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.

<p>Fibroblast growth factor homologous factors (FHFs) are noncanonical members of the fibroblast growth factor family (FGFs, FGF11-FGF14) that bind directly to voltage gated sodium channels (VGSCs), thereby regulating channel activity and consequently neuronal excitability. Mutations in FGF14 cause spinocerebellar ataxia while FGF13 is a candidate for X-linked mental retardation. Since FGF13 and FGF14 are nearly identical within their respective VGSC-interacting domains, those distinct pathological consequences have generally been attributed to regional differences in expression. I have shown that FGF13 and FGF14 have non-overlapping subcellular distributions and biological roles even in hippocampal neurons where both are prominent. While both FHFs are abundant in the axon initial segment (AIS), only FGF13 is observed within the soma and dendrites. shRNA knockdown and rescue strategies showed that FGF14 regulates axonal VGSCs, while FGF13 only affects VGSCs in the somatodendritic compartment. Thus, FGF13 and FGF14 have nonredundant roles in hippocampal neurons, with FGF14 acting as an AIS-dominant positive regulator and FGF13 serving as a somatodendritic negative regulator. Both of these FHFs also perform important non-VGSC regulatory roles. FGF14 is a novel potassium channel regulator, which binds to KCNQ2 and regulates both localization and function. FGF14 is also capable of serving as a “bridge” between VGSCs and KCNQ2 thus implicating it as a broad organizer of the AIS. FGF13, on the other hand is involved in a new form of neuronal plasticity called axon initial segment structural plasticity. Knockdown of FGF13 impairs AIS structural plasticity and reduces L-type CaV current through channels known to be important to this new form of plasticity. Both of these novel non-VGSC roles are specific to the FHF in question because FGF13 does not regulate KCNQ2 whereas FGF14 knockdown does not affect AIS position. These data imply wider roles for FHFs in neuronal regulation that may contribute to differing roles in neural disease.</p> / Dissertation