Abstracts

Rationale: Hemimegalencephaly (HMG) is a congenital brain malformation characterized by an enlarged, malformed cerebral hemisphere that often requires hemispherectomy for treatment of refractory epilepsy. We recently identified somatic mosaic mutations involving the gene encoding the serine threonine kinase AKT3: an activating point mutation (c.49G→A, creating E17K) and two trisomies of chromosome 1q containing AKT3 (Poduri et al., Neuron, 2012). Here we further characterize the E17K mutation using single cell sequencing of neurons and glia, and we report a novel germline truncating variant in AKT3 in a patient with HMG. Methods: We have identified 29 cases of HMG from which DNA from brain, leukocytes, and/or buccal cells was available. All research was conducted in accordance with the IRB of Boston Children's Hospital. We used copy number analysis of SNP data, quantitative PCR, and candidate gene sequencing. We further studied the AKT3 E17K mosaic mutation case using single cell sorting (NeuN+ vs NeuN-) and single cell sequencing to establish the relative proportions of neurons and glia that were mutation-positive. Results: (1) Having previously identified the AKT3 mosaic E17K mutation in DNA from brain tissue (and not leukocytes) in an individual with HMG, we sequenced AKT3 in our other cases. We have identified a novel AKT3 variant, W330X, in DNA obtained from a buccal cell sample in one case. This truncation does not disrupt a critical activation site (T305) but is predicted to result in the loss of the C-terminal regulatory domain, which could potentially cause activation of AKT3. Functional studies of the W330X variant are underway. (2) We further characterized the AKT3 E17K somatic mosaic mutation using single cell sorting and sequencing. Sequencing of neurons (NeuN+) and glial cells (NeuN-) confirmed the mutation is present in both cell types, at a rate of 39±7% (SE) in single NeuN positive cells (neurons) and 27±8% in single NeuN negative cells (consisting of glial and other non-neuronal cells), suggesting a de novo mutation in a progenitor cell that generates neuronal and non-neuronal cells. Conclusions: We now provide a genetic explanation for four cases of HMG involving activation of AKT3 in brain, either by duplication or activating point mutation leading to hemispheric overgrowth. Somatic mutations arising de novo in neuronal-glial progenitors in the developing brain can be responsible for HMG. Together, our data suggest that AKT3 is an important regulator of brain development and that mutations of this gene, whether germline or post-zygotic and affecting a subset of neurons and glia in one hemisphere, lead to HMG. These mutations in HMG present two models, somatic and germline, for the genetics of epileptic cortical malformations.