An integrated approach combining massive parallel sequencing and in utero-based studies to identify and characterize novel genetic determinants in Focal Cortical Dysplasia

Focal Cortical Dysplasia (FCD) belongs to the large spectrum of malformations of cortical development and represents the most common brain lesion in children with refractory epilepsy submitted to surgical treatment. Its anatomopathological presentations are variable and comprise architectural or cytoarchitectural abnormalities in the cortex, the gray-white matter junction, and subcortical white matter. A classification scheme, including clinical, imaging and neuropathological findings has been proposed by the International League Against Epilepsy (ILAE) and distinguishes three types of FCD: Type I (FCDI), Type II (FCDII) and Type III (FCDIII). Recent literature has consistently associated FCDII and Hemimegalencephaly (HMEG), a hemispheric form of FCDII, with somatic or constitutional mutations affecting mTOR pathway genes and a limited number of observations correlated somatic mutations in the SLC35A2 gene with FCDI. The fact that the majority of patients remained undiagnosed suggests that additional and unknown genetic determinants and molecular mechanisms may be implicated in the pathogenesis of these disorders. The aim of this PhD project has been to identify, using different next-generation sequencing (NGS) approaches, novel genetic causes of FCD and HMEG in a cohort of patients who underwent surgical treatment of drug-resistant epilepsy and to characterize, using an in utero electroporation-based approach in rats, a novel molecular mechanism causing HMEG. By targeted resequencing of a panel of mTOR pathway genes, we identified a novel somatic double-hit occurring in the MTOR and RPS6 genes in a patient with HMEG. Functional studies in animal models showed that both variants are pathogenic and have a synergistic effect, suggesting that the HMEG phenotype, in the patient, may result from the cumulative effect of the two mutations. Within this part of the project, we uncovered a novel mechanism for HMEG pathogenesis and identified RPS6 as a potential novel disease-related gene. By DNA methylation derived copy number profiling and targeted resequencing of regulatory and coding regions of selected genes, we identified focal copy number amplifications in noncoding regions of EGFR and PDGFRA genes in patients with FCDI and intractable epilepsy. Within this part of the project, we demonstrated a possible implication of the Receptor Tyrosine Kinase (RTK) pathway in the pathogenesis of FCDI and identified possible candidates for novel pharmacological treatments for intractable epilepsy.