Abstracts

Rationale: Hemimegalencephaly (HMEG) is a highly epileptogenic developmental brain malformation associated with enlargement of an entire cerebral hemisphere. Pathologically, cytomegalic and dysmorphic neurons, astrocytosis, dyslamination, and polymicrogyria are seen. Previous studies have suggested abnormalities in the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway in HMEG. In the present study we performed detailed immunohistochemical and genetic analyses of human HMEG tissue to further delineate involvement of this pathway. Methods: Five children with HMEG were evaluated by standard protocols and underwent resective surgery for intractable epilepsy. Double-label fluorescence immunohistochemistry studies were performed using formalin-fixed, paraffin-embedded sections (4 μM) of resected cortex. Antibodies directed against the phosphorylated forms of PDK1, AKT, tuberin, and ribosomal protein S6 were analyzed in conjunction with neuronal (MAP2) and glial (GFAP) cellular markers. DNA was isolated from specimens frozen at the time of surgery and subjected to sequence analysis using methods sensitive for detection of genetic mosaicism. Results: All specimens demonstrated increased phospho-S6 immunofluorescence in a subset of neurons, which ranged from less than 10% to nearly 100% of the total neuronal population. Varying degrees of phospho-S6 immunofluorescence were also identified in glial cells in all specimens. However, neuronal labeling with antibodies against phosphorylated proteins upstream of S6 in the PI3K/AKT/mTOR pathway was present in some patients but not others, suggesting the presence of abnormal activity at different levels of the pathway. Indeed, activating mosaic mutations in two different pathway components, the catalytic subunit of PI3K and of AKT3, were identified in patients with different immunohistochemical patterns. Conclusions: Our studies indicate that HMEG is a heterogenous condition that may be caused by mosaic activating mutations of different members of the PI3K/AKT/mTOR pathway. Different genetic forms of this malformation may also be identified by patterns of immunohistochemical labeling. These findings suggest that therapies targeting abnormal activity at different levels of the PI3K/AKT/mTOR pathway could be beneficial for children with intractable epilepsy due to HMEG.