Identification and molecular characterization of somatic mutations in MCD

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PROJECT SUMMARY Genetic mutations causing disease may be inherited, newly acquired in parental gametes and present in the zygote, or acquired as somatic events at some point in development after fertilization (post-zygotic). The burden and localization of any acquired mutation depends on when the mutation arises. As is the case for inherited genetic variation, there is accumulating evidence that somatic mutations can lead to severe tissue- specific disease. Malformations of cortical development (MCD) represent a group of disorders characterized by a range of morphological and structural abnormalities of the cerebral cortex reflecting errors in embryonic cortical development. MCD are associated with intellectual disability, as well as refractory epilepsy, and may require the surgical removal of the affected tissue. Inherited gene mutations involved in neuronal development explain only a fraction of MCD cases. These observations have led to the hypothesis that some MCD result from somatic mutations occurring in neuroglial progenitor cells give rise to abnormal cortical development. In fact, we and others have shown that in hemimegalencephaly, a severe hemispheric MCD subtype, ~30% of patients have somatic mutations in the PI3K-AKT-mTOR signaling pathway, detectable only by directly studying abnormal cortical tissue. Thus, in this project we will evaluate the hypothesis that somatic mutations disrupt embryonic cortical development and are responsible for a substantial fraction of MCD. We, with a network of collaborators, have access to a large number of resected epilepsy surgical tissue specimens. Using high-coverage next-generation sequencing of protein-coding regions in DNA extracted from abnormal brain tissue and unaffected tissue (leukocytes) from patients with three forms of MCD, we will identify somatic mutations within each patient with MCD (Aim 1). Somatic mutations confirmed to be selectively present in affected tissue will be assessed for their likelihood of being responsible for the cortical abnormality using comparisons of patterns of somatic mutations between cases and controls, phenotypic comparisons between individuals with somatic candidates in the same gene, patterns of mutations in pathologically abnormal and normal cell populations, and evaluations of the effects of the candidate mutation on cerebral cortical development in vivo (Aim 2). Finally, initial characterization of the effects and presentation of the disease- causing mutation in the cortex will be studied using in vitro assays to evaluate the effects of the mutation on key cortical developmental processes, and mutation lineage tracing to identify cell populations carrying the mutation (Aim 3). Our project brings together clinical, genetic, and neurobiological expertise and builds on the activities of the Epilepsy Phenome/Genome Project (EPGP), Epi4K, and multiple NINDS-funded initiatives in somatic genetics. These studies will: (i) provide the first detailed assessment of the role of somatic mutations across MCD subtypes, (ii) identify novel genes/pathways underlying cortical development, and (iii) establish and test in vitro and in vivo models to understand the role of genes in cortical development.