Project Summary (30 lines max; currently at 27 lines) About one-third of people with epilepsy do not respond to currently available drug regimens, though many have drug-resistant focal epilepsy amenable to surgical treatment. Among the most common epilepsy-associated structural brain lesions are Focal Cortical Dysplasias (FCDs). FCDs are malformations of cortical development where the affected neurons fail to migrate in the proper neocortex formation in utero. Somatic variants in about 10-20 genes have been reported as the underlying cause for a subset of FCDs1–13. However, we and others have shown that 60-90% of patients with FCD type I (FCD I) and FCD type II (FCD II) lack any genetic abnormality when using currently available testing methods. More strikingly, only a single gene has been identified in FCD I12 to date. One reason could be that all previous genetic studies in FCD were observational studies in candidate genes without rare variant burden testing against controls and have been performed in small (n<77), poorly characterized, cohorts. None of these studies investigated copy number variants or chromosomal alterations at the somatic level. Also, RNA dysregulation in FCD samples has not been explored yet. Here, we propose the most comprehensive genetic analysis to date of a unique cohort of FCD I and II patients with unprecedented deep clinical phenotyping. Our cohort is >13 times larger than any previously published FCD I and II cohort. Combined with controls, this cohort enables the first rare variant burden analysis for FCDs to confirm proposed and discover novel FCD-associated genes. In addition, we are the first to generate single-nucleus RNA sequencing data from the brain tissue of FCD patients to study transcriptome- level alterations associated with FCD I and II and genetic subtypes. Project hypothesis: Our comprehensive and novel approach using well-characterized brain tissues and paired blood samples from patients with epilepsy due to FCD I or FCD II will identify novel FCD causal genes and variants with clear diagnostic and therapeutic implications. Impact: A better understanding of the genetic basis of FCD etiology will lead to the introduction of novel gene-based diagnostic strategies and targeted drugs to fully manage a patient's seizures while avoiding the spectrum of side effects typically associated with current therapeutic interventions. AIM 1: Identify novel causal genes for FCD I and FCD II with germline and somatic brain variant burden. Aim 2: Identify disease-associated somatic copy number variants (CNVs) and loss-of-heterozygosity (CN-LOH) structural variants associated with FCD I and FCD II. Aim 3: Analyze resected brain tissues by single-nucleus RNA-seq (snRNA-seq) to identify cell type-specific transcriptional alterations associated with FCD histopathologies and with specific mutations.