Pathologic examination of the brain of Patient LR08-018 (p.R465W). (A) Brain size. Graph depicting the largest paediatric brain sizes (in grams) previously reported in the literature (green bars) relative to the brain size of Patient LR08-018 (with the p.R465W mutation, red bar) demonstrating that brain size for this patient is markedly larger than these patients (). The graph is an adaptation of the human brain growth diagram from the Smithsonian Institute (http://humanorigins.si.edu/human-characteristics/brains). The graph shows the periods of rapid brain growth (in orange) plus the period of decreased brain growth (in blue) followed by the plateau in brain growth. (B–E) Cerebral hemispheres. The massive brain (2313 g; approximately twice normal weight for age) was asymmetrically enlarged. The left hemisphere (B and C) weighed 941 g, and the right (D and E) weighed 1128 g. Primary fissures such as the Sylvian (sy), central (ce), postcentral (pc), and calcarine (ca) sulci were recognizable, but secondary and tertiary sulci were abnormal. Gyri appeared irregular and overall hyperconvoluted. The corpus callosum was present including genu (ge), body, and splenium (sp). The anterior commissure (ac) was present but small. White asterisks: artefactual disruption of hemispheres due to brain removal and transport. Black asterisks: torn junction of hemispheres and midbrain. All panels are at the same magnification. (F–I) Hyperconvolution and polymicrogyria in cerebral cortex. (F) Brain slice through parietal cortex showed redundant folds of cortex extending deep into white matter. (G) Histological section (haematoxylin and eosin) through the same region showed relatively sharp grey-white junctions. (H) Inferior temporal cortex exhibited features of polymicrogyria, including anomalous branching and fusion of gliotic layer 1 (GFAP immunohistochemistry). (I) Layer 1 fusion and branching were confirmed by NeuN immunohistochemistry. (J–N) Abnormal layering of cerebral cortex, and excessive white matter neurons. In foci not involved with polymicrogyria, such as right posterior perisylvian cortex, cortical layering was moderately disorganized. (J) Cortical layers were identified based on cell size and density. NeuN immunohistochemistry. (K) Layer 1 was cell-sparse and contained only small neurons. (L) Layer 4 contained typical small (granular) neurons. (M) Layer 6 neurons were particularly disorganized and maloriented. (N) Increased neurons in white matter (wm). Interestingly, neurons in this case were not strikingly enlarged or dysplastic, nor were any balloon cells present. (O–Q) Hippocampal and brainstem abnormalities of Patient LR08-018. (O) The left hippocampus was very small and gliotic, and the hippocampal sulcus (arrowhead) was open, suggesting a deficiency of perforant pathway fibres, which would normally cross the fused sulcus. GFAP immunohistochemistry. (P) Histologically, the dentate gyrus exhibited focal ‘tram-track’ splitting of the granule cell layer (arrowhead; enlarged 2× in inset), a finding usually associated with chronic epilepsy. (Q) The upper medulla showed marked asymmetry of the pyramidal tract, essentially limited to one side (arrowhead). The adjacent inferior olives were moderately hypoconvoluted (haematoxylin and eosin).