PROJECT SUMMARY Genetic mutations causing disease may be inherited, newly acquired in parental gametes and present in the zygote, or acquired at some point in development after fertilization. The burden and localization of a post- zygotically acquired mutation depends on when the mutation arises. Malformations of cortical development (MCD) are 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 refractory epilepsy as well as intellectual disability and may require the surgical removal of the affected tissue for seizure control. There is increasing recognition that post-zygotically acquired somatic mutations occurring in neuroglial progenitor cells can result in a cortical brain malformation. In the previous funding period of this grant, we made significant progress identifying and molecularly characterizing a series of pathogenic post-zygotically acquired somatic variants in the resected brain tissue of individuals with an epilepsy-associated cortical brain malformation. Most notably we identified and functionally characterized the first gene associated with focal cortical dysplasia type I, SLC35A2, and made significant advancements in the understanding of the somatic genetic landscape across MCD. The overarching objective for the next funding cycle is to continue to identify somatic variants across MCD and to functionally characterize the effects of novel variants associated with different types of MCD on cortical development. In Aim 1, we will continue to collect resected brain tissue specimens from individuals with MCD for high-depth exome or targeted gene sequencing. The goal of Aim 1 is to identify novel genes involved in MCD and to ascertain the subset of exome-negative cases for use in Aim 2. In Aim 2, we will use highly sensitive duplex sequencing to detect very low-level somatic variants and PCR-free whole-genome sequencing to detect somatic short tandem repeat variants and intermediately sized somatic copy number variants in exome-negative MCD cases. The goal of Aim 2 is to determine the contribution of these classes of somatic variants that are routinely missed due to the limitations of standard short-read exome sequencing in the overall somatic genetic risk of MCD. Finally, Aim 3 will evaluate the functional consequences of knocking out newly identified MCD genes harboring somatic loss-of-function variants on neuronal morphology, neuronal migration, and ultimately cerebral cortical development in the developing mouse brain. These studies will: (i) continue our in-depth assessment of the role of somatic mutations across MCD subtypes, (ii) identify novel genes/pathways involved in cortical development, (iii) use complementary in vitro, ex vivo, and in vivo models to understand the role of novel genes implicated cortical development, and (iv) establish biomarkers and platforms that can be used in the...