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Malformations of cortical development (MCD) are neurological conditions involving focal disruptions of cortical architecture and cellular organization that arise during embryogenesis, largely from somatic mosaic mutations, and cause intractable epilepsy. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat condition-related epilepsy. Here we report a genetic landscape from 283 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation including in utero electroporation of mice and single-nucleus RNA sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associated with distinct pathophysiological and clinical phenotypes. The unique single-cell level spatiotemporal expression patterns of mutated genes in control and patient brains indicate critical roles in excitatory neurogenic pools during brain development and in promoting neuronal hyperexcitability after birth., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmental malformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 of 66 cases (41%). Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a "two-hit" model. Mutations in the same gene cause a disease continuum from FCD to HME to bilateral brain overgrowth, reflecting the progenitor cell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non-cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy.

Focal malformations of cortical development, including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are important causes of intractable childhood epilepsy. Using targeted and exome sequencing on DNA from resected brain samples and nonbrain samples from 53 patients with FCD or HME, we identified pathogenic germline and mosaic mutations in multiple PI3K/AKT pathway genes in 9 patients, and a likely pathogenic variant in 1 additional patient. Our data confirm the association of DEPDC5 with sporadic FCD but also implicate this gene for the first time in HME. Our findings suggest that modulation of the mammalian target of rapamycin pathway may hold promise for malformation-associated epilepsy., (© 2015 American Neurological Association.)

Spontaneous pacemaker γ-aminobutyric acid (GABA) receptor-mediated synaptic activity (PGA) occurs in a subset of tissue samples from pediatric epilepsy surgery patients. In the present study, based on single-cell electrophysiological recordings from 120 cases, we describe the etiologies, cell types, and primary electrophysiological features of PGA. Cells displaying PGA occurred more frequently in the areas of greatest anatomical abnormality in cases of focal cortical dysplasia (CD), often associated with hemimegalencephaly (HME), and only rarely in non-CD etiologies. PGA was characterized by rhythmic synaptic events (5-10Hz) and was observed in normal-like, dysmorphic cytomegalic, and immature pyramidal neurons. PGA was action potential-dependent, mediated by GABAA receptors, and unaffected by antagonism of glutamate receptors. We propose that PGA is a unique electrophysiological characteristic associated with CD and HME. It could represent an abnormal signal that may contribute to epileptogenesis in malformed postnatal cortex by facilitating pyramidal neuron synchrony., (© 2013 Elsevier Inc. All rights reserved.)

Background: Rasmussen encephalitis (RE) is a rare complex inflammatory disease, primarily seen in young children, that is characterized by severe partial seizures and brain atrophy. Surgery is currently the only effective treatment option. To identify genes specifically associated with the immunopathology in RE, RNA transcripts of genes involved in inflammation and autoimmunity were measured in brain tissue from RE surgeries and compared with those in surgical specimens of cortical dysplasia (CD), a major cause of intractable pediatric epilepsy., Methods: Quantitative polymerase chain reactions measured the relative expression of 84 genes related to inflammation and autoimmunity in 12 RE specimens and in the reference group of 12 CD surgical specimens. Data were analyzed by consensus clustering using the entire dataset, and by pairwise comparison of gene expression levels between the RE and CD cohorts using the Harrell-Davis distribution-free quantile estimator method., Results: Consensus clustering identified six RE cases that were clearly distinguished from the CD cases and from other RE cases. Pairwise comparison showed that seven mRNAs encoding interferon-γ, CCL5, CCL22, CCL23, CXCL9, CXCL10, and Fas ligand were higher in the RE specimens compared with the CD specimens, whereas the mRNA encoding hypoxanthine-guanine phosphoribosyltransferase was reduced. Interferon-γ, CXCL5, CXCL9 and CXCL10 mRNA levels negatively correlated with time from seizure onset to surgery (P <0.05), whereas CCL23 and Fas ligand transcript levels positively correlated with the degree of tissue destruction and inflammation, respectively (P <0.05), as determined from magnetic resonance imaging (MRI) T2 and FLAIR images. Accumulation of CD4+ lymphocytes in leptomeninges and perivascular spaces was a prominent feature in RE specimens resected within a year of seizure onset., Conclusions: Active disease is characterized by a Th1 immune response that appears to involve both CD8+ and CD4+ T cells. Our findings suggest therapeutic intervention targeting specific chemokine/chemokine receptors may be useful in early stage RE.

Purpose of Review: Combining human genomics and molecular biology, recent studies have made pivotal progress toward understanding the cause of hemimegalencephaly (HME) and other cerebral megalencephaly syndromes. The present article highlights recent advances of the genetic cause of these conditions, and considers the role of somatic postzygotic genetic lesions in brain maldevelopment., Recent Findings: Studies over the past 12 months have identified de-novo somatic mutations as one possible cause in HME. The gene mutations involve components of the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B)-mammalian target of rapamycin (mTOR) pathway and include PIK3CA, PIK3R2, AKT3, and MTOR. These mutations were identified by comparing genomic data obtained from surgically resected brain tissue with nondiseased tissue, and by single-neuron sequencing in combination with molecular biology techniques. The association between the somatic mutations and downstream activation of the PI3K-mTOR pathway suggests that HME is a neurodevelopmental disease caused by gain-of-function activation of the PI3K-AKT-mTOR pathway., Summary: The studies reviewed suggest that somatic mutations of the PI3K-AKT-mTOR pathway limited to the brain may represent one cause of HME. Dysregulation of this pathway has possible therapeutic potential in the identification of HME. Somatic mutations may be an important yet underappreciated disease mechanism in developmental neurological diseases.

Cortical dysplasia is the most common etiology in children and the third most frequent finding in adults undergoing epilepsy neurosurgery. The new International League Against Epilepsy (ILAE) classification grades isolated cortical dysplasia into mild type I (cortical dyslamination), severe type II (dyslamination plus dysmorphic neurons and balloon cells), and dysplasia associated with other epileptogenic lesions (type III). Multilobar type II lesions present at an earlier age and with more severe epilepsy compared with focal type I abnormalities, often in the temporal lobe, and these findings are reflected in types and age of operations for cortical dysplasia. Presurgical evaluation of patients with epilepsy from cortical dysplasia can be challenging. Interictal and ictal scalp electroencephalography (EEG) accurately localizes cortical dysplasia with 50-66% accuracy. Structural magnetic resonance imaging (MRI) is negative in roughly 30% of cases, most often linked with mild type I cases. FDG-PET can be 80-90% accurate, but is not 100% sensitive. Chronic intracranial electrodes are used in about 50% of cases with cortical dysplasia, but often do not capture restricted ictal-onset zones. About 60% of patients with cortical dysplasia are seizure free after epilepsy neurosurgery, with much higher rates of becoming seizure free with complete (80%) compared with incomplete (20%) resections. The most common reason for incomplete resection is the risk of an unacceptable neurologic deficit. Future challenges include better tools in identifying subtle forms of type I cortical dysplasia, and development of adjunctive treatments from basic research for those undergoing incomplete resections., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8-40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling.

Tuberous Sclerosis Complex (TSC) and cortical dysplasia Type IIB (CDIIB) share histopathologic features that suggest similar epileptogenic mechanisms. This study compared the morphological and electrophysiological properties of cortical cells in tissue from pediatric TSC (n=20) and CDIIB (n=20) patients using whole-cell patch clamp recordings and biocytin staining. Cell types were normal-appearing and dysmorphic-cytomegalic pyramidal neurons, interneurons, and giant/balloon cells, including intermediate neuronal-glial cells. In the cortical mantle, giant/balloon cells occurred more frequently in TSC than in CDIIB cases, whereas cytomegalic pyramidal neurons were found more frequently in CDIIB. Cell morphology and membrane properties were similar in TSC and CDIIB cases. Except for giant/balloon and intermediate cells, all neuronal cell types fired action potentials and displayed spontaneous postsynaptic currents. However, the frequency of spontaneous glutamatergic postsynaptic currents in normal pyramidal neurons and interneurons was significantly lower in CDIIB compared with TSC cases and the GABAergic activity was higher in all neuronal cell types in CDIIB. Further, acutely dissociated pyramidal neurons displayed higher sensitivity to exogenous application of GABA in CDIIB compared with TSC cases. These results indicate that, in spite of similar histopathologic features and basic cell membrane properties, TSC and CDIIB display differences in the topography of abnormal cells, excitatory and inhibitory synaptic network properties, and GABA(A) receptor sensitivity. These differences support the notion that the mechanisms of epileptogenesis could differ in patients with TSC and CDIIB. Consequently, pharmacologic therapies should take these findings into consideration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Interneurons, gamma-aminobutyric acid (GABA)(A) receptor density, and subunit composition determine inhibitory function in pyramidal neurons and control excitability in cortex. Abnormalities in GABAergic cells or GABA(A) receptors could contribute to seizures in malformations of cortical development. Herein we review data obtained in resected cortex from pediatric epilepsy surgery patients with type I and type II cortical dysplasia (CD) and non-CD pathologies. Our studies found fewer interneurons immunolabeled for glutamic acid decarboxylase (GAD) in type II CD, whereas there were no changes in tissue from type I CD. GAD-labeled neurons had larger somata, and GABA transporter (VGAT and GAT1) staining showed a dense plexus surrounding cytomegalic neurons in type II CD. Functionally, neurons from type I CD tissue showed GABA currents with increased half maximal effective concentration compared to cells from the other groups. In type II CD, cytomegalic pyramidal neurons showed alterations in GABA currents, decreased sensitivity to zolpidem and zinc, and increased sensitivity to bretazenil. In addition, pyramidal neurons from type II CD displayed higher frequency of spontaneous inhibitory post synaptic currents. The GABAergic system is therefore, altered differently in cortex from type I and type II CD patients. Alterations in zolpidem, zinc, and bretazenil sensitivity and spontaneous inhibitory postsynaptic currents (IPSCs) suggest that type II CD neurons have altered GABA(A) receptor subunit composition and receive dense GABA inputs. These findings support the hypothesis that patients with type I and type II CD will respond differently to GABA receptor-mediated antiepileptic drugs and that cytomegalic neurons have features similar to immature neurons.

Tuberous sclerosis complex (TSC) and severe cortical dysplasia (CD), or CD type II according to Palmini classification, share histopathologic similarities, specifically the presence of cytomegalic neurons and balloon cells. In this study we examined the morphologic and electrophysiologic properties of cells in cortical tissue samples from pediatric patients with TSC and CD type II who underwent surgery for pharmacoresistant epilepsy. Normal-appearing pyramidal neurons from TSC and CD type II cases had similar passive membrane properties. However, the frequency of excitatory postsynaptic currents (EPSCs) was higher in neurons from TSC compared to severe CD cases, particularly the frequency of medium- and large-amplitude synaptic events. In addition, EPSCs rise and decay times were slower in normal cells from TSC compared to severe CD cases. Balloon cells were found more frequently in TSC cases, whereas cytomegalic pyramidal neurons occurred more often in CD type II cases. Both cell types were similar morphologically and electrophysiologically in TSC and severe CD. These results suggest that even though the histopathology in TSC and severe CD is similar, there are subtle differences in spontaneous synaptic activity and topographic distribution of abnormal cells. These differences may contribute to variable mechanisms of epileptogenesis in patients with TSC compared with CD type II.

Cortical dysplasia (CD) is the most common malformation of cortical development in epilepsy surgery patients. Patients with mild Palmini type I CD represent about 50% of the surgical cases, and these lesions tend to occur most often in the temporal lobe, often associated with hippocampal sclerosis. By comparison, patients with severe type II CD present at younger ages, often with multilobar extratemporal lesions, and more aggressive seizures. The presurgical evaluation in CD patients can be challenging, as no single test is 100% accurate. Based on retrospective cohort studies, the accuracy of investigations are: interictal scalp electroencephalography (EEG), 50%; ictal scalp EEG, 65%; magnetic resonance imaging (MRI), 66%; fluorodeoxyglucose-positron emission tomography (FDG-PET), 81%; and ictal single photon emission computed tomography (SPECT), 57%. Intracranial electrodes are used in about 50% of patients with CD. Contemporary series report that 62% of patients with CD are seizure free after resective neurosurgery, with higher rates for complete (77%) compared with incomplete (20%) removal of the lesion. Morbidity (<3%) and mortality (0.2%) are low for patients with CD undergoing epilepsy neurosurgery. Future challenges include the noninvasive identification of patients with CD with 100% accuracy, evaluation of long-term outcomes in surgical patients, and devising new treatments based on a better understanding of the neurobiology leading to seizures in CD tissue.

We report a case of a 5-year-old boy with intractable epilepsy who underwent therapeutic corticectomy. Histopathologic findings within the resection specimen included severe cortical dysplasia associated with abundant subpial and intraparenchymal Rosenthal fibers in a large right frontal lesion that merged into the basal ganglia. Rosenthal fiber proliferation may represent a reactive process, are frequent in pilocytic astrocytomas, and are a defining feature of Alexander disease. There was no evidence of neoplasm or leukodystrophy in this case. Genetic analysis of the specimen showed a few previously reported polymorphisms but no mutation in the GFAP gene. This case is unique among several hundred cortical resection specimens that we have studied, including numerous cases of severe cortical dysplasia.

Microgliosis is prominent in Rasmussen's encephalitis (RE), a disease with severe seizure activity. However, it is unclear if microglial activation is similar with different histopathologic substrates. Iba1-immunolabelled microglial activation was assessed in neocortex from pediatric epilepsy surgery patients with RE (n=8), cortical dysplasia (CD; n=6) and tuberous sclerosis complex (TSC; n=6). Microglial reactivity was increased, in severely affected RE areas (29% labeling) compared with minimally affected areas of RE cases (15%) and cases of TSC (14%) and CD (12%). There was no qualitative association of Iba1 immunolabelling with the presence of CD8(+) cytotoxic T-cells and no statistical association with clinical epilepsy variables, such as seizure duration or frequency. Iba1 appears to be an excellent marker for detecting extensive microglial activation in patients with RE. In addition, this study supports the notion that Iba1-labeled microglial activation is increased in patients with active RE, compared with cases of CD and TSC.

Recent findings on the clinical, electroencephalography (EEG), neuroimaging, and surgical outcomes are reviewed comparing patients with Palmini type I (mild) and type II (severe) cortical dysplasia. Resources include peer-reviewed studies on surgically treated patients and a subanalysis of the 2004 International League Against Epilepsy (ILAE) Survey of Pediatric Epilepsy Surgery. These sources were supplemented with data from University of California, Los Angeles (UCLA). Cortical dysplasia is the most frequent histopathologic substrate in children, and the second most common etiology in adult epilepsy surgery patients. Cortical dysplasia patients present with seizures at an earlier age than other surgically treated etiologies, and 33-50% have nonlocalized scalp EEG and normal magnetic resonance imaging (MRI) scans. 2-((18)F)Fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) is positive in 75-90% of cases. After complete resection, 80% of patients are seizure free compared with 20% with incomplete resections. Compared with type I, patients with type II cortical dysplasia present at younger ages, have higher seizure frequencies, and are extratemporal. Type I dysplasia is found more often in adult patients in the temporal lobe and is often MRI negative. These findings identify characteristics of patients with mild and severe cortical dysplasia that define surgically treated epilepsy syndromes. The authors discuss future challenges to identifying and treating medically refractory epilepsy patients with cortical dysplasia.

Objective: Patients with cortical dysplasia (CD) are difficult to treat because the MRI abnormality may be undetectable. This study determined whether fluorodeoxyglucose (FDG)-PET/MRI coregistration enhanced the recognition of CD in epilepsy surgery patients., Methods: Patients from 2004-2007 in whom FDG-PET/MRI coregistration was a component of the presurgical evaluation were compared with patients from 2000-2003 without this technique. For the 2004-2007 cohort, neuroimaging and clinical variables were compared between patients with mild Palmini type I and severe Palmini type II CD., Results: Compared with the 2000-2003 cohort, from 2004-2007 more CD patients were detected, most had type I CD, and fewer cases required intracranial electrodes. From 2004-2007, 85% of type I CD cases had normal non-University of California, Los Angeles (UCLA) MRI scans. UCLA MRI identified CD in 78% of patients, and 37% of type I CD cases had normal UCLA scans. EEG and neuroimaging findings were concordant in 52% of type I CD patients, compared with 89% of type II CD patients. FDG-PET scans were positive in 71% of CD cases, and type I CD patients had less hypometabolism compared with type II CD patients. Postoperative seizure freedom occurred in 82% of patients, without differences between type I and type II CD cases., Conclusions: Incorporating fluorodeoxyglucose-PET/MRI coregistration into the multimodality presurgical evaluation enhanced the noninvasive identification and successful surgical treatment of patients with cortical dysplasia (CD), especially for the 33% of patients with nonconcordant findings and those with normal MRI scans from mild type I CD.

Abnormalities in the gamma-aminobutyric acid (GABA)-ergic system could be responsible for seizures in cortical dysplasia (CD). We examined responses of pyramidal neurons to exogenous application of GABA, as well as alterations of GABAergic interneuron number and size in pediatric epilepsy surgery patients with non-CD, type I CD, and type II CD pathologies. We used the dissociated cell preparation for electrophysiology along with immunohistochemistry to identify number and size of GABAergic cells. Pyramidal neurons from type I CD tissue showed increased EC(50) and faster kinetics compared with cells from non-CD and type II CD tissue. Cytomegalic pyramidal neurons showed increased GABA peak currents and decreased peak current densities, longer kinetics, and decreased sensitivity to zolpidem and zinc compared with normal pyramidal cells from non-CD and type I CD. There were fewer but larger glutamic acid decarboxylase (GAD)-containing cells in type II CD tissue with cytomegalic neurons compared with non-CD, type I CD, and type II CD without cytomegalic neurons. In addition, GABA transporters (VGAT and GAT-1) showed increased staining surrounding cytomegalic neurons in type II CD tissue. These results indicate that there are differences in GABA(A) receptor-mediated pyramidal cell responses in type I and type II CD. Alterations in zolpidem and zinc sensitivities also suggest that cytomegalic neurons have altered GABA(A) receptor subunit composition. These findings support the hypothesis that patients with type I and type II CD will respond differently to GABA-mediated antiepileptic drugs and that cytomegalic neurons have features similar to immature neurons with prolonged GABA(A) receptor open channel times., ((c) 2008 Wiley-Liss, Inc.)