Дисертації з теми "Myoclonies"

Le syndrome de Lance-Adams est une pathologie neurologie chronique très handicapante rencontrée chez les survivants d'anoxie cérébrale. Il est caractérisé essentiellement par des myoclonies positives, d'action, multifocales ou généralisées, et des myoclonies négatives. Les mécanismes sous-tendant les myoclonies de cette pathologie sont peu connus. De multiples hypothèses ont été proposées depuis la description initiale de ce syndrome. L'étude multimodale d'une large cohorte de patients avec un syndrome de Lance-Adams a montré que les myoclonies sont générées dans le cortex cérébral, en particulier le cortex moteur (ou sensorimoteur). L'observation de l'histoire clinique de certains patients a par ailleurs permis de proposer, avec succès, une nouvelle approche thérapeutique par électroconvulsivothérapies à une patiente pharmacorésistante. Une revue extensive de la littérature a permis de faire émerger une vue d'ensemble et intégrée de cette pathologie et de mieux délimiter le profil de patients. Enfin, nous avons essayé, par différentes approches, de développer un nouveau modèle murin de myoclonies post-anoxiques, sans succès. Une amélioration de cette préparation expérimentale permettrait la réalisation de multiples explorations, notamment immunohistochimiques et électrophysiologiques, afin de mieux comprendre les mécanismes cellulaires et de réseaux à l'origine des myoclonies du syndrome de Lance-Adams
Lance-Adams syndrome is a post-anoxic disabling chronic neurological disorder. The main clinical features are action-induced multifocal or generalized positive myoclonus, and negative myoclonus. The underlying mechanisms of this disorder are poorly understood. Multiple hypotheses have been proposed since the initial description of this syndrome. The multimodal analysis of a large cohort of patients with Lance-Adams syndrome demonstrated that myoclonus originates in the cerebral cortex, particularly in the motor (or sensorimotor) cortex. Additionally, careful observation of the natural history of patients led to the successful proposal of a new therapeutic approach using electroconvulsive therapy in a pharmacoresistant patient. An extensive literature review provided an integrated overview of this pathology and helped better define the patient profile. Lastly, through various approaches, we attempted to develop a new murine model of post-anoxic myoclonus, without success. An improvement in this experimental preparation would allow for multiple explorations, particularly immunohistochemical and electrophysiological studies, to better understand the cellular and network mechanisms underlying the myoclonus in Lance-Adams syndrome

Thesis (DMed (Medicine. Internal Medicine))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Progressive Myoclonic Epilepsies (PME) are characterized by progressive neurological impairment with myoclonus, seizures and dementia. In contradistinction, Familial Adult Myoclonic Epilepsy (FAME) is characterized by a benign course with rare seizures and cortical tremor. Both conditions have neurophysiological features suggestive of a cortical origin for their myoclonus. This dissertation reports on a novel form of PME. Many of those who were affected had no or minimal progression of their illness, low seizure frequency and were cognitively intact, suggestive of non-progressive disorders linked to the FAME loci. The majority of patients had features of cortical myoclonus, with generalized spike and wave discharges on electroencephalography, enlarged evoked potentials, enhanced C reflexes, and evidence of cortical excitability with magnetic stimulation. However, there was evidence of cerebellar dysfunction both pathologically and on imaging. With regard to similar conditions, dentatorubral pallidoluysian atrophy and Unverricht-Lundborg syndrome were excluded by linkage analysis. Similarly, linkage was not present for either the FAME 1 or FAME 2 loci. This syndrome is both clinically and genetically novel, and has a nosology which is difficult to characterize, in which the condition appears to lie on the spectrum between FAME and PME. The dissociation between the pathological and radiological findings which suggest subcortical dysfunction, and the neurophysiological findings of cortical myoclonus is striking. Review of the literature associated with the neurophysiology of related conditions associated with PME and FAME suggests that: 1. The assumption that generalized forms of myoclonic disorders represent multifocal forms of focal cortical discharges is an oversimplification. 2. The dissociation between initial and later components of the evoked potential is less robust than is generally supposed, and that subcortical inputs may affect later components of the evoked potential. 3. In a high proportion of cases the latency from cortical spike discharge to myoclonic jerk obtained with jerk locked averaging is incompatible with a cortical origin for the spike discharge. 4. The proposal that myoclonus is a form of long latency reflex and that myoclonus represents a reflex arising from subclinical sensory input, is unproven.
AFRIKAANSE OPSOMMING: Progressiewe Miokloniese Epilepsie (PME) word gekenmerk deur progressiewe neurologiese agteruitgang met mioklonus, konvulsies en demensie. Daarenteen word Familiële Volwasse Miokloniese Epilepsie (FAME) gekenmerk deur 'n benigne verloop met ongereelde konvulsies en kortikale tremor. Beide entiteite het neurofisiologiese kenmerke suggestief van 'n kortikale oorsprong vir die mioklonus. Hierdie manuskrip beskryf 'n nuwe vorm van PME. Baie van die aangetaste persone toon geen of min agteruitgang van die siekte oor tyd nie, met 'n lae frekwensie van konvulsies en is kognitief intak, wat suggestief is van 'n nie-progressiewe siekte gekoppel aan die FAME loci. Die oorgrote meerderheid van pasiente het kenmerke van kortikale mioklonus gehad, met algemene spits en boog ontladings op elektroensefalografie, hoë amplitude ontlokte potensiale, versterkte C-reflekse, en tekens van kortikale eksiteerbaarheid met magnetiese stimulasie. Met neurobeelding en patologie was daar egter bewyse van serebellêre disfunksie. Soortgelyke toestande, naamlik dentatorubro-pallidoluysiese atrofie en Unverricht-Lundborg sindroom is uitgeskakel deur middel van koppelingsanalise. Koppeling met die FAME1 of FAME2 loci kon ook nie aangetoon word nie. Die sindroom is beide klinies sowel as geneties nuut en het 'n nosologie wat moeilik gekaraktiseer kan word. Dit wil voorkom of die siekte op 'n spektrum lê tussen FAME en PME. Die dissosiasie tussen die patologiese en radiologiese bevindinge, wat suggestief is van subkortikale disfunksie, en die neurofisiologiese bevindinge van kortikale mioklonus is opmerklik. ’n Oorsig van die literatuur in verband met die neurofisiologie van toestande geassosieer met PME en FAME suggesteer die volgende: 1. Die aanname dat algemene vorme van miokloniese toestande multifokale vorme van fokale kortikale ontladings verteenwoordig, is ’n oorvereenvoudiging. 2. Die dissosiasie tussen inisiële en latere komponente van die ontlokte potensiaal is minder robuust as wat algemeen aanvaar word, en subkortikale invoer mag latere komponente van die ontlokte potensiaal beïnvloed. 3. In ’n groot proporsie van gevalle is die latensie van kortikale spits ontlading tot miokloniese ruk, verkry deur “jerk locked averaging”, nie verenigbaar met met ’n kortikale oorsprong vir die spits ontlading nie. 4. Geen bewyse bestaan vir die teorie dat mioklonus ’n vorm van ’n lang latensie refleks is en dat mioklonus ’n refleks is wat ontstaan uit subkliniese sensoriese invoer nie.

La réalisation d'un mouvement volontaire implique la mise en jeu de structures cérébrales responsables des étapes de planification, de programmation, d'exécution et d'arrêt du mouvement. Par leur action via des circuits intracorticaux excitateurs et inhibiteurs, les influx sensoriels afférents générés au cours du mouvement semblent jouer un rôle primordial dans l'exécution et l'arrêt du mouvement, en limitant en particulier la survenue de mouvements parasites. Les objectifs de ce travail de thèse étaient d'étudier l'activité électrique des aires cérébrales impliquées dans le contrôle moteur et l'excitabilité du cortex moteur primaire liées aux influx sensoriels afférents à l'aide de deux outils électrophysiologiques complémentaires : l'électroencéphalographie (EEG) et la stimulation magnétique transcrânienne (SMT). Nous voulions d'une part déterminer de quelle façon l'activité du cortex sensorimoteur pouvait être modulée par les afférences sensorielles et d'autre part à quel point une modulation de cette excitabilité corticale pourrait influencer le contrôle moteur. Les rythmes EEG sont caractérisés par leur fréquence, leur amplitude, leur localisation et leur réactivité à un événement, c'est-à-dire la variation d'amplitude du signal EEG liée à un événement. Les rythmes mu et bêta, enregistrés en regard de la région centrale, sont associés au comportement moteur. La méthode des Désynchronisations et Synchronisations Liées à l'Evénement (DLE/SLE) permet l'étude de la réactivité des rythmes EEG. Elle consiste à déterminer l'évolution temporelle de la puissance du signal EEG dans une bande de fréquence donnée avant, pendant et après un événement. La DLE, qui correspond à une diminution d'amplitude du signal, serait le reflet de l'activation des neurones corticaux impliqués dans la préparation et l'exécution du mouvement. La SLE des rythmes bêta reflète quant à elle une augmentation de la puissance du signal après la fin du mouvement. Sa signification est encore discutée. Elle serait liée à la « désactivation » des neurones corticaux impliqués dans la programmation motrice mais serait également fortement dépendante des afférences sensorielles puisqu'elle disparaît sous ischémie. La SMT consiste en l'induction d'un courant électrique au niveau cérébral à partir de deux bobines délivrant un champ magnétique à la surface du scalp. Appliquée en regard du cortex moteur à une intensité supraliminaire, la SMT évoque une réponse musculaire, le potentiel évoqué moteur, dont l'amplitude est le reflet de l'état d'excitabilité de la voie corticospinale, elle-même dépendante des multiples influences (notamment sensorielles) qui s'exercent sur l'aire motrice primaire. La SMT répétitive (SMTr) permet quant à elle de modifier temporairement l'excitabilité corticale. Ainsi, une stimulation à haute fréquence du cortex moteur primaire augmente l'excitabilité de ce dernier alors qu'une stimulation basse fréquence la diminue. Dans ce travail, nous avons tout d'abord voulu déterminer si l'analyse de la SLE bêta pouvait être un bon moyen d'étude du traitement cortical des afférences sensorielles. Pour cela nous avons comparé, dans un groupe de sujets sains, la SLE bêta induite par un mouvement volontaire à des SLE bêta provoquées par différents types de stimulations périphériques. Cette étude nous a permis de conclure que les caractéristiques de la SLE bêta (amplitude et durée) sont étroitement dépendantes des afférences sensorielles et que la SLE bêta dépend du type de fibres sensorielles stimulées et de la durée de stimulation. La SLE bêta reflèterait donc une période d'inhibition corticale post-mouvement étroitement dépendante des afférences sensorielles. Elle permet donc l'étude des mécanismes corticaux de traitement des informations sensorielles liées au mouvement, mécanismes intervenant dans les processus d'intégration sensorimotrice. A la suite de ce travail mené chez le sujet sain, nous avons étudié l'intégration sensorimotrice (ISM) et l'excitabilité corticale chez des patients souffrant de myoclonies corticales. Les myoclonies corticales sont de brèves secousses musculaires provoquées par l'action et qui sont dues à une hyperexcitabilité du cortex sensorimoteur. Plusieurs arguments suggèrent des anomalies d'ISM chez ces patients. Nous avons voulu déterminer si ces anomalies impliquaient également le contrôle moteur et si elles pouvaient être détectées au cours d'un mouvement volontaire par la technique des D/SLE. Nous n'avons pas mis en évidence d'anomalie des D/SLE des rythmes bêta. En revanche, la DLE des rythmes mu, témoin de l'activation nécessaire à la programmation et l'exécution du mouvement était plus ample que les sujets témoins. Ceci confirme l'hyperexcitabilité corticale présente chez ces patients. L'étude de la SLE bêta n'a montré aucune anomalie des interactions sensorimotrices au cours du mouvement volontaire. En revanche, protocoles de SMT nous ont permis de mettre en évidence un défaut d'inhibition intracorticale et un dysfonctionnement de l'ISM à courte latence liée aux fibres extéroceptives chez les patients myocloniques. Par ailleurs, l'ISM à longue latence qui pourrait être le corrélat de la SLE bêta était normale, ce qui semble en accord avec les résultats obtenus en EEG. Toutes ces anomalies sont en faveur d'une déficience des mécanismes d'inhibition corticale, probablement responsable de l'hyperexcitabilité rapportée. Nous avons donc tenté, dans la dernière partie de ce travail, de diminuer l'excitabilité du cortex moteur primaire chez des patients souffrant de myoclonies corticales, afin de déterminer si cette diminution pouvait avoir un bénéfice clinique. Pour cela, nous avons d'abord évalué les effets de la SMTr du cortex moteur primaire et du cortex prémoteur, à basse et haute fréquence et à différentes intensités sur les courbes entrée-sortie - témoignant de l'excitabilité de la voie corticospinale - de trois muscles du membre supérieur chez des sujets sains. Cette étude nous a permis d'approfondir nos connaissances sur les effets de la stimulation magnétique répétitive sur les circuits moteurs intracorticaux en montrant notamment qu'une stimulation basse fréquence du cortex prémoteur était plus efficace qu'une stimulation du cortex moteur primaire. Les effets inhibiteurs d'une telle stimulation ont ensuite été étudiés chez une patiente souffrant de myoclonies corticales. Nous avons ainsi mis en évidence une amélioration de ses symptômes, et des bénéfices cliniques accrus après deux jours de stimulation consécutifs. Nos résultats confirment donc l'intérêt du cortex moteur/prémoteur comme cible thérapeutique de la stimulation magnétique répétitive, tout en en soulevant les limites de cette méthodologie.

Myoclonus Dystonia (MD) is an autosomal dominant disease with high but incomplete penetrance and is characterized by both involuntary myoclonic jerks and dystonic posturing. Our group has found that mutations within the epsilon sarcoglycan (SGCE) gene on chromosome 7q21 are associated with MD in 30-40% of affected individuals in 31 families studied, supporting the basis for genetic heterogeneity. Novel mutations have been found in SGCE by screening these families for point mutations and large deletions and duplications through the use of sequencing, high performance liquid chromatography (HPLC) and multi˙ligation probe amplification (MLPA) analysis. A 10cM genome wide linkage analysis of a large Canadian family provided significant LOD scores for microsatellite markers within the 18p11 region, now designated as the DYT 15 locus. Further haplotype analysis has narrowed a non-recombinant region associated with the disease phenotype to a 3.18 Mb region in this locus. Since the current understanding of Myoclonus Dystonia is poor, it is difficult to predict genes that could be responsible for MD. Sarcoglycans are essential constituents of the dystrophin-glycoprotein complex and are involved in linking the extracellular lamanin matrix to the actin filaments within the cytoplasm; therefore focus is given to the examination of potentially related structural genes that are expressed in the brain. By analyzing such candidate genes in a panel of affected individuals, we believe that a novel gene will be elucidated and provide insight into the mechanism of Myoclonus Dystonia.

Many neurologic diseases cause discrete episodic impairment. Study of the genes and mechanisms underlying these diseases has informed our understanding of the nervous system. Here we describe a novel episodic neurologic disorder, which we term familial cortical myoclonus (FCM). FCM is characterized by adult onset, slowly progressive, multifocal, cortical myoclonus, inherited as an autosomal dominant trait. On the basis of clinical, electrophysiological, and genetic data, FCM is nosologically distinct. We utilized genome-wide single nucleotide polymorphism genotyping, microsatellite linkage, and massively parallel sequencing to identify a mutation in the gene nucleolar protein 3 ( NOL3) that likely causes FCM. NOL3 is thought to bind to pro-apoptotic proteins and thereby repress apoptosis, but our extensive experimentation did not replicate these claims. In vitro, the NOL3 mutation leads to post-translational modification of NOL3 protein. We could not pinpoint the identity of the modification, but did find that this process is regulated by phosphorylation at residue T114. Finally, a proteomic screen for novel binding partners identified two candidates that modulate neuronal/astroglial differentiation. We hypothesize that the NOL3 mutation abrogates these interactions to cause FCM. This hypothesis will be tested with Nol3 mutant mice that we generated. In total, this work defines a novel episodic neurologic phenotype and the associated mutation, calls into question some of the published functions of NOL3, and presents an alternative mechanism that may explain the pathophysiology of FCM.

Myoclonic astatic epilepsy (MAE) is a rare generalised childhood epilepsy with variable but poorly described neurodevelopmental outcome. Family studies suggest a major genetic influence as up to two thirds of relatives have seizures, or electroencephalographic (EEG) abnormalities. MAE is associated with 10 different genes, yet these genes account for less than 20% of the genetic aetiology of MAE leaving the majority unexplained. The aims of this thesis were (1) describe the epilepsy and neurodevelopmental phenotype of MAE cases, (2) perform EEG studies on first degree family members for familial EEG abnormalities and compare occurrence of epileptiform features to population prevalence and (3) to collect DNA and identify MAE causative genetic variants through exome sequencing. I assembled the largest MAE cohort (n=123) to date. The epilepsy phenotype is remarkably similar to previously published cohorts. I identified a severe neurodevelopmental phenotype: intellectual disability was reported in 64.9%, autism spectrum disorder in 21.3% and attention deficit hyperactivity symptoms in 41.0%. Additionally, extremely low adaptive behavioural scores were identified in 69.4% of cases. I performed EEG studies on 38 first-degree relatives of 13 MAE families, and found an excess of epileptiform EEG features in adults (>16 years), compared to controls (P=0.05, RR 6.82). I identified likely pathogenic or candidate variants in 11 of 109 cases. This comprised known genes associated with MAE: CHD2 n=1, SYNGAP1 n=2, SLC6A1 n=1, KIAA2022 n=1; epilepsy associated genes novel for MAE: KCNB1 n=1, MECP2 n=1, KCNH5 n=1, and three new candidate genes; SMARCA2 n=1, ASH1L n=1 and CHD4 n=1. Lastly, I highlight phenotypic features which help correlate with known and novel specific gene associations, discuss that MAE is a phenotypic and genetic nosological bridge between genetic generalised epilepsy and epileptic encephalopathy, and discussion applications and future directions leading on from this project.

Myoclonus dystonia (DYT11, OMIM 159900) (MD) is a movement disorder characterized by bilateral alcohol responsive myoclonic jerks often seen in combination with dystonia. MD is inherited as an autosomal dominant trait with reduced penetrance upon maternal transmission. Patients with this disorder are not diminished in their intellectual capacity and have a normal life span. In 2001, mutations in the epsilon-sarcoglycan gene on human chromosome 7q21 were implicated in causing this disorder. Our laboratory identified a 2nd locus on chromosome 18p11 that co-segregates with this disorder, however, a disease causing mutation has not been identified. To establish the function of epsilon-sarcoglycan within the mammalian brain, I generated a conditional knock-out (CKO) mouse of Sgce and utilized bacterial recombineering to generate a cassette that contained a "floxed" exon 1 of epsilon-sarcoglycan. ES cells that had correctly incorporated the CKO targeting cassette were selected and used to generate 3 chimeric male mice by blastocyst injection or morula aggregation. Once germline transmission of the CKO allele has been established, these mice will be bred to Cre expressing mice to generate an sgcenull, and recapitulate the phenotype of MD patients. In addition, I also developed an in vitro model of MD using RNAi directed against epsilon-sarcoglycan in the mouse neuroblastoma cell line, N1E-115. Silencing of epsilon-sarcoglycan in this cell line resulted in a decrease in the neuronal nitric oxide synthase (nNos) and an increase in cell proliferation. This data will provide insight and direction during the characterization of the sgcenull mice.

Myoclonus is a complex disorder of rapid repetitive muscle jerks that can occur in proximal or distal appendicular or axial muscles. It can be of cortical, sub-cortical or spinal cord origin; part of progressive and severely disabling epilepsy syndromes, basal ganglia conditions, and physiological or even functional (psychogenic)1. A systematic review of the literature shows the knowledge gap of the genetic causes of myoclonus in South Africa with 25 identified publications from Africa of which eleven were from South Africa. Publications varied from case studies to case series and included four publications with cortical myoclonic tremor (CMT) and two with North Sea Progressive Myoclonic Epilepsy, two with subcortical myoclonus and case studies with rare cases of individuals with myoclonic disorders. In this publication the study of myoclonus in three different settings is presented. In the first: cortical myoclonic tremor (CMT), a rapid distal form of myoclonus, resembling tremor, with neurophysiological evidence of cortical origin. The study researched a South African family with Familial Cortical Myoclonic Tremor with Epilepsy (FCMTE). The first part of this study showed the median onset of cortical tremor 16 was and that of epilepsy was 42 years; patients were stable with long term follow up after 30 years without evidence of progressive ataxia or cognitive impairment. The second part of the study presents the discovery of the genetic mutation causing this condition: a pentanucleotide repeat expansion in the intronic region of the STARD7 gene. This mutation was also found in families with FCMTE2 with a similar phenotype and followed on work showing pentanucleotide repeat expansion mutations in other forms of FCMTE in different genetic locations. The second setting proved a new mutation, a premature stop mutation p.L275X, in the epsilon-sarcoglycan gene causing subcortical origin, Myoclonus Dystonia Syndrome (MDS) in a three generation South African family with mild phenotype differences in the clinical presentation: myoclonus and dystonia in the same appendicular body part as well as truncal. Two of the affected individuals studied underwent Deep Brain Stimulation surgery of the Globus Pallidum with significant sustained improvement in the motor and non-motor features of MDS recorded and confirmed by a blinded rater. In the third setting, two patients with sporadic Paroxysmal Non-kinesigenic dyskinesia (PNKD) with the complex phenomenology of episodic dystonia, myoclonus and chorea of South African origin is presented. Both patients underwent successful DBS of the pallidum with long-term outcomes presented. Although these two individuals were not tested for the known myofibrillogenesis regulator-1 (MR-1) mutation they represent two cases of this rare disorder from South African setting and prove the successful use of DBS treatment.
Thesis (PhD)--University of Pretoria, 2021.
Neurology
PhD
Unrestricted

Mutations in the EFHC1 gene, encoding a novel microtubule binding protein, have been linked to juvenile myoclonic epilepsy. EFHC1 has been proposed to regulate cell division by controlling mitotic spindle organization and cerebral cortex lamination by modulating neuroblast migration. To understand EFHC1 function in vivo we generated knock-out Drosophila for the fly homolog Defhc1. We found that the NMJ synapse of Defhc1 mutants display an increased number of satellite boutons and increased spontaneous neurotransmitter release. Defhc1 binds to microtubules in vitro and overlaps in vivo with axonal and synaptic microtubules. Elimination of Defhc1 from synaptic terminals reduces the number of microtubule loops, whose presence correlates with halted bouton division, suggesting that Defhc1 is a negative regulator of bouton division. These results suggest that Defhc1 functions as an inhibitor of neurite growth by finely tuning the microtubule cytoskeleton dynamics and that EFHC1-dependent JME may result from augmented spontaneous neurotransmitter release due to overgrowth of neuronal processes.
Mutazioni nel gene EFHC1, che codifica per una proteina in grado di legarsi ai microtubuli, sono state correlate con l’insorgenza dell’Epilessia Mioclonica Giovanile (JME). Il gene EFHC1 è stato proposto come regolatore della divisione cellulare attraverso il controllo dell’organizzazione del fuso mitotico e come modulatore della migrazione dei neuroblasti nella corteccia cerebrale. Per comprendere in vivo la funzione del gene EFHC1 abbiamo generato il mutante knock-out per il gene omologo Defhc1 in Drosophila. Le sinapsi di giunzioni neuromuscolari (NMJ) di larve mutanti per Defhc1 mostrano un maggior numero di bottoni satellite e l’aumento del rilascio spontaneo di neurotrasmettitore. Esperimenti in vitro hanno dimostrato che la proteina Defhc1 si lega ai microtubuli e che in vivo colocalizza con i microtubuli sinaptici e assonali. In seguito all’eliminazione di Defhc1 dalle terminazioni sinaptiche è stata osservata una diminuzione del numero di loops formati dai microtubuli, la cui presenza è correlata con il blocco della divisione dei bottoni sinaptici, suggerendo che il gene Defhc1 possa essere un regolatore negativo della divisione dei bottoni sinaptici. Questi risultati suggeriscono che Defhc1, attraverso una fine regolazione della dinamicità dei microtubuli del citoscheletro, agisca da inibitore della crescita delle terminazioni sinaptiche tramite e che la JME dipendente da mutazione di EFHC1 potrebbe dipendere da un aumento del rilascio spontaneo di neurotrasmettitore conseguente all’eccessiva crescita sinaptica.

Orientadores: Li Li Min, Fernando Cendes
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A epilepsia mioclônica juvenil é o subtipo mais frequente entre as epilepsias generalizadas idiopáticas. Apresenta bom prognóstico e fácil controle em relação às crises epilépticas. O nosso objetivo foi a análise do tálamo combinada com métodos funcional e estruturais de neuroimagem. Estudamos vinte e um pacientes (treze do sexo feminino com média de idade 30 ± 9 anos) com epilepsia mioclônica juvenil do ambulatório de epilepsia dos Hospital das Clínicas da UNICAMP em Campinas. O grupo controle foi constitúído de dezoito voluntários hígidos (onze do sexo feminino) com média de idade 35 ± 16 anos. Todos foram submetidos ao protocolo de pesquisa incluindo ressonância magnética. Além da ressonância diagnóstica, foi realizada uma sequência em 3D para investigação estrutural e a colocação de um voxel único de (2x2x2cm3) em tálamo direito para a ressonância por espectroscopia de prótons. A morfometria baseada em voxel e análise da forma demonstraram as anormalidades talâmicas restritas em porção anterior e inferior. A espectroscopia evidenciou uma redução da relação de N-acetil aspartato e creatina. Existe alteração morfofuncional do tálamo e este padrão de comprometimento estrutural e funcional do tálamo é sugestivo da participação desta estrutura em uma rede neural na epilepsia mioclônica juvenil
Abstract: Juvenile myoclonic epilepsy is a common disease found among the group of all idiopathic generalized epilepsies. It shows a good prognosis, and seizures can be controlled easily. The objective of the present study was to make a comparison between thalamic analysis and functional and structural neuroimaging. We selected twenty-one patients (thirteen females) with mean age 30 ± 9 years old from our clinic in Hospital das Clinicas da UNICAMP, in (Campinas). Eighteen healthy volunteers (eleven females) with mean age 35 ± 6 years old were selected for the control group,. All subjects were submitted to a structured protocol of research including magnetic resonance. A 3D sequence with a single voxel (2x2x2cm3) on right thalamus was also performed for spectroscopy. Voxel based morphometry and shape analysis demonstrated thalamic abnormalities in anterior and inferior portions. Brain spectroscopy showed a decrease in NAA/Cr ratio. There is a dysfunction of the thalamus, and this pattern of structural abnormality suggests that the thalamus is involved in the neural network associated with myoclonic epilepsy
Doutorado
Neurociencias
Doutor em Fisiopatologia Medica

Orientadores: Marilisa Mantovani Guerreiro, Maria Augusta Montenegro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A Epilepsia Mioclônica Grave da Infância ou Síndrome de Dravet (SDr) e a Epilepsia Miclônico-Astática ou Síndrome de Doose (SDo) são epilepsias raras da infância, que cursam com crises epilépticas refratárias, nas quais há predomínio das mioclonias, e que podem levar à deterioração do desenvolvimento neuropsicomotor. Em aproximadamente 2/3 dos pacientes com SDr encontra-se mutação da subunidade alfa do canal de sódio (SCN1A). Nosso objetivo neste estudo foi de avaliar as características clínicas e eletrencefalográficas na SDr e SDo. Avaliamos 14 pacientes com SDr, com idade de início das crises entre três e 18 meses (média: 9 ± 4 meses), sendo quatro do sexo masculino e 10 do sexo feminino. Avaliamos também 13 pacientes com Sdo, com idade de início das crises entre dois e seis anos (média: 3,02 ± 1,22 anos), sendo sete do sexo masculino e seis do sexo feminino. Em todos os pacientes foram realizados exame neurológico tradicional, EEG (2 a 8 exames por paciente), RM crânio e Escala de Vineland, que estabelece o nível de maturidade social e psicomotora da criança, para permitir a avaliação do comportamento adaptativo nas áreas relacionadas à comunicação, atividade de vida diária, socialização e habilidades motoras fina e grossa. Foram encontradas alterações no exame neurológico em todos os pacientes com SDr e quatro pacientes com SDo. Ocorreu deterioração do comportamento adaptativo em todos os pacientes com SDr e oito pacientes com SDo. Nosso estudo encontrou ataxia, sinais leves de liberação piramidal, hiperatividade, espectro autístico nos pacientes com SDr e SDo. Foi observada lentificação da atividade de base e atividade epileptiforme no EEG em dez pacientes com SDr e ritmo de Doose em nove pacientes com SDo, além de atividade epileptiforme em todos os pacientes com SDo. Observamos alterações epileptiformes focais e generalizadas na SDr e alterações epileptiformes predominantemente generalizadas na SDo. A pesquisa da mutação da subunidade alfa do canal de sódio (SCN1A) foi realizada em 10 pacientes com SDr e em 10 pacientes com SDo e foi encontrada a mutação em seis pacientes com SDr, sendo normal nos demais. Nas SDr e SDo os nossos dados concordaram com os achados da literatura em relação aos achados clínicos, exame neurológico, EEG, exames de imagem, genotipagem e tratamento; no entanto, foi discordante da literatura quanto à distribuição quanto ao sexo na SDr, o que pode ser justificado pela nossa casuística modesta. A aplicação da Escala de Vineland foi dado inovador nos estudos da SDr e SDo. Acreditamos que nossa pesquisa permitiu melhor caracterização dessas duas síndromes epilépticas e mostrou resultados semelhantes aos estudos europeus, onde foram originalmente descritos
Abstract: Severe Myoclonic Epilepsy of Infancy or Dravet syndrome (SMEI) and Mioclonic-Astatic Epilepsy or Doose Syndrome (MAE) are rare childhood epilepsies, which occur with refractory seizures, where there is a predominance of myoclonic seizures. Deterioration of the neuropsychomotor development usually occurs. Approximately two thirds of patients of the patients with SMEI may have mutation of the alpha subunit of the sodium channel (SCN1A). Our objective was to evaluate the clinical and electroencephalographic features of SMEI and MAE. We evaluated 14 patients with seizure onset between three and 18 months of age with SMEI (mean age: 9 ± 4 months). Four were male and 10 female. We also evaluated 13 patients with seizure onset between two and six years with MAE (mean age: 3, 02 ± 1, 22 years). Seven were male and six female. We performed traditional neurological examination, EEG (2-8 exams per patient), cerebral MRI and Vineland Scale, that establishes the level of psychomotor and social maturity of the child to allow an assessment of the adaptive behavior in areas related to communication, activity of daily living, socialization and fine and gross motor skills. Our study found changes in the neurological examination of 14 patients with SMEI and four patients with MAE. We observed slowing of background activity and epileptiform activity in 10 patients with SMEI. Doose rhythm was detected in nine patients with MAE and epileptiform activity in all patients with MAE. Deterioration of adaptive behavior occurred in all patients with SMEI and eight patients with MAE. Our study showed ataxia, mild signs of pyramidal liberation, hyperactivity and autistic spectrum in patients with SMEI and MAE. We observed focal and generalized epileptiform discharges in patients with SMEI and disturbance was predominantly generalized in patients with MAE. The study of mutation of the alpha subunit of the sodium channel (SCN1A) was performed in 10 patients with SMEI and 10 patients with MAE and the mutation was found in six patients with SMEI and the remaining examination was normal. In SMEI and EMA our data are in keeping with the findings of the literature regarding clinical, neurological examination, EEG, cerebral MRI, genotyping and treatment; except for gender distribution in the SMEI, which can be attributed to the small study group. The assessment with Vineland scale brought a new light to the understanding of SMEI and MAE. We believe that our research has enabled better characterization of these two epileptic syndromes, showing that the results are similar to those from Europe, where they were originally described
Mestrado
Neurologia
Mestre em Ciências Médicas

Myoclonus dystonia (MD) is a rare autosomal-dominant combined dystonia movement disorder characterised by quick, involuntary muscle jerks (myoclonus) paired with sustained muscular contraction (dystonia). Although known to be genetically heterogeneous, the most common genetic factor is mutations within SGCE, the gene encoding ε-sarcoglycan, accounting for approximately 45% of cases. Previous linkage analyses conducted on a family displaying inherited MD without SGCE mutations lead to the identification of another critical region, DYT15. Preliminary data suggested that mutations within the long non-coding RNA (lncRNA) CD108131, found within the DYT15 locus, resulted in decreased expression of both the SGCE transcript, as well as the SGCE protein. Validation of the remaining variants of interest yielded no new candidate genes. A low coverage area coinciding with the entire sequence of TMEM200C was discovered, however subsequent sequencing data revealed no potential disease-causing variants. Therefore, to further characterise the relationship between CD108131 and SGCE suggested by the preliminary data, a CRISPR-Cas9 knockout was developed in HEK293 cells using a double-cut strategy that allowed for complete excision of the CD108131 gene. Stable CD108131 knockout mutant cell lines were examined for differences in gene expression. QRT-PCR analysis was conducted and revealed a significant decrease in SGCE expression in the absence of CD108131. Additionally, expression also trended towards a decrease for ZBTB14, however ARHGAP28 and RPPH1 were not significantly altered. This data demonstrates that the lncRNA CD108131 is likely to have a regulatory effect on SGCE, and perhaps ZBTB14, transcription.

In this PhD thesis I summarize my research into the genetics and pathophysiology of progressive myoclonus epilepsy (PME) associated with mutations in GOSR2. This disorder is characterized by early disease onset with ataxia around 3 years of age, followed by development of cortical myoclonus, generalized epilepsy and a rapid deterioration of motor function. Upon beginning my PhD, only one homozygous GOSR2 mutation – c.430G > T (p.G144W) – had been shown to cause PME. Furthermore, because GOSR2 encodes a Golgi SNARE protein (termed Membrin) that mediates ER-to-Golgi trafficking in every cell of the human body, it was an unresolved mystery how this mutation gives rise to a largely selective neuronal disorder. I first describe my discovery of the novel c.491-493delAGA (p.K164del) GOSR2 mutation in a PME patient who also carried the previously described c.430G > T variant in the compound heterozygous state. Overall, the clinical phenotype of this patient was remarkably consistent with previous cases, although her disease course appeared milder. My finding thus expanded the phenotypes and genotypes linked to this disorder, thus providing an additional tool to investigate the underlying disease mechanisms. In the subsequent chapters I summarize our attempts to unravel why the nervous system is selectively affected in GOSR2-PME. To this end I examined how pathogenic Membrin mutations impacted ER-to-Golgi trafficking in patient-derived fibroblasts, and developed novel Drosophila models of GOSR2-PME to study neuronal pathophysiology. Intriguingly, while ER-to-Golgi trafficking was remarkably preserved in G144W mutant Membrin fibroblasts, neuronal integrity was severely disturbed in GOSR2- PME model Drosophila, where dendrites were significantly shorter. Neurons have special secretory demands owing to their very large surface area, and hence appear selectively vulnerable to partial loss of function mutations in Membrin. Thus, the results presented in this thesis provide a possible explanation for the nervous system specificity of GOSR2-PME.

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

Orientador: Iscia Teresinha Lopes-Cendes
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os genes LGI1 e EFHC1 não codificam canais iônicos; entretanto, afetam indiretamente a corrente nesses canais em síndromes epilépticas determinadas geneticamente. O gene LGI1 (do inglês Leucine-rich, glioma inactivated gene 1) está relacionado à epilepsia parcial autossômica dominante com sintomas auditivos. Recentemente, observou-se a interação de LGI1 com ADAM22 em um complexo que possivelmente regula a transmissão sináptica. A forma mutante da LGI1 é incapaz de se ligar à ADAM22, o que fortalece a hipótese de um mecanismo relacionado à perda de função nesta síndrome epiléptica. O gene EFHC1 (do inglês EF-hand domain C-terminal containing 1) foi encontrado mutado em algumas famílias com epilepsia mioclônica juvenil. Células transfectadas com EFHC1 apresentam maior taxa de apoptose, o que indica uma possível participação em morte celular programada. Clonamos e expressamos as proteínas humanas LGI1 em sua forma inteira (AA 1-557) e a porção C-terminal (AA 224-557; LGI1C), também chamada de domínio epitempina; as formas N-terminal (AA 78-364; EFHC1N), que compreende dois domínios DM10, e C-terminal (AA 403-640; EFHC1C) de EFHC1, formada por um domínio DM10 e um EF-hand putativo. Diversas fusões foram testadas com a proteína LGI1, seja inteira (GST, Trx, SUMO), seja a porção C-terminal (GST, Trx, NusA, MBP e SUMO), em diferentes cepas de Escherichia coli. Obteve-se proteína solúvel com NusALGI1C e MBP-LGI1C; entretanto, só foi possível a captura por cromatografia de afinidade à amilose com a fusão à MBP, na presença ou não da chaperonina GroEL. Apesar do protocolo de purificação de MBP-LGI1C ter sido estabelecido, a clivagem da cauda não foi eficiente, além de apresentar baixo rendimento. Espalhamento de luz dinâmico e SAXS mostraram que a proteína fusionada apresentava-se em um estado de forte agregação. As porções N- e C-terminal de EFHC1 foram clonadas com a fusão SUMO, apresentando alta solubilidade em bactéria. Essas construções foram capturadas em cromatografia de afinidade a níquel e submetidas à clivagem da cauda: obteve-se rendimento próximo de 100% com EFHC1C, enquanto que com a EFHC1N não houve reação. EFHC1C clivada foi separada da cauda e passou por mais um passo de purificação, para polimento. Espectroscopia de dicroísmo circular mostrou que esta porção é composta principalmente por a-hélices e a temperatura de transição foi determinada em 54,5ºC, na presença ou ausência de agente redutor DTT, indicando alta estabilidade. Espalhamento de luz dinâmico e SAXS mostraram que a proteína está monodispersa e modelagem computacional econstituindo o envelope da EFHC1C por SAXS indica uma forma prolata. Em relação à EFHC1N, observou-se a presença de duas populações distintas de proteínas por SAXS e por filtração em gel
Abstract: Non-ion channel genes, as LGI1 and EFHC1, have been shown to indirectly affect ion channel currents in genetically determined epilepsy syndromes. LGI1 (Leucine-rich, glioma inactivated gene 1) is linked to a rare form of partial epilepsy (autosomal dominant partial epilepsy with auditory features, ADPEAF). Recently, LGI1 protein was associated with ADAM22 in a complex that regulates synaptic transmission. The mutated form of LGI1 is incapable of binding to ADAM22, leading to a loss of function mechanism causing ADPEAF. EFHC1 is mutated in some families with juvenile myoclonic epilepsy (JME). It has been observed that EFHC1 transfected cells have a higher rate of apoptosis; therefore, it seems that EFHC1 protein could be involved in programmed cell death. We have successfully cloned and expressed the full form (AA 1-557) and C-terminal epitempin domain of human LGI1 (AA 224-557); and the N-terminus (AA 78-364; named EFHC1N), comprasing two DM10 domains in tandem, and the C-terminus portion of human EFHC1 (AA 403-640; named EFHC1C), comprising one DM10 domain and the EF-hand motif. Several fusion constructs were tested with full LGI1 (GST, Trx and SUMO) and C-terminal half (GST, Trx, NusA, MBP and SUMO) in different Escherichia coli strains. Soluble protein was obtained with NusA-LGI1C and MBP-LGI1C, but only MBP-LGI1C was captured by affinity amilose in the presence or absence of chaperonine GroEL. Despite the fact that we suceffuly established a purification protocol for MBP-LGI1C, tag cleavage presented low yield. Dinamic light scattering and SAXS showed that LGI1C fused to MBP was strongly aggregated. Furthermore, the N- and C-terminal of EFHC1 were cloned with SUMO fusion and showed high solubility in bacteria. These constructions were captured by nickel affinity chromatography and submitted to cleavage reaction. Near complete cleavage was achieved with EFHC1C, but no cleavage was obtained with EFHC1N. SUMO tag was separeted from EFHC1Cand a final purification step was performed. Circular dichroism spectroscopy showed that EFHC1C is composed mainly by a-helices and transition temperature in the presence or absence of reducing agent DTT was 54.5ºC, indicating high stability. Dynamic light scattering and SAXS showed that EFHC1C is in a monodisperse state and presents a prolate shape. EFHC1N presents two different populations of proteins, as determined by SAXS and size exclusion chromatography
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular

The primary purpose of this study was the identification of patients with JME, in order to identify suitable families for studying the nature of the linkage of the syndrome of JME to the HLA (Human Leukocyte Antigen) locus. This search for suitable candidate families necessitated examination of a large number of individuals with a range of IGE syndromes, and patients were classified into these various syndromes. Patients with JME were thereafter investigated further. The study could thus be broken up into three sections: i) Determination of the various syndromes of IGE. ii) Characterisation of JME. iii) Linkage Studies on JME patients.

A Epilepsia Mioclônica Juvenil (EMJ) é uma epilepsia generalizada idiopática geralmente associada à ausência de alterações estruturais. Estudos neuropsicológicos, com um número restrito de paradigmas, sugerem que indivíduos com EMJ apresentam pior desempenho nos testes que avaliam funções executivas. De maneira complementar, a descrição da personalidade nos pacientes com EMJ corrobora os estudos neuropsicológicos, uma vez que relata maior exacerbação da impulsividade nestes pacientes, o que refletiria uma possível disfunção de lobo frontal. Estes estudos baseiam-se ou em observações clínicas ou nos critérios de classificação categorial do DSM-IV. Não há até o momento, estudos sobre traços de personalidade assim como a correlação com a presença de disfunção executiva e traços de personalidade impulsivos nos pacientes com EMJ. Além disso, o impacto da EMJ sobre o funcionamento social não foi estudado. Os objetivos deste estudo foram: 1. verificar se as funções atencionais e executivas encontram-se prejudicadas; 2. verificar se existem diferentes níveis de comprometimento das funções executivas e atencionais; 3. verificar se há alteração dos traços de personalidade, através de instrumento objetivo; 4. verificar se existem prejuízos da adequação social; 5. verificar a correlação entre o desempenho nas funções executivas e atencionais e a expressão de traços de personalidade relacionados a um pior controle de impulsos; 6. verificar a correlação entre as pontuações obtidas nos testes neuropsicológicos avaliadores das funções executivas e atencionais e os escores em adequação social e; 7. verificar se as variáveis clínicas da epilepsia se correlacionam com as pontuações obtidas nos testes neuropsicológicos, a expressão de traços de personalidade relacionados a um pior controle de impulsos e com os escores em adequação social em pacientes com EMJ. Para tanto, foram avaliados 42 pacientes com EMJ através de: a) bateria compreensiva de testes neuropsicológicos avaliadores de funções executivas e atencionais; b) questionário padronizado de avaliação de traços de personalidade (ITC) e; c) escala padronizada de avaliação da adequação social (EAS). Os desempenhos e escores nos testes, questionário e escala dos pacientes com EMJ foram comparados a um grupo de 42 sujeitos controle, sem diagnóstico psiquiátrico ou neurológico, pareados por idade, escolaridade e nível sócio-econômico. Os pacientes com EMJ tiveram piores desempenhos que os controles em testes de atenção imediata, controle mental, atenção seletiva e sustentada, flexibilidade mental, controle inibitório, fluência verbal, formação de conceitos, manutenção de metas, fluência verbal e memória verbal a curto prazo. Quanto à gravidade da disfunção executiva, observou-se que 83,33% apresentaram disfunção executiva moderada ou grave. Os pacientes com EMJ também apresentaram maior expressão dos traços de personalidade impulsivos e pior adequação social em comparação com o grupo controle. Disfunção executiva/ atencional se correlacionaram com o pior controle dos impulsos, porém não com o pior funcionamento social. Houve correlação entre a frequencia de crises e a presença de transtornos psiquiátricos com o pior desempenho executivo/atencional, com a maior expressão de traços impulsivos e com a pior adequação social. A maior duração da epilepsia e a idade de início precoce tiveram relação com a disfunção executiva e personalidade, respectivamente. Na análise categorial entre os grupos de fácil e difícil controle, pacientes refratários apresentaram pior disfunção executiva e uma presença ainda mais expressiva dos traços de personalidade impulsivos. Nosso estudo demonstra a presença de disfunção atencional e executiva nos pacientes com EMJ, além da presença de traços de personalidade impulsivos. O presente estudo identificou a presença de pior funcionamento social destes pacientes. Além disso, verificamos a existência de dois grupos distintos de pacientes, sendo que pacientes mais refratários apresentam-se globalmente comprometidos. Estes achados sugerem que há uma necessidade de melhor caracterização fenotípica dos pacientes com EMJ a fim de incluir endofenótipos visto que nossos resultados demonstram uma possível existência de grupos distintos de pacientes com EMJ.
Juvenile Myoclonic Epilepsy (JME) is an idiopathic generalized epilepsy usually associated with absence of structural changes. Neuropsychological studies in patients with JME, using a limited number of paradigms, show worse performance on tests assessing executive functions. In addition, the description of personality traits in patients with JME corroborates neuropsychological studies, reporting failure of impulse control in these patients, which reflects a possible frontal lobe dysfunction. These studies are either based on clinical observations or on the categorical classification criteria of DSM-IV. To moment, the correlation between executive dysfunction and impulsive personality traits in patients with JME, has not been performed, as well as neither an objective study of the social adjustment of this population. The objectives of this study were to: 1. Investigate if attentional and executive functions are impaired; 2. Investigate if there are different levels of impairment in attentional and executive functions 3. Investigate if there are alterations in personality traits using an objective instrument 4. Investigate if there is impairment of social functioning 5. Investigate if there are correlations between performance on attentional and executive functions and the expressions of personality traits related to poor impulse control, 6. Verify if there are correlations between scores on neuropsychological tests of attentional and executive functions and social functioning 7. Investigate if there are correlations between clinical variables of epilepsy and neuropsychological performance, expression of impulsive personality traits and social functioning. We evaluated 42 patients with JME by: a) a comprehensive battery of neuropsychological tests of attentional and executive functions b) a standardized assessment of personality traits (TCI) and c) a standardized scale for assessing social functioning (SAS-SR). The performances and scores on tests, of patients with JME were compared to a group of 42 control subjects without neurological or psychiatric diagnosis, matched for age, education and socioeconomic status. Patients with JME showed worse performance than controls on tests of attentional span, working memory, inhibitory control, concept formation, maintenance of goals, verbal fluency and immediate verbal memory. We also observed that 83.33% of the patients with JME had moderate or severe executive dysfunction. Patients with JME showed higher expression of personality traits associated with an impaired impulse control and worse social functioning, when compared with the control group. Attentional/ executive dysfunction was correlated with poor impulse control, but not with worse social functioning. We found correlations between the frequency of seizures and the presence of psychiatric disorders with attentional and executive dysfunction, with the highest expression of impulsive traits and with worsened social functioning. Longest duration of epilepsy and the early age of onset were respectively associated with executive dysfunction and personality. In the category analysis between groups of patients easy and difficult to control seizures, refractory patients had worse executive dysfunction with an even a greater presence of impulsive personality traits. Our study demonstrates the presence of attentional and executive dysfunction in patients with JME, as well as the presence of impulsive personality traits. Moreover, this study identified the presence of poor social functioning in these patients. We also note the existence of two distinct groups of patients, were more refractory patients appear to present broader impairment. These findings suggest that there is a need for better phenotypic characterization of patients with JME to include diverse phenotypes since our results suggest a possible existence of distinct groups of patients with JME.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

Glycine receptor antibodies have been identified in a few patients with progressive encephalomyelitis with rigidity and myoclonus (PERM), a highly disabling disorder characterised by rigidity, spasm and brainstem symptomatology. The clinical characteristics of patients with glycine receptor antibodies have not yet been fully described and it is not clear whether GlyR-Abs are pathogenic or just an epiphenomenon. This study examined the clinical features and immunotherapy responses of 45 patients; characterised the GlyR-Ab pathogenicity, subunit specificity and binding to different brain region in vitro, and examined mice injected with GlyR-Abs to model the disease in vivo. Most of the patients were classified as PERM but some patients had symptomatology beyond the classical motor manifestations and there were four patients with tumours (thymomas and lymphomas). GlyR-Ab titres were varied in serum and CSF, but there was intrathecal synthesis in the six patients with suitable samples. Most patients were very disabled but almost all showed excellent responses to immunotherapies. The antibodies were mainly IgG1 and IgG3 subclasses, activated complement on glycine receptor-transfected HEK cells at room temperature, and caused internalisation and lysosomal degradation of the glycine receptors at 37°C. GlyR-Abs bound to rodent spinal cord and brainstem co-localising with monoclonal antibodies to GlyRα1 on the surface of neurons. GlyR-IgG injected intra-peritoneally led to impairment in forced walking ability, sensorimotor function and coordination. Analysis of the brain showed that animals injected with patients' IgG, but not control IgG, had antibodies bound to the brainstem, spinal cord, cerebellum and caudate, co-localising with GlyRα1 monoclonal antibody. Intra-cerebroventricular injection of GlyR-IgG caused an anxiety-like behaviour in mice but no evident motor disturbances. These results provide the first evidence of in vitro and in vivo pathogenicity of the GlyR-Abs, supporting the use of long term immunosuppression in these patients to provide them with a good prognosis.

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Objetivo: As bases neuroanatômicas e as anormalidades bioquímicas subjacentes à epilepsia mioclônica juvenil (EMJ) não são totalmente conhecidas. Apesar de o tálamo atuar na sincronização de diversas regiões do córtex cerebral durante uma crise, há evidências sugerindo que nem todos os neurônios corticais são afetados de forma homogênea. Compreender a participação destas redes neuronais específicas na EMJ pode esclarecer alguns de seus mecanismos fisiopatológicos. O objetivo deste estudo foi investigar as diferenças metabólicas e estruturais cerebrais entre pacientes com EMJ e controles normais. Métodos: Todos os pacientes possuíam o diagnóstico de EMJ baseado em história e semiologia das crises, eletrencefalografia (EEG), vídeo-EEG e neuroimagem por ressonância magnética (RM) convencional normal, conforme os critérios da Comissão de Classificação e Terminologia da International League Against Epilepsy, 1989. Sessenta pacientes com EMJ foram submetidos a protocolos de espectroscopia de prótons e morfometria baseada em voxels (VBM) por RM de 1,5 T. O grupo controle foi constituído por 30 voluntários saudáveis, pareados por sexo, idade e dominância manual. Este estudo foi realizado após aprovação do comitê de ética da instituição e obtenção de consentimento informado, por escrito, de todos os participantes. Resultados: Demonstrou-se redução da razão de N-acetilaspartato/Creatina (NAA/Cr) dos pacientes com EMJ em relação ao grupo controle nos córtices frontal, pré-frontal e no tálamo. Observou-se diferença na razão do complexo glutamato-glutamina (GLX)/Cr nos córtices frontal, pré-frontal, ínsula, corpo estriado e cíngulo posterior entre os dois grupos. Análise por regressão múltipla nos pacientes com EMJ demonstrou maior correlação funcional entre o tálamo e o córtex pré-frontal. Também foi encontrada correlação negativa entre NAA/Cr e a duração da epilepsia. Análise estrutural quantitativa por VBM demonstrou redução do volume da substância cinzenta no tálamo, ínsula e cerebelo bilateralmente e aumento do volume do córtex frontal nos pacientes. Conclusões: O comprometimento de algumas regiões cerebrais nestes pacientes sugere envolvimento de um circuito tálamo-cortical específico na fisiopatologia desta síndrome, considerada “generalizada”. Reduções em NAA podem representar perda ou lesão de neurônios ou axônios bem como disfunções metabólicas, enquanto o GLX é considerado um neurotransmissor excitatório, envolvido na patogênese das crises epilépticas. As anormalidades estruturais encontradas reforçam a existência de uma rede ictogênica anátomo-funcional específica na EMJ e o conceito de ‘system epilepsies’.
Purpose: The neuroanatomical basis and the neurochemical abnormalities that underlie juvenile myoclonic epilepsy (JME) are not fully understood. While the thalamus plays a central role in synchronization of widespread regions of the cerebral cortex during a seizure, emerging evidence suggests that all cortical neurons may not be homogeneously involved. The purpose of this study was to investigate the cerebral metabolic and structural differences between JME patients and normal controls. Methods: All patients had a JME diagnosis based on seizure history and semiology, EEG recording, normal magnetic resonance neuroimaging (MRI) and video-EEG according to the Commission on Classification and Terminology of the International League Against Epilepsy, 1989. Sixty JME patients (JME-P) were submitted to 1.5 T MRI multi-voxel proton spectroscopy and voxel-based morphometry (VBM). The control group consisted of 30 age and sex-matched healthy volunteers. The Institutional Ethics Committee approved the study, and informed consent was obtained from all participants. Results: Group analysis demonstrated lower N-acetyl-aspartate/Creatine (NAA/Cr) ratio among patients compared to controls on prefrontal, frontal cortices and thalamus. Patients had a statistically significant difference in glutamate-glutamine complex (GLX)/Cr on prefrontal and frontal cortices, insula, striatum and posterior cingulate gyrus. When evaluating the relationship among the various components of this epileptic network among JME-P, the strongest correlation occurred between thalamus and prefrontal cortex and a significant negative correlation between NAA/Cr and duration of epilepsy was found. Also, VBM demonstrated significantly reduced gray matter volume (GMV) in thalami, insula cortices and cerebellar hemispheres bilaterally; while significantly increased GMV was observed in right superior frontal, orbitofrontal and medial frontal gyri among JME-P when compared to controls. Conclusions: The identification of a specific network of neurochemical dysfunction and slight structural abnormalities in patients with JME, with diverse involvement of particular structures within the thalamocortical circuitry, suggests that cortical hyperexcitability in JME is not necessarily diffuse, supporting the knowledge that the focal/generalized distinction of epileptogenesis should be reconsidered and reinforcing the concept of ‘system epilepsies’.
TEDE
BV UNIFESP: Teses e dissertações

As epilepsias generalizadas idiopáticas (EGIs) associam-se a controle satisfatório de crises e a exames de neuroimagem convencionais normais. Métodos de neuroimagem avançada, como DTI (diffusion tensor imaging) e VBM (voxel based morphometry), permitiram melhor compreensão dos mecanismos envolvidos no comportamento clínico das EGIs. O objetivo do estudo foi avaliar diferenças clínicas entre pacientes com EGI não refratária e refratária, assim como avaliar as diferenças entre pacientes com EGI não refratária, refratária e indivíduos saudáveis através de ressonância por DTI e VBM. Avaliamos 40 pacientes com características clínicas e eletrencefalográficas de EGI, sendo 22 pacientes com EGI não refratária (GNR) e 18 pacientes com EGI refratária (GR). Participaram do estudo 20 indivíduos saudáveis, os quais compuseram o grupo controle (GC). O grupo GR apresentava maior número de pacientes usuários de fármacos benzodiazepínicos (p=0,01) e de fármacos antiepilépticos não-valproato (p=0,02). Pacientes do grupo GR também utilizavam doses maiores de VPA que os pacientes do grupo GNR (p=0,03) e recebiam maior carga total média de fármacos antiepilépticos (p=0,04). Observou-se, em relação aos 16 feixes e tratos avaliados nos índices de DTI (AF, DM, DR, DA) que houve diferença estatística do grupo GNR em relação ao GC em duas áreas do índice AF (anisotropia fracional), seis áreas do índice DM (difusividade média), seis áreas do índice DR (difusividade radial) e seis áreas do índice DA (difusividade axial), assim como houve diferença estatística do grupo GR em relação ao GC em duas áreas do índice AF, sete áreas do índice DM, seis áreas do índice DR e três áreas do índice DA. Entre as 94 regiões estudadas por VBM, observou-se redução volumétrica estatística em nove áreas de interesse no GNR quando em comparação ao GC e em sete áreas de interesse no GR quando em comparação ao GC. Não se observaram diferenças entre os grupos GNR e GR nos parâmetros avaliados por DTI ou por VBM. Como esperado, observamos que pacientes com EGI refratária mais frequentemente utilizam fármacos antiepilépticos de segunda linha ou não habituais a este tipo de epilepsia. O estudo permitiu concluir que o comprometimento encefálico nas EGIs analisadas é difuso e envolve áreas habitualmente não associadas a estas epilepsias, como o hipocampo e outras áreas temporais, e que os achados imagenológicos não se associam à refratariedade clínica dos pacientes
Generalized idiopathic epilepsies (IGEs) are usually associated with good seizure control and normal conventional neuroimaging exams. Advanced neuroimaging methods, such as DTI (diffusion tensor imaging) and VBM (voxel based morphometry) have provided a better understanding of the IGEs. This study´s primary objective was to evaluate clinical diferences between refractory and non-refractory IGEs, and to compare advanced MRI methods (DTI and VBM) findings in refractory and non-refractory IGE patients. Forty IGE patients were divided in two groups: 22 non-refratory (NRG) patients and 18 refractory (RG) patients. Twenty healthy subjects were enrolled as a control group (CG). RG patients received benzodiazepines (p=0,01) and non-valproate antiepileptic drugs (p=0,02) more often than NRG patients. RG group also received a higher mean total of antiepileptic drug load (p=0,04) than NRG group. Regarding neuroimaging methods, DTI index analysis (FA, MD, RD, AD) statiscally demonstrated that NRG group had two compromised areas on FA (fractional anisotropy) index, six areas on MD (mean diffusivity) index, six areas on RD (radial diffusivity) index and six areas on AD (axial diffusivity) index, when compared to CG. On RG group, DTI index analysis statiscally demonstrated that this group had two compromised areas on FA index, seven areas on MD index, six areas on RD index and three areas on AD index, when compared to CG, of 16 analyzed areas of interest. VBM analysis of 94 regions of interest showed reduced volumes in nine areas in the NRG group when compared to CG and in seven areas of interest in the RG group when compared to CG. We found no differences on DTI and VBM parameters comparing NRG and RG groups. As expected, refractory IGE patients received second line or non-usual antiepileptic drugs for this epilepsy type more often than non-refractory patients. We concluded that brain involvement´s in IGEs is diffuse and affects areas usually not related to this epilepsy type, such as the hipocampus and other temporal areas. Advanced neuroimaging findings in IGEs were not associated with clinical refractoriness

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One important information for the classification of epilepsy is the cortical localization of the discharges source. Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy (IGE) that typically presents generalized tonic-clonic, myoclonic, or absence seizures, or a combination of these. In typical cases of JME, the seizures are usually bilateral and symmetric, and EEG shows generalized interictal epileptiform discharges and a generalized seizure pattern that also is bilaterally synchronous. Despite of the generalized pattern of this type of epilepsy, there are some electroencephalographic and clinical features that suggest focal origin for the discharges. In this work, EEG recordings of six patients were analyzed in order to find evidences for this cortical origin in JME. The analysis of the signals was based on independent component analysis (ICA) for separating epileptiform discharges from artifacts and other brain sources; then the discharge components were used to spatially localize its source. In the six patients the dipole sources were localized mainly in the frontal region, what suggests an important participation of the frontal lobe for this kind of epilepsy.
Uma informação importante para a classificação da epilepsia é a localização cortical das suas fontes de descargas. A epilepsia mioclônica juvenil (EMJ) é uma epilepsia generalizada idiopática (EIG), que tipicamente apresenta crises tônico-clônicas, mioclônicas , crises de ausênica ou uma combinação destas. Em casos típicos de EMJ, as crises são geralmente bilaterais e simétricas, e o EEG mostra descargas epileptiformes interictais generalizadas em um padrão geralmente sincrônico. A despeito dos padrões generalizados deste tipo de epilepsia, há algumas características eletroencefalográficas e clínicas que sugerem uma origem focal para estas descargas. Neste trabalho, os registros de EEG de seis pacientes foram analisados, afim de encontrar evidências para uma origem cortical em EMJ. O processamento dos sinais foi baseado na técnica de análise de componentes independentes (ICA), com a finalidade de separar descargas epileptiformes de artefatos e de outras fontes cerebrais. Após esse processo, as componentes de descargas foram usadas para localizar espacialmente suas fontes. Em seis pacientes, as fontes dipolo foram localizadas principalmente nas regiões frontais, o que sugere uma importante participação do lobo frontal para esse tipo de epilepsia.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O tremor congênito em suínos possui diversas etiologias, inclusive os pestivirus. O objetivo deste trabalho foi avaliar se o vírus da diarreia viral bovina (BVDV) é um dos agentes etiológicos da enfermidade. Para tal, foi realizada inoculação de dez fêmeas suínas gestantes com BVDV-2 em dois diferentes modelos experimentais, sendo o primeiro a inoculação oronasal das fêmeas (Grupo 1; n=4), e o segundo a inoculação fetal intrauterina (Grupo 2; n=4). O terceiro grupo (Grupo 3; n=2) foi o controle. As marrãs e os fetos foram desafiados aos 45 dias de gestação com BVDV-2. Foram colhidas amostras sangue de todos os leitões nascidos para obtenção de sangue total e soro, para determinação dos títulos de anticorpos pela virusneutralização (VN) e detecção de RNA viral pela técnica de RTPCR. Um terço dos neonatos foram eutanasiados ao terceiro dia de idade, e deles coletaram-se fragmentos de encéfalo, tronco encefálico e medula espinhal para avaliação anatomohistopatológica e RT-PCR. Os leitões que permaneceram vivos foram avaliados clinicamente todos os dias, e foi realizada colheita de sangue periodicamente durante 35 dias, as quais foram submetidas à sorologia (VN) e RTPCR. Os leitões de ambos os grupos não apresentaram sinais clínicos neurológicos e nasceram com ausência de vírus no sangue e nos órgãos. Os leitões do Grupo 1 não apresentaram anticorpos contra o BVDV-2 ao nascimento, que posteriormente foram adquiridos por transferência passiva materna. Ao contrário, os leitões do Grupo 2 nasceram com altos títulos de anticorpos contra o agente, que permaneceram altos até o término do período experimental. Microscopicamente, não foram observadas alterações dignas de nota. Macroscopicamente, observou-se que 29,5% do total de leitões abatidos dos grupos infectados nasceram com baixa relação entre cérebro e cerebelo, o que pode ser indicativo de hipoplasia cerebelar. Desta forma, concluiu-se que o BVDV não parece ser um agente etiológico para o tremor congênito suíno.
Congenital tremor in pigs has several etiologies, including pestiviruses. The objective of this study was to evaluate whether bovine viral diarrhea virus (BVDV) is one of the etiological agents of this disease. Ten pregnant gilts were inoculated with BVDV-2 in two different experimental models, the first being the oronasal inoculation of the females (group 1; n=4), and the second was the intrauterine fetal inoculation (group 2; n=4). The third group (group 3; n=2) constituted the control group. Gilts and fetuses were challenged at 45 days of gestation with strain BVDV-2 SV 280. Blood samples were collected from all piglets born to obtain whole blood and serum for determination of antibody titers by virus neutralization (VN) and detection of viral RNA by the RT-PCR technique. One third of the neonates were euthanized at the third day of age, and fragments of brain, cerebellum, brain stem and spinal cord were collected for anatomopathological and RT-PCR evaluation. The piglets that remained alive were clinically evaluated every day, and blood samples were collected periodically for 35 days, which were submitted to serology (VN) and RT-PCR. The piglets of both groups showed no clinical neurological signs and were born without virus in the blood and organs. Group 1 piglets did not present antibodies against BVDV-2 at birth, which were acquired by passive maternal transfer. In contrast, Group 2 piglets were born with high antibody titers against the agent, which remained high until the end of the experimental period. Microscopically, no noticeable changes were observed. Macroscopically, it was observed that 29.5% of the total piglets slaughtered from the infected groups were born with a low ratio between brain and cerebellum, which may be indicative of cerebellar hypoplasia. Thus, it was concluded that BVDV does not appear to be an etiological agent for congenital pig tremor.
409435/2016-3
2016/02982-3,