical features: docile temperament, large ears, prominent jaw and ligamentous laxity. The individual 3660 presents hyperactivity, poor contact with eyes, large ears, prominent jaw, pectus excavatum, macroorchidism and little communication. Information about the disease helped the families of both individuals with FXS to understand the condition, the prognosis and about the recurrence risk. We found a FXS prevalence of 0.84% in our sample, although relatively low, it is in the range of incidence of diagnosed cases in other populations that report mostly incidences ranging from 0 to 3%. We partially attribute the percentage found due to the inclusion criteria used in our study. We conclude that the protocol for molecular screening used in our study proved to be efficient and appropriate to the reality of Maranhão, constituting an auxiliary tool to be applied in the routine assessment of patients with MR, with great benefits for the state.

O retardo mental (RM) representa um problema de saúde pública mundial ainda negligenciado no Brasil e, em especial nas regiões mais pobres como o Nordeste. A síndrome do X frágil (SXF) é uma das formas mais estudadas de RM hereditário em seres humanos. Esta doença monogênica, de herança ligada ao X dominante, é decorrente de uma mutação no exon 1 do gene FMR1, localizado na região Xq27.3. A mutação no FMR1 se caracteriza pelo aumento de repetições de trinucleotídios CGG em tandem na região 5 UTR desse gene, sendo a expansão dessas trincas o principal evento mutacional responsável pela SXF. De maneira geral, os fenótipos cognitivos de indivíduos do sexo masculino com a síndrome incluem deficiência intelectual de moderada à grave. No presente trabalho, realizamos um estudo transversal da SXF em indivíduos portadores de retardo mental de causa desconhecida, engajados em Programas de Educação Especial e em instituições psiquiátricas de São Luís-MA, rastreando amplificações de sequências trinucleotídicas no gene FMR1. A amostra foi composta por 238 indivíduos do sexo masculino, não aparentados, na faixa etária de 4 a 60 anos (média = 21 9 anos). O DNA dos participantes foi obtido a partir de 5 mL de sangue coletados em tubos com anti-coagulante EDTA e a análise molecular da região gênica de interesse foi realizada através da reação em cadeia da polimerase, utilizando-se três primers. Dentre os indivíduos triados quanto à presença de mutações no gene FMR1, apenas um apresentou um resultado inconclusivo e 2 (0,84%) foram positivos para a SXF, sendo que um deles (3503) apresentou mais de 200 repetições CGG no locus FRAXA e o outro indivíduo (3660) apresentou uma deleção de ~197 pb envolvendo parte das repetições CGG e uma região proximal às repetições CGG. Ambos possuíam história familiar de RM ligado ao X. No indivíduo 3503 observamos as seguintes características clínicas: temperamento dócil, orelhas grandes, mandíbula proeminente e flacidez ligamentar. O indivíduo 3660 apresentava hiperatividade, contato pobre com os olhos, orelhas grandes, mandíbula proeminente, pectus excavatum, macroorquidismo e pouca comunicação. O esclarecimento sobre a doença oferecido às famílias de ambos contribuiu sobremaneira para o entendimento da condição, do prognóstico e dos riscos de recorrência. A prevalência da SXF em nossa amostra, 0,84%, embora relativamente baixa, encontra-se na faixa de incidência de casos diagnosticados em outras populações que, em sua maioria, relatam incidências variando de 0 a 3%. Em parte, atribuímos o percentual encontrado aos critérios de inclusão utilizados em nosso estudo. Concluímos que o protocolo de triagem molecular utilizado em nosso estudo se mostrou eficiente e adequado para a realidade do Maranhão, podendo constituir uma ferramenta auxiliar a ser aplicada na avaliação de rotina dos portadores de RM, com grandes benefícios para o Estado. / Mental retardation (MR) is considered a global public health problem in Brazil and it is still ignored mainly in poor regions like Northeast Brazil. The fragile X syndrome (FXS) is one of the most common heritable disease in humans. it is a monogenic disease with X-linked dominant inheritance due to a mutation in exon 1 of the FMR1 gene, located at Xq27.3 region. The mutation in FMR1 is characterized by the increase in number of CGG repeats in the 5 'UTR of the gene. This expansion of CGG triplets in the first exon of the FMR1 gene is the main mutational event responsible for FXS. In general, the cognitive phenotypes of males with this syndrome include intellectual disabilities from moderate to severe. In this work, we conducted a cross-sectional study of FXS in individuals with MR of unknown cause, in Especial Education Programs and Psyquiatric Instituitions in São Luís-MA, by screening for amplifications of trinucleotide sequences within the FMR1 gene. The sample consisted of 238 unrelated males, which ages were from 4 to 60 years (mean = 21 9 years). The DNA of all individuals was obtained from 5 mL of peripheral blood which was colected in EDTA-anticoagulated tubes. The molecular analysis of the genetic region of interest was performed by polimerase chain reaction using three primers. Of the individuals screened for the presence of the mutation in the FMR1 gene, only one was inconclusive and two (0.84%) were positive for FXS. One (3503) presented more than 200 CGG repeats in FRAXA locus, and the other (3660) presented with a ~ 197 bp deletion involving part of CGG repeats and a proximal region to the CGG repeats. Both of these individuals have family history of X-linked Mental Retardation. The individual 3503 has the following clinical features: docile temperament, large ears, prominent jaw and ligamentous laxity. The individual 3660 presents hyperactivity, poor contact with eyes, large ears, prominent jaw, pectus excavatum, macroorchidism and little communication. Information about the disease helped the families of both individuals with FXS to understand the condition, the prognosis and about the recurrence risk. We found a FXS prevalence of 0.84% in our sample, although relatively low, it is in the range of incidence of diagnosed cases in other populations that report mostly incidences ranging from 0 to 3%. We partially attribute the percentage found due to the inclusion criteria used in our study. We conclude that the protocol for molecular screening used in our study proved to be efficient and appropriate to the reality of Maranhão, constituting an auxiliary tool to be applied in the routine assessment of patients with MR, with great benefits for the state.

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No. of bitstreams: 2 Tese - Fabiana Gonçalves dos Reis - 2017.pdf: 5970849 bytes, checksum: e7504d09f87c5499323f6d7c66e10a0f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Neurological disorders are a group of conditions that manifest early in the development of the child, so that delayed neuropsychomotor development (ADNPM) and intellectual disability (ID) can impair cognitive, language, motor and social behavior. The etiology of ID is quite heterogeneous and prenatal, perinatal and postnatal factors are associated with an increased risk of this deficiency. However, 30 to 50% of the cases remain with the unknown etiology. In this context, the main objective of this study was to identify submicroscopic genomic alterations (<5Mb) by means of microarray chromosomal analysis (CMA) in patients with clinical indication of ADNPM and/or ID, sent by attending physicians of the state public health network of Goiás. We analyzed 149 cases of both sexes. Of the total number of patients, 47 had the diagnosis clarified using cytogenetics by G banding. Of 102 patients with an incomplete diagnosis, 72 agreed to participate in the present study and, therefore, performed the CMA. The elucidation of the diagnosis by CMA was possible in 22 patients. Among the results obtained, three rare cases were selected to compose this thesis. The first case is from a patient in whom a de novo microduplication of 0.45 Mb in the 5q35.2q35.3 region containing the NSD1 gene was identified. The effect of the dosing of this gene has been related to Sotos Syndrome and its inverted phenotype. The second case shows the molecular detection of an absent allele on the X chromosome and the presence of 28 CGG repeats in FMR1 gene in the present allele. The CMA showed that the patient had a de novo microdeletion of 4.176 Mb in the Xq27.3-q28 region that affected 34 genes, including five genes (TMEM185A, TMEM257, FMR1, IDS, and FMR2) that were directly correlated with ID phenotypes and neurological disorders. The third case is a de novo microdeletion of 1.59 Mb in the 1p32.3 region involving the DHCR24 gene, which causes a gene dosage effect influencing the activation of enzymes that cause desmosterolosis, which is a desmosterol conversion disorder in cholesterol. Thus, the results of this thesis showed the relevance of the use of the CMA technology to diagnose patients with clinical signs of ADNPM and/or ID that presented karyotype without alterations, evidencing the importance of this technology for public health. / Os distúrbios neurológicos constituem um grupo de condições que se manifestam precocemente durante o desenvolvimento da criança, de forma que o atraso do desenvolvimento neuropsicomotor (ADNPM) e a deficiência intelectual (DI) podem acarretar prejuízo às funções cognitivas, de linguagem, motricidade e comportamento social. A etiologia da DI é bastante heterogênea e fatores pré-natais, perinatais e pós-natais estão associados ao aumento do risco dessa deficiência. No entanto, 30 a 50% dos casos permanecem com a etiologia desconhecida. Neste contexto, o objetivo principal deste estudo foi identificar alterações genômicas submicroscópicas (<5Mb) por meio da análise cromossômica por microarranjos (CMA) em pacientes com indicação clínica de ADNPM e/ou DI, encaminhados por médicos assistentes da rede pública de saúde do Estado de Goiás. Foram analisados 149 casos, de ambos os sexos. Do total de pacientes, 47 tiveram o diagnóstico esclarecido utilizando-se a citogenética por bandeamento G. Dos 102 com diagnóstico não concluído, 72 concordaram em participar da presente pesquisa e, portanto, realizaram o CMA. A elucidação do diagnóstico pelo CMA foi possível em 22 pacientes. Dentre os resultados obtidos, foram selecionados três casos raros para compor esta tese. O primeiro caso é de um paciente em que foi identificada uma microduplicação de novo de 0,45 Mb na região 5q35.2q35.3, contendo o gene NSD1. O efeito da dosagem deste gene tem sido relacionado à síndrome de Sotos e ao seu fenótipo invertido. O segundo caso apresenta a detecção molecular de um alelo ausente no cromossomo X e a presença de 28 repetições CGG no gene FMR1 no alelo presente. O CMA mostrou que a paciente tem uma microdeleção de novo de 4,176 Mb na região Xq27.3-q28 que afetou 34 genes, dentre estes, cinco genes (TMEM185A, TMEM257, FMR1, IDS, and FMR2) que foram correlacionados diretamente com os fenótipos de DI e distúrbios neurológicos. O terceiro caso é de uma microdeleção de novo de 1,59 Mb na região 1p32.3 envolvendo o gene DHCR24, que acarreta um efeito de dosagem gênica influenciando na ativação de enzimas que causam a desmosterolose, que é um transtorno de conversão do desmosterol em colesterol. Assim, os resultados desta tese mostraram a relevância da utilização da tecnologia do CMA para diagnosticar pacientes com indicação clínica de ADNPM e/ou DI que apresentaram cariótipo sem alterações, evidenciando a importância desta tecnologia para a saúde pública.