Functional and structural connectivity in patients with focal epilepsy

Orientadores: Fernando Cendes, Ana Carolina CoanTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Introdução: Estudos recentes demonstram que as epilepsias são doenças relacionadas a alterações de redes neuronais. Técnicas de neuroimagem funcional e de difusão aliadas a avançados métodos de pós-processamento computacional permitem avaliações de conectividade funcional e estrutural do cérebro fornecendo informações sobre os padrões organizacionais das redes associadas. Este estudo visa avaliar a conectividade estrutural e funcional em pacientes com epilepsias focais, caracterizando as alterações de pacientes com epilepsia de lobo temporal mesial (ELTM) associada à esclerose hipocampal (EH) e comparando com outras epilepsias focais. Métodos: Instrumentações de software foram desenvolvidas para as análises realizadas: 1- de conectividade estrutural e 2- de conectividade funcional. Para avaliação da conectividade estrutural, foram selecionados três grupos de pacientes (ELTM-EH, ELT-não lesional [NL] e epilepsia de lobo frontal associada a displasia cortical focal [ELF-DCF]) e um grupo controle. Metodologia de tractografia semiautomática avaliou a anisotropia fracionada (FA), difusividade radial (RD) e axial (AD) de quatro fascículos: 1-uncinado; 2-fórnix; 3-fronto-occiptal inferior; 4-cíngulo. As análises de conectividade funcional foram realizadas comparando pacientes de ELTM-EH com lateralização à direita (D-ELTM) e à esquerda (E-ELTM) e um grupo controle. Setenta regiões de interesse representando 12 redes funcionais foram usadas para análise de criação das matrizes de adjacência. O segundo nível foi realizado comparando pacientes e controles. Resultados: Análise macroestrutural de substancia branca (SB) mostrou alterações (principalmente ipsilaterais) para ELTM-EH e ELF-DCF. A análise microstrutural mostrou alterações em parâmetros de difusão para os mesmos grupos. Os pacientes ELT-NL não apresentaram alterações em nenhuma das análises de integridade estrutural. Comparados ao grupo controle, os grupos D-ELTM e E-ELTM apresentaram alterações de conectividade funcional. Das 12 redes, apenas auditory e visual não apresentaram alterações em ambos os grupos. Para D-ELTM, a anterior salience e a sensorimotor também foram preservadas. Pacientes de E-ELTM apresentaram alterações mais difusas, afetando os dois hemisférios de forma mais evidente. Discussão e conclusão: Pacientes com epilepsias focais apresentam alterações de conectividade funcional e estrutural. Pacientes com ELTM-EH apresentaram piores resultados de alterações estruturais e vasta rede de alterações quando comparados a frontais ou não lesionais. Adicionalmente, apresentam complexa rede de alterações funcionais. Pacientes com E-ELTM apresentaram pior padrão de alterações funcionais comparados aos com D-ELTM. O desenvolvimento de toolboxes para as modalidades metodológicas propostas possibilitaram padronização e melhor eficiência da análise dos dadosAbstract: Introduction: Recent studies has shown that epilepsies are diseases related to neuro networks alterations. Functional and diffusion neuroimaging techniques explored by advanced computational methodologies, allows the functional and structural brain connectivity evaluation providing information regarding the brain networks behavior. This project aim to evaluate the functional and the structural connectivity in patients with temporal lobe epilepsy comparing its alteration patterns with other focal epilepsies. Methods: We developed software resources to perform the analysis: 1- Structural connectivity and 2- functional connectivity. For structural evaluations, three groups of patients were included (mesial temporal lobe epilepsy [MTLE] associated to hipocampal sclerosis [HS], non-lesional temporal lobe epilepsy [TLE-NL] and frontal lobe epilepsy associated to focal cortical dysplasia [FLE-FCD]) and a control group. We performed a semi-automatic tractography procedure to evaluate the fractional anisotropy (FA), the radial diffusivity (RD) and the axial diffusivity (AD) of four anatomic relevant fasciculi: uncinate, body of fornix, inferior fronto-occipital and body of cingulum. We performed functional connectivity analysis comparing patients with left and right MTLE-HS and controls. We used 70 regions of interest (ROIs) from 12 functional networks to compute the connectivity adjacency matrices and performed a second level analysis to compare patients and controls groups. Results: The macrostructural white matter (WM) analysis showed alterations (manly ipsilateral) on MTLE-HS and FLE-FCD. The microstructural WM analysis presented alterations on diffusion parameters for the same groups. Patients with TLE-NL showed no changes for both structural analysis. Compared to the control group, the R-MTLE and L-MTLE groups showed functional connectivity alterations. From the 12 studied networks, only the auditory and the visual networks were preserved on both groups. For the R-MTLE patients, the anterior salience and the sensorimotor networks were also not affected. Patients with L-MTLE showed more diffuse alterations, more evidently affecting both hemispheres. Discussion and conclusion: The study and the proposal methodology were effective for the identifications and characterization of functional and structural connectivity alterations in patients with focal epilepsies. The MTLE-HS showed worse widespread structural alterations compared to the FLE and TLE-NL. Additionally, they presented complex and widespread functional networks abnormalities. Patients with L-MTLE demonstrated worse and more bilaterally affected pattern of alterations when compared to R-MTLE. The development of toolboxes to perform the proposal methodology enabled the standardization and high data analysis efficiency, throughout clear proceduresDoutoradoFisiopatologia MédicaDoutor em Ciência

The Molecular Genetic Investigation of Epilepsy of Infancy with Migrating Focal Seizures

Epilepsy of infancy with migrating focal seizures (EIMFS) is characterised by the onset of frequent focal seizures in the first 6 months of life, a typical migratory EEG pattern and severe developmental delay. In this thesis, I report a cohort of patients with EIMFS, delineate clinical features and investigate the molecular genetic basis of this syndrome. In 2012, heterozygous mutations in the sodium-gated potassium channel KCNT1, were described in patients with EIMFS. Using a variety of genetic techniques, I have identified 12 patients with mutations in this gene. Four are novel, previously unreported mutations. Functional investigations, including protein homology modelling and electrophysiology in a xenopus oocyte model showed that all novel KCNT1 variants were gain-of-function mutations. In addition, I describe a new genetic cause of EIMFS. Within my cohort, I identified a consanguineous family with two affected children. Autozygosity mapping and whole exome sequencing revealed a novel, homozygous mutation in SLC12A5. SLC12A5 encodes KCC2, the neuronal potassium chloride co-transporter that determines the direction and polarity of GABA-mediated signalling. Through international collaboration, I found a second family with two affected children harbouring compound heterozygous SLC12A5 mutations. All three SLC12A5 variants were investigated using an overexpression HEK293 cell model. Immunoblotting and immunohistochemistry revealed decreased cell surface expression of mutant KCC2. Electrophysiology experiments showed a depolarization of the chloride reversal potential and a delayed response to chloride loading. Taken together, these results indicate that loss of KCC2 function is likely to result in abnormal neuronal inhibition in this form of EIMFS. The genetic heterogeneity in EIMFS is strong evidence that a wide variety of different pathogenic mechanisms can result in the severe epilepsy and abnormal neurodevelopment observed in this condition. Further elucidation of causative genes in both animal and cell models is needed to identify novel therapeutic targets for this devastating disorder