Dissertations / Theses on the topic 'X linked mental retardation'

In order to determine the proportion of MR due to fragile X, CGG repeat analysis was carried out on DNA from 723 mentally retarded patients in S.E. Scotland. This resulted in the identification of 8 new cases of fragile X syndrome (˜1% of the total number of referrals for developmental delay). This developmentally delayed cohort was also compared to normal and high IQ cohorts to address the possibility of a relationship between CGG repeat number and IQ variation in the general population. Two complicated prenatal diagnostic cases were studied in detail to answer important practical questions concerning the nature and timing of repeat expansion. Well over 100 other XLMR conditions have been described, of which approximately 50% have been regionally mapped. Some of these are non-specific forms of XLMR in which MR is the only manifestation; others are XLMR syndromes with a characteristic and well defined phenotype. A family with non-specific XLMR is described in which the proband presented as a non dysmorphic male with unexplained MR, for exclusion of fragile X syndrome. He had a CGG allele in the normal range and the MR segregating in the family was shown not to be linked to FMRI. Subsequent analysis of polymorphic X-linked markers showed linkage to Xq21. A new XLMR syndrome is represented by a large family with severe MR and spastic diplegia which was previously thought to be MASA syndrome, a condition caused by mutations in the LICAM gene in distal Xq28. Negative LOD scores with Xq28 markers led to a search for linkage to other regions; this was found in Xp22 where several other XLMR loci are located, although the clinical features of this family differ from those of other XLMR syndromes and conditions in Xp22.

The Indonesian archipelago comprises more than 17,000 islands, inhabited by ~200 million people constituting more than 350 recognizable ethnic and tribal groups which can be classified into two broad ethno-linguistic groups [the Austronesian (AN) and non-Austronesian (NAN) speaking peoples] and 3 physical anthropology groups (Deutero Malay, Proto Malay and Papuan). The origins of these groups are of considerable anthropological interest. The anthropology of Indonesia is extremely complex and still controversial. The present populations of Indonesia show very great diversity. The data presented below result from an investigation of the Fragile X A syndrome and the size and distribution of alleles at fragile sites on the X chromosome among Javanese males with developmental disability (DD) and unselected males from 10 major Indonesian ethnic groups. The Fragile X syndrome is caused by expansion of a CGG trinucleotide repeat array in the 5' untranslated region of the FMR-1 gene at Xq27.3. Normal X chromosomes have between 6-54 CGG trinucleotide repeats, whereas premutation alleles have 55-230 and full mutation alleles more than 230 repeats. In a study of predominantly Caucasian males with intellectual disability, the prevalence of Fragile X syndrome is estimated to be approximately 1:4,000. FRAXE mental retardation syndrome is caused by an expansion of a GCC trinucleotide repeat in the 5'UTR of FMR2 gene located 600 kb telomeric to FMR1. The prevalence of FMR2 is 1-2 per 100,000 live births. FMR2 common alleles consist of 11-30 GGC repeats; intermediate alleles between 31-60 GCC repeats; premutation alleles with 61-200 repeats and full mutation alleles have over 200 repeats with attendant methylation of the repeat array The first Indonesian screening program aimed at determining the presence and prevalence of fragile XA syndrome among individuals with mild DD (IQ above 50) from special schools (N=205) and isolated areas (N=50) of Java was undertaken in 1994-1996 by cytogenetic and molecular studies. In this first study 4 fragile X positive children were found among 255 males with DD. The estimated prevalence of fragile-X in males with mild DD from special schools was 1.95% (5/205) and the overall prevalence was 1.57% (4/255). The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes were determined by PCR in 254 Fragile XA-negative Javanese male children with DD. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with DD compared to that in equivalent Caucasian populations. The trimodal distribution of Indonesian FMR1 alleles (29, 30 and 36 repeats) is largely in agreement with findings from other Asian populations). This provides supportive evidence that the origin of Indonesians could be the same as that of the Chinese and Japanese. Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals' FMR1 alleles in the 'grey zone' (35-52 repeats). The identification of 16 unrelated individuals with a (CGG)36 allele that also contains a (CGG)6 segment [(CGG)9AGG(CGG)9AGG(CGG)6 AGG(CGG)9 or 9A9A6A9 pattern] is in agreement with earlier observations in the Japanese population. It is proposed that this FMR1 array pattern may be specific for Asian populations and that Javanese and Japanese populations may have arisen from a single progenitor population. The presence of pure 25, 33 and 34 CGGs in FMR1 alleles with 36, 44 and 45 repeats respectively, suggests that these may represent alleles at high risk for instability and may therefore be at early stages of expansion to a premutation. The lack of the characteristic (CGG)6 in all three alleles with ?? 25 pure CGG arrays suggests that the most common Asian 36 repeat allele is not predisposed to slippage expansion. Seven of the 8 alleles with 36 CGG repeats could be sequenced. Seven of 36 CGG repeats FMR1 alleles from the Hiri population has been sequenced and 4 alleles indicated 9A9A6A9 pattern, 1 sample with 10A25 pattern Two of the remaining alleles showed 12A6A6A9 structure, which consisted of a tandem duplication of the (CGG)6 segment. The presence of a tandem duplication of (CGG)6 segments has never been reported in any other population. The other major findings of this study are that FRAXE syndrome is a rare cause of developmental disability in this predominantly-Javanese population. The most common FMR2 (GCC)20 allele in this selected Asian population is significantly longer than that previously reported for Caucasian populations. There was a weak correlation between the overall length of the FMR1 and FMR2 repeat arrays within the normal range (Spearman's Rank Correlation = 0.130, p-value=0.042) in the Indonesian population, which have been no previous associations reported for alleles within the normal range. One approach to studying the origins of the human populations is to study the genetic structure of polymorphic alleles such as those at the FMR1 locus and its linked microsatellite markers DXS548 and FRAXAC1. Length polymorphisms of the FMR1 gene (CGG)n repeat array, DXS548 and FRAXAC1 were studied in a total of 1,008 unselected males from 10 different Indonesian ethnic groups. FMR1 alleles were identified ranging from 8 to 57 CGG repeats. The most common CGG repeat allele was 29 (45.6%) followed by 30 (27.4%) and 36 repeats (8.0%). One hundred and forty four grey zone (3-52 CGG) alleles were found in the study population. Four people of the same ethnic group from an isolated island in Eastern Indonesia (Hiri, Ternate), a representative of the NAN ethnolinguistic group, had CGG repeat lengths of 55-57. The prevalence of these alleles is estimated to be 3.3% (4/120) in the population of Hiri or 0.4% (4/1008) of whole Indonesian population. Thirteen different alleles were found at the DXS548 locus, of which allele numbers 7 [194 bp] (44.1%), 6.5 [195bp] (43.5%) and 6 [196bp] (7.5%) are the most common. Seven rare alleles, some of which have not been previously found in Asian peoples were also identified (190, 191,192, 193, 197,198, 199, 202, 204 and 206) and accounted for 3.9% of the total. The odd number alleles were dominantly found in this study whereas almost none found in Caucasian. The finding of many "odd numbered" alleles DXS548 has never been found in other Asian population and has only been documented extremely rarely in Caucasians and Africans. Five different alleles of FRAXAC1 identified with alleles D [106 bp] (62.2%) and C [108bp] (35.6%) accounting for 97.8% of FRAXAC1 alleles in the population. Three rare alleles (104, 110, 112 bp = 2.2%) were identified that have not been previously found in other Asian populations (1-3). There is a striking linkage disequilibrium of FMR1 alleles with FRAXAC1 (p=0.0001), 88% of 29 (CGG)n repeats alleles associated with FRAXAC1 allele D (106bp) versus only 17% with the 30 (CGG)n repeat alleles, which is in agreement with other studies. The value of D' was calculated to be 0.7. The longer alleles of both DXS548 and FRAXAC1 were found mostly in the NAN ethnolinguistic group. Moreover the Irian Jaya people also showed a higher percentage of people with 30 CGG repeats and the 108 bp FRAXAC1. The Eastern Indonesian NAN groups demonstrate a different genetic background probably due to the contribution of Melanesian peoples. The Analysis of Molecular Variance (AMOVA) identified that the vast majority of genetic diversity occurs within, rather than between, ethnic groups. These data are consistent with a model where there is sufficient migration (~20 per generation) between populations to minimise differentiation of population through genetic drift. The results obtained are consistent with three clusters of populations that share similar allele frequencies at the fragile X locus. The most clearly defined cluster is based in the east of Indonesia and includes the two Irian populations, Minahasans and Hiri. A surprising finding was that the Minahasan who are Deutero-Malay in origin and physical appearance are genetically closer to the Irianese. This may reflect the admixture of Melanesian alleles or other eastern Indonesian alleles as a result of their geographic location in that part of Indonesia. The second major cluster is largely based in the west of the country and is composed of the following Deutero-Malay populations; Javanese, Balinese, Acehnese but which also includes people from Ternate (not including those from Hiri). Using Delta Mu and Nei's genetic distance for FMR1 locus in this study the Javanese were shown to have the closest distance to Balinese which is consistent with anthropological data and with published data. The third group is a "western and central" group composed of Bimanese, Dayak and Sundanese who share some features of the western and eastern clusters but mostly resemble the western Indonesian populations. Bima is located in the lesser Sunda in between west Indonesia and east Indonesia. The Bimanese are of mixed Deutero & Proto Malay origin that is consistent with their geographic location. The Bataks are distinctive and sit somewhat apart in this scheme. In this study, Bataks were found not to resemble the other Proto-Malay group studied (the Dayak). The Dayaks were found to have fewer alleles than the Bataks at FRAXAC1 and DXS548. In all four methods of calculating genetic distance Bataks showed a large genetic distance to almost all other ethnic groups. There are differences in allele frequency between east and west Indonesia as well as other Asian nations, but the genetic similarities between these groups are also very impressive. The findings from this study are consistent with other genetic anthropological evidence that the people of Indonesia have the same origin as North-east Asian groups. This model is referred to as the "express train from Taiwan" in which the Austronesian speakers are proposed to have radiated from Taiwan bringing the Malayo-Polynesian language group to the Philippines, Borneo and Sulawesi around 5000-4500 B.P.E. However Richards et al.(1998) have used the diversity in the mtDNA D Loop to propose an alternative to the "express train" model. The "two train7quot; model proposes that the Austronesian languages originated within eastern Indonesia during the Pleistocene era and spread through Melanesia and into the remote Pacific within the past 6,000 years. Unfortunately the high migration rates between population groups that were demonstrated in this thesis and the known migration patterns of populations through Indonesia preclude determining whether the observed allelic heterogeneity is a function of the original population or due to the admixture of several gene pools in more recent times.

Mental retardation (MR) affects 23% of the population; those due to X linked mutations commonly result in moderate to severe MR. The OPHN1 gene (Ophn1 in mice) has been implicated in X linked mental retardation (XLMR) and encodes the RhoGAP protein, oligophrenin 1. Loss of function mutations alter Rho GTPase dependent signalling pathways and result in altered actin cytoskeleton dynamics which are important in dendritic spine structure, the site of neurotransmission. Here, using electrophysiological recordings combined with intracellular staining techniques and dendritic morphological analysis, I characterise synaptic (dys)function in neocortical and hippocampal neurons from the Ophn1 mouse model of MR. This study demonstrates an excitatory synaptic deficit in neocortical neurons combined with region specific changes in dendritic spine morphology. Inhibitory transmission was normal in both neocortical and hippocampal neurons. Kainate induced gamma oscillations were unaltered whereas spontaneous oscillations were reduced which lead to changes in synaptic function in CA3. Morphometric analysis showed ventriculomegaly in Ophn1 deficient mice that was associated with reduced cortical thickness. This study shows the loss of several previously reported phenotypes, including, altered inhibitory transmission, gamma oscillations and vesicle dynamics. Their loss, but preservation of morphological deficits, suggests that the model may be susceptible to genetic drift.

Twenty-one genes are known that when mutated cause non-syndromic X-linked mental retardation (NS-XLMR), a condition in which learning disability is the only consistent clinical feature among affected individuals. The principal aim of this project was to identify further NS-XLMR genes. To achieve this aim we characterized X chromosomal breakpoints in rare patients with apparently balanced X;autosome translocations and learning disability to identify disrupted candidate NS-XLMR genes. In patient LPW, a female with a translocation involving chromosomes X and 8, we identified a small 650 kb duplication at the X chromosome breakpoint that was likely to be responsible for the patient’s learning disability. In patient AGO819, the CLCN5 gene was found to be disrupted by the X chromosomal breakpoint. As CLCN5 is a gene mutated in a familial renal disease not associated with learning disability then expression analysis of genes adjacent to CLCN5 was performed. A position effect disrupting the expression of a neighbouring gene, CCNB3, was detected indicating that the expression of one or more genes surrounding the X chromosome breakpoint was disrupted. One of these genes, KIAA1202, has recently been reported to be mutated in familial learning disability. In a third patient with a balanced translocation, KD, no gene was found disrupted by the X chromosome breakpoint and further analysis indicated that this patient’s learning disability was likely to be related to abnormalities at the autosomal breakpoint. The project was concluded by characterizing two mutations identified in DLG3. We proved that the genomic mutations disrupted splicing of DLG3 at the mRNA level, confirming that the mutations were likely to be disease-causing in the two families.

Mental retardation (MR) is a handicap with severe implications not only for thosethat suffer from this disability, but also for their families, society and the welfaresystems which support them. A large proportion of these individuals are afflictedwith the X-linked form of the condition. To date a total of 87 genes have beenimplicated in the pathogenesis of X-linked mental retardation (XLMR).

The primary objective of the studies presented here was to identify candidate genes for nonsyndromic X-linked mental retardation (NS-XLMR) in Xp11.2 by molecular characterisation of the Xp11.2 breakpoints in five rare female individuals with balanced X;autosome translocations and mental retardation. The hypothesis underlying this positional cloning strategy was that genes disrupted by the X chromosome breakpoints are candidate genes for NS-XLMR. Five Xp11.2 and three autosomal translocation breakpoints were mapped by fluorescence in situ hybridisation (FISH) using large insert genomic clones generated by the Human Genome Project as probes. In silico analysis of genomic sequences at the X breakpoint loci was used to identify genes potentially disrupted by the translocations. In collaboration with Dr Vera Kalscheuer’s group in Berlin, a putative diacylglycerol kinase XM_066534 was found to be disrupted by a t(X;22), and its involvement in NS-XLMR evaluated. Restriction mapping by southern blot hybridisation analysis was undertaken to fine map the X breakpoints of two translocations, and RT-PCR analysis used to investigate expression of genes at the X breakpoint loci in translocation cell lines. One t(X;16) was found to disrupt expression of the zinc finger gene ZNF81. This is the second example of a balanced X;autosome translocation associated with mental retardation which disrupts this gene, providing further evidence that mutations in ZNF81 cause mental retardation. The X breakpoint of a second t(X;16) was localised to a 9 kb interval between two partial cDNAs. Further work on these sequences led to the cloning and characterisation of two novel genes expressed in brain, WNK3, a putative protein kinase, and CXorf17 and its mouse ortholog, orf34. Characterisation of CXorf17 and orf34 facilitated the identification of a novel, chordate-specific gene family which encodes several putative transmembrane proteins that have no sequence homology to any previously annotated proteins, known functional domains or sequence motifs.

Mental retardation is the most common brain disease. One of the first genes identified in X-linked mental retardation (XLMR) was the OPHN-1 gene. Mutation of this gene has been described in patients with moderate to severe cognitive impairments. MR is characterized by reduced cognitive function with or without other clinical features, thus providing a direct approach to study the neurobiology of cognition and pathogenesis of MR. I propose in this thesis to clarify the underlying mechanisms responsible for the learning impairments. My first approach was to investigate the functioning of a neuronal population using extracellular recording of fast oscillations which are thought to underlie higher cognitive performance. I showed that \(Ophn-1\) null mice displayed weaker gamma oscillations. Thereafter, Investigation of the synaptic properties of CA3 pyramidal neurons using the patch-clamp technique has been undertaken. I have shown reduced inputs of excitatory and inhibitory neurotransmission to CA3 pyramidal neurons accompanied with reduced frequency dependent facilitation of the inhibitory neurotransmission at 33Hz. Finally, a reduction in readily releasable pool size in inhibitory synapses of CA3 area was unravelled. This defect explained the reduction of frequency of sIPSCs and consequently the reduction in gamma oscillations power in Ophn-1\(^{-/y}\) slices.

Este trabalho teve o objetivo de estimar a frequência de deficiência mental causada por mutações no cromossomo X entre pacientes do sexo masculino, que constituem casos isolados de deficiência mental. A estratégia adotada foi a determinação do padrão de inativação do cromossomo X nas mães dos afetados, com base (a) nas indicações de que desvios extremos do padrão casual de inativação do cromossomo X têm alta probabilidade de estar relacionados à presença de mutações do cromossomo X e (b) na observação de que a frequência desses desvios está significantemente aumentada em mulheres certamente portadoras de mutações que causam deficiência mental de herança ligada ao X. A vantagem seletiva das células que possuem o alelo não mutado no cromossomo X ativo é uma explicação para tais desvios extremos da inativação do cromossomo X, raramente encontrados na população geral. Selecionamos 115 meninos portadores de deficiência mental moderada a grave associada a outros sinais clínicos, não característicos de síndrome conhecida e que tinham cariótipos normais e teste negativo para a síndrome do cromossomo X frágil; suas genitoras concordaram com a participação no estudo. Esses pacientes foram encaminhados ao Serviço de Aconselhamento Genético do Laboratório de Genética Humana, Departamento de Genética e Biologia Evolutiva, por diferentes serviços médicos, para diagnóstico e orientação quanto a riscos de recorrência na família. O padrão de inativação do cromossomo X nas mães dos afetados foi investigado, com base na metilação diferencial dos alelos do gene AR (Androgen Receptor gene) , no cromossomo X ativo e inativo. As mães de 100 desses meninos se revelaram heterozigóticas quanto à repetição polimórfica CAG do gene AR, requisito do teste para determinar o padrão de inativação do cromossomo X. Onze mulheres (11%) apresentaram desvios extremos do padrão de inativação do X (≥ 98:2), frequência significativamente maior (P = 0.0001; teste exato de Fisher) do que aquela que a literatura registra, em estudo utilizando o mesmo ensaio, entre mulheres adultas da população geral (0,017; IC 95% = 0,007 0,034). A raridade de desvios tão extremos na população geral permite admitir que as mães dos afetados que apresentam tais desvios sejam portadoras de mutação no cromossomo X, que causa a deficiência mental em seus filhos. Sendo assim, estimamos em 11% a frequência de deficiência mental em nossa amostra de 100 meninos casos isolados de deficiência mental (IC 95% = 0,056 0,188), sem incluir a síndrome do X frágil, responsável por 2,5% a 3% da deficiência mental no sexo masculino. Essa nossa estimativa para a proporção de deficiência mental moderada grave ligada ao X entre indivíduos do sexo masculino com DM é da mesma ordem de grandeza daquelas relatadas na literatura, baseadas (a) na frequência da síndrome do X frágil em coortes de homens com deficiência mental e entre famílias com deficiência mental de herança ligada ao X ou (b) nas inferências da prevalência de deficiência mental e de deficiência mental causada por mutações no cromossomo X na população geral masculina. Entretanto, a frequência por nós determinada deve ser uma subestimativa, considerando que os desvios extremos do padrão de inativação ocorrem em apenas um terço das portadoras obrigatórias de mutações que causam deficiência mental com herança ligada ao X. Com base nos resultados deste estudo, consideramos indicada a avaliação do padrão de inativação do cromossomo X em mães de indivíduos do sexo masculino, casos isolados de deficiência mental. A detecção de desvio extremo da inativação deve ser considerada indicativa de deficiência mental de herança ligada ao X, constituindo subsídio para o aconselhamento genético da família e podendo levar á identificação da mutação causadora da deficiência mental.
Nearly a third of obligate carriers of mutations causative of X-linked mental retardation (XLMR) have been reported to have extreme X-inactivation skewing in peripheral blood cells, compared to their non-carrier relatives. Selective advantage of cells with the non-mutated allele on the active X chromosome would explain this skewing. Based on these findings, we used the pattern of X-inactivation in mothers of mentally retarded boys, as a parameter to evaluate the frequency of XLMR among non-familial cases. To determine the X-inactivation pattern in these women, we investigated the methylation status of the AR (Androgen Receptor) alleles in blood cells. We selected 115 boys with moderate to severe mental retardation of unknown cause, who had normal karyotypes and tested negative for fragile X syndrome; the mothers of 100 of these boys were found to be heterozygous for the polymorphic CAG repeat of the AR gene, a requisite of the X-inactivation assay. Eleven women (11%) had extremely skewed X-inactivation (≥ 98:2), a frequency significantly higher (P = 0.0001; Fisher exact test) than the frequency reported for adult women from the general population (1.7%; 95% CI = 0.007 0,034). Assuming that every mother with extremely skewed X-inactivation is a carrier of an X-chromosome mutation that causes mental retardation in her son, the frequency of XLMR in our sample of 100 boys is 11% (95% CI = 0,056 0,188), the fragile X syndrome being excluded. Although these figures are quite in agreement with previous estimations of the frequency of XLMR among mentally retarded men, they might be an underestimation, when it is taken into account that only about a third of obligate carriers of XLMR mutations have highly skewed X inactivation.

Estudamos uma família com cinco homens (dois falecidos) afetados por deficiência mental (DM) não-sindrômica em duas gerações, num padrão de herança ligada ao cromossomo X. A análise do padrão de inativação do cromossomo X, com base na metilação do gene AR, evidenciou que a mulher portadora obrigatória tinha desvio completo de inativação nos leucócitos, uma característica freqüente em portadoras de mutações do cromossomo X relacionadas com DM. Para o mapeamento da DM, genotipamos 28 locos de microssatélites ao longo do cromossomo X e delimitamos um segmento de cerca de 32 Mb, entre os marcadores DXS986 e DXS8067, compartilhado pelos afetados e pela portadora obrigatória, mas não pelo homem normal ou pelas possíveis portadoras que não tinham desvio do padrão de inativação do cromossomo X. Na busca do gene mutado, analisamos, por seqüenciamento direto, genes mapeados no intervalo compartilhado e já relacionados a DM ou que tivessem expressão em cérebro e leucócitos. Nos afetados e na portadora obrigatória, encontramos a mutação c.845C→T no gene ACSL4, que resulta na substituição do aminoácido histidina, conservado na família de sintetases de acil-CoA humanas e em diversos outros organismos, por tirosina (p.H323Y da isoforma cérebro-específica). Tratando-se de mutação que altera um aminoácido evolutivamente conservado em gene já relacionado com DM, que segregava com a DM na família, não tendo sido encontrada em amostra controle de 160 indivíduos do sexo masculino, concluímos que era a causa da DM na família. Mutações de ponto no gene ACSL4 foram relacionadas com a DM não sindrômica em três famílias descritas na literatura. O gene ACSL4 codifica a acil-coA sintetase 4 da família das sintetases de cadeia longa, que catalisa a formação de ésteres acil-coA a partir de ácidos graxos de cadeia longa. Sua expressão já foi documentada em vários tecidos, incluindo o cérebro e dados recentes mostraram que a proteína é essencial para a formação normal de espinhos dendríticos. A nova mutação do gene ACSL4 que descrevemos como causa de DM vem reforçar a relação alterações desse gene e a DM de herança ligada ao X. O padrão de inativação do X totalmente desviado foi mais uma vez observado em mulher portadora da mutação, indicando a importância da expressão desse gene em leucócitos. A presença de dificuldades de aprendizado na portadora da mutação concorda com o observado nas três famílias da literatura em que o estudo das portadoras foi relatado, indicando o efeito de mutações do gene ACSL4 sobre a função intelectual mesmo em heterozigose. A ausência de correlação entre o padrão de inativação do cromossomo X em células do sangue periférico e o comprometimento intelectual foi confirmada. Na família estudada, a identificação da mutação permitiu o aconselhamento genético.
We studied a family with five men (two of them deceased) affected by nonsyndromic mental retardation in two generations, in a pattern of X-linked inheritance (MRX). The study aimed at identifying the causative mutation. The obligate female carrier showed completely skewed inactivation of the X chromosome, based on the methylation status of the AR gene in peripheral blood in leukocytes, a common feature in carriers of X-linked mutations that cause mental retardation. We genotyped 28 microsatellite loci mapped throughout the X chromosome and delimited a 32 Mb segment, between markers DXS986 and DXS8067, that was shared by the affected males and obligate carrier, but was not present in a normal man or in two women who did not show skewed X-inactivation. We searched for the causative mutation by sequencing genes mapped to this candidate interval that had been associated with MR and/or were expressed in brain and leukocytes. In the affected men and obligate carrier, we found a c.845C→T mutation in the ACSL4 gene, resulting in the amino acid tyrosine substituting for a histidine (p.H323Y in brain isoform), which is conserved in the acyl-CoA synthetase family in humans and others organisms. This mutation was not found in a control sample of 160 men. Previously, point mutations in the ACSL4 gene had been identified as the cause of MRX in three families. ACSL4 encodes the acyl-CoA synthetase long-chain family member 4, which catalyzes the formation of acyl-CoA esters from long-chain fatty acids. It is expressed in several tissues, and in brain it is essential for the normal formation of dendritic spines. The novel mutation here described confirmed the causal association of ACSL4 mutations with non-syndromic mental retardation. The completely skewed Xinactivation, also observed in the previously described carriers, supported a functional role for this gene in peripheral blood leukocytes. The intellectual impairment present in the carrier in the family here reported is in accordance with previous findings pointing to the effect on intellectual abilities of ACSL4 mutations in heterozygosis. The absence of correlation between the pattern of X-inactivation in leukocytes and mental status was confirmed.

Includes abstract.
Includes bibliographical references (leaves 127-134).
Alpha thalassaemia X-linked mental retardation (ATR-X) syndrome is a rare, X-linked intellectual disability syndrome with an estimated prevalence in the range of 1-9/1 000 000. The prevalence in South Africa (SA) is unknown; however in Cape Town there is one extended family with seven males who were clinically, and later molecularly, diagnosed with this condition. Due to the identification of the mutation in this family, carrier and prenatal testing is available. However, since the announcement in 2007 that testing is available, no individuals have presented themselves for their carrier status to be determined. The aim of this study was to investigate the reasons why females in this family have not presented for carrier testing.

Neste trabalho utilizamos diferentes abordagens para o estudo de genes associados com desenvolvimento e funcionamento do SNC assim como com doenças neurológicas: 1) uma das abordagens consistiu na identificação do alelo associado a uma forma de retardo mental sindrômico com herança recessiva ligada ao cromossomo X, síndrome de Snyder-Robinson, em uma família Brasileira. Utilizando as estratégias de estudo de ligação genética e análise de genes candidatos, identificamos a segunda mutação patogênica no gene SMS (que codifica a enzima espermina sintase) associada à síndrome de Snyder-Robinson. A identificação dessa mutação contribuiu para: delinear e expandir o espectro clínico da síndrome, evidenciar domínios importantes para o funcionamento da proteína espermina sintase, comprovar a importância dessa proteína nos processos cognitivos, e também possibilitar um aconselhamento genético preciso para membros da família; 2) outra abordagem consistiu em analisar (triar mutação) o gene codificador da proteína colibistina (ARHGEF9), a qual está envolvida em sinaptogênese inibitória, em pacientes Brasileiros portadores de hiperecplexia (6 pacientes) e em pacientes portadores de retardo mental associado com epilepsia (22 pacientes). Não identificamos nenhuma alteração patogênica no gene ARHGEF nos 28 pacientes estudados; contudo, o número de pacientes analisados foi muito pequeno. Julgamos que a análise de um número maior de pacientes com essas doenças neurológicas pode vir a revelar novas mutações deletérias em ARHGEF9; 3) a última abordagem consistiu no estudo funcional da proteína colibistina. Com o objetivo de identificar outras proteínas que interagem com a colibistina humana utilizamos o sistema de duplo-híbrido em leveduras e experimentos de co-imunoprecipitação in vitro e in vivo. Identificamos a proteína eIF3-p40 interagindo com a proteína colibistina e também com a proteína gefirina (a qual, por sua vez, também interage com colibistina e está envolvida com funcionamento de sinapses inibitórias). A proteína eIF3-p40 é uma das subunidades do complexo do fator 3 de iniciação de tradução protéica em eucariotos (eIF3). Essas interações ligam as proteínas colibistina e a gefirina à maquinaria de tradução protéica, revelando uma provável nova função dessas proteínas no controle da tradução em sítios pós-sinápticos inibitórios.
In this work we have used different approaches to the study of genes associated with CNS development and function as well as with neurological diseases: 1) one study involved the identification of the allele associated with an X-linked recessive sindromic form of mental retardation, Snyder-Robinson syndrome, in a Brazilian family. Using genetic linkage analysis and candidate gene strategy, we identified the second pathogenic mutation in the SMS gene (that encodes the spermine synthase enzyme) associated with the Snyder-Robinson syndrome. The identification of this mutation contributed to: the delineation and expansion of the clinical spectrum of the syndrome, highlight important domains for spermine synthase protein functioning, demonstrate the importance of this protein in cognitive processes, and also a precise genetic counseling for family members; 2) a second study involved the mutation screening of ARHGEF9, gene encoding the collybistin protein, which is involved in inhibitory synaptogenesis, in Brazilian patients with hyperekplexia (6 patients) and in patients with mental retardation associated with epilepsy (22 patients). We did not identify any pathogenic alteration in the ARHGEF9, gene in the 28 studied patients, but the number of patients analysed was very small. However, the possibility remains that additional mutations in ARHGEF9, may contribute to other cases of hyperekplexia and mental retardation associated with epilepsy; 3) the last study involved the functional analysis of collybistin protein. In order to identify other proteins that interact with human collybistin, we used the yeast two-hybrid system and in vitro e in vivo co-immunoprecipitation experiments. We identified the eIF3-p40 protein as collybistin and gephyrin (another protein involved in the function of inhibitory synapses that also interacts with collybistin) binding partner. The eIF3-p40 protein is one of the subunits of the eukaryotic initiation factor 3 complex (eIF3). These interactions link the collybistin and gephyrin proteins to the protein translation machinery, revealing a putative new role of these proteins in the translation control at inhibitory postsynaptic sites.

Em trabalho anterior, identificamos a mutação c.382C8594;T no gene UBE2A, localizado em Xq24 e codificador de enzima conjugadora de ubiquitina, como causa de nova síndrome de deficiência mental (DM) de herança ligada ao cromossomo X. Foi a primeira descrição de mutação nesse gene e a primeira associação de mutação em gene que codifica conjugase de ubiquitina com patologia humana. Neste trabalho, focalizamos o gene UBE2A quanto a expressão dos transcritos alternativos, função das isoformas por eles codificadas e o efeito da mutação c.382C 8594; T como causa de deficiência mental (DM). No Capítulo I, revisamos os aspectos genéticos da DM, dando ênfase à herança ligada ao cromossomo X, principal causa de DM herdada e resumimos o estudo que levou à identificação da mutação em UBE2A como causa de quadro sindrômico de DM. Revisamos o papel da via de ubiquitinação de proteínas e das enzimas que participam do processo, em especial as conjugases de ubiquitina. Levantamos evidências na literatura que não deixam dúvida sobre a importância da via de ubiquitinação no sistema nervoso, tanto em processos de neurodesenvolvimento como neurodegeneração. No Capítulo II, avaliamos a contribuição de mutações em UBE2A como causa de DM. Apresentamos os resultados do sequenciamento direto da região codificadora do gene UBE2A em afetados de 23 famílias em que a DM segrega ligada à segmento que inclui Xq24, onde está localizado o gene UBE2A. Uma dessas famílias foi averiguada no Serviço de Aconselhamento Genético do Laboratório de Genética Humana do Departamento de Genética e Biologia Evolutiva, Instituto de Biociência, USP (LGH-IB/USP), coordenado pelo Dr. Paulo A. Otto e pela Dra. Angela Vianna Morgante. As demais 22 famílias pertencem ao banco de amostras do Consórcio Europeu de Deficiência Mental (European Mental Retardation Consortium EURO-MRX). A triagem foi também realizada em um indivíduo afetado por DM sindrômica que compartilha características clínicas com nossos pacientes. Como acontece com a maioria dos genes do cromossomo X, o gene UBE2A não parece ser responsável por parcela significativa dos casos de DM, já que novas mutações em UBE2A não foram detectadas nessa triagem. Avaliamos o efeito da mutação c.382C 8594; T nos níveis da transcrição e da tradução em homem afetado e em mulher portadora. A presença da mutação que leva a um códon de parada prematura não resultou na degradação do RNA, que detectamos nas células do afetado. Já na mulher portadora, apenas o transcrito normal foi detectado, de acordo com nossos dados anteriores que mostraram desvio completo no padrão de inativação do cromossomo X nas portadoras da mutação, tendo um mesmo cromossomo X ativo nas células do sangue. A vantagem proliferativa das células em que o cromossomo X com alelo mutado estava inativo deve ter levado a esse padrão de inativação desviado do casual, evidenciando o efeito deletério da mutação. Entretanto, o mecanismo pelo qual a mutação afeta a via de UBE2A permanece interrogado. A proteína UBE2A alterada foi encontrada em baixa quantidade nas células do paciente, o que pode ser o resultado de síntese prejudicada ou de degradação pós-traducão. Independente do mecanismo responsável, o fato de apenas uma pequena quantidade da proteína mutada ter sido encontrada, nos permite afirmar que, nas células desses indivíduos, há perda de função de UBE2A. Devemos, contudo, considerar que a proteína mutada é sintetizada e que, no caso de a menor quantidade dever-se à degradação pós-tradução, esse processo pode prejudicar a homeostase celular e contribuir para o quadro clínico. O capítulo II focaliza os transcritos alternativos de UBE2A. Diversos bancos de dados apontam para a existência de três transcritos alternativos do gene UBE2A humano, mas não há trabalho científico que caracterize os tecidos em que os transcritos são expressos ou a função das proteínas por eles codificadas. A mutação c.382C 8594; T localiza-se no éxon 6 do gene, comum a todos os transcritos, de forma que, no caso de eles codificarem proteínas funcionais, a mutação comprometeria três proteínas, e não apenas uma. Demonstramos que os três transcritos de UBE2A são xpressos em leucócitos, pré-adipócitos, placenta, córtex cerebral e hipocampo humanos. Detectamos também os três transcritos nas células de sangue e pré-adipócitos de um de nossos pacientes portador da mutação c.382C 8594; T. Embora os bancos de dados apontem para a existência de apenas um transcrito de UBE2A em camundongos, identificamos um transcrito alternativo correspondente ao transcrito alternativo 3 humano. Este foi detectado inclusive em camundongos nocaute quanto ao gene UBE2A o processo de geração do animal nocaute foi realizado por recombinação homóloga em que o cassete de neomicina foi inserido no éxon 1 do gene, de maneira que não eliminou a existência do transcrito correspondente ao transcrito 3 humano, que utiliza uma 5 UTR alternativa localizada no íntron 3. Entretanto, as proteínas codificadas pelos transcritos alternativos não foram detectadas nos extratos protéicos analisados humanos e de camundongo. Esse resultado poderia ser explicado pela falta de especificidade do anticorpo utilizado ou por essas isoformas representarem pequena parcela do pool de proteínas da célula. Os anticorpos comerciais anti-RAD6 e anti-HR6A/HR6B foram produzidos após imunização de coelhos com a porção N-terminal da isoforma 1. Seria, portanto, possível que não fossem capazes de detectar as isoformas 2 e 3, em que o segmento utilizado para a produção dos anticorpos está total ou parcialmente ausente. No caso de as proteínas estarem pouco representadas na célula, experimentos de co-imunoprecipitação auxiliariam na identificação dessas isoformas nos extratos protéicos. Entretanto, para a detecção de todas as isoformas de UBE2A seria necessário anticorpo que reconhecesse a porção C-terminal de UBE2A. Em 2009, duas novas mutações em UBE2A foram descritas em estudo colaborativo realizado no Welcome Trust Sanger Institute, Hinxton, Cambridge, Reino Unido, após sequenciamento em larga escala de aproximadamente 700 genes do cromossomo X de cerca de 200 indivíduos com DM de herança ligada ao X. Ambas as mutações c.215C 8594; T e c.328C 8594; G eram do tipo missense. Não foram fornecidas informações quanto ao quadro clínico dos portadores dessas mutações e também não foi esclarecido porque apenas a alteração c.215C 8594; T que resulta na troca do resíduo de fenilalanina da posição 72 por um resíduo de serina (F72S) foi considerada pelos autores como possivelmente patogênica. Nossos estudos in vitro, apresentados no Capítulo III, sugerem que ambas as alterações afetam a função de UBE2A. Em 2010, foram publicados dois trabalhos associando novas alterações em UBE2A a quadro de DM. Honda e col. (2010) descreveram uma microdeleção em Xq24 que inclui UBE2A e outros oito genes, em um menino com DM e características também presentes em nossos pacientes. Budny e col. (2010) descreveram mutações missense em UBE2A em duas famílias em que segregava quadro de DM sindrômica semelhante ao de nossos pacientes. Por comunicação pessoal de Arjan de Brouwer (Departamento de Genética Humana da Universidade Radboud, Nijmegen, Holanda), soubemos da existência de três outras microdeleções de segmentos do cromossomo X que incluem UBE2A, em pacientes do sexo masculino, não aparentados. Comparamos as características clínicas de nossos pacientes com as dos portadores das microdeleções em Xq24 e com aquelas dos portadores de mutações missense em UBE2A. A DM grave e o comprometimento significativo ou ausência de fala são comuns a todos. Outras características como baixa estatura, sinófris, boca grande e lábios finos com comissuras voltadas para baixo, pescoço curto e largo, implantação baixa de cabelos na nuca, mamilos espaçados, pênis pequeno, hirsutismo generalizado e a ocorrência de convulsões parecem predominar. Entretanto, enquanto a microcefalia aparece em dois dos três portadores de microdeleções avaliados, a macrocefalia parece predominar no grupo em que ocorrem as mutações de ponto. No Capítulo III, abordamos estudos funcionais in vivo e in vitro para avaliar se as isoformas alternativas de UBE2A compartilham suas funções de conjugase de ubiquitina e compreender o efeito da mutação c.382C8594;T na função de UBE2A. Buscamos estabelecer modelo celular para avaliar o efeito da mutação na formação de neuritos. Trabalho previamente publicado havia demonstrado que a diferenciação neuronal de células PC12 concomitantemente com a inibição parcial do mRNA de UBE2B (parálogo de UBE2A) resultava na redução de 20-30% do comprimento de neuritos. Entretanto, nossos ensaios de diferenciação de pré-adipócitos não responderam nossas questões sobre o efeito da mutação na formação de neuritos, pois não conseguimos obter, nas células do controle ou nas do paciente, a densidade de neuritos descrita anteriormente na diferenciação de pré-adipócitos. Diferentemente das células PC12, de origem ectodérmica, os pré-adipócitos tem origem mesodérmica, o que dificulta sua diferenciação em linhagem derivada de outro folheto germinativo. A elevada conservação entre as proteínas ortólogas UBE2A e UBE2B humanas e RAD6 de levedura e a observação de que ambas as parálogas humanas são capazes de complementar os fenótipos apresentados pela linhagem 916;rad6 de Saccharomyces cerevisiae nos levou a considerar a linhagem de levedura 916;rad6 como modelo para nossos estudos funcionais. Também, avaliamos a capacidade das isoformas 2 e 3 e da isoforma Q128X de UBE2A para ubiquitinar histonas H2A in vitro, conforme previamente descrito para a isoforma UBE2A/1. Os resultados dos ensaios in vivo indicam que apenas a expressão do transcito 1 de UBE2A é capaz de complementar os fenótipos apresentados pela linhagem 916;rad6 de S. cerevisiae. A expressão dos transcritos 2 ou 3 não resulta na restituição do fenótipo de sensibilidade à UV - a expressão gera certa toxicidade, agravada quando as células são cultivadas a 37o C. Entretanto, as isoformas por eles codificadas não parecem ser estáveis na levedura: assim como nos tecidos humanos testados, não conseguimos detectá-las nos extratos protéicos das leveduras que expressavam esses transcritos. A expressão do transcrito 1 contendo a mutação c.382CT revelou que a isoforma UBE2A/Q128X, por sua vez, é estável na linhagem 916;rad6, porém, além de não restituir o fenótipo de sensibilidade à UV, foi, dentre as isoformas de UBE2A, a mais tóxica. Os fenótipos de toxicidade não foram observados após expressão em linhagem selvagem de S. cerevisiae. Esses resultados indicam que as isoformas 2 e 3 de UBE2A não apresentam atividade de conjugase de ubiquitina e que são, aparentemente, degradadas imediatamente após sua expressão em levedura. O fato de o fenótipo de toxicidade ser agravado, em condições de choque térmico, apóia a hipótese de degradação dessas isoformas, em levedura. A degradação pode ser resultado da ausência de parceiro que permita sua estabilidade, mas a ausência das isoformas também em extratos protéicos de tecidos humanos sugere que o mesmo processo de degradação ocorra em mamíferos. Segundo a classificação das E2, as diversas conjugases de ubiquitina têm em comum o domínio UBC altamente conservado e as variações observadas consistem em inserções ou extensões C-terminais, mas nunca deleções, como ocorre nas isoformas 2 e 3 de UBE2A. Os transcritos alternativos teriam, assim, função regulatória. Os ensaios in vitro confirmaram a capacidade de UBE2A/1 ubiquitinar histonas H2A. Os ensaios com UBE2A/2 e UBE2A/3 não foram conclusivos, uma vez que a incapacidade de ubiquitinação de histonas que observamos pode ter consequência da renaturação in vitro, que pode ter ocorrido prejudicando sua função. Entretanto a obtenção das isoformas puras nos permitiu verificar que, caso a isoforma 2 estivesse presente nos extratos de levedura, ela seria reconhecida pelo anticorpo anti-RAD6. Verificamos que a proteína mutada UBE2A/Q128X é capaz de interagir com a E1, da qual recebe a molécula de ubiquitina, mas não é capaz de transferi-la para a histona. O segmento C-terminal ausente nessa isoforma é, portanto, importante nesse processo. Os ensaios in vitro despertaram nossa atenção para o fenômeno de autoubiquitinação de UBE2A, possível mecanismo de autorregulação previamente considerado na literatura. O fato de alguns trabalhos sugerirem que as E2 atuem também como dímeros in vivo e in vitro e a elevada conservação entre as parálogas humanas UBE2A e UBE2B nos levaram a considerar a possibilidade de mecanismo de regulação recíproca. Dessa maneira, a degradação de UBE2A/Q128X nas células do paciente poderia ser dependente de UBE2B. A reduzida capacidade de autoubiquitinação da isoforma mutada dificultaria sua degradação e tornaria necessária a atividade da paráloga. Isso explicaria porque ela é estável quando expressa na linhagem de levedura 916;rad6, mas não nas células do paciente. A presença de RAD6 estaria diretamente relacionada à ausência de toxicidade após a expressão de UBE2A/Q128X em linhagem selvagem a degradação da isoforma mutada está ocorrendo nessas células. A não viabilidade de camundongo duplo-nocaute quanto as parálogas UBE2A e UBE2B não permite testar a estabilidade da isoforma mutada em células de mamíferos. Observamos, de fato, que a inibição do proteassoma nas células do paciente leva ao acúmulo dessa proteína. A presença da mutação c.382C8594;T nas células do paciente parece resultar no fenótipo de DM devido à perda de função de UBE2A: a isoforma mutada não restitui o fenótipo de sensibilidade à UV de S. cerevisiae e não foi capaz de ubiquitinar histonas H2A in vitro. Além disso, indivíduos com microdeleções de UBE2A apresentam fenótipo semelhante ao de nossos pacientes. Por outro lado, a presença da proteína mutada que necessitaria de UBE2B para ser degradada pode caracterizar um ganho tóxico de função - comprometeria a função de ambas as parálogas. É possível que os dois mecanismos contribuam para o quadro clínico. Os dados dos ensaios in vivo e in vitro abrem caminhos de investigação do processo de regulação de UBE2A e UBE2B no nível da proteína, sugerindo a autoubiquitinação e a ubiquitinaão recíproca como possíveis mecanismos reguladores, que podem explicar a conservação das duas parálogas de RAD6 em mamíferos.
We have previously described a nonsense mutation (c.382C8594;T) in the UBE2A gene, at Xq24, which encodes a ubiquitin conjugating enzyme (E2), as the cause of a new X-linked mental retardation syndrome. The predicted protein lacks the 25 C-terminal amino acid residues conserved in vertebrates and in Drosophila. This was the first description of a mutation in a ubiquitin conjugating enzyme gene causative of a human disease. In the present work, we focused on the UBE2A gene, its alternative transcripts and isoforms, and the effect of the c.382C8594;T mutation. We screened for UBE2A mutations 23 males presenting X-linked mental retardation (XLMR), previously mapped to the interval encompassing this gene, and one isolated case, who shared clinical features with our previously described patients. No mutations were detected in this selected series of patients suggesting that mutations in UBE2A is not a common cause of XLMR, similarly to the majority of the XLMR genes hereto described. Very recently four Xq24 microdeletions encompassing UBE2A and three missence mutations were found by other groups in mentally retarded males that shared several clinical features with our patients. Comparing these and our patients, a clinical picture emerges of mental retardation associated with severe speech impairment, present in all of them. Short stature, large mouth with downturned corners and thin lips, short and broad neck, low posterior hairline, widely spaced nipples, marked generalized hirsutism and seizures are common features. However, microcephaly was observed only in patients carrying UBE2A deletions, while carriers of missense or nonsense mutations showed macrocephaly. We evaluated the effect of the UBE2A c.382C8594;T mutation on transcription and translation. This mutation affects the last UBE2A exon and, as expected, does not lead to nonsense mediated RNA decay, demonstrated by the presence of UBE2A mRNA in leucocytes of an affected male. However, only a small amount of the mutated protein was detected in the patients cells, suggesting the loss of UBE2A function as the cause of the syndrome. The posttranslational degradation of the mutated protein could also disturb the cellular homeostasis, a gain of function that remained a possibility. The detrimental effect of the c.382C8594;T mutation was further supported by the presence of only the normal transcript in leucocytes of a heterozygous woman, who had completely skewed X inactivation, thus pointing to the selective advantage of lymphocytes carrying the normal allele on the active X chromosome. Our search in DNA and protein sequence databases suggested that the UBE2A gene produces three alternative transcripts all classified as protein coding. These three tanscripts contain the mutation site (c.382C8594;T). We showed that all three UBE2A transcripts are expressed in human leucocytes, adipocytes, placenta, cerebral cortex and hippocampus. We also detected an alternative transcript in murine, which corresponds to the human transcript 3. This alternative transcript was present in all murine tissues analyzed, including samples from a UBE2A knockout mouse. However, we failed to detect the proteins encoded by the alternative transcripts. This could result from low affinity of the used commercial antibody to the isoforms. Alternatively, a small amount of these proteins in the pool of cellular proteins, might have not been detected by Western blotting. We performed in vivo and in vitro assays to address the role of the alternative UBE2A isoforms, and to evaluate the effect of c.382C-T mutation on UBE2A function. Taking into account the high amino acid conservation between the human UBE2A and the Saccharomyces cerevisiae ortholog RAD6, we used a 916;rad6 yeast strain to verify whether UBE2A alternative and mutated isoforms were able to complement its UV-sensitivity phenotype, as previously demosntrated for UBE2A isoform 1. We also performed in vitro assays to evaluate their ubiquitination activity towards histone H2A, a known in vitro substrate of RAD6 and UBE2A. Only UBE2A isoform 1 could rescue the UV sensitivity phenotype of the knockout yeast strain. The expression of the alternative isoforms 2 and 3 was partially toxic to this yeast strain, and toxicity increased under heat shock conditions. However, these two isoforms do not seem to be stable in yeast cells: as in human tissues, we failed to detect UBE2A isoforms 2 and 3 in yeast cells expressing the corresponding transcripts. The mutant isoform was stable in yeast, but was unable to rescue the UV-sensitivity phenotype, its expression resulting in severe toxicity to the 916;rad6 strain. On the other hand, toxicity was not observed when the mutant UBE2A isoform was expressed in wild type yeast. These findings suggest that isoforms 2 and 3 do not have ubiquitin conjugating activity and, apparently, are degraded immediately after translation. The fact that toxicity is enhanced when these isoforms are expressed under heat shock conditions supports Degradation hypothesis. The degradation could also be due to the absence of a functional partner, in yeast, that could contribute to their stability. Since the alternative isoforms were not detected in the human tissues analyzed, the degradation might occur in human cells as well. E2 enzymes share a catalytic domain and variations among them consist of insertions or terminal extensions, never deletions. Both isoforms 2 and 3 would have deletions of the catalytic domain, suggesting that they are not functional. A regulatory role for these transcripts is a possibility. Our in vitro assays confirmed that UBE2A isoform 1 is capable of histone H2A ubiquitination. The assays for isoforms 2 and 3 were inconclusive, since their lack of ubiquitin conjugating activity could be caused by incorrect in vitro refolding, required because the proteins were obtained from bacterial inclusion bodies after heterologous expression. The mutated protein, however, was able to interact with the ubiquitin molecule, but failed to transfer it to histones, thus pointing to the importance of the C-terminal segment in this process. Our in vitro assays stongly suggested that UBE2A autoubiquitination occur, an activity previously considered a possible E2 regulatory mechanism. Since there is evidence that some E2s form functional dimers, we hypothesized that, due to their high amino acid conservation, UBE2A and its paralog UBE2B might form heterodimers in vivo, as a mutual regulating mechanism. Under this hypothesis, the degradation of the mutated protein could be UBE2B dependent. The reduced autoubiquitination capacity of the mutated isoform could impair its degradation, and require the participation or the paralog. This would explain why the mutated protein was stable in the 916;rad6 yeast strain, but not in the patient´s cells with a functional UBE2B. Following the same reasoning, in wild type yeast, the presence of RAD6 would explain the absence of the mutated protein and toxicity. The non-viability of the double (UBE2A and UBE2B) knockout cells prevented testing whether the mutated protein was stable in the absence of its paralog. However, proteasome inhibition in cultured cells from one of our patients resulted in accumulation of the mutated protein, confirming its degradion via the ubiquitin-proteasome pathway. In conclusion, the UBE2A c.382C8594;T mutation seems to lead to mental retardation in our patients due to loss of UBE2A function: the mutated isoform is unable to rescue the UV-sensitivity phenotype of 916;rad6 yeast or to ubiquitinate histones in vitro. In addition, patients carrying UBE2A deletions share clinical manifestations with our patients. On the other hand, the possibility remains of a clinical effect of the requirement of UBE2B for degrading the mutated UBE2A. Our data suggest reciprocal ubiquitination in addition to autoubiquitination as UBE2A and UBE2B regulatory mechanism that would explain the conservation of the two paralog genes in mammals.

La synapse est le lieu de communication entre les neurones à l'origine de nos capacités cognitives. Les mutations des gènes codant pour des protéines synaptiques sont responsables des maladies neurodéveloppementales appelées synaptopathies, recouvrant un large spectre de pathologies, de la déficience intellectuelle aux troubles du spectre autistique. Cependant, il est actuellement établi que les neurones ne sont pas les seuls acteurs au niveau de la synapse. Les astrocytes jouent également un rôle essentiel dans la mise en place du réseau neuronal et le fonctionnement de la synapse. Ils assurent aussi l'homéostasie ionique synaptique et sont capables de sécréter des glio-transmetteurs qui modulent l'activité synaptique. Oligophrénine-1 (OPHN1) est un gène associé à la déficience intellectuelle liée à l'X chez l'Homme. OPHN1 est une protéine synaptique dont les fonctions neuronales sont bien connues. La protéine peut directement interagir avec le cytosquelette d'actine et joue un rôle dans la formation et la maturation des épines dendritiques. Cette protéine est aussi exprimée dans les astrocytes mais sa fonction astrocytaire n'est pas connue. A l'aide d'un modèle KO de souris pour Ophn1, nous avons mis en évidence les conséquences de l'absence d'Ophn1 dans les astrocytes. Nous avons démontré que la délétion d'OPHN1 altère la migration et la morphologie des astrocytes in vitro. Sachant qu'Ophn1 est capable d'inactiver la GTPase RhoA, nous avons utilisé un inhibiteur de la voie RhoA/ROCK pour retrouver un phénotype de migration normal. In vivo nous avons choisi un modèle de cicatrisation gliale cortical afin de pouvoir observer la migration et la morphologie des astrocytes au niveau de la cicatrice. Nous avons observé que la délétion d'Ophn1 altérait la cicatrisation gliale et que les astrocytes à proximité de la cicatrice était moins ramifiés. L'ensemble de ces résultats nous permet de constater que les astrocytes sont altérés dans notre modèle murin de déficience intellectuelle liée à l'X. De plus, le KO conditionel astrocytaire mis en place nous permettra à l'avenir d'étudier les conséquences de la perte d'OPHN1 uniquement dans les astrocytes, et de comprendre la contribution astrocytaire dans la physiopathologie de cette maladie neuro-développementale
The synapse mediates the inter-neuron communication that forms the basis of all cognitive activity. Mutations in genes encoding for synaptic proteins are responsible for neurodevelopmental disorders called synaptopathies, covering a large clinical spectrum from intellectual disability (ID) to autism spectrum disorders. However it is currently established that neurons are not the only active cells at the synapse. Astrocytes play as well an essential role for its development and functioning. They maintain synaptic ionic homeostasis and are capable of secreting gliotransmitters, which can modulate synaptic activity. Oligophrenin-1 gene (OPHN1) was identified and associated with X-linked human ID. OPHN1 is a synaptic protein, which neuronal function is well known. It can directly interact with the actin-cytoskeleton and plays a role in the formation and maturation of dendritic spines. This protein is also expressed in astrocytes but its astrocytic function is still unknown. Using the Ophn1 KO mouse model, we were able to highlight in vitro the consequences induced by its deletion in astrocytes. We demonstrated that the absence of OPHN1 disturbed astrocytic migration and morphology in vitro. Since Ophn1 negatively regulates RhoA GTPase, we used an inhibitor of the RhoA/ROCK pathway to rescue the phenotype back to control. In vivo we took advantage of the cortical glial scar formation to observ astrocytic migration and morphology in KO mice. We found that Ophn1 deletion disrupted glial scar formation and that astrocytes near the wound were less ramified. Taken together, these results show that astrocytes are altered in our mouse model of X-linked ID. Moreover the development of an astrocytic conditional KO for Ophn1 will allow us to study the consequences of astrocytic loss of OPHN1 and determine the astrocytic contribution in the pathophysiology of this neurodevelopmental disease

Le syndrome de l’X-fragile est la forme la plus fréquente de déficience intellectuelle héréditaire liée au chromosome X. Cette maladie résulte de la mutation du gène FMR1 localisé sur le chromosome X. La protéine correspondante, FMRP, est absente chez les patients atteints de la maladie. Il faut noter ici qu’il existe un modèle murin mimant la pathologie humaine. Ainsi dans ces animaux qui n’expriment pas la protéine FMRP, les neurones présentent des anomalies architecturales de la synapse entraînant d’importants dysfonctionnements dans la transmission et la plasticité synaptique qui sont à l’origine des déficits intellectuels observés chez les patients atteints du syndrome de l’X-fragile. FMRP joue donc un rôle majeur dans la genèse et la maturation des épines dendritiques. Une des fonctions de FMRP est de lier de nombreux ARNm, de les transporter et d’inhiber leur traduction jusqu’à la synapse. Pour accomplir ses fonctions, FMRP interagit avec de nombreux partenaires cellulaires et ses interactions sont finement régulées par différentes modifications post-traductionnelles. Nous avons montré in vivo que la protéine FMRP est un substrat d’une nouvelle modification, la sumoylation. Nous avons également montré que la sumoylation de FMRP est impliquée dans le maintien de l’architecture synaptique et participe à la régulation de la transmission synaptique. Et enfin, nous avons montré que la sumoylation de FMRP permet sa dissociation avec ses partenaires protéiques au sein des complexes ribonucléoprotéiques se trouvant à la base des épines dendritiques. Les ARNm réprimés par FMRP au sein de ces complexes sont ainsi libérés puis traduits
Fragile X Syndrome is the most frequent inherited cause of intellectual disability in children and is caused by the lack of the mRNA-binding Fragile-X Mental Retardation Protein (FMRP) expression. FMRP plays a role in the activity-dependent targeting and translation of specific mRNAs in dendrites. The absence of FMRP expression in neurons leads to an abnormal neuronal morphology with increased spine length and density. FMRP is therefore playing key roles both in neuronal development and synaptic plasticity. However, the molecular mechanisms underlying the functional regulation of FMRP-mediated mRNA trafficking, translation and subsequent protein synthesis are still largely unknown. My host laboratory has recently discovered that FMRP is sumoylated in vivo. Sumoylation is a post-translational modification that consists in the covalent conjugation of the protein SUMO to specific lysine residues of target proteins. To start unraveling the functional consequences of FMRP sumoylation, I studied first the spine morphology of the WT and FMRP Knock Out mice that recapitulated the human disease. Morphological analysis of fmr1-KO neurons transfected with the WT form of FMRP restores the correct mature spine morphology whereas the non-sumoylatable protein failed to do so. Moreover the non-sumoylatable form of FMRP acts as a dominant negative on WT neurons so confirming the important role of FMRP sumoylation in its function. We report here that FMRP sumoylation is required for the control of spine morphology

Mutations in the ATRX gene are responsible for the alpha-thalassemia mental retardation (ATR-X) syndrome. ATRX is a putative global transcription regulator and chromatin remodelling protein. The goal of this research is to characterize interactions ATRX has with other proteins involved in transcription regulation, and identify domains in ATRX that may be responsible for these interactions. Several stable NIH 3T3 tet-off cell lines have been established that contain a human ATRX transgene. In addition, ATRX, PML, and Daxx appear to co-localize in nuclear bundles, suggesting they may act together transiently, or in a complex, in a regulatory role. A domain has been identified on ATRX that appears to target the protein to nuclear bundles, and interact with PML and Daxx. ATRX patient mutations appear to alter these interactions. This work attempts to elucidate cellular functions of ATRX, in hopes of establishing a better understanding of the neuropathology of this complex disease.

Thesis (Ph. D.)--Dept. of Paediatrics, Faculty of Medicine, University of Adelaide, 1991.
Typescript (Photocopy). Includes published papers co-authored by the author at the end of volume 2. Includes bibliographical references (leaves 195-237 of vol. 1).

Le syndrome de l’X-fragile (FXS) représente la forme la plus fréquente de retard mental héréditaire. Ce syndrome est dû à l’absence de la protéine Fragile X Mental Retardation (FMRP), une protéine de liaison aux ARN impliquée dans plusieurs aspects du métabolisme des ARNm. L’absence de FMRP chez les patients FXS et dans le modèle souris de la pathologie conduit à des altérations de la morphologie des épines dendritiques des neurones corticaux et à des altérations de la plasticité synaptique. La synthèse des protéines synaptiques, qui est essentielle à la plasticité synaptique, est altérée dans les neurones de souris FXS. Ainsi, FMRP, grâce à ses propriétés de liaison aux ARN, apparaît comme un acteur clé du contrôle de la traduction synaptique. Le rôle exact de FMRP dans ce processus demeure incompris. Cette thèse a visé à mieux comprendre le rôle moléculaire de FMRP dans le métabolisme des ARNm neuronaux et en particulier dans le transport et la traduction des ARNm. Plusieurs aspects du métabolisme des ARNm ont été abordés. Dans un premier temps, nous avons étudié le lien possible entre FMRP et la régulation de la traduction par les micro-ARN [. . . ] Dans un deuxième temps, nous avons entrepris de caractériser les cibles ARNm neuronales de FMRP en relation avec le phénotype dendritique du FXS […] Dans la troisième partie de ce travail, nous avons étudié la localisation intracellulaire de FMRP avec elle-même (dimérisation) et avec ses interacteurs (FXR1, FXR2, CYFIP1/Sra1, CYFIP2) en cellules vivantes en utilisant la méthode de complémentation de fluorescence bimoléculaire (BiFC) […] Enfin, nous avons étudié le rôle du paralogue de FMRP, FXR1P, une protéine de liaison aux ARN importante pour le développement normal des muscles squelettiques et du coeur [. . . ]
Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is due to the lack of the expression of Fragile X Mental Retardation protein (FMRP). FMRP is a RNA-binding protein involved in several steps of RNA metabolism. Data from fragile X patients and from the fragile X mouse model revealed abnormalities of the dendritic spines and of the synaptic plasticity. Synaptic protein synthesis, which underlies synaptic plasticity, is altered in the fragile X mouse model. Thus, FMRP, due to its RNA binding properties, appears as a key actor in the control of local mRNA translation in dendrites. The exact role of FMRP in this process remains however unclear. This thesis aimed at characterizing the exact molecular function of FMRP in neuronal mRNA metabolism and in particular in the transport and local translation of mRNAs. In a first part, we studied the previously proposed link between FMRP and translation regulation by miRNAs. [. . . ] In a second part, we characterized some neuronal mRNA target of FMRP relevant to the dendritic phenotype of FXS. […]In a third part, we studied the intracellular localization of FMRP with itself (dimerization) and its interactors (FXR1, FXR2, CYFIP1/Sra1, CYFIP2) in living cells using the bi-molecular fluorescence complementation assay (BiFC) […] Finally, we studied the role of FMRP paralogue, FXR1P, an RNA binding protein important for heart and skeletal muscle development [. . . ]

Le syndrome de l'X Fragile, qui constitue la forme la plus fréquente de retard mental héréditaire, est du à l'absence de FMRP, une protéine de liaison à l'ARN qui régulerait la traduction au niveau synaptique. Afin de mieux comprendre le role de FMRP, nous avons réalisé un criblage double-hybride dans la levure pour identifier certains de ses interacteurs. Deux protéines ont été isolées, CYFIP1 et CYFIP2 (Cytoplasmic FMRP Interacting Proteins 1/2). Ces deux protéines cytoplasmiques sont hautement homologues mais interagissent différement avec les deux autres protéines de la famille FXR, FXR1P et FXR2P. Le site de liaison à CYFIP recouvre le site d'homo- et d'hétérodimérisation de FMRP, suggérant que la liaison à CYFIP pourrait moduler l'activité de FMRP. D'autre part, l'interaction entre CYFIP1 et Rac1 a été démontrée précédemment. Rac1, une protéine de la famille des Rho GTPase, est un des principaux régulateurs de la réorganisation du cytosquelette d'actine et joue un role clé dans la maturation et la maintenance des épines dendritiques, structures synaptiques riches en actine anormalement développées chez les patients X-Fragile et dans les souris invalidées pour FMRP. De nombreux gènes des voies Rho/Rac étant impliqués dans des retards mentaux, Rac1, CYFIP1 et FMRP pourraient participer à une voie commune contrôlant la morphogénèse synaptique et le fonctionnement cognitif. Pour valider cette hypothèse in vivo, nous avons choisi Drosophila melanogaster comme organisme modèle. Nous avons découvert que CYFIP y est très exprimé dans le système nerveux embryonnaire, et s'accumule notamment dans les axones centraux et aux jonctions neuro-musculaires (JNM). Des mutations de CYFIP causent des défauts de croissance, de ramification et de connexion des axones et conduisent à une morphologie anormale des synapses des JNM. Ainsi, l'absence de CYFIP provoque des défauts similaires à ceux précedemment décrits chez les mutants dFMR1 et/ou dRac1. L'analyse des interactions génétiques et biochimiques entre ces trois protéines suggère que, lorsque la voie est activée, dRac1 inhibe CYFIP qui régule à son tour négativement dFMR1
Fragile X Syndrome is the most frequent form of hereditary mental retardation and caused by the absence of FMRP, an RNA binding protein that seems to regulate local protein translation at synapses. To better understand the physiological function of FMRP, we conducted a yeast two-hybrid screen to determine interacting proteins. We identified CYFIP1 and CYFIP2 (Cytoplasmic FMRP Interacting Proteins 1/2), two highly homologous cytoplasmic proteins, which show a different pattern of interaction with the two FMRP-related proteins FXR1P and FXR2P. The CYFIP binding site of FMRP overlaps with its homo- and heteromerisation domain, suggesting that binding to CYFIP may modulate FMRP function. Importantly, CYFIP1 has been previously reported to interact with Rac1. Rac1, a Rho GTPase, is a key regulator of actin cytoskeleton remodelling with a well-established role in maturation and maintenance of dendritic spines, which are actin-rich synaptic structures that are abnormally developed in Fragile X patients and FMRP null mice. Since several genes of Rac/Rho signalling pathways are implicated in mental retardation, our work suggested that Rac1, CYFIP and FMRP work in a common pathway determining synapse morphogenesis and cognitive function. To address this hypothesis in vivo, we have chosen the fruitfly Drosophila melanogaster as a genetic model organism. Drosophila CYFIP, a previously undescribed gene, is highly expressed in the embryonic nervous system, where it strongly accumulates in central axons and at the neuromuscular junction (NMJ). CYFIP mutations induce defects in axon growth, branching and pathfinding and result in abnormal synapse morphology at the neuromuscular junction. Hence, loss of CYFIP involves defects that have been previously described in dFMR1 and/or dRac1 mutants. Analyses of biochemical and genetic interactions amongst these three proteins suggest that upon activation, dRac1 acts antagonistically on CYFIP, which in turn negatively regulates dFMR1

Mestrado em Biologia Molecular e Celular
The impact of the Fragile Mental Retardation-1 (FMR1) gene CGG repeat number in the female reproductive function is well established. Carriers of a CGG repeat number between 55 and 200, designated a premutation, are prone to develop primary ovarian insufficiency or early menopause. Yet, an impact on the reproductive function in carriers of “normal” genotypes and sub-genotypes (CGG<54) is controversial. The presence of AGG in normal-sized alleles confers stability, hampering the expansion of the repeat number in future generations. To the best of our knowledge testing the influence of the AGG number and pattern on the female reproductive function has never been endeavored. Herein, the ovarian reserve markers were correlated with CGG number as well as AGG number and pattern, in female carriers of FMR1 normal-sized alleles. Our cohort comprised 50 healthy young females, candidates for oocyte donation. Considering AGG number and pattern are not routinely determined different methodologies were implemented: 1) Triplet-Primed Polymerase Chain Reaction; 2) Sanger sequencing; and 3) Restriction Fragment-Length Analysis. A projection of the association between the CGG repeat values and the hormonal levels, by multivariate analysis, was performed, considering the FMR1 new “normal” sub-genotypes previously defined. The hormonal levels associated with the different samples were not sufficient to discriminate the sub-genotypes, indicating that the individualization of the samples classified by sub-genotype was not possible. Resorting to a mathematical formula that determines the allelic score, taking into account total allele size, and AGG number and pattern. After statistical analysis, it was possible to divide the samples into two groups: a first called an equivalent group and a second called an opposite group. The equivalent group is composed mainly of samples carrying alleles in the normal FMR1 sub-genotype and the opposite, where most of the samples have an FMR1 low/normal sub-genotype. In the equivalent group, a positive and significant correlation was observed between the number of antral follicles and the hormonal levels: prolactin and luteinizing hormone (LH). Thus, it is possible to predict the largest number of antral follicles produced combining the levels of prolactin and LH. These results actually confirm prior publications as the low/normal sub-genotype has been previously associated with a diminished ovarian reserve. Overall, this study confirms the association of the FMR1 CGG repetitive region in the female reproductive function and suggests that the stability of the alleles is a determining factor for the ovarian response success.
A relação entre o número de repetições CGG do gene Fragile Mental Retardation-1 (FMR1) e a função reprodutiva em mulheres não é uma novidade. Está descrito que as portadoras de alelos com um número de repetições CGG entre 55 e 200, designados por pré-mutação, têm uma predisposição para desenvolver insuficiência ovárica primária ou menopausa precoce. Porém, a existência de risco de diminuição da função reprodutiva nas mulheres, com genótipos considerados “normais” (CGG<54), e respetivos subgenótipos ainda não é clara. Sabe-se que a presença de interrupções AGG confere a esses alelos uma maior estabilidade, impedindo a expansão do número de repetições CGG para um tamanho considerado patogénico. A forma como o número e o padrão de interrupções AGG poderá influenciar a função reprodutiva feminina, nunca foi estudada. No presente trabalho, os marcadores de reserva ovárica foram correlacionados com o número de repetições CGG e perfil das interrupções AGG. A população em estudo incluiu 50 mulheres jovens e saudáveis, candidatas à doação de oócitos. Dado que o número e o padrão das interrupções AGG não são determinados por rotina, foi então necessário implementar a sua análise, recorrendo a diferentes metodologias: 1) Triplet-Primed Polymerase Chain Reaction; 2) Sanger sequencing; and 3) Restriction Fragment-Length Analysis. Foi realizada uma projeção da associação entre o número de repetições CGG e os níveis hormonais, através de uma análise multivariável, considerando os novos subgenótipos "normais" FMR1 previamente definidos. Os níveis hormonais associados às diferentes amostras não foram suficientes para discriminar subgenótipos, indicando que a individualização das amostras classificadas por sub-genótipos não era possível. Recorrendo a uma fórmula matemática que determina a pontuação alélica, tendo em consideração o tamanho total do alelo, e o número e o padrão de AGG. Após análise estatística foi possível dividir as amostras em dois grupos: um primeiro designado por grupo equivalente e um segundo designado por grupo oposto. O grupo equivalente, que é composto principalmente por amostras que possuem alelos do subgenótipo “normal” FMR1, e o oposto, onde a maioria das amostras possui subgenótipo “normal/baixo” FMR1. No grupo equivalente, observou-se correlação positiva e significativa entre número de folículos antrais e os níveis hormonais: prolactina e hormona luteinizante (LH). Assim, é possível prever o número de folículos antrais produzidos combinando os níveis de prolactina e LH. Estes resultados confirmam publicações anteriores, já que o sub-genótipo “normal/baixo” foi anteriormente associado a uma diminuição da reserva ovárica. No geral, este estudo confirma a associação da região repetitiva CGG do FMR1 na função reprodutiva feminina e sugere que a estabilidade dos alelos é um fator determinante para o sucesso da resposta ovárica.

Le syndrome du X fragile, première cause de retard mental héréditaire, est une maladie monogénique liée au chromosome X. Le syndrome affecte environ un homme sur 4000 et une femme sur 6000 dans la population générale. Il est causé par l'inactivation du gène Fragile Mental Retardation 1 (FMR1) entraînant l'absence de la Fragile X Mental Retardation Protein (FMRP). Celle-ci est une protéine de liaison à l'ARN ayant pour rôle présumé de coordonner le devenir et la traduction d'un grand nombre d'ARN messagers (ARNm). L'absence de FMRP provoquerait une dérégulation subtile du transport des ARNm, conduisant à une altération de la synthèse protéique locale nécessaire à la connexité synaptique, entraînant ainsi le retard mental. Il est accepté que la FMRP possède des signaux de localisation nucléaire et d'exportation cytoplasmique (Nuclear Localisation Signal et Nuclear Export Signal ; NLS et NES) permettant à la protéine de pénétrer dans le noyau et supposément d'en ressortir. Cependant, les anticorps disponibles dans le passé ne permettent pas d'étudier la localisation et le rôle de FMRP dans le noyau. Grâce à de nouveaux anticorps monospécifiques développés dans le laboratoire, nous avons pu étudier la compartimentalisation de sous-populations de la protéine FMRP. Je développerai donc ici brièvement le devenir de la FMRP cytoplasmique (cFMRP) dans les neurones, et je caractériserai la FMRP nucléaire (nFMRP), que de nombreux laboratoires ont recherché durant de nombreuses années, et qui serait constituée par des isoformes particulières de la protéine FMRP qui se localiseraient dans les corps de Cajal, structures décrites il y a plus d'un siècle par Santiago Ramon y Cajal. Les données présentées ici soulèvent le doute sur le modèle de trafic nucléo-cytoplasmique de la FMRP, suggéré sur la base de rares travaux. La découverte de la nFMRP pourrait avoir d'importantes implications dans le domaine du Syndrome du chromosome X Fragile en ouvrant un champ nouveau d'étude sur le rôle nucléaire de la FMRP dans les cellules, et donc sur les conséquences de son absence chez les patients.
Fragile X syndrome, a monogenic disease linked to the chromosome X, is the first cause of inherited mental retardation. The syndrome affects about one out of 4000 man, and one out of 6000 woman. Fragile X is caused by the inactivation of the Fragile X Mental retardation (FMR1) gene, leading to the absence of its product, the Fragile X Mental Retardation Protein (FMRP). The absence of FMRP, an RNA binding protein, is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. It is accepted that FMRP possesses a nuclear localisation signal (NLS), and a nuclear export signal (NES), allowing the protein to enter the nucleus, and possibly to exit from it as well. However, available antibodies do not allow to study the nuclear localisation of FMRP. Thanks to a new generation of monospecific antibodies developed in our laboratory, we were able to study the cytoplasmic and the nuclear distribution of FMRP. I will therefore shortly develop the fate of cytoplasmic FMRP (cFMRP) in neurons, and I will characterise the nuclear FMRP (nFMRP) that has been sought after for many years. nFMRP consists in particular nuclear FMRP isoforms that localize to Cajal bodies, structures described more than a century ago by the famous neuroscientist Santiago Ramon y Cajal. Data presented here also raise doubts on the nucleocytoplasmic traficking model, which relies on very few evidence. The discovery of nFMRP could have great implication in the Fragile X domain, opening a whole new field of investigation on the role of FMRP in the cell nucleus, and therefore on the consequences of its absence in patients.

Thesis (Ph.D. in Clinical Science) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 59-67). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Le syndrome de l’X Fragile est la cause héréditaire la plus commune des retards mentaux qui touche environ 1 sur 4000 garçons et 1 sur 8000 femmes. Les patients ont en moyenne un IQ inférieur à 75, en raison de défauts de transmission et de plasticité neuronale. Ce syndrome est causé par la perte de fonction de la protéine « Fragile X Mental Retardation Protein » (FMRP) via des mécanismes mal connus. Comprendre le rôle de FMRP dans la plasticité neuronale est très important pour traiter cette maladies. Cependant, étant donnée la difficulté d’étudier la protéine chez les humains pour des raisons éthiques, la maladie a été explorée en plusieurs aspects sur des modèles animaux simples, tels que Drosophila melanogaster. Ceci permet d’utiliser des approches génétiques, de biologie cellulaire et moléculaire et électrophysiologiques. La jonction neuromusculaire de la drosophile (JNM) représente une structure très accessible et un modèle classique de plasticité neuronale. Les animaux mutants pour la protéine FMRP, connue comme régulatrice de la traduction, ont des JNM très ramifiées par rapport aux animaux sauvages. Ce phénotype ressemble beaucoup aux longues et immatures épines dendritiques des patients atteints du syndrome de l'X fragile. Ainsi, le phénotype basique de la carence en FMRP peut être modélisé avec la JNM de la drosophile. Plusieurs études soulignent le rôle du remodelage du cytosquelette d'actine dans le développement des axones et dans la formation des synapses. Le complexe WAVE est connu pour réguler le complexe actin nucleator Arp2/3 et favorise ainsi le remodelage du cytosquelette. Deux événements cruciaux sont donc nécessaires pour le développement et la modulation de la fonction de JNM: remodelage du cytosquelette d'actine et la synthèse des protéines locales. De plus, les données acquises au laboratoire suggèrent une interférence entre ces deux processus. L’orthologue de FMRP chez la drosophile (dFMR1) representerait donc un pont, en interagissant directement avec le complexe WAVE/SCAR, qui induit le remodelage du cytosquelette d'actine, et avec des ARNm, qui contrôle la traduction locale. A l'intérieur du complexe, la protéine WAVE interagit directement avec HSPC300. Dans ma première année de thèse, j'ai participé à la caractérisation de HSPC300, la plus petite sous-unité du complexe. Nous avons déterminé le rôle de HSPC300 in vivo, caractérisé HSPC300 chez la drosophile et définir son rôle dans la connectivité neuronale. HSPC300 est une protéine de 8 KD hautement enrichie dans le système nerveux, comme observé par un anticorps produit dans la maison. En générant des mutants HSPC300 perte et gain de fonction, nous avons observé que cette petite protéine est essentielle pour la stabilité du complexe WAVE et pour la morphogenèse de JNM. La perte de HSPC300 entraîne des graves défauts des axones et des JNM. Ces défauts sont sauvés en exprimant spécifiquement HSPC300 dans le tissu neural. Ces données impliquent HSPC300 comme un élément indispensable du complexe WAVE, et une protéine cruciale pour le développement du système nerveux (Qurashi et al. 2007). Nous avons précédemment montré que dFMR1 interagit biochimiquement avec CYFIP, un autre membre du complexe WAVE, et on a constaté que les deux protéines affectent la croissance JNM dans des sens opposés. DFMR1 supprime la croissance synaptique alors que CYFIP est nécessaire pour la croissance des synapses. La perte de dFMR1 induit une exagération de la ramification et de la croissance, tandis que la perte de CYFIP donne lieu à des synapses courtes mais extrêmement greffés. En outre, des expériences de dosage ont prouvé que dFMR1 et CYFIP agissent ensemble pour le contrôle de la croissance de JNM et travaillent de façon antagoniste. Durant ma deuxième année, j'ai réalisé un projet en collaboration avec les Pr. Zhang (Chine) et Hassan (VIB, Belgique) et visant à cartographier les régions d’interaction. Nous avons généré des mouches portant une mutation ponctuelle intragénique de dFMR1 obtenu par mutagenèse à l’EMS. Un crible par double hybride montre que des mutations intragéniques spécifiques empêchent FMRP d'interagir avec CYFIP. Nous avons ainsi montré que l’extrémité N-terminale de FMRP est essentielle pour l'interaction avec CYFIP. J'ai analysé le phénotype de JNM en utilisant dix mutants intragéniques dFMR1 et j’ai défini in vivo le domaine requis pour l’interaction FMRP-CYFIP. J'ai montré que les mutants dFMR1 qui maintiennent l'interaction biochimique avec CYFIP, maintiennent aussi l'interaction génétique; par conséquent, la surexpression de CYFIP sauve la surexpression de ces formes de dFMR1 mutantes. Chez les mouches porteuses d'une mutation qui abolit l’interaction biochimique dFMR1-CYFIP, la surexpression de CYFIP ne réprime plus le phénotype de gain de la fonction dFMR1. La co-surexpression crée un phénotype d’une croissance exagérée de JNM, qui n'est pas obtenu par un simple CYFIP ou dFMR1 surexpression. Toutes ces données prouvent in vivo que dFMR1 interagit avec CYFIP par l'intermédiaire de l’extrémité N-terminale de la protéine dFMR1. Leur interaction a un rôle important pour un contrôle précis de la croissance synaptique et pour le remodelage du cytosquelette d'actine. Cette étude est publiée dans le Journal of Neuroscience en Mars 2008 (Reeve et al. 2008). Durant ma troisième année de doctorat, j'ai étudié le rôle de CYFIP dans le remodelage du cytosquelette d'actine chez les photorécepteurs de la drosophile. En utilisant le système binaire UAS/Gal4 nous avons obtenu des mutants conditionnels CYFIP, temps et tissu spécifiques. Nous avons révélé le rôle de CYFIP dans des cellules précises: les cellules photoréceptrices et les cellules pigmentaires. Nous avons également révélé la conséquence directe du défaut de nucléation de l'actine qui est observée dans les jonctions cellule-cellule (Galy et al, in prep). Plusieurs groupes ont suggéré que FMRP a une activité comme répresseur traductionnel. Même si le mode de cette activité est encore un mystère, certains chercheurs ont fourni des preuves montrant que FMRP interagit avec plusieurs composants du complexe RISC. Au cours de ma quatrième année de thèse, j'ai étudié l'interaction FMRP avec RISC en collaboration avec le Pr Bozzetti, en Italie (Specchia et al, in prep). Dans tous les projets auxquels j'ai participé jusqu'à présent, je vise à faire la lumière sur les voies FMRP et WAVE. Nous avions l'intention de déchiffrer les voies contrôlant le remodelage du cytosquelette d'actine et la synthèse des protéines locales dans JNMs chez la mouche. Dans la présente thèse, tous les résultats sont discutés en détail
Fragile X syndrome is the most common inherited cause of mental retardation affecting approximately 1 in 4000 males and 1 in 7000 females. Average IQ score of the patients is under 75 as a result of neural wiring and synaptic plasticity defects due to loss of functional Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA binding protein and a translational regulator highly abundant in the nervous system. We intend to explore underlying mechanisms of these defects using Drosophila (fruit fly) model organism. Several studies point out the role of actin cytoskeleton remodeling in synapse architecture and function. WAVE (/SCAR) complex stimulates the Arp2/3 complex actin nucleator and thereby promote cytoskeleton remodeling. Current data suggest that the crosstalk between actin cytoskeleton remodeling and local protein synthesis is essential for development and modulation of synapses. During my thesis, I have focused onto this topic and have the tackled three aspects of it: Characterization of the A) WAVE complex and its role in synaptic plasticity, B) the interaction between FMRP and WAVE complex, C) the actin cytoskeleton remodeling during morphogenesis. WAVE protein directly interacts with HSPC300, the smallest subunit of WAVE complex. Using targeted expression we characterize Drosophila HSPC300 and define its role in neural wiring. HSPC300 is highly enriched in the nervous system. Its mutation causes severe axonal/synaptic defects that are rescued by specific HSPC300 expression in the neural tissue. We conclude HSPC300 is an indispensable component of WAVE complex and an essential protein for nervous system development. We have previously shown that FMRP biochemically and genetically interacts with CYFIP, another member of the WAVE complex. To further characterize these interactions FMRP point mutations were generated in collaboration with two different labs. Particular mutants lose CYFIP binding. Using the intragenic mutants we point crucial FMRP residues for CYFIP interaction in vivo. FMRP is a negative regulator of neural growth. Here we describe nature of the FMRP-CYFIP genetic interaction in the context of neuromuscular synapses. We further assess CYFIP role on actin cytoskeleton in neatly organized fly eye and mostly actin-based photoreceptors. Making use of Drosophila genetics we define the spatiotemporal requirement of CYFIP for nervous system development. In all the projects I have been involved so far; I aimed at shedding light on FMRP and WAVE complex interaction. We intend to decipher these proteins and how their interactions regulate actin cytoskeleton remodeling, local protein synthesis and consequently neural connectivity using fly nervous system. I discuss these issues in the presented thesis

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-25T21:04:26Z No. of bitstreams: 1 Marcela Kelly Astete Gómez Estudo dos alelos da região....pdf: 1187772 bytes, checksum: d35081c0e79dae64122fa5e5a15c60c3 (MD5)
Made available in DSpace on 2012-07-25T21:04:26Z (GMT). No. of bitstreams: 1 Marcela Kelly Astete Gómez Estudo dos alelos da região....pdf: 1187772 bytes, checksum: d35081c0e79dae64122fa5e5a15c60c3 (MD5) Previous issue date: 2011
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
A Síndrome do X-Frágil (SXF) é a principal causa hereditária de deficiência mental de herança dominante ligada ao cromossomo X. Em 1991, o gene FMR1 (Fragile X Mental Retardation 1) foi descoberto como responsável pela SXF. De acordo com o número de repetições, os alelos se dividem em: (a) alelo normal compreendendo 6 a 55 repetições (b) pré-mutado: 61 a 200 repetições e (c) mutado: com >200 repetições. Os alelos que apresentam 45 a 60 repetições são considerados como zona intermediária ou gray-zone. A base molecular dessa doença é bastante peculiar quando comparada com os padrões típicos observados em outras desordens de etiologia genética. As pré-mutações podem agregar-se de forma silenciosa por muitas gerações de uma família antes de se expandir para a mutação completa, levando aos sinais clínicos da doença. Na Bahia existem poucas pesquisas envolvendo esta doença, consequentemente, é desconhecida sua frequência nessa região. Portanto, este trabalho tem como objetivo detectar a frequência das classes alélicas do gene FMR1 em uma amostra de indivíduos do sexo masculino da população geral de Salvador-BA. Este estudo foi realizado em 511 homens provenientes de outro amplo estudo desenvolvido pelo Instituto de Saúde Coletiva (ISC) da Universidade Federal da Bahia (UFBA) sendo utilizada a técnica da PCR e posterior classificação dos alelos baseando-se na relação entre o número de repetições CGG e o tamanho do fragmento obtido na PCR. Dos 511 homens analisados no presente estudo observou-se predominância de alelos normais pertencentes à classe 2 (11 a 26 repetições CGG) em 73,70% dos indivíduos analisados, seguido da classe 3 (27 a 40 repetições CGG) em 25,10% dos indivíduos. Apenas 1,20% foram incluídos na classe 1 (<10 repetições CGG) e nenhum alelo foi encontrado nas classes 4 (41 a 60 repetições CGG), classe 5 ( >60 repetições CGG) e classe 6 (>200 repetições CGG). Este é o primeiro estudo utilizando a técnica da PCR para detecção dos alelos do gene FMR1 em uma população geral de Salvador-BA, podendo direcionar futuros trabalhos envolvendo o gene FMR1 tanto para o estado da Bahia, quanto para a região Nordeste e também minimizar deficiências existentes em termos de diagnóstico da SXF na cidade de Salvador-BA.
The Fragile X syndrome (FXS) is the leading cause of inherited mental deficiency (MD) of dominant inheritance linked to X chromosome. In 1991, the FMR1 gene (Fragile X Mental Retardation 1) was discovered as responsible for FXS. According to the number of repetitions, the alleles are divided in: (a) normal stable allele comprising 6 to 55 repetitions; (b) premutation: 61 to 200 repetitions and (c) mutant with> 200 repeats. The molecular basis of this disease is quite unusual when compared with the typical patterns seen in other disorders of genetic etiology. The pre-change can add up so silent for many generations of a family before they expand to full mutation, leading to clinical signs of disease. In Bahia there are few studies involving this disease, therefore, its frequency is unknown in this region. So, this study aims to detect the frequency of allelic classes of FMR1 gene in a sample of males from the general population of Salvador-BA city. This study was conducted on 511 samples using the PCR technique and subsequent classification of alleles based on the number of CGG repeats and the size of the fragments in PCR. Of the 511 individuals examined was found to predominate among the normal alleles the class 2 (11 to 26 repetitions) with 73,70% followed alleles analyzed in Class 3 (27 to 40 repetitions) with 25,10%, and Class 1 (<10 repetitions) with only one 1,20% . No allele was found in class 4 (41 to 60 repetitions), which corresponds to gray zone and class 5 (> 60 repetitions) for the premutation and class 6 (>200 repetitions) the corresponds full mutation.This is the first study using the PCR technique for detection of alleles of the FMR1 gene in a general population of Salvador, Bahia, and may direct future studies involving the FMR1 gene for both the state of Bahia, the Northeast and to also minimize deficiencies existing in the diagnosis of FXS in Salvador, Bahia.

As alterações cromossômicas estruturais associadas a fenótipos clínicos oferecem a oportunidade de identificação de genes cujas mutações possam estar determinando essas patologias, tendo em vista a possibilidade de que esses genes podem ter sido alterados pelas quebras ou ter o número de cópias modificado. Um número cada vez maior de evidências aponta para a participação de certas seqüências do genoma na formação de rearranjos cromossômicos recorrentes e não recorrentes. Neste trabalho, estudamos duas deleções cromossômicas detectadas em indivíduos com retardo mental associado a sinais clínicos. O objetivo foi determinar que mecanismos originaram esses rearranjos e como a perda ou quebra dos segmentos cromossômicos está relacionada com o fenótipo dos portadores. A caracterização das seqüências nos pontos de quebra e junção desses rearranjos é fundamental para a compreensão dos mecanismos de formação das alterações cromossômicas. A delimitação precisa dos segmentos deletados é necessária para a correlação com o quadro clínico. Para isso, este trabalho aliou o estudo cromossômico por hibridação in situ fluorescente (FISH) à análise do DNA.
Structural chromosomal alterations related to clinical phenotypes bring the opportunity to identify gene mutations determining the pathologies, because the causative genes may have been disrupted by the breaks or may have an altered number of copies. The delimitation of the segments involved in the chromosomal rearrangements is necessary for these genotype-phenotype correlations. The characterization of breakpoint and junction sequences in these chromosome alterations enables the identification of mechanisms originating them, and evidence has been produced pointing to the participation of particular genomic sequences in their formation. In this work, we studied two chromosomal deletions in patients with syndromic mental retardation, combining chromosomal analysis by fluorescent in situ hybridization (FISH) to DNA analysis. Our aim was to determine the mechanisms that originated these aberrations and how they were involved with the clinical phenotypes.

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.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-18T13:16:28Z No. of bitstreams: 1 Roberto Carlos Gomes da Silva.pdf: 3176847 bytes, checksum: b508a4a3eb30a62abd86ea9330f277e4 (MD5)
Made available in DSpace on 2016-08-18T13:16:28Z (GMT). No. of bitstreams: 1 Roberto Carlos Gomes da Silva.pdf: 3176847 bytes, checksum: b508a4a3eb30a62abd86ea9330f277e4 (MD5) Previous issue date: 2008-04-09
Since DNA structure was described, several studies have been carried out in genetics that promoted a revolution in the practice of medicine. Human syndromes that were practically undiagnosed became easily diagnosed with molecular tools. However, most of the genetic diseases remain under diagnostic obscurity, increasing health concerns for affected people and public demand for preventive health care such as the case of Fragile-X Syndrome, the most common heritable form of mental retardation in humans. FXS is caused by an expansion of CGG repeat sequence in the promoter region of FMR1 gene, located in Xq27.3. Both men and women are affected by FXS and pre-mutation can expand to a full mutation in the next generation. Under full mutation status ( 200 repeats) the gene is silenced and FMRP protein is not produced causing mental retardation, speech delay, and behavior problems, the most frequent symptoms in FXS. Prevalence of FXS is estimated in 1:4000 and 1:8000 and of carriers in the general population as 1:813 and 1:259 for men and women, respectively. Because of FXS potential to affect subsequent generations it is crucial to properly diagnose the syndrome. Laboratory analysis of DNA from FXS, using PCR or Southern Blotting, allows reaching the diagnosis in 99% of cases carrying mutated genes. However, to date the Brazilian Public Health System does not recognize the molecular methods to reach complete diagnostic in FXS. Early diagnose would allow fore more appropriate and efficient therapy approaches, favoring satisfactory development of all affected people, minimizing their suffering and the burden on their families, increasing, on the other hand, their quality of life which should go beyond survival.
A partir da descrição da estrutura do DNA, várias pesquisas foram desenvolvidas na área da Genética, promovendo uma revolução na prática da medicina. Síndromes, antes difíceis ou até impossíveis de serem detectadas, com tecnologia e ferramentas moleculares, tornaram-se facilmente diagnosticadas. Entretanto, diversas doenças ainda persistem na obscuridade de diagnóstico e geram problemas de saúde pública como é o caso da Síndrome do X-Frágil (SXF), que é a causa mais comum de retardo mental masculino herdado, e que consiste na expansão do número de cópias de uma seqüência de bases CGG do DNA no gene FMR1, localizado no cromossomo Xq27.3. A SXF afeta tanto homens quanto mulheres e a pré-mutação poderá expandir-se à mutação completa nas próximas gerações. Os portadores da pré-mutação continuam produzindo a proteína FMRP e os portadores da mutação completa são afetados pela SXF, pois o gene FMR1 é silenciado, e a proteína não é produzida, causando retardo mental, problemas de linguagem e de comportamento. A prevalência da SXF é estimada em 1:4000 e 1:8000, e para portadores na população em geral é 1:813 e 1:259 para homens e mulheres respectivamente. A importância do reconhecimento clínico e diagnóstico da SXF vem do fato de que as gerações futuras poderão estar comprometidas. O estudo do DNA para X-frágil pela PCR e Southern blotting permite determinar com segurança superior a 99% quem é portador da pré-mutação do gene FMR1 e quem possui a mutação completa. Entretanto, o SUS não reconhece os métodos moleculares, apesar do diagnóstico permitir intervenções terapêuticas, com respostas bastante eficientes, favorecendo o desenvolvimento de modo integral das pessoas afetadas, minimizando seu sofrimento e de seus familiares, uma vez que a qualidade de vida deve ir além da sobrevivência.

La Fragile X Mental Retardation Protein (FMRP) est une protéine régulant la traduction locale de nombreux ARNm dans le cerveau. Elle est absente dans le Syndrome de l'X Fragile (SXF), première cause de retard mental héréditaire. J'ai étudié le rôle de FMRP dans la plasticité structurelle des cellules granulaires (CG) produites à l'âge adulte dans le bulbe olfactif (BO) en réponse à un apprentissage chez la souris. L'apprentissage perceptif (AP) induit de profonds changements structurels des nouveaux neurones du BO adulte. En absence de FMRP dans les CG nouvellement générées, l'apprentissage et la plasticité induite par cet apprentissage sont abolis. αCamKII est une cible traductionnelle connue de FMRP impliquée dans la plasticité synaptique et structurelle. En absence de l'ARNm de αCamKII dans les neurites, l'AP et la plasticité structurelle associée ne sont pas permis. De plus, l'AP augmente la traduction locale de αCamKII de façon dépendante de FMRP. De manière inattendue, αCamKII est présente dans 50% des CG. La plasticité structurelle des CG induite par l'apprentissage ne se produit que dans les CG qui expriment αCamKII (αCamKII+). De manière intéressante, l'AP active les deux populations de manière similaire. Ces résultats révèlent un rôle nouveau de la traduction locale dans la plasticité structurelle induite par l'apprentissage. De plus, l'AP produit des effets différents sur les deux populations que nous avons identifiées, qui pourtant participent probablement toutes deux à l'AP
The Fragile X Mental Retardation Protein (FMRP) is a major regulator of local translation in neurons. It is absent in the Fragile X Syndrom (FXS), which is the main cause of inherited intellectual deficiency. I studied the role of FMRP in structural plasticity of adult-born granule cells (abGC) of the mouse olfactory bulb (OB) in response to learning. Perceptual learning (PL) induces profound structural changes in abGC. In absence of FMRP in adult-born neurons, learning and associated structural plasticity are prevented. αCamKII is a well known translational target of FMRP, which is involved in synaptic and structural plasticity. In absence of αCamKII mRNA in neurites, PL and associated structural plasticity are abolished. Besides, PL increases the dendritic local translation of αCamKII in an FMRP-dependent manner. Unexpectedly, αCamKII is present in 50% of the total GC population of the OB. Learning-associated structural plasticity occurs only in αCamKII expressing GC. Interestingly, PL activates similarly both populations. These results reveal a new role for local translation in learning-induced structural plasticity. Moreover, PL induces different effects in the two subpopulations we identified, which probably both participate to PL

Ce travail contribue à l'établissement de la carte physique de la région q13-q22 du chromosome X humain. Nous avons rassemblé huit translocations X ; Autosomes (t(X;A)) équilibrées chez des femmes présentant des troubles de la fonction ovarienne. Dans une première partie, nous définissons huit nouveaux points de cassure dans la région Xq13-q22. Ces balises sont tout d'abord isolées par hybridation somatique interspécifique à partir des t(X;A) ; elles sont ensuite localisées par rapport aux marqueurs de la région proximale des bras longs du chromosome X humain. En regroupant nos t(X;A) avec des délétions de la région Xq21 publiées précédemment, nous proposons un panel de réarrangements qui subdivise la région Xq21 en 21 segments, soit plus d'une borne par mégabase d'adn. Cette collection constitue donc un bon canevas pour la construction d'une carte physique, génétique et fonctionnelle détaillée de la région q21 du chromosome X humain. La deuxième partie de ce travail consiste en la caractérisation moléculaire fine des points de cassure sur l'X pour les deux t(X;A) présentées dans cette étude qui sont associées avec des pathologies récessives liées à l'X. D'une part, chez la patiente TDo, atteinte de choroïdérémie et porteuse d'une t(X;7)(q21;p12), nous confirmons la localisation du point de cassure sur l'X dans le gène CHM, entre le troisième et le quatrième exon. D'autre part, le point de cassure sur le chromosome X chez la patiente PMI, porteuse d'une t(X;13)(q13;q31) en association avec un retard mental, a permis le clonage positionnel d'un gène qui pourrait être un bon candidat pour l'un des nombreux retards mentaux non spécifiques liés à l'X que compte la région proximale des bras longs du chromosome X humain

Learning and memory formation are based on the mechanisms of synaptic plasticity. Synaptic plasticity modulates development and strengthening of new neuronal connections, or synapses, upon learning and is based on local protein synthesis next to activated neuronal spines. Thousands of mRNA species are transported from the soma of neuron to dendrites, in order to be translated on demand. The mechanisms of this mRNA transport remain poorly understood. Mutations of Fragile X-mental retardation protein (FMRP) cause a spectrum of mental retardation disorders. Among other functions, FMRP mediates signal-mediated mRNA transport and local translation in dendrites. In multiple attempts to understand how FMRP is implicated in mRNA transport, there were identified few motor protein candidates. Besides controversy in the literature, none of these motor proteins was demonstrated to bind directly and transport FMRP. In my PhD studies, I employed in vitro reconstitution assays, coupled to Total Internal Reflection (TIRF) microscopy, to test, which of the proposed candidate motors can transport FMRP along the microtubules and whether FMRP can co-transport mRNA molecules. In order to understand the biochemistry of FMRP-mediated mRNA transport, I have purified and tested motor proteins from three Kinesin subfamilies. In this PhD thesis, I am reporting that FMRP binds directly to and is transported by Kinesin-2 motor (KIF3A/C heterodimer), but not by the other tested motors. Mutational analysis of FMRP suggests that its C-terminal region plays the biggest role in Kinesin-2 binding, and that this interaction does not depend on the RGG box region, known to recognise the G-quadruplex structure of FMRP’s mRNA targets. These results suggest that mRNA and motor binding by FMRP are not mutually exclusive, and thus FMRP must be capable to mediate mRNA transport. I also show that KIF3A/C motor binds several mRNA targets, with and without G-quadruplex structure, and that G-quadruplex mRNA competes with FMRP for motor binding. These results raise many questions that I address in the Discussion part of the thesis. This work is the first of its kind, to my knowledge, to systematically test kinesin motor proteins for direct interaction with FMRP and to reconstruct an FMRP transport complex. I conclude that FMRP binds directly to the Kinesin-2 motor and that this complex moves processively along the microtubules. This complex is still missing its cargo, mRNA, which will be investigated beyond the scope of this PhD thesis. I analyse the speeds of used kinesin motors and compare them to the literature. In the end, I discuss possible reasons why FMRP was not binding the G-quadruplex mRNA in my experimental conditions and outline the caveats of in vitro reconstitution assays.
El aprendizaje y la formación de la memoria se basan en los mecanismos de plasticidad sináptica. La plasticidad sináptica modula el desarrollo y el fortalecimiento de nuevas conexiones neuronales, o sinapsis, al aprender y se basa en la síntesis local de proteínas al lado de las espinas neuronales activadas. Miles de especies de ARNm son transportadas desde el soma de la neurona a las dendritas, para ser traducidas bajo demanda. Los mecanismos de este transporte de ARNm siguen siendo poco conocidos. Las mutaciones de la proteína Fragile X-mental Retardation (FMRP) causan un espectro de trastornos de retraso mental. Entre otras funciones, FMRP media el transporte de ARNm inducido por señal y la traducción local en las dendritas. En múltiples intentos de comprender cómo FMRP está implicada en el transporte de ARNm, se identificaron algunos candidatos de proteínas motoras. Además de la controversia en la bibliografía, no se demostró que ninguna de estas proteínas se uniera directamente y transportara FMRP. En mis estudios de doctorado, he utilizado ensayos de reconstitución in vitro, junto con la microscopía de reflexión interna total (TIRF), para probar cuál de los motores candidatos propuestos puede transportar FMRP a lo largo de los microtúbulos y si FMRP puede co-transportar moléculas de ARNm. Para comprender la bioquímica del transporte de ARNm mediado por FMRP, he purificado y probado proteínas motoras de tres subfamilias de kinesina. En esta tesis doctoral, presento que FMRP se une directamente y es transportada por el motor Kinesin-2 (heterodímero KIF3A/C), pero no por los otros motores probados. El análisis de mutaciones de FMRP sugiere que su región C-terminal juega el papel más importante en la unión con Kinesin-2, y que esta interacción no depende de la región de RGG box, conocida por identificar la estructura de G.quadruplexde de los ARNm diana de FMRP. Estos resultados sugieren que FMRP es capaz de unirse al motor y transportar ARNm simultáneamente. También muestro una observación de que el motor KIF3A/C se une a varios ARNms, con y sin estructura de G-quadruplex, y que el ARNm con G-quadruplex compite con FMRP por la interacción con la proteína motora. Estos resultados plantean muchas preguntas que abordo en la parte de Discusión de esta tesis. Este trabajo es el primero de su tipo, en mi conocimiento, para probar sistemáticamente la interaccion directa de FMRP con las proteínas motoras de kinesina y para reconstruir un complejo de transporte de FMRP. Concluyo que FMRP se une directamente al motor Kinesin-2 y que este complejo se mueve procesivamente a lo largo de los microtúbulos. A este complejo todavía le falta su carga, ARNm, que se investigará más allá del alcance de esta tesis doctoral. Analizo las velocidades de los motores de kinesina utilizados ​​y los comparo con la bibliografía. Al final, discuto las posibles razones de por qué FMRP no se estaba uniendo el ARNm de G-quadruplex en mis condiciones experimentales y describo los escollos en utiliza de los ensayos de reconstitución in vitro.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro
O retardo mental (RM) é caracterizado por um funcionamento intelectual significantemente abaixo da média (QI<70). A prevalência de RM varia entre estudos epidemiológicos, sendo estimada em 2-3% da população mundial, constituindo assim, um dos mais importantes problemas de saúde pública. Há um consenso geral de que o RM é mais comum no sexo masculino, um achado atribuído às numerosas mutações nos genes encontrados no cromossomo X, levando ao retardo mental ligado ao X (RMLX). Dentre os genes presentes no cromossomo X, o Jumonji AT-rich interactive domain IC (JARID1C) foi recentemente identificado como um potencial candidato etiológico do RM, quando mutado. O JARID1C codifica uma proteína que atua como uma desmetilase da lisina 4 da histona H3 (H3K4), imprescindível para a regulação epigenética. Tão recente como a identificação do gene JARID1C, é a descoberta de que mudanças no número de cópias de sequências de DNA, caracterizadas por microdeleções e microduplicações, poderiam ser consideradas como razões funcionalmente importantes de RMLX. Atualmente, cerca de 5-10% dos casos de RM em homens são reconhecidos por ocorrerem devido a estas variações do número de cópias no cromossomo X. Neste estudo, investigamos mutações no gene JARID1C, através do rastreamento dos éxons 9, 11, 12, 13, 15 e 16, em 121 homens de famílias com RM provavelmente ligado ao X. Paralelamente, realizamos a análise da variação do número de cópias em 16 genes localizados no cromossomo X através da técnica de MLPA no mesmo grupo de pacientes. Esta metodologia consiste em uma amplificação múltipla que detecta variações no número de cópias de até 50 sequências diferentes de DNA genômico, sendo capaz de distinguir sequências que diferem em apenas um nucleotídeo. O DNA genômico foi extraído a partir de sangue periférico e as amostras foram amplificadas pela técnica de PCR, seguida da análise por sequenciamento direto. Foram identificadas três variantes na sequência do gene JARID1C entre os pacientes analisados: a variante intrônica 2243+11 G>T, que esteve presente em 67% dos pacientes, a variante silenciosa c.1794C>G e a mutação inédita nonsense c.2172C>A, ambas presentes em 0,82% dos indivíduos investigados. A análise através do MLPA revelou uma duplicação em um dos pacientes envolvendo as sondas referentes ao gene GDI1 e ao gene HUWE1. Este trabalho expande o estudo de mutações no gene JARID1C para a população brasileira ereforça a importância da triagem de mutações neste gene em homens portadores de RM familiar de origem idiopática, assim como, é primeiro relato científico relativo à investigação de variações no número de cópias de genes localizados no cromossomo X em homens brasileiros com RM, através da técnica de MLPA.
Mental retardation (MR) is defined as a disability characterized by significant below average intellectual functioning (IQ>70). The prevalence of MR varies between epidemiological studies, estimated at 2-3% of the population, thus constituting a major public health problem. There is a general consensus that MR is more common in males, a finding attributed, in part, to mutations in the genes located on the X chromosome, leading to an X-linked mental retardation (XLMR). Among all the genes present on X chromosome, Jumonji AT-rich interactive domain IC (JARID1C) was recently identified as aetiologic potential candidate of MR, when mutated. The JARID1C gene encodes a protein that acts as a histone demethylase specific for histone 3 lysine 4 (H3K4) and it is indispensable for the epigenetic regulation. As recently as the identification of the JARID1C gene, it is the discovery that changes in the number of copies of DNA sequences, characterized by microdeletions and microduplications, could be regarded as functionally important reasons to XLMR. Currently, about 5-10% of men MR cases are known to occur due to these variations in the number of copies of chromosome X. In this study we investigated mutations in the JARID1C gene by screening of exons 9, 11, 12, 13, 15 and 16 in 121 patients from families with X-linked MR. At the same time we analyzed the variation in the number of copies in 16 genes located in X chromosome through the MLPA technique. This metodology consists of a multiplex amplification that detects variations in the number of copies up to 50 different genomic DNA sequences, being able to distinguish sequences that differ by only one nucleotide. Genomic DNA was extracted from peripheral blood and the samples were amplified by PCR followed by direct sequencing analysis. We identified three sequence variants among 121 patients. The intronic variant c.2243 +11 G> T, which was present in 67% of patients analyzed, the silent variant c.1794C> G and the novel nonsense mutation c.2172C> A, which was present in 0,82% of patients analyzed. The MLPA analysis revealed that the patient 58 exhibited a duplication involving probes for the GDI1 gene and the HUWE1 gene, resulting in an increase in the number of copies of this gene. This work expands the study of mutations in the JARID1C gene for the Brazilian population and reinforces the importance of screening for mutations in this gene in men with idiopathic mental retardation, and it is the first scientific report on the investigation of variations in the number of copies in genes located on chromosome X in Brazilian men with MR using the MLPA technique.

Orientador: Paulo Dalgalarrondo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: As síndromes genéticas de Williams (SW), do X-Frágil (SXF) e de Prader-Willi (SPW) apresentam déficit cognitivo geral que varia do grau leve ao moderado. Apesar de compartilharem rebaixamento da inteligência como um todo, prejuízos e potencialidades em habilidades cognitivas específicas dessas síndromes são amplamente descritos na literatura internacional, mas não tão enfaticamente no Brasil. Este estudo teve por objetivo investigar, descrever e comparar o perfil cognitivo de crianças e adolescentes com SW, SXF e SPW. Trinta e quatro crianças e adolescentes de seis a 16 anos, de ambos os sexos, com diagnóstico confirmado para a SW (n = 10), a SXF (n = 13) e a SPW (n = 11), pacientes dos ambulatórios de Psiquiatria da Criança ou Adolescente e/ou Genética Geral II do Hospital de Clínicas (HC) da Unicamp participaram deste estudo. Os sujeitos foram avaliados em suas funções cognitivas por meio da Escala de Inteligência Wechsler para Crianças (WISC-III). Dados sócio-culturais, exames citogenéticos e os sintomas e diagnósticos psiquiátricos associados aos participantes foram coletados nos prontuários médicos e também por meio de entrevistas com os responsáveis por cada criança ou adolescente. O QI total (QIT), o QI verbal (QIV), o QI de execução (QIE) e os escores ponderados de cada subteste da escala WISC-III, assim como os dados pessoais dos sujeitos de pesquisa, foram transpostos para o software estatístico SPSS, versão 17 para Windows. Não foram encontradas diferenças significativas em relação à idade, à classe social e ao tipo de escola que cada participante freqüenta entre as três síndromes. Por outro lado, houve diferença significativa quanto ao gênero dos participantes (p < 0,05). Foram encontradas diferenças significativas em relação ao QIV, aos subtestes verbais Informação, Vocabulário, Compreensão e em relação aos subtestes de execução Cubos e Armar Objetos. O teste post hoc de comparações múltiplas de Dunn (? = 0,05) apontou um escore significativamente superior nos subtestes de linguagem verbal e no QIV para o grupo com SW e um escore significativamente superior nos subtestes visuo-espaciais para o grupo com SPW. Estes resultados dão suporte à noção do perfil cognitivo específico para estas síndromes genéticas, constituído por "picos e vales" de rendimento, apesar do déficit intelectual geral destas condições, contrariando a concepção do fator g em crianças e adolescentes com SW, SXF e SPW
Abstract: Genetic syndromes such as Williams (WS), Fragile X (FXS) and Prader-Willi syndrome (PWS) present general cognitive impairment ranging from mild to moderate. Despite sharing a diminishment of intelligence as a whole, strengths and weakness in specific cognitive abilities of each syndrome are well described in international literature, but not so emphatically in Brazil. This study aimed to investigate, describe and compare the cognitive profile of children and adolescents with WS, FXS and PWS. Thirty-four children and adolescents, aged between 6 and 16, of both genders, with a confirmed diagnosis of either WS (n = 10), FXS (n = 13) or PWS (n = 11), from the outpatient clinics of Child and Adolescent Psychiatry or from the General Genetics II clinic, located at the Hospital das Clinicas (HC), participated in this study. The subjects cognitive functions were evaluated using the Wechsler Intelligence Scale for Children (WISC-III). Socio-cultural, cytogenetic tests and associated psychiatric symptoms and diagnoses were collected from the participants medical records and through interviews with those responsible for each child or adolescent. The Full-Scale IQ (FSIQ), Verbal IQ (VIQ), Performance IQ of (PIQ) and the standard scores of each subtest in the WISC-III scale, as well as the personal data of research subjects, were entered in version 17 of the SPSS statistical software for Windows. No significant differences were found between the three syndromes regarding age, social class or the type of school (private or public) each participant attends. On the other hand, there were significant differences in gender of the participants (p <0.05). Significant differences were found with respect to VIQ, and the verbal subtests Information, Vocabulary and Comprehension, and also in relation to the performance subtests Block Design and Object Assembly. The Dunn's multiple comparison test showed a significantly higher score on the verbal subtests and VIQ for the group with WS and a significantly higher score on the visuospatial subtests for the group with PWS. These results support the notion that there are specific cognitive profiles for these genetic syndromes, consisting of "peaks and valleys" in performance, despite the general intellectual deficit of these conditions, contrary to the g factor concept in children and adolescents with WS, FXS and PWS
Mestrado
Saude da Criança e do Adolescente
Mestre em Ciências

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand
Mental retardation (MR) is estimated to affect ~2-3% of the general population and may result from genetic causes and/or environmental causes. X-linked mental retardation (XLMR) is a heterogeneous group of disorders with a broad range of phenotypes and can be classified into syndromic XLMR (S-XLMR) and nonsyndromic XLMR (NS-XLMR) types. A number of X-linked genes have been identified that are associated with the different forms of XLMR. In an attempt to refine the diagnostic service to patients with XLMR, the Division of Human Genetics, Molecular Laboratory at the National Health Laboratory Service (NHLS) has investigated a number of associated X-linked genes. The main objective of this project was to investigate three genes, FMR2, XNP and ARX, associated with NS-XLMR and their contribution to XLMR in the South African (SA) population of MR males. Patients from different ethnic groups, referred to the Division of Human Genetics for fragile X MR syndrome that tested negative for the FMR1 expansion mutation were investigated for mutations in these genes. In addition, a cohort of Black institutionalized males was also investigated. The FMR2 expansion mutation responsible for fragile X E syndrome was not identified in 1194 FMR1 expansion negative MR male patients. FMR2 allele distribution analysis showed that a GCC repeat size of 15 was common in the MR cohort, accounting for 42% of alleles identified. From a total of 210 FMR1 expansion negative MR male subjects screened for mutations in a hotspot region (exons 7, 8 and 9) of the XNP gene, none was found to have a mutation in this region. Two patients from a cohort of 868 FMR1 expansion negative MR males were found to have a mutation in the ARX gene – one patient tested positive for the common 24 bp duplication mutation and a second patient appeared to have a deletion in the region amplified. These results indicate that the FMR2, XNP and ARX genes do not contribute significantly to MR in the SA population. As a result of this study, routine DNA testing for the FMR2 expansion, mutation screening in the hotspot region of the XNP gene and screening for the common 24 bp duplication mutation in the ARX gene in FMR1 expansion negative MR male subjects will not be implemented. A retrospective analysis was also done on 1862 probands referred to the Molecular Genetics Diagnostic service from 1992 to 2009 for fragile X MR syndrome testing. The FMR1 full expansion mutation was detected in 6.2% of probands, higher than reported worldwide figures. FMR1 allele distribution analysis in a cohort of 1184 FMR1 expansion negative MR males showed that 29 CGG repeats was the most frequent repeat size observed, accounting for 32.6% of all alleles in the cohort. The analysis of FMR1 alleles in MR males shows a similar distribution between different ethnic groups and compares well with other reported studies. This study reinforces the presence of fragile X MR syndrome in the SA Black population. Molecular investigations were also undertaken on 3 patients clinically suspected to have X-linked -thalassaemia mental retardation syndrome (ATR-X) and extended family members. Mutations were identified in each of the patients – two patients were found to have a novel mutation in the XNP gene and the third patient had a common XNP mutation. As a result, carrier testing and prenatal diagnosis was made possible in these families. A large family positive for the FMR1 expansion causing fragile X A MR syndrome was investigated. As an incidental finding, 2 females were found to be compound heterozygotes for 2 FMR1 alleles. Extended family members were tested and their FMR1 status was determined. Haplotype analysis was used to track the high-risk X chromosome in the family. As a result of this investigation, females at risk for premature ovarian failure and fragile X tremor ataxia syndrome have been identified. The approach to testing genes implicated in NS-XLMR has to be refined to allow for a cheaper and more efficient alternative. The use of newer techniques such as CGH microarray and MLPA has allowed for better detection of mutations. Delineating the causes of MR and their molecular and cellular consequences will assist families but also provide insight into the mechanisms that are required for the normal development of cognitive functions in humans.