Malformations of cortical development (MOD) are the most common cause of intractable epilepsy in children. Focal cortical dysplasia (FCD) and hemimegalencephaly (HMEG) affect restricted brain regions. FCD and HMEG are sporadic disorders and have no identified cause. FCD and HMEG share similar histological features including cells exhibiting cytomegaly and abnormal morphology called balloon cells (BCs). We have shown that there is cell selective activation of the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 pathway in FCD and HMEG as evidenced by expression of phosphorylated ribosomal S6 protein (phospho-S6) only in BCs. We have also identified increased activity of the Wnt/beta-catenin pathway in FCD and HMEG. We propose that FCD and HMEG result from related pathogenic mechanisms and that BCs are specifically generated by somatic gene mutations leading to activation of PI3K-AKT-TOR- p70S6kinase-ribosomal S6 or Wnt/beta-catenin cascades. To test this hypothesis, we will first demonstrate immunohistochemically that the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 and Wnt/beta-catenin cascades are activated in BCs compared with cytomegalic neurons and astrocytes in FCD and HMEG. Second, we will use single cell gene expression analysis to identify altered expression of candidate genes that modulate the TOR and Wnt pathways as well as to investigate other candidate gene pathways that may contribute to increased cell size in FCD and HMEG. Third, we will use 4 mutation analysis strategies to define whole gene, multi-exon, single exon, or point mutations that may be causative in FCD and HMEG. Single nucleotide polymorphism (SNP) array analysis will permit a whole genome approach to define candidate gene loci. Loss of heterozygosity and multiplex-ligation dependent probe analysis (MLPA) assays will detect whole gene and exonic deletions. We have developed a technique to sequence genes from cDNA isolated from single microdissected cells or from genomic DNA isolated from pooled cells so that we can directly sequence at least 20 candidate genes that modulate the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 and Wnt/beta-catenin pathways. These studies provide a targeted, pathway directed strategy to identify altered protein expression, gene transcription, and gene sequence that lead to the formation of FCD and HMEG during brain development.