Dissertations / Theses on the topic 'Fragile X syndrome; X-linked'

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.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Education (School Psychology)." Discipline: Education; Department/School: Education.

A series of empirical studies is presented that examine the contribution of Fragile X Mental Retardation 1 (FMR1) gene expression to the structure and function of the visual system. This contribution is documented using a histological approach in human and nonhuman primate tissue in conjunction with psychophysical testing of Fragile X Syndrome affected patients who are lacking FMR1 expression.
In the first set of experiments, immunohistological studies of unaffected human and primate brain tissue were carried out to reveal the staining pattern for Fragile X Mental Retardation Protein (FMRP), the protein product of the FMR1 gene, within the two main subcortical pathways at the level of the lateral geniculate nucleus (LGN). FMRP is expressed in significantly greater quantity within the magnocellular (M) neurons of the LGN when compared to levels obtained from the parvocellular (P) neurons. This finding suggests that M neurons depend on FMRP to greater extent than P neurons for determining their normal structure and function. A subsequent histological analysis of the LGN from a FXS affected individual revealed atypical LGN composed of small-sized neurons that were more P- than M-like. This result supports the notion that with the lack of FMR1 expression as occurs in FXS, the impact is greatest to M neuron morphology.
A second set of experiments explored the idea that the M neuron pathology in FXS results in a functional deficit for processing of visual information carried by this pathway. Detection thresholds for stimuli known to probe either M or P-pathway integrity were obtained from individuals affected by FXS as well as age- and developmental-matched control participants. In support of this hypothesis, FXS affected individuals displayed significantly elevated thresholds for M-but not P-specific achromatic visual stimuli. The selectivity of this deficit was verified in a consequent experiment that evaluated colour vision, a visual attribute known to be exclusively processed by the P-pathway. Affected individuals did not differ significantly from developmental-matched control participants in their ability to detect chromatic stimuli. Finally, the effect of the M pathway deficit on cortical visual function was assessed. Results of these experiments reveal that the thresholds for detection of coherent motion, but not form, are significantly elevated in the FXS group. This finding suggests that the parietal (dorsal) visual stream, the major cortical recipient of input from the M pathway, is detrimentally affected in FXS.
A third experiment examines the extent to which the M pathway deficit impacts on cortical visual functioning by employing stimuli of varying complexity that probe the parietal (dorsal) and temporal (ventral) visual streams separately. Results suggest that FXS affected individuals have a pervasive deficit in their ability to detect both simple and more complex forms of motion. In contrast, these same individuals have normal detection thresholds for simple form stimuli. However, with more complex form stimuli affected individuals have significant elevations in threshold. Taken together these results support the notion that the M pathway deficit is amplified at higher levels of visual processing and further, that FXS affected individuals have difficulties integrating all early visual information.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第8739号
論農博第1951号
新制||農||691(附属図書館)
学位論文||H6||N2760(農学部図書室)
UT51-94-Z490
(主査)教授 小田 順一, 教授 左右 田健次, 教授 駒野 徹
学位規則第4条第2項該当

Le but de ce travail est l'étude de la connectivité anatomique et fonctionnelle desréseaux neuronaux et le développement des nouveaux outils à cet effet. Car le dernieraspect est une préoccupation majeure de la neuroscience actuelle, nous avonsdeveloppé d'abord un nouveau traceur virale permettant la reconstruction neuronale.Nous avons ensuite appliqué cet et d'autres techniques pour sonder les défauts deconnectivité neuronale dans le syndrome de l'X fragile.Dans la première partie, nous avons discuté les avantages et inconvénients d'unetechnique émergente en utilisant un nouveau type de vecteur viral qui permet uneunique application pour l’étude du cerveau.Dans la deuxième partie, nous avons développé, au départ de ce vecteur viral, unenouvelle variante de faciliter le traçage et reconstruction des caractéristiquesmorphologiques de neurones. Nous avons montré la force de cette varianteantérograde du virus de la rage recombinant glycoprotéine supprimé pour lareconstruction de calcul de toutes les caractéristiques morphologiques clés deneurones: les dendrites, épines, les axones longs envergure dans tous les terminaux ducerveau et les boutons.Dans la troisième partie, nous avons examiné les modifications dans la connectivitédes structures cérébrales dans le syndrome du X fragile (FXS). FXS est le retardmental héréditaire la plus fréquente et la forme génétique la plus fréquente del'autisme, ce qui conduit à l'apprentissage et de la mémoire des déficits, lescomportements répétitifs, des convulsions et une hypersensibilité à des stimulisensoriels (visuels). Une des hypothèses éminents dans le domaine de l'autismesuppose une phénotype de hyper-connectivité locale mais de hypo-connectivité pourles connexions longue portée. Pour tester cette hypothèse dans un modèle de sourisFXS nous avons utilisé l'imagerie par résonance magnétique, pour balayer la totalitédu cerveau et de mesurer la connectivité anatomique et fonctionnel. Cela nous apermis d'identifier des altérations de connectivité dans plusieurs domains. Après nous8avons utilisé des traceurs viraux pour explorer un de ceux domains plus detaillé. Enutilisant le virus de la rage rétrograde à quantifier le nombre de neurones projetantvers ces zones, nous avons confirmé une connectivité d'entrée modifié pour le cortexvisuel primaire, ce qui pourrait contribuer au traitement visuel altéré de l'information.Nous avons découvert une connectivité réduite à longue portée globale anatomique etfonctionnelle entre plusieurs régions du cerveau, l'identification FXS comme unepathologie de la connectivité neuronale, ce qui pourrait expliquer les difficultés deplusieurs stratégies de sauvetage visant des cibles moléculaires sont actuellementconfrontés
The goal of this work was the investigation of the anatomical and functionalconnectivity of neuronal networks and the development of novel tools for thispurpose. Since the latter aspect is a major focus of current neuroscience, we firstsought a novel viral tracer enabling sparse neuronal reconstruction and neuronclassification. We then applied this and other techniques to probe neuronalconnectivity defects in Fragile X Syndrome.In the first part we discussed the merits and drawbacks of a emergingtechnique using a new type of viral vector that allows in a unique manner mapping ofthe input of a given brain area.In the second part we developed, departing from this viral vector, a newvariant to facilitate the tracing and reconstructing of morphologic features of neurons.We showed the strength of this anterograde variant of the recombinant glycoproteindeletedrabies virus for computational reconstruction of all key morphologicalfeatures of neurons: dendrites, spines, long-ranging axons throughout the brain andbouton terminals.In the third part we examined alterations in the wiring of brain structures inthe Fragile X Syndrome (FXS). FXS is the most common inherited mental retardationand most frequent genetic form of autism, leading to learning and memory deficits,repetitive behavior, seizures and hypersensitivity to sensory (e.g. visual) stimuli. Oneof the eminent hypotheses in the autism field assumes a local hyper- connectivityphenotype but hypo-connectivity for long-ranging connections. To test this hypothesisin a FXS mouse model we used magnetic resonance imaging, to scan the entire brainand measure the anatomical and functional connectivity. This allowed us to identifyconnectivity alterations in several areas that we further explored using viral tracers.Using retrograde rabies virus to count the number of neurons projecting to such areaswe confirmed an altered input connectivity to the primary visual cortex, which couldcontribute to the altered visual information processing. We discovered an overallreduced anatomical and functional long-range connectivity between several brainareas, identifying FXS as pathology of neuronal connectivity, which might explain thedifficulties several rescue strategies aiming at molecular targets are currently facing

Le Syndrome de l'X Fragile (FXS) est la forme héréditaire majoritaire dedéficience intellectuelle et la cause monogénique de l'autisme. Le FXS est causé par unemutation du gène Fragile X Mental Retardation 1 (Fmr1), qui entraîne son inactivationet l'absence d’expression de la protéine codée: Fragile X Mental Retardation Protein(FMRP). FMRP est une protéine de liaison à l’ARN, impliquée dans la régulation de lasynthèse protéiques à la synapse. Un rôle central est attribué au sous-type 5 desrécepteurs métabotropiques au glutamate du groupe I (mGluR5) dans laphysiopathologie du FXS. En effet, une réponse exagérée suite à l'activation de mGluR5pourrait expliquer le dysfonctionnement synaptique dans ce syndrôme. Bien que denombreux travaux aient mis l'accent sur la dérégulation de la synthèse des protéinessynaptiques comme une conséquence de cette signalisation accrue du mGluR5, il y aaussi un équilibre altéré dans l'association de mGluR5 avec les différentes isoformes desprotéines Homer, partenaires de densité post-synaptique (PSD) du mGluR5. Bien qu'uneabondante littérature décrit l'association mGluR5/Homer, les conséquences de laperturbation de cette interaction dans le contexte du FXS sont peu connues. Parconséquent, l'objectif de ma thèse était d'étudier les conséquences de la perturbation del’interaction mGluR5/Homer au niveau des propriétés et des fonctions de mGluR5, tellesque l'expression durant le développement, l'expression de surface et le ciblageaxonal/dendritique, l’internalisation déclenchée par l'agoniste, les dynamiques desurface, et la modulation des courants NMDAR induite par mGluR5.Dans un premier temps, nous avons étudié l’expression de surface de mGluR5dans des neurones hippocampiques in vitro issus de souris sauvages et Fmr1 KO, par destechniques d’immunofluorescence et de biotinylation. Nous avons constaté que mGluR5est plus exprimé à la surface neuronale et est différemment distribué dans les dendrites etles axones des neurones Fmr1 KO. Puis, nous avons démontré que cette altérationd’expression et de ciblage est une conséquence directe de l’altération de l’interactionmGluR5/Homer. Nous avons aussi observé que mGluR5, indépendamment del’altération de l’interaction mGluR5/Homer, ne subit pas d’internalisation suite sonactivation soutenue par DHPG dans les neurones Fmr1 KO.Dans la seconde partie de mon étude, nous avons étudié les conséquences de laperturbation de l’interaction mGluR5/Homer dans les dynamiques de surface de mGluR5et par conséquent pour la fonction du NMDAR dans les neurones Fmr1 KO. Par destechniques d'imagerie et de pistage moléculaire, nous avons constaté que l’altération ducomplexe mGluR5/Homer augmente spécifiquement la diffusion latérale à la synapsedes neurones hippocampiques Fmr1 KO in vitro.La mobilité élevée du mGluR5 conduit à une probabilité accrue d'une interactionphysique transitoire avec NMDAR dans la PSD du Fmr1 KO.Cette interaction altère la modulation, induite par mGluR5, des courantsNMDAR. En effet, en utilisant des enregistrements en patch-clamp de neuronespyramidaux de CA1 sur tranches couplés à la stimulation des fibres collatérales deSchaffer, nous avons constaté que les courants excitateurs post-synaptiques induits parNMDAR (NMDAR-EPSCs) présentent des amplitudes plus faibles dans les neuronesFmr1 KO. De plus, l'expression post-synaptique de mGluR5, induite par la dépression àlong-terme de NMDAR-EPSCs est réduite dans les neurones Fmr1 KO. Finalement,nous avons démontré que ces défauts des courants NMDAR sont dépendants de laperturbation de l’interaction mGluR5/Homer et altèrent les dynamiques de mGluR5.Cette étude pourrait avoir des conséquences dans le traitement desdysfonctionnements synaptiques du mGluR5 dans le FXS, en ciblant l’interactionmGluR5/Homer, et offre de nouvelles suggestions pour corriger la signalisationdéfectueuse sous-jacente aux troubles du spectre autistique
Fragile X Syndrome (FXS) is the most common inherited form of intellectualdisability and autism. FXS is caused by a mutation in the fragile X mental retardation 1(Fmr1) gene which leads to the lack of the encoded FMRP protein. FMRP is an RNAbinding protein involved in protein synthesis regulation at synapses. Many evidencessuggest a central role of the Group-I metabotropic glutamate receptor subtype 5(mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling responsefollowing mGluR5 activation may underlie synaptic dysfunction in this disorder.Although much work has focused on the dysregulation of synaptic protein synthesis as aconsequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there isalso an altered balance of mGluR5 association with Homer scaffolding proteins, whichare postsynaptic density (PSD) partners of mGluR5. Although an extensive literaturedescribes the mGluR5/Homer association, very little is known about the consequences ofthe disruption of this interaction in the FXS context. Therefore, the goal of my thesis wasto study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout(KO) mouse model of FXS in terms of properties and functions of mGluR5, such asexpression during development, surface expression and axonal/dendritic targeting,agonist-induced internalization, surface dynamics and mGluR5-mediated modulation ofNMDA receptor (NMDAR) currents.In a first set of experiments we investigated the mGluR5 surface expression incultured hippocampal neurons from WT and Fmr1 KO mice by usingimmunofluorescence techniques and biotinylation assay. We found that mGluR5 wasmore expressed on the neuronal surface and was differently distributed in dendrites andaxons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were adirect consequence of the mGluR5/Homer crosstalk disruption. We demonstrated thatthe altered expression and targeting of mGluR5 were critically dependent onmGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergointernalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons.This latter phenotype, however, was not dependent on the disruption of themGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homercrosstalk disruption contributes to the pathophysiology of FXS altering expression andtargeting of mGluR5 on the surface of Fmr1 KO neurons.In the second part of my study we investigated the consequences of the disruptedmGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently forNMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging andsingle-molecule tracking, we found that mGluR5/Homer crosstalk disruption specificallyincreased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampalneurons. The higher mGluR5 mobility resulted in an increased probability of transientphysical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered themGluR5-mediated modulation of NMDAR currents as evidenced by the two followingchanges. First, using patch-clamp recordings from CA1 pyramidal neurons, we foundthat NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked bySchaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second,the postsynaptic expression of mGluR5 mediated long term depression (LTD) ofNMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that thesedefects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalkdisruption and altered mGluR5 dynamics.Altogether, our results show that mGluR5/Homer disruption contributes to themGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for thetreatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homerinteraction and provide new suggestions to correct the defective signaling underlyingcognitive impairment and autism

Mon projet de thèse vise à élucider les conséquences physiopathologiques d'une mutation faux sens sur le gène FMR1 identifiée chez des patients atteints du syndrome du X fragile (SXF). Le SXF est la forme la plus fréquente de déficience intellectuelle (DI) héréditaire et l'une des principales causes monogéniques connues d’autisme pour laquelle il n'existe pas de traitement efficace. Cette maladie est généralement causée par une expansion anormale du triplet CGG dans le 5' UTR du gène FMR1, entraînant son extinction transcriptionnelle et en conséquence, la perte d'expression de la protéine FMRP. FMRP est une protéine liant les ARNm et les transportant dans des granules le long des dendrites jusqu’à la base des synapses actives pour permettre la régulation de la traduction locale. Une des caractéristiques principales du SXF est la surabondance de protrusions dendritiques immatures qui conduisent à des déficits de transmission et de plasticité synaptiques. Plusieurs mutations faux sens du gène FMR1 ont été récemment identifiées chez les patients atteints de SXF. Trois patients sans lien de parenté, dont une femme, présentaient la mutation R138Q dans FMRP. Il est intéressant de noter que cette mutation se localise près d'un des sites actifs de la SUMOylation de FMRP, le résidu de lysine 130 (K130). Nous avons démontré que la SUMOylation de FMRP en réponse à l’activation des récepteurs mGlu5R est responsable de sa dissociation des granules d'ARNm, permettant ainsi la libération et la traduction locale de ses cibles d'ARNm. En retour, les épines dendritiques surnuméraires sont éliminées et/ou maturées. Nous avons donc émis l'hypothèse que la mutation R138Q pourrait modifier la régulation de la SUMOylation de FMRP et par conséquent, sa fonction synaptique, participant ainsi à l'étiologie du SXF chez ces patients. À cette fin, nous avons généré un modèle de souris knock-in (R138Q-KI) portant la mutation humaine FMRP-R138Q. Lors de ma thèse, j'ai caractérisé ce nouveau modèle de souris sur le plan moléculaire, cellulaire, électrophysiologique et comportemental
My PhD project aims to unravel the pathophysiological consequences of an FMR1 missense mutation identified in Fragile X Syndrome (FXS) patients. FXS is the most frequent form of inherited Intellectual Disability (ID) and a leading monogenic cause of autism for which there are no effective therapies available. This disorder is typically caused by an abnormal expansion of CGG repeats within the 5’ UTR of the FMR1 gene, resulting in its transcriptional silencing and consequently, the loss-of-expression of the FMRP protein. FMRP is an RNA-binding protein that transports mRNAs in granules along dendrites to the base of active synapses for local translation in an activity-dependent manner. A hallmark of FXS is the hyper-abundance of immature dendritic protrusions, leading to synaptic transmission and plasticity deficits. Several FMR1 missense mutations were recently identified in FXS patients. Among them, three unrelated patients presented the same R138Q mutation, including one female. Interestingly, this mutation localizes close to one of the active SUMO sites of FMRP, the lysine 130 (K130) residue. We showed that the mGlu5R-dependent sumoylation of FMRP triggers its dissociation from mRNA granules, allowing the release and local translation of its mRNA targets which in turn, regulate spine maturation and elimination. We thus hypothesized that the R138Q mutation may alter the mGlu5R-dependent FMRP sumoylation and consequently, its synaptic function, thereby participating in the aetiology of FXS in these patients. To this purpose, we generated an Fmr1 R138Q knock-in (R138Q-KI) mouse model. Here, I present the results obtained during my PhD on the characterization of this novel mouse model at the molecular, cellular, electrophysiological and behavioural levels

Thesis (M.A.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Oct. 5, 2009). "... in partial fulfillment of the requirements for the degree of Master of Arts in the School of Education Early Childhood, Intervention and Literacy." Discipline: Education; Department/School: Education.

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

In the present study, performance on a range of mathematical reasoning and number processing tasks was assessed across two syndrome groups for which numerical ability is under-researched: Fragile X syndrome and Down syndrome. Given the paucity of current research, it was unknown whether all aspects of arithmetic and number processing would be globally affected across groups or whether there would be syndrome specific proficiencies and deficiencies. Statistical analysis revealed that males with fragile X syndrome performed significantly worse on all tasks even when performance was compared to typically developing children of a similar developmental level. However, when performance was compared to children with Down syndrome differing profiles emerged, with greater weaknesses by the fragile X syndrome males on specific tasks requiring mental arithmetic and basic numeracy skills. The importance of using syndrome specific information in the assessment of math disabilities and the design of early educational interventions are discussed.

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).

Faces provide important information necessary for social communication. The current study aimed to evaluate Event-Related Brain Potentials (ERPs) as a method of exploring face processing abilities in fragile X syndrome (FXS), a genetic disorder where social deficits lie at the core of the cognitive phenotype. Neural changes were investigated in three children with FXS across various conditions such as upright vs. inverted faces, intact faces vs. faces with no eyes as well as faces vs. cars. Relative to chronological age matched controls, children with FXS displayed greater N170 amplitudes and shorter latency peaks across conditions. In addition, the FXS group showed right hemispheric specialization for both face and non-face stimuli. Heightened electrophysiological responses in FXS are discussed in the context of reported hyper-sensitivity and arousal.

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

Cette étude explore les réponses évoquées, l'activité intrinsèque et spontanée de deux populations neuronales différentes dans la région du cerveau correspondant à la patte arrière des souris. Dans cet article, nous nous sommes concentrés sur un modèle murin du syndrome de l'X fragile (SXF), qui est la forme la plus commune de syndrome de retard mental héréditaire et une cause fréquente de troubles du spectre autistique (TSA). SXF est un trouble à gène unique (Fmr1), qui peut être modélisé de manière fiable par un modèle murin transgénique : la souris Fmr1-/y déficiente pour le gène codant Fmr1. L'hyperexcitabilité des réseaux néocorticaux et l'hypersensibilité aux stimuli sensoriels sont des caractéristiques importantes du SXF et des TSA.Ceci est directement lié à un changement du nombre de synapses locales, de canaux ioniques, de l'excitabilité membranaire et de la connectivité des circuits de cellules individuelles. Précédemment, nous avons identifié un défaut dans les canaux ioniques, comme pouvant contribuer à ces phénotypes. Nous avons testé cette hypothèse comme un mécanisme contribuant aux défauts de traitement sensoriel chez les souris Fmr1-/y. Le cortex somatosensoriel primaire de la souris (S1) traite différentes informations sensorielles et constitue la plus grande zone du néocortex, soulignant l'importance de la modalité sensorielle pour le comportement des rongeurs. Nos connaissances concernant le traitement de l'information dans S1 proviennent d'études du cortex en tonneaux lié aux moustaches, mais le traitement des entrées sensorielles des pattes postérieures est mal compris. Par l’utilisation de la technique d’enregistrement de cellule entière par patch clamp in vivo, nous avons classes les cellules en répondeurs supraliminaires (cellules qui répondaient aux stimulations de la patte arrière avec un potentiel d'action), les répondeurs subliminaires (les cellules qui répondaient sans déclencher un potentiel d'action) et les cellules non répondeuses qui ne présentaient aucune réponse. Puis, nous avons comparé les réponses évoquées sub et supraliminaires, les propriétés intrinsèques et l’activité spontanée des neurones pyramidaux de la couche 2/3 (L2/3) de la region S1 de la patte arrière (S1-HP) d’animaux anesthésiés sauvage (WT) et Fmr1-/y. Nous avons identifié des altérations de réponse spontanée, intrinsèque et évoquée chez les souris Fmr1-/y. L’application d’un ouvreur de canaux ioniques BKCa a restauré certaines de ces propriétés altérées chez les souris Fmr1-/y
This study explores the evoked responses, intrinsic and spontaneous activity of two different neuronal populations in the hind paw region of the primary somatosensory cortex (S1) of mice. Initially, we explored information processing in these neurons under normal physiological conditions, and subsequently in a mouse model of Fragile X Syndrome (FXS). FXS is the most common form of inherited mental retardation syndrome and a frequent cause of autism spectrum disorders (ASD). FXS is a single gene (Fmr1) disorder, which can be reliably modeled by a mutant mouse model, the Fmr1 knockout (Fmr1-/y) mouse. Hyperexcitability of neocortical networks and hypersensibility to sensory stimuli are prominent features of FXS and ASD. We previously established a strong causal link between a channelopathy, hyperexcitability of neurons in the primary sensory region of the neocortex and sensory hypersensitivity in this mouse model. In the current study, we extended these findings, by conducting a detailed exploration of the processing of tactile sensory information (evoked by hind paw stimulation) in the neocortex of these mice.Most of our knowledge regarding information processing in S1 comes from studies of the whisker-related barrel cortex (which processes tactile-related sensory information derived from the whiskers), yet the processing of sensory inputs from the hind-paws is poorly understood. Using in vivo whole-cell patch-clamp recordings, we classified the cells into suprathreshold responders (the cells which responded to the hind-paw stimulations with an action potential), subthreshold responders (the cells responded without eliciting an action potential) and non-responder cells (neurons which did not show any response). We then compared the evoked sub- and supra-threshold responses, intrinsic properties, and spontaneous activity of layer (L) 2/3 pyramidal neurons of the S1 hind-paw (S1-HP) region of anaesthetized wild type (WT) and Fmr1-/y mice. We identified spontaneous, intrinsic and evoked response alterations in Fmr1-/y mice. We probed possible mechanisms contributing to this sensory impairment in Fmr1-/y mice. Finally, we tested the possibility of correcting pathophysiological alterations in these neurons using specific pharmacological agents targeting the ion channel defects described previously by our team

Abstract Fragile X syndrome, the most common inherited form of mental retardation syndrome, is caused by an expansion of the CGG trinucleotide repeat in the 5' UTR of the FMR1 gene, with concurrent hypermethylation of the region, which represses FMR1 expression. The syndrome is associated with the folate-sensitive chromosomal fragile site at Xq27.3 (FRAXA), where the gene responsible for the syndrome was first localized by linkage analysis using RFLP markers. In this study the linkage relationships of the RFLP markersat Xq27-28 and the characteristics of the CGG repeat expansion were investigated in northern Finnish fragile X families and molecular diagnostic methods were applied in order to improve diagnosis of the syndrome. Furthermore, the origin of fragile X mutations in the northern part of Finland was studied by haplotype analysis. Linkage studies were performed in 34 northern Finnish fragile X families/pedigrees using a total of 15 RFLPs (defining 11 loci). A refined genetic map around FRAXA including five RFLP markers having recombination fractions of 0.04 or less with FRAXA was obtained in an international study of 112 affected families, containing linkage data on twelve northern Finnish families. Linkage analysis significantly improved carrier detection in fragile X families compared with previous cytogenetic methods used in diagnosis. The most efficient RFLP-based protocol for carrier detection was proposed, which is based on use of the most adjacent markers and a minimum number of restriction enzymes. CGG repeat expansion of the FMR1 gene was investigated in original families collected for linkage studies and additional new ones. Large CGG repeat expansions (Δ > 500 bp) with concomitant methylation of the adjacent CpG island, i.e. full mutations, were found to be associated with mental retardation completely in males, but only 50% of the females having a full mutation were mentally impaired. Premutations (Δ < 700 bp) were found in healthy carriers. There was a size range of Δ = 500 to 700 bp, where the expansions could be either abnormally methylated or non-methylated, and it appeared that methylation is more important in determining the phenotype than the exact size of an expansion. Instability of the enlarged CGG repeats was detected, leading preferentially to size increases in successive generations. The instability of premutations was found to be stronger and the size increases larger in maternal than in paternal transmissions, and transition to a full mutation occurred only in female transmissions. In addition, the size of a maternal premutation was shown to have an important influence on the risk of its transition to a full mutation when transmitted. The critical premutation size leading invariably to full mutation in the offspring was found to be between Δ = 175 to 200 bp. In one of the studied families a rare contraction of a paternal premutation to a normal CGG repeat number in one of the daughters and further in her son was detected. Direct mutation analysis including measurement of the CGG repeat size and hypermethylation allowed unambiguous diagnosis of carriers and affected individuals in most cases. Haplotype analysis using two tightly linked microsatellite markers flanking the CGG repeat mutation was performed in 60 unrelated northern and eastern Finnish fragile X families. A significant difference was found in allelic and haplotypic distributions between normal X and fragile X chromosomes. A single haplotype, which was present only in 8% of the normal X chromosomes, accounted for 80% of the fragile X chromosomes. This enrichment of one fra(X) mutation in the Finnish population suggests founder effect.

Fragile X syndrome (FXS) is the most commonly inherited form of intellectual disability as well as a leading genetic cause of autism spectrum disorder. It is typically the result of a trinucleotide repeat expansion in the Fmr1 gene which leads to loss of the encoded protein, fragile X mental retardation protein (FMRP). Animal model studies over the past twenty years, mainly focusing on the Fmr1 knockout (KO) mouse, have uncovered several cellular and behavioural phenotypes associated with the loss of FMRP. Seminal work using the Fmr1 KO mouse found that metabotropic glutamate receptor mediated long-term depression (mGluR-LTD) in the hippocampus is both exaggerated (Huber et al., 2002) and independent of new protein synthesis (Nosyreva & Huber, 2006). These findings, together with studies focusing on other brain regions including the prefrontal cortex (Zhao et al., 2005) and amygdala (Suvrathan et al., 2010), have contributed to the ‘mGluR theory of FXS’ (Bear et al., 2004) which suggests that group 1 metabotropic receptor function is exaggerated in FXS. The development of genetically modified rats allows the modelling of FXS in an animal model with more complex cognitive and social behaviours than has been previously available. It also provides an opportunity for comparison of phenotypes across mammalian species that result from FMRP deletion. While the study of Fmr1 rats can significantly contribute to our understanding of FXS, we must first confirm the assumption that cellular phenotypes are conserved across mouse and rat models. In this thesis, we first aimed to test if the key cellular and synaptic phenotypes that contribute to the ‘mGluR theory of FXS’ are conserved in both the hippocampus and amygdala of Fmr1 KO rats. In agreement with mouse studies, we found mGluR-LTD was both enhanced and independent of new protein synthesis in Fmr1 KO rats. Similarly, group 1 mGluR long-term potentiation (LTP) was significantly decreased at both cortical and thalamic inputs to the lateral amygdala. Secondly, we investigated mPFC intrinsic excitability and synaptic plasticity in Fmr1 KO rats. The mPFC plays a key role in several of the cognitive functions that are affected in fragile X patients including attention, cognitive flexibility and anxiety (Goto et al., 2010). The regulation of mPFC plasticity and intrinsic excitability has also been associated with mGluR signalling. Here we found that intralaminar LTP in the mPFC showed an age-dependent deficit in Fmr1 KO rats. The mPFC also provides top down control of several cortical and subcortical regions through long-range connectivity. One pathway of interest in the study of FXS is mPFC-amygdala connectivity which is associated with fear learning and anxiety behaviours (Burgos- Robles et al., 2009). Using retrograde tracing, we showed layer 5 pyramidal neurons that provide long-range connections to the basal amygdala were intrinsically hypoexcitable in Fmr1 KO rats. This phenotype could possibly be explained through homeostatic changes in the axon initial segment which regulates neuronal excitability. This work provides the first evidence for conservation of cellular phenotypes associated with the loss of FMRP in mice and rats which will be key in the interpretation of future studies using Fmr1 KO rats. We also provide evidence of deficits in mPFC long-range connectivity to the basal amygdala, a pathway that is associated with FXS relevant behaviours. Together this highlights how study of the rat model of FXS can complement existing studies of Fmr1 KO mice as well as provide new insights into the pathophysiology resulting from the loss of FMRP. Some of this work was published in Till et al., 2015.

Fragile X syndrome (FXS) is the leading cause of inherited mental retardation and developmental delay. In the vast majority of cases, this X-linked disorder is due to a CGG expansion in the 5' untranslated region of the fmr-1 gene and the resulting decreased expression of its associated protein, FMRP (Fragile X associated Mental Retardation Protein). FXS is characterized by a number of cognitive, behavioural, anatomical, and biological abnormalities. This monogenic disorder provides a unique opportunity to study the consequence of a mutation in a single gene on the development and proper functioning of the CNS.
Histological work on FMRP expression in the monkey lateral geniculate nucleus (LGN) has revealed differential staining in the magnocellular and parvocellular layers, with increased expression in the magnocellular layers (Kogan, Boutet, Cornish, Zangenehpour, & Mullen, 2004). In individuals with fragile X, this differential expression pattern is correlated at the behavioural level with impairments in the M but not the P-visual pathway processing. These findings by Kogan and colleagues, led to the hypothesis that brain regions that express high levels of FMRP are particularly susceptible to its reduced expression, as occurs in FXS. It was therefore of interest to extend this work to determine the pattern of FMRP expression throughout the monkey brain, with the aim of identifying the brain structures most susceptible to reduced expression of the fmr-1 gene product.
The current focus on the role of FMRP in RNA translation and neuronal maturation makes it timely to assemble the extant information on how reduced expression of the fmr-1 gene leads to neuronal dysmorphology. The first section of this manuscript offers a summary of recent genetic, neuroanatomical, and behavioural studies of fragile X syndrome, and provides potential mechanisms to account for the pleiotropic phenotype of this disorder. The following section presents a detailed account of the FMRP expression profile in the monkey brain, and reveals the striking correlation between the expression of the protein and behavioural deficits associated with its reduced expression, as occurs in FXS.
The last chapter of this manuscript offers insight into future trends in FXS research. A number of electrophysiological and behavioural studies point to a particular involvement of the metabotropic glutamatergic system in FXS, with a preeminent role for metabotropic glutamate receptor type V (mGluR5). The involvement of this receptor in FXS and the potential therapeutic implications of pharmacological regulation of this receptor will be discussed.
There is a body of work pointing to the remarkable behavioural similarities between FXS and autism. While FXS is a single-gene disorder, autism is associated with a number of genes, which have not yet been precisely identified. The final section of this manuscript delineates the neuroanatomical, behavioural, and linguistic overlap, as well as the differences between the two conditions. Finally, this section affords some insight as to how FXS, a single-gene disorder, may assist us in our understanding of autism.
Keywords: Fragile X Syndrome (FXS), fmr-1 mutation, lateral geniculate nucleus (LGN), anterior cingulated cortex (ACC), dentate gyros (DG), superior frontal gyrus (SFG), cerebellum, metabotropic glutamate receptor type V (mGluR5), postsynaptic density protein 95 (PSD-95), dendritic spine, autism
Abbreviations: fragile X syndrome (FXS), long-term potentiation (LTP), long-term depression (LTD), RNA-binding protein (RBP), lateral geniculate nucleus (LGN), anterior cingulated cortex (ACC), dentate gyrus (DG), superior frontal gyrus (SFG), metabotropic glutamate receptor type V (mGluR5), postsynaptic density protein 95 (PSD-95), region of interest (ROI), deep cerebellar nucleus (DCN).

Raising a child with a pervasive developmental disorder (PDD), particularly that of Fragile X Syndrome (FXS), is challenging, as it comes with parental stressors for both mothers and fathers. Research on these stressors has been limited to only the stressors that mothers of children with a PDD experience and has failed to thoroughly examine the experiences and stressors of fathers of children with a PDD, particularly that of FXS. Using Hill's ABC-X family stress theory, this quantitative research study investigated the effects of marital satisfaction due to the amount of shared childcare responsibilities and parental stress among the mothers and fathers of children diagnosed with FXS. This study also examined whether significant differences exist among these parents, who were recruited through the use of flyers, notices, and handouts that were randomly passed out to parents at the FXS Alliance of Texas located in the southwest region of Texas. Participants for this study were 128 parents of children with FXS, each of whom completed a demographic questionnaire, the Kansas Marital Satisfaction Scale, and The Sharing of Childcare Responsibilities Scale and Parental Stress Level Scale. An independent samples t test and multiple linear regression statistical analysis was employed. The results of the study indicated that parental stress associated with the amount of shared childcare responsibilities accounted for a significant degree of the variance in marital satisfaction. Yet the study did not find a significant mean difference in the level of parental stress that was experienced uniquely across gender. Potential social changes may include future development and improvements in treatment, therapeutic approaches, and predicted outcomes in efforts to enhance parental stress interventions so as to improve stress-related outcomes for parents of children with FXS.

Mise en contexte : Le syndrome du X fragile (SXF) résulte de la perte d’expression de la protéine FMRP. L’absence de FMRP est responsable d’une série de perturbations signalétiques, notamment une hyperactivation de la voie MAPK/ERK. La lovastatine, un médicament hypocholestérolémiant, possède comme effet pléiotrope la capacité d’inhiber la voie MAPK/ERK et a permis de corriger certains phénotypes pathologiques clés du modèle murin du SXF, mettant en lumière son potentiel thérapeutique chez l’humain. Ainsi, nous avons réalisé en 2013 une étude ouverte visant à étudier l’effet d’un traitement de 12 semaines à la lovastatine sur les troubles cognitifs et comportementaux des enfants et des adultes avec SXF. La plupart des individus ont présenté des améliorations cognitives et comportementales, telles qu’évaluées par les échelles cliniques Vineland-II Adaptive Behavior Scale (VABS-II) et Aberrant Behavior Checklist-Community (ABC-C), respectivement. Ces échelles remplies par les tuteurs et les soignants sont toutefois évaluateur-dépendantes et sujettes à l’effet expérimentateur. Ces variables parasites, qui s’ajoutent à l’effet placebo inhérent à la conception ouverte de l’essai thérapeutique, peuvent ainsi avoir faussé l’évaluation de la réponse au traitement. Nous avons donc étudié si les cascades signalétiques des plaquettes sanguines peuvent être utilisées comme biomarqueurs objectifs pour surveiller la réponse au traitement. Méthode : Des échantillons sanguins des 15 individus SXF ayant participé à l’essai clinique ont été recueillis au début et à la fin de l’étude afin d’évaluer par Western Blot l’effet in vivo de la lovastatine sur l’activité de ERK dans les plaquettes sanguines, et ainsi de pouvoir corréler les réponses biologiques et cliniques. L’état de phosphorylation de ERK a également été étudié dans les plaquettes d’une cohorte contrôle. Résultats : Nos résultats démontrent une augmentation significative de près du double de la phosphorylation basale de ERK dans les plaquettes sanguines des individus avec SXF en comparaison avec les sujets contrôles (p=0,002). De plus, nous avons observé une normalisation de la phosphorylation de ERK chez 13 des 15 individus SXF après le traitement de 12 semaines à la lovastatine (p=0,007). Notre étude fournit ainsi les premières évidences d’un effet bénéfique de la lovastatine dans le SXF chez l’humain. Nous avons également démontré que les changements de la phosphorylation de ERK étaient partiellement corrélés à la réponse clinique, et ce, pour le score total et les scores des sous-domaines ‘socialisation’ et ‘compétences de la vie quotidienne’ de l’échelle VABS-II (p=0,003). Conclusion : De façon générale, ces résultats suggèrent que les cascades signalétiques plaquettaires peuvent être utilisées comme biomarqueurs pour évaluer de façon objective la réponse au traitement lors de futurs essais thérapeutiques.
Abstract: Background: Fragile X syndrome (FXS) results from loss of FMRP expression, which causes several signaling dysregulations, including the hyperactivation of the Mitogen-activated protein kinase (MAPK)/Extracellular signal-regulated kinase (ERK) pathway. Lovastatin, a drug used for the treatment of hypercholesterolemia, pleiotropically inhibits the MAPK/ERK cascade and has successfully corrected key pathological phenotypes in the FXS mouse model, underscoring its ‘disease-modifying’ potential. Thereby, we conducted in 2013 the first open-label clinical trial investigating the effect of a 12-week lovastatin regimen on cognitive and behavioral disabilities in FXS. Most individuals presented subtle positive cognitive changes as assessed by the Vineland-II Adaptive Behavior Scale (VABS-II) as well as behavior improvements using the widely used scale Aberrant Behavior Checklist-Community (ABC-C). The latter two scales are filled up by caregivers making them rater-dependent and prone to observer-expectancy effect. This might result in a placebo effect which is inherent to the open-label design of the trial. We therefore investigated whether blood platelets’ signaling cascades may be used as objective biomarkers to monitor treatment response. Methods: Blood samples were gathered from 15 FXS individuals during the trial in order to evaluate by quantitative Western Blotting the in vivo effect of lovastatin on ERK activity in blood platelets, and to correlate clinical and biological responses. The basal phosphorylation status of ERK was also assessed in platelets from a control cohort. Results: Our results showed a more than two-fold significant increase in FXS blood platelet basal ERK phosphorylation as compared to controls (p=0.002). Of note, we found that this hyperphosphorylation was normalized following the 12-week lovastatin trial (p=0.007) in 13 of the 15 FXS individuals enrolled in the trial. This represents the first evidence for a beneficial effect of lovastatin in human FXS. The extent of changes in ERK phosphorylation was also found to partly correlate with the clinical response scales’ scores, especially for the VABS-II. Indeed, the composite total score and the ‘daily living skills’ as well as the ‘socialization’ subscales scores of the VABS-II were correlated with the biological response (p=0.03). In comparison, no correlation was observed with the ABC-C scale. Conclusion: Broadly, these results suggest that platelets’ signaling cascades could be used as biomarkers to objectively assess treatment response during future clinical trials.

Résumé: Mémoire présenté à la Faculté de médecine et des sciences de la santé en vue de l’obtention du diplôme de maître sciences (M.Sc.) en biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4. Le document présent est un mémoire par article, lequel explorera la fonction d'une protéine liant l'ARN, FMRP, dans la différenciation cellulaire. Cette protéine joue un rôle de premier plan puisque son absence conduit à des anomalies développementales durant la neurogenèse et à une plasticité synaptique déficiente. Ces anomalies sont observées chez la souris KO pour le gène FMR1, mais également dans le cerveau des individus avec le syndrome du X fragile (SXF). Ces derniers dont le gène FMR1 est muté présentent une déficience intellectuelle (DI). Puisque la DI est la principale manifestation du SXF, et que les neurones « normaux » expriment FMRP à des niveaux supérieures à ceux des autres tissus corporels, son rôle a presqu'exclusivement été étudié dans les cellules neuronales. Pourtant, FMRP est une protéine hautement conservée et est exprimée dans presque toutes les cellules du corps. Logiquement, FMRP devrait jouer un rôle important dans tous les tissus l’exprimant à des niveaux de base, bien que son absence dans les tissus ne se manifeste pas cliniquement. Cette polyvalence fonctionnelle est encore plus probable de par le fait que plusieurs ARNm différents ont la possibilité d’interagir avec FMRP puisqu’elle identifie ses cibles par reconnaissance de motifs. L'étude présentée se fonde sur l'hypothèse que FMRP effectue des fonctions de base critiques au développement de tous types de tissus humains, et non seulement dans les neurones. L’hypothèse sera davantage développée. Puis, un modèle innovateur de la différenciation cellulaire non-neuronal sera présenté pour l'étude de FMRP. L'enquête sur la distribution subcellulaire et les interactions dynamiques de cette protéine sera détaillée durant les différents changements morphologiques de la spécialisation et de la maturation cellulaire. Les résultats des expériences seront analysés en profondeur. Puis, un retour sur l'hypothèse en guise des résultats expérimentaux permettra de constater que FMRP semble bien jouer un rôle durant la différenciation cellulaire non-neuronale. Ce rôle est intimement lié à la réorganisation du cytosquelette et à la synthèse protéique locale, régulée dans les complexes mRNPs composés de FMRP et ses cibles d'ARNm qui sont régulés, stabilisés et transportés vers les régions en maturation. Ultimement, plusieurs éléments indiquent que FMRP doit interagir correctement avec de nombreuses molécules, décrites dans ce mémoire, afin de permettre aux cellules de se spécialiser et d'acquérir les caractéristiques désirées au cours d’une différenciation normale.
Abstract: The present article-based memoire will explore the involvement of an important RNA-binding protein, FMRP, in cellular differentiation. This protein is well-known for the developmental anomalies during neurogenesis as well as the loss of synaptic plasticity which occur when its gene, FMR1, is mutated. Since FMRP expression is most pronounced in neurons and because the absence of its expression results in the intellectual deficiency known as the Fragile X Syndrome, FMRP has nearly always been studied in neurons alone. However, FM RP is a highly conserved protein, expressed ubiquitously across the body. Additionally, its influence in cells can be vast since its motif - based RNA recognition renders it capable of binding a variety of transcripts. Logically speaking, FMRP should play a role of first rate importance in the other tissues where it is present. The clinical manifestation of its impact in those tissues is likely to be lessened only by the lower levels of FMRP expressed in the average human cell. The main hypothesis of the current study is that FMRP performs critical but basic functions involved in the development of all human tissues where it is present at basal levels, rather than exerting an impact limited to the nervous system alone. The hypothesis will be further elaborated. An innovative non-neuronal model will be presented for the study of FMRP throughout the differentiation process. The behaviour and dynamic interactions of FMRP during cell specialization and morphological maturation will be investigated. An in-depth analysis of the experimental results will follow. Returning to the hypothesis of the study with these results at hand, it will be concluded that FMRP does indeed appear to play a major role in the differentiation of non-neuronal cells. In fact, FMRP's function seems to be closely linked to cytoskeletal reorganization, as well as local protein synthesis through the formation of mRNP complexes with its target mRNAs, which are stabilized, regulated and transported towards the active areas of the cell in differentiation. Ultimately, it is clear that proper interaction between FMRP and certain types of molecules, described in this memoire, is required for cells to specialize and acquire the characteristics of mature cells through normal differentiation.

The dissertation is divided into two separate experiments that explore the effects of visual-spatial learning on PSD-95 dorsal hippocampal expression. Specifically, the aim of these studies was to explore the effect of learning an assay, the Hebb-Williams mazes, on the protein expression of PSD-95 in Fmr1 KO mice. PSD-95 is an important scaffolding protein hypothesized to be involved in learning and memory. In cellular models of Fragile X Syndrome it has been shown to be dysregulated but it has never been measured following behavioural learning. Establishment of a deficit using an ecologically valid behavioural assay could lead to the development of novel interventions. Study one employed a subset of the Hebb-Williams mazes of various levels of difficulty to evaluate PSD-95 protein expression in Fmrp intact and Fmr1 KO mice following learning. The results revealed significant increases in PSD-95 protein expression in control runners when compared to Fmr1 KO mice. There was a negative correlation between PSD-95 protein levels and mean total errors on the mazes meaning that as expression was increased, errors were decreased. The goals of study two were to reverse the molecular and behavioural deficits using pharmacological antagonist treatment shown to be effective in cellular models of Fragile X Syndrome. Fmr1 KO mice were treated with either saline or 20 mg/kg of a metabotropic glutamate receptor antagonist, 2-Methyl-6-(phenylethynyl) pyridine (MPEP). Relative to saline treated controls, drug treated Fmr1 KO mice made fewer errors on the same subset of Hebb-Williams mazes used in study one. Latency to complete these mazes did not differ between groups, indicating that MPEP treatment does not adversely affect motor functioning. Protein assessment revealed that PSD-95 was selectively rescued in MPEP treated mice and not saline controls. Similar to study one, a negative correlation between PSD-95 protein levels and mean total errors was observed. When taken together, these studies indicate that protein deficits are associated with a deficit of learning that can be reversed with a selective glutamate receptor antagonist. One of the strengths of the Hebb-Williams mazes is that performance is measurable without floor or ceiling effects, which plague other common behavioural assays. These data further suggest that pharmacological antagonist treatments may be promising in correcting the learning deficits in human Fragile X Syndrome patients.

This dissertation describes separate but related studies that explore visual spatial learning and memory in Fragile X Syndrome. Across all studies, either the performance of individuals affected by FXS and/or fmr1 KO mice was compared to comparison controls on seven H-W mazes of increasing difficulty levels. Study one employed the traditional configuration of the H-W mazes to evaluate performance variables that include latency to complete the maze and number of the errors. The results of study 1 revealed significant differences in performance for both FXS groups as compared to mental age-matched comparison individuals and wild type mice, respectively. In contrast to the FXS group, performance of the comparison group improved as indicated by significantly fewer errors across trials. A similar pattern of results was observed when latency across trials was analyzed. Taken together, the results of study one support the hypothesis that a selective deficit in spatial learning and memory characteristic of the FXS phenotype can be observed in the murine model of FXS, if equivalent tasks are employed in testing humans and mice. Study two expanded on these findings by adding landmarks to the maze environment to evaluate how these may impact spatial learning and memory in fmr1 KO mice. Contrary to our hypotheses, landmarks significantly impaired wild type control performance. In addition, results revealed that the performance of the fmr1 KO mice generally did not differ between landmark and non-landmark tasks, indicating that the presence of landmarks neither enhanced nor hindered mouse performance. Lastly, study three entailed a more in-depth behavior analysis of maze navigation performance for FXS individuals from study 1. Consistent with the hypotheses and findings from study 1, results revealed significant differences in performance variables between individuals, with FXS participants generally performing worse than the comparison group participants. Taken together, the results of study 3 generally supported the hypothesis that there was greater impairment in performance for individuals affected by FXS as compared to controls. This impairment was evident in the pattern of pathways taken to solve H-W mazes, consistent with the notion that affected individuals employed different behavioral strategies.

Fragile-X syndrome (FXS) is the most common form of inherited intellectual disability (ID), representing a considerable burden of health in our society. FXS is caused by repression of the transcription of one gene, Fmr1. Normally, expression of the Fmr1 gene leads to the production of one type of protein, the Fragile-X Mental Retardation Protein (FMRP). At the cellular level, FXS is caused by a lack of FMRP. The fact that mice and humans possess a nearly identical Fmr1 gene has permitted the generation of a mouse model of FXS using modern transgenesis techniques (Fmr1 knockout (KO) mice). The study of the behavior of Fmr1 KO mice was expected to quickly reveal ID with subsequent elucidation of the syndrome’s neurobiological underpinnings. Unfortunately, the manifestation of presumed ID (defined as significant impairments in intellectual and adaptive functioning) at the behavioral and neurobiological levels in Fmr1 KO mice has been surprisingly elusive. How repression of Fmr1 gene expression affects the human brain to produce ID is unclear. The dentate gyrus (DG) subfield of the hippocampus is a region of the brain that is associated with learning and emotion, exhibits marked structural and functional plasticity, and was unexplored in Fmr1 KO mice prior to the work presented in this thesis. Our overarching hypothesis is that lack of expression of the Fmr1 gene deleteriously alters structural and functional plasticity in the mammalian DG, and impairs aspects of learning and emotion associated with this brain region. Chapter 1 introduces topics such as FXS, the hippocampus, plasticity and the mouse model of FXS. Specific hypotheses are listed at the end of chapter 1. Chapters 2 and 3 are manuscripts written for publication in peer-reviewed journals. The bulk of the data relating to the testing of the specific hypotheses are presented in these chapters. Chapter 4 is a general discussion that seeks to place the results presented in the thesis into context within the literature, and also identifies important future directions. The thesis concludes with a new model posited for the pathophysiology of FXS.

Challenging behaviours (CBs) are a common issue amongst individuals with Fragile X Syndrome (FXS). The aim of the present thesis was to further understanding of this issue, through exploring physiological factors which may have a motivative influence upon the operant learning of CBs in this group. Analysis of prior literature highlighted that CBs were most commonly negatively reinforced amongst males with FXS. This may reflect an elevated motivation to escape from stressors, associated with atypical stimulus-bound arousal. Accordingly, prior data suggest autonomic hyperarousal and a systematic literature review suggested that the hypothalamic-pituitary-adrenal axis may be implicated in males with FXS. However, the relationship between arousal and escape-maintained CB had not previously been explored, and so was addressed through two empirical studies. In the initial study, CBs were observed in a natural environment, alongside explorations of circadian rhythmicity of salivary arousal measures (cortisol and α-amylase) in boys with FXS and unaffected siblings. Whilst between-group differences were apparent in arousal measures, there were no associations with observed behaviours in the FXS group. In a subsequent study, behaviour and physiological responding were measured in response to a structured demand assessment, amongst individuals with intellectual disability and males with FXS. Despite between-group differences in behaviour, no differences in physiological responding or physiology-behaviour relationships were observed. A final exploratory study of parental reports of the behavioural and emotional timecourse of instances of CBs was conducted, in order to guide future research. Together the results suggest that initial hypotheses were overly simplistic and that a broad range of aspects of the FXS phenotype must be accounted for when explaining CBs in this group. Implications for future research and practice are discussed.

Cell cultures were examined for percentage of fragile sites seen in straight-stained, Q-stained and reverse fluorescent-stained preparations. In all cases, percentage of fragile site expression was decreased when compared to straight-stained preparations. However, fragile sites seen in Q- and RF-stain could be identified as on X chromosomes.

FMRP est une protéine cytoplasmique possédant des domaines consensus de liaison à l'ARN. Dans tous les tissus ou types cellulaires étudiés jusqu'à présent, elle est majoritairement retrouvée associée aux polysomes en se liant à l'ARN messager. FMRP jouerait donc un rôle dans le métabolisme de l'ARN. Toutefois, sa fonction précise reste encore inconnue. Notre hypothèse est que l'étude des plaquettes sanguines, où la synthèse protéique est très faible, pourrait permettre de découvrir une nouvelle fonction pour FMRP qui est habituellement camouflée par son importante association avec les polyribosomes dans les autres types cellulaires. Mon objectif de recherche est d'étudier la localisation subcellulaire de FMRP dans les plaquettes quiescentes et activées afin de mieux comprendre sa fonction. Différentes approches biochimiques et biophysiques ont été utilisées afin d'étudier la distribution subcellulaire de FMRP dans les plaquettes sanguines. Nous avons confirmé par des immunobuvardages faits sur des extraits totaux de plaquettes que la protéine détectée se présente sous forme d'isoformes comparables aux autres cellules. La technique de bombe à l'azote montre une localisation cytoplasmique de FMRP. Alors qu'en immunofluorescence, nous observons une colocalisation de FMRP avec des protéines impliquées dans le métabolisme de l'ARN. Toutefois, les résultats de centrifugations différentielles de plaquettes quiescentes suggèrent que FMRP possède une distribution subcellulaire différente de celle observée dans les autres types cellulaires. Elle a un coefficient de sédimentation entre 6-10S alors que celui observé dans les autres types cellulaires varient entre 150-500S. Bien que FMRP soit retrouvée dans la fraction soluble dans les plaquettes quiescentes, ce type cellulaire est néanmoins métaboliquement peu actif. L'activation des plaquettes par des agonistes enclenche une myriade de mécanismes. Suivant l'activation, FMRP est redistribuée dans la fraction du cytosquelette. La concentration ionique affecte cette distribution, suggérant une association protéine-protéine et/ou protéine-ARN.En conclusion, le profil d'expression de FMRP semble distinct dans les plaquettes et leur activation modifierait dynamiquement cette distribution. FMRP semble donc avoir une fonction dans les plaquettes puisqu'elle est affectée par le processus d'activation. L'étude de l'association au cytosquelette pourrait s'avérer une voie intéressante dans la détermination de la fonction de FMRP dans les plaquettes.

Cette thèse part de la proposition d\'une rencontre possible entre psychanalyse et génétique médicale par le biais des soins offerts aux enfants porteurs de syndromes génétiques, notamment le syndrome de l\'X fragile. Nous avons trouvé dans les recherches en épigénétique une voie de rapprochement de ces différents champs du savoir. L\'idée selon laquelle l\'environnement est capable de modifier l\'expression des gènes représente la rupture d\'un certain déterminisme génétique autrefois accepté, et ouvre un espace où penser la singularité. Notre travail propose d\'élargir le concept d\'environnement, en y considérant la relation de l\'enfant avec l\'Autre, lieu du langage, comme opérateur de marques sur son corps : marques symboliques, constituées dès le tout début de la rencontre de l\'infans et de ceux qui s\'occupent de lui. C\'est justement dans cet espace d\'échange avec l\'Autre qu\'a lieu l\'émergence d\'un sujet. Nous avons opté pour les concepts de sujet et de transfert pour soutenir l\'articulation de la clinique psychanalytique et de la génétique médicale en ce qui concerne le traitement. Nous avons donc exposé trois cas cliniques issus de notre pratique, d\'enfants traversés par le diagnostic de l\'X fragile afin d\'illustrer de quelle manière les conceptions de sujet et de transfert se reflètent dans la clinique. Tenant compte que la psychothérapie est également prise comme objet d\'étude de l\'épigénétique, et qu\'elle est donc considérée comme un environnement capable de provoquer, voire de renverser des marques épigénétiques, l\'enjeu de notre travail repose sur la proposition suivante : et pourquoi pas la psychanalyse également ? La psychothérapie psychanalytique, ancrée sur le transfert, ne peut-elle pas, elle aussi, laisser des marques sur le petit patient
The current thesis assumes a possible encounter between psychoanalysis and medical genetics based on the treatment applied to children carrying genetic syndromes such as the Fragile X Syndrome. Epigenetic studies are a way to approximate different knowledge fields. The assumption that the environment is able to change gene expression strays from the genetic determinism we once believed and opens the way for us to reason about singularity. The proposition in the present study lies on expanding the concept of environment, by taking into consideration the relation between the child and the Other in the environment in question, as well as the place of language as the operator marking the childs body. These symbolic marks start emerging in the first encounter between the infans and caregivers. The subject emerges precisely 3 within an environment of exchanges that is set with the Other. The concepts of subject and transference were chosen to support the treatment articulation between psychoanalytic clinic and medical genetics. Thus, the present study reports three clinical cases followed by the authors, which involved children diagnosed with fragile X syndrome. These cases illustrate how the aforementioned concepts affect the clinical practice. Since psychotherapy has also been taken as the object of epigenetic studies, and as it is considered an environment able to cause, and even reverse, epigenetic marks, the current study relies on the following proposition: why not psychoanalysis as well? Can the psychoanalytic psychotherapy, anchored in the concept of transference, leave marks on the little patient too?

The purpose of this study was to explore what contributes to resilience in females diagnosed with Fragile X Syndrome. Fragile X Syndrome can be defined as an inherited (genetic) condition that causes mental impairment, attention deficit and hyperactivity, anxiety and unstable mood, autistic behaviours, hyper-extensible joints, and seizures. I became aware of Fragile X Syndrome during my time as a live-in caretaker to an adolescent female who was diagnosed with Fragile X Syndrome. Because she coped with her disability so resiliently, I was encouraged to explore what contributes to resilience in females diagnosed with Fragile X Syndrome. I followed a qualitative approach, anchored in the interpretivist paradigm. This means that I tried to understand the resilience of females diagnosed with Fragile X Syndrome through the meanings that the participants in my study assigned to them. Furthermore, I worked from a transformative paradigm, which meant that I was interested in changing the traditionally negative ways in which females diagnosed with Fragile X Syndrome are seen. I followed a multiple case study approach, which included four case studies. I conveniently selected the first participant, but realised that convenience sampling was not very credible for a qualitative case study. An Advisory Panel was then used to purposefully recruit three more participants. In order to explore what contributed to their resilience, I made use of interviews, observations, and visual data collection. I also interviewed adults (e.g. parents, teachers and consulting psychologists) who were significantly involved in the lives of my participants. My findings suggest that resilience in females with Fragile X Syndrome is rooted in protective processes within the individual as well as within her family and environment. Because my findings do not point to one specific resource, my study underscores newer understandings of resilience as an Eco systemic transaction. Most of the resilience-promoting resources noted by the participants in my study as contributing to their resilience have been identified as resilience-promoting in previous studies. Although the themes that emerged in my study have been reported in resilience previously, I make a contribution to theory because I link traditional resilience-promoting resources to resilience in females diagnosed with Fragile X Syndrome. Peer support was previously reported as a resilience-promoting resource, but in my study I noticed that the main source of peer support came from peers who were also disabled. Furthermore, my study transforms how we see females diagnosed with Fragile X Syndrome. This transformation encourages communities and families to work together towards resilience in females diagnosed with Fragile X Syndrome.
Ph.D, North-West University, Vaal Triangle Campus, 2011