Dissertations / Theses on the topic 'Developmental and epileptic encephalopathy'

12 month embargo; Available online 9 November 2016
SCN8A encephalopathy is a severe, early-onset epilepsy disorder resulting from de novo gain-of-function mutations in the voltage-gated sodium channel Na(v)1.6. To identify the effects of this disorder on mRNA expression, RNA-seq was performed on brain tissue from a knock-in mouse expressing the patient mutation p.Asn1768Asp (N1768D). RNA was isolated from forebrain, cerebellum, and brainstem both before and after seizure onset, and from age-matched wildtype littermates. Altered transcript profiles were observed only in forebrain and only after seizures. The abundance of 50 transcripts increased more than 3-fold and 15 transcripts decreased more than 3 fold after seizures. The elevated transcripts included two anti-convulsant neuropeptides and more than a dozen genes involved in reactive astrocytosis and response to neuronal damage. There was no change in the level of transcripts encoding other voltage-gated sodium, potassium or calcium channels. Reactive astrocytosis was observed in the hippocampus of mutant mice after seizures. There is considerable overlap between the genes affected in this genetic model of epilepsy and those altered by chemically induced seizures, traumatic brain injury, ischemia, and inflammation. The data support the view that gain-of-function mutations of SCN8A lead to pathogenic alterations in brain function contributing to encephalopathy.

Razionale e Obiettivi: Lo spettro clinico dell’epilessia dovuta a mutazione del gene PCDH19 è molto variabile, essendo riportati sia pazienti con epilessia farmacoresistente, disabilità intellettuale (ID) e disturbo dello spettro autistico (ASD) sia pazienti con buon controllo delle crisi e normale sviluppo cognitivo. Scopo di questo studio analizzare un’ampia popolazione di pazienti con mutazione PCDH19 al fine di definire il fenotipo elettro-clinico, la correlazione genotipo-fenotipo, definire l’outcome a lungo termine e identificare i fattori prognostici, e gli aspetti clinici importanti per la diagnosi differenziale con la sindrome di Dravet (DS). La tramissione x-linked, ha permesso di ipotizzare una differente espressione genica dei geni che codificano per gli enzimi AKR1C1-3, coinvolti nel metabolismo degli steroidi, con conseguente riduzione dei livelli plasmatici di allopregnanolone e questo studio si propone si verificare tale ipotesi mediante misurazione dei neurosteroidi nelle pazienticon mutazione PCDH19. Metodi: Sono stati retrospettivamente collezionati i dati genetici, clinici ed EEG di 61 pazienti con epilessia dovuta a mutazione del gene PCDH19, provenienti da 15 centri italiani. I pazienti sono stati divisi in due gruppi a seconda dell’outcome e sono state analizzate le seguenti variabili: mutazione, età di esordio, età al follow-up, tipo di crisi, occorrenza di stato epilettico, anomalie EEG. La ROC analisi è stata utilizzata per verificare la presenza di una eventuale età di riduzione della frequenza delle crisi. 15 pazienti con mutazione PCDH19 sono stati confrontati con 19 pazienti con DS Fisher's exact test o Student's t-test. Per verificare la riduzione di allopregnanolone, è stato condotto uno studio caso-controllo prospettico (12 pazienti-15 controlli). L’analisi dei neurosteroidi è stata effettuata sia in condizioni basali che dopo stimolo con ACTH, mediante spettroscopia. Risultati: All’ultimo follow-up (mediana 12 anni; range 1.9-42.1), 13 pazienti (21,3%) avevano crisi settimanali, mensili, 78,8% crisi annuali, e 12 pazienti (19.7%) erano seizure-free da almeno 2 anni. La curva ROC ha mostrato una riduzione delle crisi dopo 10.5 anni (sensibilità 81.0%; specificità 70.0%). 36 pazienti avevano ID, 31 pazienti ASD. L’ètà d’esordio dell’epilessia più precoce è risultata l’unico fattore predittivo di ID (p=0.05) e ASD (p<0.014). Al contrario non sono stati identificati fattori predittivi dell’outcome dell’epilessia. L’età di esordio più precoce è risultata essere un fattore clinico utile per la diagnosi differenziale con la DS (5.0+2.1 vs 11.2+7.0 mesi; p<0.05) così come la latenza tra la prima e la seconda crisi (10.1+13.6 vs 2.2+2.1 mesi; p<0.05). Dall’analisi dei livelli plasmatici dei neurosteroidi è risultata una ridotta produzione nei pazienti rispetto ai controlli, confermata anche dopo il test con ACTH. Conclusioni: Questo studio ha premesso di identificare l’età di esodio precoce quale indicatore prognostico di un outcome peggiore caratterizzato da epilessia farmacoresistente e disabilità cognitiva. La riduzione della frequenza delle crisi con la pubertà ben correla con l’ipotesi della down-regulation dei neurosteroidi che è stata confermata con l’analisi dei livelli plasmatici. Questo risultato apre a nuove possibilità terapeutiche, essendo i neurosteroidi e in particolare l’allopregnanolone un modulatore allosterico del recettore GABA-A e quindi modulatore dell’eccitabilità neuronale.
Objective: PCDH19-related epilepsy is an epileptic syndrome, arising within the first three years of life and characterized by clustered and fever-induced seizures. In most of cases it is associated with Intellectual Disability (ID) and autistic features. Aim of this study is to analyze a large Italian population with PCDH19-related epilepsy in order to better define the epileptic phenotype, identify genotype-phenotype correlation, predicting factors for outcome, and markers for differential diagnosis with Dravet Syndrome (DS). The unusual, gender reversed X-chromosome inheritance of PCDH19-FE led also to speculate that genes with different expression between the two sexes may play a role in the pathogenesis and that AKR1C1-3 genes, could be dysregulated, thus resulting in allopegnanolone reduced blood levels. Methods: We retrospectively collected genetic, clinical and EEG data of 61 patients affected by PCDH19-related Epilepsy, coming from 15 Italian hospitals. We stratified patients into two groups according the outcome. We analysed the following variables: mutation type, age at onset, age at study, seizure type, occurrence of status epilepticus, EEG abnormalities, cognitive and behavioural disorders. ROC curve analysis was performed in order to discriminate the age at which seizures could decrease in frequency. A group of 15 patients with PCDH19-related epilepsy were compared with 19 patients with DS. Comparisons were performed with Fisher's exact test or Student's t-test. In order to ascertain allopregnanolone deficiency, we performed a prospective case-control study. We enrolled 12 patients affected by PCDH19-related epilepsy and 15 controls, age-and sex-matched. Controls were recruited among subjects evaluated for praecox puberty or hyperandrogenism. In both groups blood samples were taken at basal (T0) and 60 min after (ACTH) administration (T1). Quantitative analysis of neuroactive steroids in serum was performed by liquid chromatography-electrospray tandem mass spectrometry. Results: At last follow-up (median 12 years; range 1.9-42.1), 13 patients (21.3%) had monthlyweekly seizures, 78.7% annual seizures/clusters or less frequent. Twelve patients (19.7%) were seizure-free since > 2 years. ROC analysis showed a significant decreasing of seizure frequency after the age of 10.5 years (sensitivity 81.0%; specificity 70.0%). Thirty-six patients (59.0%) had ID and behavioral disturbances. ID was moderate-severe in almost half of them. Autistic spectrum disorder was present in 31 patients. An earlier age at epilepsy onset resulted the only predictor factor for ID (p=0.05) and autistic spectrum disorder (p<0.014). Conversely, age at onset was not a predictor factor for seizure outcome (p<0.214). Epilepsy onset was earlier in DS (5.0+2.1 vs 11.2+7.0 months; p<0.05). The second seizure/cluster occurred after a longer latency in PCDH19- related epilepsy rather than in DS (10.1+13.6 vs 2.2+2.1 months; p<0.05). All neuroactive steroids resulted down produced in patients with PCDH19-related epilepsy rather than controls and this data was confirmed after ACTH stimulus. Conclusions: We found that an earlier age at epilepsy onset is linked with a significant risk for ID and autistic spectrum disorder. The decreasing of seizure frequency after the age of 10.5 years supports the hypothesis of a down-regulation of neurosteroid-metabolizing enzymes and allopregnanolone deficiency in PCDH19-related epilepsy. We also documented a down regulation of all steroidogenesis in PCDH19-related epilepsy. Particularly we found allopregnanolone and pregnenolone sulfate deficiency. Allopregnanolone is a GABA-A receptor modulator influencing the neuronal excitability, thus representing a realistic therapeutic target for PCDH19-related epilepsy. We failed to identify any genotype-phenotype correlation considering the site and type of PCDH19 mutations. We were able to find out some distinctive features, which could address the diagnosis towards DS or PCDH19-related epilepsy, since first manifestation. These considerations suggest to definitively considering PCDH19 gene as cause of a proper epileptic phenotype.

Abstract Novel genetic aetiologies for epileptic encephalopathies and movement disorders have been discovered by using next-generation sequencing methods. The phenotypic and genotypic variability in these conditions is very wide. The aim of this study was to discover novel genetic causes and phenotypes of childhood-onset drug-resistant epilepsy and epileptic or developmental encephalopathies that occur separately or together with movement disorders, and familial movement disorders. Furthermore, the use of whole-exome sequencing (WES) as a diagnostic tool in clinical practice was evaluated. Altogether, 12 children with undefined aetiology, who fulfilled the inclusion criteria, were included in the study. GABRG2 gene was identified as a genetic cause of epileptic encephalopathies. Novel GABRG2-associated phenotypes included progressive neurodegeneration, epilepsy in infancy with migrating focal seizures, and autism spectrum disorder. New pathogenic variants, GABRG2 p.P282T and p.S306F, were discovered. The pathogenic NACC1 variant caused focal epilepsy, developmental disability, bilateral cataracts, and dysautonomia. The novel phenotype associated with the NACC1 p.R298W variant included hyperkinetic movement disorder. SAMD9L was found to be the genetic cause for the familial movement disorder. The phenotype associated with the novel SAMD9L p.I891T variant was very variable. Neuroradiological findings included cerebellar atrophy and periventricular white matter changes. After publication of these results, SAMD9L was reported to be one of the most common genetic aetiologies of childhood bone marrow failure and myelodysplastic syndrome. The pathogenic homozygous MTR variant was found to cause early-onset epileptic encephalopathy that occurred together with movement disorder and haematological disturbances. Drug resistant seizures responded to cofactor and vitamin treatments. Whole-exome sequencing for 10 patients with drug-resistant epilepsy or epileptic or developmental encephalopathy provided a genetic diagnosis for two patients (20%). This study confirmed that, for epileptic encephalopathies and movement disorders in which the genetic causes and phenotypes are heterogeneous and sometimes treatable, WES is a useful tool for diagnostics and in the search for novel aetiologies, which might turn out to be more common than expected
Tiivistelmä Uusien sekvensointimenetelmien käyttöönotto on mahdollistanut epileptisten enkefalopatioiden ja liikehäiriöiden uusien geneettisten syiden löytymisen. Näissä sairausryhmissä geenien ja ilmiasujen vaihtelevuus on suurta. Tutkimuksen tarkoituksena oli löytää uusia geneettisiä syitä ja ilmiasuja lapsuusiällä alkavissa vaikeahoitoisissa epilepsioissa ja epileptisissä tai kehityksellisissä joko itsenäisesti tai yhdessä liikehäiriön kanssa esiintyvissä enkefalopatioissa sekä perheittäin esiintyvissä liikehäiriöissä. Lisäksi selvitettiin eksomisekvensoinnin käyttökelpoisuutta kliinisessä diagnostiikassa näiden potilasryhmien kohdalla. Tutkimukseen osallistui yhteensä 12 sisäänottokriteerit täyttävää lasta, joiden sairauden syy oli jäänyt tuntemattomaksi. GABRG2-geenin mutaatiot aiheuttivat epileptisiä enkefalopatioita, joiden uutena ilmiasuna oli etenevä taudinkuva, johon liittyivät aivojen rappeutuminen, migroiva imeväisiän paikallisalkuinen epilepsia sekä autismikirjon häiriö. Tutkimuksessa löydettiin uusia GABRG2-mutaatioita: p.P282T ja p.S306F. NACC1-geenin mutaatio aiheutti epilepsian, kehitysvammaisuuden, molemminpuolisen kaihin ja autonomisen hermoston toiminnan häiriön. Hyperkineettinen liikehäiriö oli uusi NACC1 p.R298W -mutaatioon liittyvä ilmiasu. SAMD9L-geenin mutaatio aiheutti perheessä esiintyvän liikehäiriön. Neurologinen ja hematologinen ilmiasu olivat hyvin vaihtelevia. Aivojen kuvantamislöydöksiin sisältyi pikkuaivojen rappeutumista ja valkoisen aivoaineen muutoksia aivokammioiden ympärillä. Näiden tutkimustulosten julkaisemisen jälkeen SAMD9L-geenin mutaatioiden on todettu olevan yksi yleisimmistä perinnöllisistä luuytimen vajaatoiminnan ja myelodysplasian syistä. Homotsygoottinen MTR-geenin mutaatio aiheutti varhain alkaneen epileptisen enkefalopatian, liikehäiriön ja hematologisen häiriön. Kofaktori- ja vitamiini hoidot vähensivät epileptisiä kohtauksia, joihin tavanomainen lääkitys ei tehonnut. Geneettiset syyt ja ilmiasut ovat epileptisissä enkefalopatioissa ja liikehäiriöissä hyvin vaihtelevia, ja osaan on olemassa spesifi hoito. Eksomisekvensointi on käyttökelpoinen diagnostiikan ja uusien geneettisten syiden etsimisen apuna. Tässä tutkimuksessa eksomisekvensoinnin avulla kymmenestä potilaasta kahdelle (20%) saatiin varmistettua geneettinen diagnoosi

Objectives: To fully re-evaluate patients with early-onset epilepsy and intellectual disability with neurological, neurophysiological and neuropsychological examination in order to contribute to expanding the phenotypic spectrum of known epileptic encephalopathy (EE)-related genes and to identify novel genetic defects underlying EEs. Methods: We recruited patients with epilepsy and intellectual disability (ID) referring to our Epilepsy Centre. Patients underwent full clinical and neurophysiologic evaluation. When possible they underwent neuroradiologic investigations. Selected cases also underwent genetic analysis. Results: We recruited 200 patients (109 M, 91 F; mean age 36 years old). Mean age at epilepsy onset was 4 years old. The degree of ID was borderline in 4.5% of patients, mild in 25%, moderate in 38% and severe in 32.5%. EEG showed epileptiform abnormalities in 79.5% of patients. One hundred and thirty-one patients out of the 200 recruited (65.5%) did not have an aetiological diagnosis. All the patients underwent full clinical reassessment and when necessary they performed neuroradiologic and genetic investigations as well. We identified 35 patients with a genetic aetiology. In 8 cases a structural brain lesion was observed. In 33 patients, a genetic aetiology was identified. In 2 patients with drug-resistant seizures video-EEG allowed the identification of non-epileptic seizures, and in one patient we discontinued anti-epileptic drugs. In these patients, the aetiological diagnosis was made after 30 years (range 9-60 years) from the disease onset. Conclusions: In a population of 200 adult patients with epilepsy and ID, an aetiological cause was identified in 45 patients after 30 years from the disease onset. Aetiological diagnosis, especially if genetic, has significant positive implications for patients, even if it has been made after years from the beginning of the disease. Benefits include better-focused antiepileptic drug (AED) choice, sparing of further unnecessary investigations and improved knowledge of comorbidities.

Background. Non-epileptic attack disorder (NEAD) is one of the most common differential diagnoses to epilepsy. Due to the impact of misdiagnosis, research has focused on improving differential diagnosis by identifying factors distinguishing the two populations. These factors, though non-specific and common place comprise much of the understanding of the aetiology of NEAD. Theories which adequately explain the processes by which attacks develop and are maintained are lacking. Although it is agreed that psychological processes underpin NEAD, therapeutic approaches targeting specific processes are under developed. In light of the limitations of currently employed structural approaches, a functional approach may improve understanding of possible mechanisms underpinning NEAD development and maintenance. Aim. This study aimed to use Multiple Sequential Functional Analysis to explore whether behavioural principles of learning, applied to detailed life histories, can be used to understand the developmental pathway of non-epileptic attacks. Method. Three adult participants were recruited from outpatient Neurology clinics in the East Midlands, UK. Clinical interviews were conducted using a biographical format to collate detailed information around all aspects of the participant’s histories, current situation, and non-epileptic attacks. To improve the hypotheses made, interview data was triangulated with data from an interview with a relative and a file review. The MSFA was conducted according to the principles of radical behaviourism and applied functional analysis. Data was utilised in the analysis based on the pragmatic truth criterion of functional contextualism. Results. The results are three detailed functional analytic case studies that track the development of non-epileptic attacks for each participant from formative experiences to their current attack experiences. The results demonstrate that functional analytic principles can be used to understand the developmental pathway of NEAD in these three adults. Though the participants had very different experiences and presentations, an across-case analysis identifies that attacks have similar functional values for these people. Issues including avoiding/reducing stress and emotional suppression appear to be important factors in the development and maintenance of the behaviour. Discussion. The findings that non-epileptic attacks hold functional value for this group of people, supports the theorised roles of avoidance and secondary gain in the developmental process. The findings have important implications for future research. A strength of the present methodology is that it identifies subtle differences in the learning histories, which has implications for the development of assessment and treatment approaches for those with NEAD.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第7060号
医博第1956号
新制||医||681(附属図書館)
UT51-98-C173
京都大学大学院医学研究科病理系専攻
(主査)教授 川口 三郎, 教授 柴崎 浩, 教授 芹川 忠夫
学位規則第4条第1項該当

L’épilepsie avec crises focales migrantes du nourrisson se caractérise par l’apparition de crises focales avant 6 mois qui s’intensifient jusqu’à une phase d’orage où il y a des crises migrantes. Les mutations gain-defonction du gène KCNT1 sont les principales mutations responsables de cette épilepsie. Nous nous sommes intéressés à une cohorte de patients ayant une mutation du gène KCNT1 et cette épilepsie afin de mieux comprendre ce syndrome en vue de le modéliser. D’abord, nous avons précisé la clinique de ces patients, notamment le mauvais devenir à long terme, la forte mortalité, la microcéphalie et la présence de symptômes extra-neurologiques. Puis, nous avons déterminé, via l’étude des EEG ictaux, que les crises migrantes n’étaient pas chaotiques mais correspondaient plutôt à un type de propagation et nous avons identifié des marqueurs spécifiques de cette épilepsie. Ensuite, nous avons montré que la majorité des mutations KCNT1 semblaient se regrouper dans des "points chauds" et qu’il n’y avait pas de corrélation génotype-phénotype stricte. Finalement, nous avons modélisé cette épilepsie à l’échelle microscopique et mésoscopique. Les résultats préliminaires montraient une baisse de l’excitation, une chute de l’inhibition et l’implication du GABA dépolarisant. Nous discutons ensuite des différents aspects de nos travaux à la lumière de la littérature et décrivons les perspectives ouvertes par cette thèse d’un point de vue fondamental, clinique et physiologique
Epilepsy in infancy with migrating focal seizures is characterized by focal seizures beginning before 6 months that intensify to a stormy phase where so-called migrating focal seizures appear. The gain-of-function mutations of the KCNT1 gene are the main causes of this epilepsy. We focused on a cohort of patients with a KCNT1 mutations and this epilepsy to better understand this syndrome in order to model it. First, we specified the clinic for these patients, including long-term poor outcomes, high mortality, microcephaly and the presence of extra-neurological symptoms. Then, we determined, through the study of ictal EEGs, that migrating seizures were not chaotic but rather corresponded to a type of propagation and we have identified specific markers of this epilepsy. Then, we showed that the majority of KCNT1 mutations appeared to cluster in "hot spots" and that there was no strict genotypephenotype correlation. Finally, we modelled this epilepsy at microscopic and mesoscopic levels. Preliminary results showed a decrease in excitation, a fall in inhibition and involvement of depolarizing GABA. We then discuss the different aspects of our work in the light of the literature and describe the perspectives opened by this thesis from a fundamental, clinical and physiological point of views

Anomalies de structure du génome et Déficience Intellectuelle : Recherche des gènes candidats de Déficience intellectuelle en utilisant l'analyse chromosomique sur microréseau d'ADN pangénomique 180K et l'analyse chromosomique sur microréseau d'ADN de haute résolution 1M ciblée des gènes candidats de Déficience intellectuelle. L'analyse chromosomique sur microréseau d'ADN (ACM) de haute résolution est une innovation technologique puissante afin de détecter les aberrations chromosomiques concernant les variations du nombre de copies. En utilisant l'ACM 180K, l'ACM 1M et la PCR quantitative, nous avons identifié les 5 variations du nombre de copies (CNV) intragéniques pathogènes de novo impliquant les gènes : RUNX1T1, KIAA1468, FABP7, ZEB2 (syndrome de Mowat-Wilson) et ANKRD11 (syndrome de KBG). Les 5 patients ayant une DI et une dysmorphie faciale. L'ACM 180K a révélé une délétion d'une taille de 92 kb emportant le gène KIAA1468 candidat pour le syndrome de West chez un enfant de 3 ans présentant une DI sévère et une encéphalopathie épileptique infantile précoce. Le criblage des mutations du gène KIAA1468 a été réalisé chez 35 patients atteints de syndrome de West. Un variant intronique c.2761-7 T>C et un variant faux-sens hérité de la mère c.3547 G>A avec signification clinque inconnue ont été identifiés. En utilisant des approches par l'ACM 1M de haute résolution chez 45 patients atteints de DI idiopathique modérée à sévère, un seul CNV causal a été identifié, une délétion intragénique d'une taille de 28.37 kb du gène ZEB2. Notre étude confirme une fréquence très faible des délétions/duplications intragéniques avec la détection d'une seule aberration chromosomique (1/45). En conclusion, si la fréquence des mutations ponctuelles est élevée, nous avons également souligné l'application de la technique de séquençage à haut débit avec un rendement diagnostique jusqu'à 45%-55% des cas de DI sévère idiopathique chez lesquels aucun CNV n'a été détecté sur ACM
Chromosomal structural abnormalities and Intellectual Disability : In search of intellectual disability candidate genes by using pangenomic comparative genomic hybridization 180 K and high resolution comparative genomic hybridization 1M targeting intellectual disability candidate gene.High resolution microarray-based comparative genomic hybridization (a-CGH) has been a powerful technical innovation in order to detect submicroscopic chromosomal aberrations related to copy number variations. By using a-CGH 180K, 1M high resolution a-CGH and quantitative PCR, we have identified 5 pathogenic intragenic copy number variations (CNVs) de novo : RUNX1T1, KIAA1468, FABP7, ZEB2 (Mowat-Wilson syndrome) and ANKRD11 (KBG syndrome). All five patients had intellectual disability (ID) and facial dysmorphism. Interestingly, a-CGH 180K has revealed a 92 kb deletion of a candidate gene KIAA1468 for West syndrome in a 3 year-old boy with severe ID and early infantile epileptic encephalopathy. Mutational screening for candidate gene KIAA1468 was performed in 35 patients with West syndrome. An intronic variant c.2761-7 T>C and a non synonymous maternally inherited variant c.3547 G>A with unknown clinical significance were identified. By using 1M high-resolution a-CGH approach in 45 patients presenting moderate to severe idiopathic ID, only one causal CNV was identified, a 28.37 kb intragenic ZEB2 deletion. Our study has confirmed the low frequency of intragenic deletion/duplication with the detection of only one chromosomal aberration (1/45). In conclusion, providing that the high frequency of intragenic point mutation, we also stressed the application of next-generation sequencing technology with 45-55% diagnostic yield in patients with idiopathic severe ID in case of no apparent CNV(s) on high-resolution a-CGH

Les Encéphalopathies Épileptiques Précoces sont des pathologies rares et sévères caractérisées par des crises fréquentes commençant dans les trois premiers mois de vie accompagnées d’un EEG intercritique altéré et un pronostic très défavorable. Au cours de la caractérisation génétique d’une cohorte de 402 patients, nous avons mis en évidence une délétion de 19,9 kb localisée en Xp11.23 chez un garçon et 34 mutations de novo du gène KCNQ2. La première partie de mon projet a consisté en l’étude de la pathogénicité de la délétion Xp11.23, qui implique trois gènes dont WDR45. Les mutations de WRD45 ont été décrites dans une dégénérescence neuronale avec accumulation de fer et presque exclusivement chez des patients de sexe féminin. Le diagnostic initial, chez ce patient, montre une IRM normale avec un phénotype d'EEP et l'accumulation de fer a été détectée à partir de 5 ans. Ce travail m’a permis de décrire le premier patient atteint d’EEP porteur d’une délétion de WDR45. La deuxième partie de mon projet a concerné le gène KCNQ2. Nos résultats ont montré que les mutations sont impliquées dans deux mécanismes physiopathologiques, une délocalisation subcellulaire et un gain de fonction. Ces résultats ouvrent de nouvelles perspectives en terme de compréhension de la pathologie et de thérapies qui peuvent être proposées. Une dernière partie de ce projet a consisté en l’élaboration de nouveaux modèles in vitro, j’ai mis au point des lignées stables exprimant KCNQ2 qui permettront le criblage de molécules thérapeutiques à haut-débit, ainsi que des progéniteurs neuronaux différenciés à partir de cellules iPS issues de la reprogrammation de fibroblastes de patients
Early onset epileptic encephalopathies are rare and severe disorders, characterized by frequent motor seizures occurring before three months of age associated with an altered interictal EEG pattern. The prognosis is poor. During the course of the genetic characterization of a cohort of 402 EOEE patients, we identified a de novo deletion located at Xp11.23 in a male patient and 34 KCNQ2 de novo mutations. The first part of my project consisted in the study of the pathogenicity of the Xp11.23 deletion that encompasses three genes including WDR45. Mutations in the WDR45 gene been have recently identified in patients suffering from neurodegeneration with brain iron accumulation. WDR45 mutations have been almost exclusively found in females. Our patient with the Xp11.23 deletion presented a normal MRI and the EOEE phenotype was predominant. Iron accumulation began only at 5 years. My work reveals that deletions of WDR45 are viable in males and can be diagnosed as EOEE. The second part of my project was aimed at the functional study of two KCNQ2 gene mutations. During this work, my results showed that those mutations were involved in new pathological mechanisms, namely a mislocalization or gain of function. Those results provide new perspectives in term of disease knowledge and therapy. The last part of my project consisted in the development of two new in vitro models for the study of KCNQ2 mutations: stable cell lines expressing the Kv7.2 channel for high-throughput screening of drugs and the production of neurons from induced pluripotent stem cells arising from reprogrammed patient fibroblasts

Mutations in genes that cause transcriptional dysregulation, such as genes that encode DNA and RNA-binding proteins (RNABPs), are a well-described cause of neurodevelopmental syndromes such as autism and epilepsy. Heterozygous de novo mutations involving the gene HNRNPU, which encodes the heterogeneous nuclear ribonuclear protein U, have been implicated in a neurodevelopmental syndrome most commonly characterized by epileptic encephalopathy. Although hnRNP U is a highly-abundant and ubiquitously-expressed DNA- and RNA-binding protein involved in a variety of important nuclear processes—most notably gene expression regulation—the role it plays in neurological disease is unclear and has yet to be studied. The work presented here examines a precision medicine approach for epilepsies thought to have a transcriptomic basis, starting with a thorough neurophysiological characterization of a heterozygous loss-of-function Hnrnpu mouse model (Hnrnpu+/113DEL), followed by a comprehensive and region-specific single-cell transcriptomic study, and finally the validation of implicated brain regions. Characterization of the Hnrnpu+/113DEL mouse line revealed an increased susceptibility to seizures in Hnrnpu+/113DEL mice, along with an increased perinatal mortality, global developmental delay and gait abnormalities. Gene expression profiling, including bulk RNA-sequencing of neocortex and single cell RNA-sequencing of both neocortex and hippocampus, revealed widespread, yet modest, dysregulation of gene expression that was largely inversely correlated to gene-length, and involved important, neurodevelopmental disease genes. In particular, pyramidal neurons of the subiculum displayed greater transcriptional burden upon heterozygous loss of Hnrnpu, with the known epilepsy gene Mef2c as a clear outlier showing greater than 50% reduction in expression. Follow-up investigation into whether this region- and cell-type specific gene dysregulation correlated to differences in neuronal function using c-Fos immunostaining, revealed an overall decrease in neuronal activity within the ventral subiculum in Hnrnpu+/113DEL mice. In summary, our data validates the presence of neurodevelopmental defects upon heterozygous loss of Hnrnpu and supports the notion of transcriptional dysregulation as a likely contributing factor to hnRNP U-related disease, possibly through the dysfunction of subiculum-derived excitatory neurons. Future studies evaluating the relationship between reduced activity within the ventral subiculum and hnRNP U disease phenotypes are an important next step, and may serve as the basis for targeted therapeutic discovery.

Children with focal intractable epilepsy caused by MCD, FCD and TSC are in a high risk of development of cognitive delay, as a result of both drug resistant epilepsy and genetically determined abnormal structure of the neuronal networks. Epilepsy surgery represents an established and safe treatment method of focal drug resistant epilepsy, and increases the chances for these patients to be rid of epileptic seizures, anti-epileptic medication and cognitive comorbidities. Current data on genetic background of focal MCD and FCD and their comorbidities provide space to expand the diagnostic process in epilepsy surgery candidates. However, available information on genetic causes of MCD and FCD do not allow us to infer prognostic estimates on chances of seizure freedom and optimal cognitive development. Future studies should elucidate these uncertainties.

Girls Clustering Epilepsy is the second most common developmental and epileptic encephalopathy. GCE is due to variants in the X chromosome gene PCDH19 and is underpinned by cellular mosaicism due to X-chromosome inactivation (XCI) in females or somatic variant in males. The hallmark feature is that seizures occur in clusters and mainly affect females. At the time of thesis submission, GCE was re-named “X-linked clustering epilepsy” (XCE) to accommodate the growing number of affected male cases. Seizures typically present as generalized tonic-clonic and/or focal, which may evolve to bilateral, tonic-clonic. The clinical profile includes variable cognitive impairment and psychiatric features. The prevalence of these comorbidities and cause of the variable clinical expressivity is unknown. We performed a systematic review and meta-analysis to identify the comorbidities associated with PCDH19 variants and examine phenotype- and genotype-phenotype associations. Data from 38 peer-reviewed original articles were used and included 271 individual cases. We found that seizure onset ≤ 12 months was significantly associated (p = 4.127 x 10-7) with more severe intellectual disability compared with onset > 12 months. We identified two recurrent variants p.Asn340Ser and p.Tyr366Leufs*10, occurring in 25 (20 unrelated) and 30 (11 unrelated) cases, respectively. PCDH19 variants were associated with psychiatric comorbidities in approximately 60% females, 80% affected mosaic males, and reported in nine hemizygous males. Executive dysfunction, and hyperactive, autistic, and obsessive-compulsive features were most frequently associated with PCDH19 variants. We developed a PCDH19 survey to systematically examine the comorbidities identified in our review using standardized neuropsychiatric assessments. The survey was completed by 122/186 (66%) participants diagnosed with GCE or with a confirmed likely pathogenic PCDH19 variant. Executive functions were measured using the Behavior Rating Inventory of Executive Function. Psychiatric comorbidities were assessed via the Social Responsiveness Scale or Social Communication Questionnaire, the Strengths and Difficulties Questionnaire, and the Dimensional Obsessive-Compulsive Scale. Genetic, seizure, and developmental information were also collected. Of the 112 evaluated participants (15 males), there were 70 unique variants. Thirty-five variants were novel and included a newly identified recurrent variant Ile781Asnfs*3. There were no phenotypic differences between published and unpublished cases. Seizures occurred in clusters in 94% individuals, with seizures resolving in 28% at an average age of 17.5 years. Developmental delay prior to seizure onset occurred in 18% of our cohort. Executive dysfunction and autism spectrum disorder (ASD) occurred in approximately 60% of individuals. The ASD profile included features of attention-deficit hyperactivity disorder. Obsessive-compulsive symptomology was observed in 21% individuals. There were no phenotypic differences between heterozygous females and mosaic males. We describe a mosaic male and two hemizygous males with atypical clinical profiles. Earlier seizure onset age and increased number of seizures within a cluster were associated with more severe clinical outcomes. No clinical profile was observed for transmitting males. The penetrance of GCE is incomplete; estimated to be around 80-90%, and might be explained by cellular interference. Cellular interference postulates that the coexistence of PCDH19 wild-type and variant cells would be pathogenic, whereas a homogenous cell population would be tolerated; an idea supported by the presence of asymptomatic PCDH19-negative hemizygous and symptomatic PCDH19-mosaic males. The cellular interference hypothesis was tested through analyses of GCE penetrant and non-penetrant female fibroblast cell lines using assays to determine XCI patterns and relative PCDH19 cDNA expression. Specifically, we hypothesized that XCI and PCDH19 cDNA expression will be skewed towards complete wild-type or variant expression in non-penetrant females. We have shown that XCI patterns do not correlate with relative PCDH19 cDNA expression in fibroblasts, thus invalidating use of this assay to infer PCDH19 expression. No clear association was observed between penetrance in XCE and the degree of variant and wild-type PCDH19 mRNA expression in skin fibroblasts. Although we were able to identify three non-penetrant females with 100% wild-type PCDH19 expression, we were unable to provide support for the mechanism of cellular interference through our finding clinical phenotypes in individuals with markedly skewed XCI. Neuropsychiatric disorders can be very responsive to early intervention; therefore, a better understanding of these comorbidities may help to inform treatment and ultimately lead to better developmental outcomes for individuals affected by GCE. We show that both seizure onset age and activity are associated with clinical outcomes. Clinicians can use this information to inform prognosis and provide targeted intervention and guidance for patients and their families.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Le syndrome de West (SW), communément appelé spasmes infantiles (SI), est un trouble épileptique généralement caractérisé par la triade de spasmes infantiles, un modèle d'électroencéphalogramme (EEG) pathognomonique appelé hypsarythmie, et la régression du développement. Alors que des études précédentes ont été en mesure d'obtenir une réponse relativement adéquate par rapport au contrôle des spasmes et la résolution d’hypsarythmie, elles n’ont pas réussi à fournir des options thérapeutiques décisives à l’égard des séquelles neurodéveloppementales souvent associées aux SI. Notre étude, sur laquelle est basée cette thèse, est la première à utiliser un traitement complémentaire aux médicaments antiépileptiques conventionnels, avec l'intention d'améliorer les résultats neurodéveloppementaux de cette population. Les patients recrutés dans notre essai clinique randomisé (ECR) original ont suivi un protocole de traitement standardisé composé de vigabatrin (VGB) comme traitement de première intention pendant deux semaines, suivi de l'hormone corticotrope (ACTH) chez les non-répondeurs pour une période de deux autres semaines, et le topiramate dans les cas réfractaires. En plus, les patients ont été randomisés pour recevoir soit le traitement expérimental, flunarizine, soit un placebo, pendant six mois. Notre ECR multicentrique consistait à recruter et évaluer 68 patients, la plupart suivis à 8 différentes visites sur une période de cinq ans afin de précisément évaluer leurs progrès neurodéveloppementaux. Notre essai clinique a généré trois études principales qui forment le coeur de cette thèse. Dans une première étude, les données cliniques et cognitives des deux premières années d’évaluation ont été analysées. Les résultats cliniques à court terme indiquent un taux élevé de cessation de spasmes et de l’hypsarythmie. De plus, cette étude rapporte les premiers résultats cognitifs mesurés par le Bayley Scales of Infant Development (BSID) et le Vineland Adaptive Behavior Scale (VABS). Notre deuxième étude a essentiellement fourni des données cognitives à plus long terme, 5 ans après le début de son initiation. Les réponses cognitives ont été mesurées par le BSID, le VABS, et aussi par le Stanford-Binet Intelligence Scale (SB5) chez les patients ayant un fonctionnement cognitif plus élevé. Une amélioration significative et progressive des fonctions cognitives a été observée, indépendamment de la thérapie adjuvante. Des facteurs de risque cognitifs à long terme ont également été révélés dans cette étude. Notre dernière étude a essayé d’élucider la relation entre les SI et les troubles du spectre autistique (TSA). Un test de dépistage avec le Checklist for Autism in Toddlers (CHAT) a été effectué à 24 mois, et un diagnostic a été obtenu par moyen du Autism Diagnostic Observation Schedule (ADOS) à 30 et 60 mois. L’ADOS a évalué 44 patients, dont 10 ont été diagnostiqués avec TSA. Une description des facteurs de risque associés aux TSA ont été présentés dans cet article. Enfin, basé sur nos résultats et les informations à ce sujet dans la littérature, nous avons tenté d'élucider les caractéristiques physiopathologiques de la maladie. Une description des mécanismes biologiques sous-jacents impliqués dans le syndrome de West et des traitements cibles associés ont été présentés. Bien que le traitement complémentaire, le flunarizine ne se soit pas avéré être avantageux pour notre cohorte, notre protocole de traitement a tout de même été en mesure de démontrer des résultats cliniques et cognitifs supérieurs dans le sous-groupe de patients avec SI dont l’étiologie est inconnue. Ces résultats, ainsi que l’identification de nouveaux facteurs de risque neurodéveloppementaux potentiels, pourraient être utilisés cliniquement afin d’améliorer le diagnostic et le suivi médical des patients atteints du syndrome de West.
West syndrome (WS), commonly referred to as infantile spasms (IS), is an epileptic disorder usually characterized by the triad of infantile spasms, a pathognomonic electroencephalogram (EEG) pattern called hypsarrhythmia, and developmental regression. While previous treatment studies were able to achieve relatively adequate spasm control and hypsarrhythmia resolution in this population of patients, they have failed to provide conclusive and definite therapeutic options aimed at improving the poor cognitive outcome often associated to IS. Our study, on which this thesis is based, was the first to use an add-on treatment to conventional antiepileptic drugs, with the intent to improve long-term cognitive outcome in this population. Patients recruited in our original randomized clinical trial (RCT) followed a standardized treatment protocol consisting of vigabatrin (VGB) as first-line treatment for two weeks, followed by adrenocorticotropic hormone (ACTH) in non-responders for another two-week period, and topiramate in refractory cases. In addition, patients were randomized to either receive placebo or flunarizine adjunct therapy for six months. Our multi-centric RCT recruited and evaluated 68 patients, most of which were followed at 8 different time points over a five-year period, to precisely evaluate their neurodevelopmental progress. Our clinical trial generated three main studies which comprise the core of this thesis. In a first study, clinical and cognitive data from the first two years were analyzed. Spasm arrest and hypsarrhythmia resolution were the short-term clinical endpoint measures, while the Vineland Adaptive Behavior Scale (VABS) and Bayley Scales of Infant Development (BSID) were used as cognitive outcome measures at 2 years. This first study most importantly reports on the superior short-term clinical response rate achieved in our study population. Preliminary cognitive results were also presented in this work. Our second study essentially presented long-term cognitive data 5 years after the start of the study. Cognitive outcome measures were similar to those used at two years with the addition of the Stanford-Binet Intelligence Scale, Fifth Edition (SB5) for higher functioning patients. Most IS patients, particularly those with no known etiology, displayed a significant and progressive improvement of cognitive functions, irrespective of adjunctive therapy. Risk factors of long term poor cognitive outcome were also revealed in this study. Our last study tried to understand the relationship between IS and autism spectrum disorders (ASD). Autism was initially screened by means of the Checklist for Autism in Toddlers (CHAT) at 24 months, and formally assessed at the 30-and 60-month follow-up visits using the Autism Diagnostic Observation Schedule (ADOS). ADOS was performed in 44 patients, 10 of which were diagnosed with ASD. A description of risk factors associated with an ASD outcome in the IS population were presented in this article. Finally, based on our study results and in conjunction with literature information on the topic, we attempted to elucidate the pathophysiological characteristics of the disorder. A conceivable description of the underlying biological mechanisms implicated in West syndrome and associated target treatments were presented. Although our complementary treatment, flunarizine, did not prove to be beneficial in our cohort, our treatment protocol was nonetheless able to demonstrate superior clinical and cognitive outcomes in patients with unknown etiologies. These findings, as well as the identification of new potential neurodevelopmental risk factors, could be used clinically to improve the diagnosis and medical follow-up of patients with West syndrome.

Neurodevelopmental disorders develop over time and are characterized by a wide variety of mental, behavioral, and physical phenotypes. The categorization of neurodevelopmental disorders encompasses a broad range of conditions including intellectual disability, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy, schizophrenia, bipolar disorder, and epilepsy, among others. Diagnostic classifications of neurodevelopmental disorders are complicated by comorbidities among these neurodevelopmental disorders, unidentified causal genes, and growing evidence of shared genetic risk factors.

We sought to identify the genetic underpinnings of a variety of neurodevelopmental disorders, with a particular emphasis on the epilepsies, by employing next–generation sequencing to thoroughly interrogate genetic variation in the human genome/exome. First, we investigated four families presenting with a seemingly identical and previously undescribed neurodevelopmental disorder characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. These families all exhibited an apparent autosomal recessive pattern of inheritance. Second, we investigated a heterogeneous cohort of ∼60 undiagnosed patients, the majority of whom suffered from severe neurodevelopmental disorders with a suspected genetic etiology. Third, we investigated 264 patients with epileptic encephalopathies — severe childhood epilepsy disorders — looking specifically at infantile spasms and Lennox–Gastaut syndrome. Finally, we investigated ∼40 large multiplex epilepsy families with complex phenotypic constellations and unclear modes of inheritance. The studied neurodevelopmental disorders exhibited a range of genetic complexity, from clear Mendelian disorders to common complex disorders, resulting in varying degrees of success in the identification of clearly causal genetic variants.

In the first project, we successfully identified the disease–causing gene. We show that recessive mutations in ASNS (encoding asparagine synthetase) are responsible for this previously undescribed neurodevelopmental disorder. We also characterized the causal mutations in vitro and studied Asns–deficient mice that mimicked aspects of the patient phenotype. This work describes ASNS deficiency as a novel neurodevelopmental disorder, identifies three distinct causal mutations in the ASNS gene, and indicates that asparagine synthesis is essential for the proper development and function of the brain.

In the second project, we exome sequenced 62 undiagnosed patients and their unaffected biological parents (trios). By analyzing all identified variants that were annotated as putatively functional and observed as a novel genotype in the probands (not observed in the unaffected parents or controls), we obtained a genetic diagnosis for 32% (20/62) of these patients. Additionally, we identify strong candidate variants in 31% (13/42) of the undiagnosed cases. We also present additional analysis methods for moving beyond traditional screens, e.g., considering only securely implicated genes, or subjecting qualifying variants from any gene to two unique analysis approaches. This work adds to the growing evidence for the utility of diagnostic exome sequencing, increases patient sizes for rare neurodevelopmental disorders (enabling more detailed analyses of the phenotypic spectrum), and proposes novel analysis approaches which will likely become beneficial as the number of sequenced undiagnosed patients grows.

In the third project, we again employ a trio–based exome sequencing design to investigate the role of de novo mutations in two classical forms of epileptic encephalopathy. We find a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 x 10–3, likelihood analysis). We provide clear statistical evidence for two novel genes associated with epileptic encephalopathy — GABRB3 and ALG13. Together with the 15 well–established epileptic encephalopathy genes, we statistically confirm the association of an additional ten putative epileptic encephalopathy genes. We show that only ∼12% of epileptic encephalopathy patients in our cohort are explained by de novo mutations in one of these 24 genes, highlighting the extreme locus heterogeneity of the epileptic encephalopathies.

Finally, we investigated multiplex epilepsy families to uncover novel epilepsy susceptibility factors. Candidate variants emerging from sequencing within discovery families were further assessed by cosegregation testing, variant association testing in a case–control cohort, and gene–based resequencing in a cohort of additional multiplex epilepsy families. Despite employing multiple approaches, we did not identify any clear genetic associations with epilepsy. This work has, however, identified a set of candidates that may include real risk factors for epilepsy; the most promising of these is the MYCBP2 gene. This work emphasizes the extremely high locus and allelic heterogeneity of the epilepsies and demonstrates that very large sample sizes are needed to uncover novel genetic risk factors.

Collectively, this body of work has securely implicated three novel neurodevelopmental disease genes that inform the underlying pathology of these disorders. Furthermore, in the final three studies, this work has highlighted additional candidate variants and genes that may ultimately be validated as disease–causing as sample sizes increase.

Dissertation

Epilepsy affects approximately 0,5-1% of children. Epileptic seizures originate in and propagate along certain neural pathways involved in physiological processes of cognition. Consequently, cognitive impairment frequently accompanies epilepsy in childhood and contributes to diminished quality of life of these patients.The main goal of this PhD thesis was to study multiple aspects of cognitive impairment in children suffering from intractable focal epilepsy. In the first and primary study, we described for the first time the negative impact of quasi- periodic epileptiform discharges in sleep (termed "hurdles" in our work) on cognitive functions in children with focal structural epilepsy. We have also shown that epileptiform activity in sleep has a more prominent negative impact on cognitive functions than epileptiform activity in wake. Although "hurdles" are by definition generalized, they do not predict worse outcomes of epilepsy surgery, compared to controls. In the second study, we analyzed the relationship between the extent of epileptogenic zone, functional brain plasticity (evaluated by fMRI) and cognitive dysfunction in children with drug resistant temporal epilepsy. Comparing patients with isolated focal cortical dysplasia (FCD) and patients with combined pathology (FCD and hippocampal...

The catalytically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), acts as a coenzyme in a variety of different enzymatic reactions. Organisms which are not able to synthesize PLP de novo acquire B6 vitamers from nutrients and interconvert them through a salvage pathway, which involves pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PDXK). PNPOx converts pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to PLP, using flavinmononucleotide (FMN) as coenzyme. Both Escherichia coli and human PNPOx are homodimers and, although these enzymes share only 39% of sequence identity, have very similar structural and functional properties. PNPOx plays a crucial role in the regulation of PLP metabolism. It has been proposed that PLP inhibits the catalytic activity of both E. coli and human PNPOx by binding at the active site and acting as a competitive inhibitor. However, PLP can also bind tightly at a secondary site. Our kinetics characterisation suggests that PLP inhibition results from binding of this vitamer at an allosteric site, in both E. coli and human enzymes. This interpretation was confirmed by the analysis of mutated forms of E. coli PNPOx, in which PLP binding at the active site is impaired. Crystallographic studies carried out by other authors on the E. coli PNPOx indicated a possible location of the secondary PLP binding site in two surface pockets of the protein, but site-directed mutants of amino acid residues putatively critical for this interaction showed that this hypothesis is wrong. Molecular docking analyses identified a possible alternative PLP binding site, which is a cleft on the protein surface mainly delimited by arginine residues and located near the subunit interface. Characterisation of mutant forms of this site and crystallographic studies suggested that this might be the allosteric PLP binding site. Concerning human PNPOx, it is known that missense mutations in the gene encoding this enzyme lead to the onset of a rare neurological disease, the neonatal epileptic encephalopathy (NEE); however, the molecular reason of most PNPOx mutations remains to be established. We expressed PNPOx mutants as recombinant proteins in E. coli, purified and characterised them with respect to structural and functional properties, in order to better understand the molecular basis of the disease. The other key enzyme involved in the salvage pathway is PDXK, which converts pyridoxal (PL), pyridoxamine (PM) and pyridoxine (PN) into PLP, PMP and PNP, respectively. In Drosophila, mutations in the dPdxk gene encoding PDXK cause chromosome aberrations (CABs) and increase glucose content in larval haemolymph. This observation suggests that PDXK mutations in humans may be involved in diseases such as cancer and diabetes. We analysed the effect of the expression of four PDXK human variants in Drosophila dPdxk mutants: three of them (D87H, V128I and H246Q) are listed in databases, and one (A243G) was found in a genetic screening of patients with gestational diabetes. None of the variants was able to completely rescue CABs and glucose content. Our biochemical analysis revealed reduced catalytic activity and different affinity of these variants for PLP precursors. Overall, our findings suggest that, when PLP levels are reduced by the presence of these PDXK variants, cancer and diabetes risk may be increased.

Dissertation submitted to the Faculty of Health Sciences, School of Therapeutic Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Occupational Therapy Johannesburg, 2016
This study aimed to determine the developmental outcomes for infants between the ages of five and 16 months who had and had not received induced hypothermia at Chris Hani Baragwanath Academic Hospital (CHBAH). Forty-three infants diagnosed with HIE II and HIE III were assessed using the Bayley Scales of Infant and Toddler Development – III (Bayley-III) at baseline and were reassessed three to nine months later having received intervention from the transdisciplinary rehabilitation team at CHBAH. The majority of infants (78.3%) who had and had not received induced hypothermia both presented with typical developmental outcomes at baseline with 21.7% having moderate to severe dysfunction. On reassessment (31 infants), the developmental outcomes deteriorated for both groups; with more infants in the group which had received hypothermia falling into the at risk category. In spite of this, the majority of infants (in both groups) that were reassessed were overall developmentally on par. This study highlights the importance of on-going monitoring of infants with HIE, receiving home programmes and the need for therapeutic intervention after the age of one year with a focus on specific developmental issues. Standardized assessments are essential to facilitate more precise and effective intervention programmes.
MT2016

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Occupational Therapy Johannesburg, 2019
Hypoxic ischaemic encephalopathy (HIE) is the occurrence of impaired neurological function in a new-born, associated with asphyxia at birth. It may be considered mild, moderate or severe depending on the presence or absence of various clinical signs. The incidence of HIE in South Africa ranges from 0.4 – 3.7 per 1000 live births. In the moderate and severe form, HIE is known to cause functional motor deficits, cognitive deficits, intellectual impairment, language, learning and executive skills limitations and/or social impairments. This study was a quantitative cross-sectional study investigating the motor outcomes, as measured by the Peabody Developmental Motor Scales: second edition (PDMS-2), of 28 children with various severities of HIE. The severity of the HIE was measured by the Thompson HIE score. Participants were between the ages of nine and twenty-four months and attended the Mowbray Maternity Hospital Neurodevelopmental High-Risk Clinic. There were thirteen participants with mild HIE, seven participants with moderate HIE and eight participants with severe HIE. Demographic and perinatal factors were comparable across groups. Results of the study showed that all participants functioned within the normal range for all subtests of the PDMS-2. The mild and moderate HIE groups were comparable in all areas and therefore were combined and compared to the severe group. This comparison showed that the severe HIE group performed worse in all subtests with small to large effect sizes. It is therefore important for occupational therapists to ensure that children with severe HIE, according to the Thompson HIE score receive comprehensive assessment and follow up treatment. This assessment and treatment should focus on fine motor development, particularly VMI.
MT 2020

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
The West syndrome is an epileptic encephalopathy in the child's first year of life, corresponding to 1.4% of the epileptic syndromes in pediatric age. It is characterized by the triad: 1) epileptic spasms, 2) hypsarrhythmic tracing on the electroencephalogram (EEG) and 3) neurodevelopment regression. It affects both sexes, with a higher incidence in males. The syndrome's etiology is diverse, including genetic, structural, metabolic, infectious, immunological and still unknown causes, which is why its diagnostic complexity is not negligible. Neonatal hypoxic-ischemic encephalopathy is one of the most frequent etiologies, but extensive genetic, structural and metabolic investigation is often necessary for a correct etiological diagnosis. The epileptic spasms accompanying this pathology are sometimes brief and subtle and are often confused with abdominal cramps or episodes of gastroesophageal reflux, which delays the recognition of this syndrome. At diagnosis, it is possible to document ictal and interictal epileptic activity on the EEG that occurs in an early period of brain maturation, which is why it is the cause of significant cognitive impairment. The prognosis is variable, according to the etiology, the age of onset, the age of diagnosis, the therapy instituted and the response to therapy.In about two thirds of cases, the prognosis of West Syndrome is reserved, with progression to Lennox Gastaut syndrome and/or association with Autism Spectrum Disorder. The identification of the etiology can play a crucial role, since it provides information that should be considered in the selection of the most effective antiepileptic therapy. Some etiologies respond better to specific medications and may show a worse response with others. The unfavorable prognostic impact of this encephalopathy will depend, among other factors, fundamentally on the etiology, on an early diagnosis of the encephalopathy and will certainly benefit from a therapeutic approach as directed as possible.
O Síndrome de West é uma encefalopatia epilética do primeiro ano de vida da criança, correspondendo a 1,4% dos síndromes epiléticos, em idade pediátrica. Caracteriza-se pela tríada de espasmos epiléticos, traçado hipsarrítmico no eletroencefalograma (EEG) e paragem/regressão do neurodesenvolvimento. Atinge ambos os sexos, com maior incidência no sexo masculino. A etiologia do síndrome é diversa, incluindo causas genéticas, estruturais, metabólicas, infeciosas, imunológicas ou ainda desconhecida, pelo que lhe está associado uma complexidade diagnóstica não negligenciável. A encefalopatia hipóxico-isquémica neonatal é uma das etiologias mais frequentes, mas é muitas vezes necessária extensa investigação genética, estrutural e metabólica para um correto diagnóstico etiológico. Os espasmos infantis que acompanham esta patologia apresentam-se por vezes breves e subtis, sendo frequentemente confundidos com cólicas abdominais ou episódios de refluxo gastroesofágico, o que leva a algum atraso na identificação deste síndrome. Ao diagnóstico, é possível documentar atividade epilética ictal e interictal no EEG que ocorre num período precoce de maturação cerebral, sendo, por isso, causa de importante atraso no neurodesenvolvimento. O prognóstico é variável, de acordo com a etiologia, a idade de início e de diagnóstico, a terapêutica instituída e sua resposta. Em cerca de dois terços dos casos, o prognóstico do Síndrome de West é reservado, com evolução para síndrome Lennox Gastaut e/ou associação a perturbação do espectro do autismo. A identificação da etiologia pode desempenhar um papel crucial, uma vez que fornece informação que pode e deve ser considerada na seleção da terapêutica antiepilética mais eficaz. Algumas etiologias respondem melhor a medicações específicas e poderão mostrar pior resposta com outras. O impacto prognóstico desfavorável desta encefalopatia dependerá, entre outros factores, fundamentalmente da etiologia, do diagnóstico precoce da encefalopatia, beneficiando certamente de uma abordagem terapêutica o mais dirigida possível.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Occupational Therapy. Johannesburg, 2013
This study determined outcomes for motor developmental delay in infants, 12-14 months, diagnosed with HIE II and III, at Chris Hani Baragwanath Academic Hospital. Twenty nine infants diagnosed with HIE II and nine infants diagnosed with HIE III were assessed using the Peabody Development Motor Scale- 2, at their corrected age. Demographic, antenatal and perinatal factors similar to those in other studies were found for this sample. Infants with HIE III had significantly more developmental delay (p=0.01) than infants with HIE II. Fifty two percent of infants with HIE II had no delay while a 100% of infants with HIE III presented with disability. A greater percentage of infants had delay in fine motor skills. Infants with severe and moderate disabilities were receiving intervention whereas those mild disabilities were often missed in screening clinics. It is vital to ensure these infants are assessed and followed up to remediate difficulties as soon as they arise.

Les encéphalopathies épileptogènes sont des maladies graves de l’enfance associant une épilepsie, souvent réfractaire, et un retard de développement. Les mécanismes sous-tendant ces maladies sont peu connus. Cependant, nous postulons que ces épilepsies puissent être causées par une dysfonction du réseau inhibiteur. En effet, des défauts de migration ou de maturation des interneurones GABAergiques (INs) corticaux induisent l’épilepsie, tant chez l’humain que chez la souris. Dans le but d’étudier les causes génétiques des encéphalopathies épileptogènes sporadiques inexpliquées, le laboratoire de la Dre Rossignol a procédé au séquençage d’exome entier d’une cohorte d’enfants atteints. Cela a permis d’identifier, chez un patient, une nouvelle mutation de novo, possiblement pathogène, dans le gène MYO9b. MYO9b est impliqué dans la migration de cellules immunitaires et cancéreuses et est exprimée durant le développement cérébral. Nous émettons l’hypothèse voulant que MYO9b puisse être importante pour la migration des INs corticaux. Les résultats présentés dans ce mémoire démontrent que Myo9b est exprimé dès le stade embryonnaire par les progéniteurs des INs corticaux et que son expression se restreint aux INs dans le cortex mature. De plus, nous démontrons que la répression ex vivo de Myo9b sélectivement dans les INs au sein de tranches corticales organotypiques embryonnaires mène à des défauts morphologiques majeurs de ces cellules en migration. En effet, ces cellules présentent une morphologie multipolaire et des neurites rostraux plus longs et plus complexes. Ces changements morphologiques pourraient avoir un impact majeur sur la migration des INs et ainsi perturber le développement des réseaux inhibiteurs.
Epileptic encephalopathies are early-onset diseases characterized by refractory epilepsy with developmental delay. To identify the underlying genetic cause of these disorders, Dr Rossignol’s laboratory has performed whole-exome sequencing in children with sporadic epileptic encephalopathies. They have identified a novel de novo mutation in the MYO9B gene in one patient. Myo9b is known to regulate cell migration in the immune system and in cancer cells. It is expressed in the developing rodent brain, but its roles during brain development are largely unknown. Recent evidence suggests that epilepsy can be caused by an imbalance between inhibition and excitation in cortical circuits. Indeed, defects in the development or the functions of cortical GABAergic interneurons (INs) have been associated with epilepsy in human and in mouse models. Therefore, we postulated that Myo9b might play a role in the development of INs. In this thesis, I show that Myo9b is expressed in INs from early embryonic ages to post-natal ages. Furthermore, I demonstrate that the ex vivo downregulation of Myo9b in INs in cortical embryonic organotypic cultures causes morphological defects in migrating INs, including aberrant polarization of these cells. These morphological changes might result in aberrant IN migration, which would be expected to perturb the cortical inhibitory-excitatory ratio.