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Exome-wide sequencing studies recently described PTPN11 as a novel brain somatic epilepsy gene. In contrast, germline mutations of PTPN11 are known to cause Noonan syndrome, a multisystem disorder characterized by abnormal facial features, developmental delay, and sporadically, also brain tumors. Herein, we performed a deep phenotype-genotype analysis of a comprehensive series of ganglioglioma (GG) with brain somatic alterations of the PTPN11/KRAS/NF1 genes compared to GG with common MAP-Kinase signaling pathway alterations, i.e., BRAFV600E. Seventy-two GG were submitted to whole exome sequencing and genotyping and 84 low grade epilepsy associated tumors (LEAT) to DNA-methylation analysis. In 28 tumours, both analyses were available from the same sample. Clinical data were retrieved from hospital files including disease onset, age at surgery, brain localization, and seizure outcome. A comprehensive histopathology staining panel was available in all cases. We identified eight GG with PTPN11 alterations, copy number variant (CNV) gains of chromosome 12, and the commonality of additional CNV gains in NF1, KRAS, FGFR4 and RHEB, as well as BRAFV600E alterations. Histopathology revealed an atypical glio-neuronal phenotype with subarachnoidal tumor spread and large, pleomorphic, and multinuclear cellular features. Only three out of eight patients with GG and PTPN11/KRAS/NF1 alterations were free of disabling-seizures 2 years after surgery (38% had Engel I). This was remarkably different from our series of GG with only BRAFV600E mutations (85% had Engel I). Unsupervised cluster analysis of DNA methylation arrays separated these tumours from well-established LEAT categories. Our data point to a subgroup of GG with cellular atypia in glial and neuronal cell components, adverse postsurgical outcome, and genetically characterized by complex alterations in PTPN11 and other RAS-/MAP-Kinase and/or mTOR signaling pathways. These findings need prospective validation in clinical practice as they argue for an adaptation of the WHO grading system in developmental, glio-neuronal tumors associated with early onset focal epilepsy.

An increasing number of epilepsies are being attributed to variants in genes with epigenetic functions. The products of these genes include factors that regulate the structure and function of chromatin and the placing, reading and removal of epigenetic marks, as well as other epigenetic processes. In this Review, we provide an overview of the various epigenetic processes, structuring our discussion around five function-based categories: DNA methylation, histone modifications, histone-DNA crosstalk, non-coding RNAs and chromatin remodelling. We provide background information on each category, describing the general mechanism by which each process leads to altered gene expression. We also highlight key clinical and mechanistic aspects, providing examples of genes that strongly associate with epilepsy within each class. We consider the practical applications of these findings, including tissue-based and biofluid-based diagnostics and precision medicine-based treatments. We conclude that variants in epigenetic genes are increasingly found to be causally involved in the epilepsies, with implications for disease mechanisms, treatments and diagnostics.

Ongoing challenges in diagnosing focal cortical dysplasia (FCD) mandate continuous research and consensus agreement to improve disease definition and classification. An International League Against Epilepsy (ILAE) Task Force (TF) reviewed the FCD classification of 2011 to identify existing gaps and provide a timely update. The following methodology was applied to achieve this goal: a survey of published literature indexed with ((Focal Cortical Dysplasia) AND (epilepsy)) between 01/01/2012 and 06/30/2021 (n = 1349) in PubMed identified the knowledge gained since 2012 and new developments in the field. An online survey consulted the ILAE community about the current use of the FCD classification scheme with 367 people answering. The TF performed an iterative clinico-pathological and genetic agreement study to objectively measure the diagnostic gap in blood/brain samples from 22 patients suspicious for FCD and submitted to epilepsy surgery. The literature confirmed new molecular-genetic characterizations involving the mechanistic Target Of Rapamycin (mTOR) pathway in FCD type II (FCDII), and SLC35A2 in mild malformations of cortical development (mMCDs) with oligodendroglial hyperplasia (MOGHE). The electro-clinical-imaging phenotypes and surgical outcomes were better defined and validated for FCDII. Little new information was acquired on clinical, histopathological, or genetic characteristics of FCD type I (FCDI) and FCD type III (FCDIII). The survey identified mMCDs, FCDI, and genetic characterization as fields for improvement in an updated classification. Our iterative clinico-pathological and genetic agreement study confirmed the importance of immunohistochemical staining, neuroimaging, and genetic tests to improve the diagnostic yield. The TF proposes to include mMCDs, MOGHE, and "no definite FCD on histopathology" as new categories in the updated FCD classification. The histopathological classification can be further augmented by advanced neuroimaging and genetic studies to comprehensively diagnose FCD subtypes; these different levels should then be integrated into a multi-layered diagnostic scheme. This update may help to foster multidisciplinary efforts toward a better understanding of FCD and the development of novel targeted treatment options.

Introduction: MOGHE is defined as mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Approximately half of patients with histopathologically confirmed MOGHE carry a brain somatic variant in the SLC35A2 gene encoding a UDP-galactose transporter. Previous research showed D-galactose supplementation results in clinical improvement in patients with a congenital disorder of glycosylation due to germline variants in SLC35A2. We aimed to evaluate the effects of D-galactose supplementation in patients with histopathologically confirmed MOGHE, with uncontrolled seizures or cognitive impairment and epileptiform activity at the EEG after epilepsy surgery (NCT04833322). Methods: Patients were orally supplemented with D-galactose for six months in doses up to 1.5 g/kg/day, monitored for seizure frequency including 24-hour-video-EEG recording, cognition and behavioral scores, i.e., WISC, BRIEF-2, SNAP-IV, SCQ and quality of life measures, before and 6 months after treatment. Global response was defined by >50% improvement of seizure frequency and/or cognition and behavior (Clinical Global Impression of “much improved” or better). Results. Twelve patients (aged 5-28 years) were included from three different centers. Neurosurgical tissue samples were available in all patients and revealed a brain somatic variant in SLC35A2 in six patients (non-present in blood). After six months of D-galactose supplementation a 50% reduction or higher of seizure frequency was achieved in 3/6 patients, with an improvement at EEG in 2/5 patients. One patient became seizure-free. An improvement of cognitive/behavioral features encompassing impulsivity (mean SNAP-IV -3.19 [-0.84; -5.6]), social communication (mean SCQ -2.08 [-0.63;-4.90]) and executive function (BRIEF-2 inhibit -5.2 [-1.23; -9.2]) was observed. Global responder rate was 9/12 (6/6 in SLC35A2-positive). Two patients presented gastrointestinal discomfort, solved after dose spacing or dose reduction. Conclusion: Supplementation with D-galactose in patients with MOGHE is safe and well tolerated. Although the efficacy data warrant larger studies, it might build a rationale for precision medicine after epilepsy surgery.

Objective Focal cortical dysplasia (FCD) Type 1 and its three subtypes have yet not been fully characterized at the clinical, anatomopathological, and molecular level (International League Against Epilepsy [ILAE] FCD classification from 2011). We aimed to describe the clinical phenotype of patients with histopathologically confirmed FCD1A obtained from a single epilepsy center between 2002 and 2016. Methods Medical records were retrieved from the hospital's archive. Results from electroencephalography (EEG) video recordings, neuroimaging, and histopathology were reevaluated. Magnetic resonance imaging (MRI) post-processing was retrospectively performed in nine patients. DNA methylation studies were carried out from archival surgical brain tissue in 11 patients. Results Nineteen children with a histopathological diagnosis of FCD1A were included. The average onset of epilepsy was 0.9 years (range 0.2-10 years). All children had severe cognitive impairment and one third had mild motor deficits, yet fine finger movements were preserved in all patients. All patients had daily seizures, being drug resistant from disease onset. Interictal electroencephalography revealed bilateral multi-regional epileptiform discharges. Interictal status epilepticus was observed in 8 and countless subclinical seizures in 11 patients. Regional continuous irregular slow waves were of higher lateralizing and localizing yield than spikes. Posterior background rhythms were normal in 16 of 19 children. Neuroimaging showed unilateral multilobar hypoplasia and increased T2-FLAIR signals of the white matter in 18 of 19 patients. All children underwent tailored multilobar resections, with seizure freedom achieved in 47% (Engel class I). There was no case with frontal involvement without involvement of the posterior quadrant by MRI and histopathology. DNA methylation profiling distinguished FCD1A samples from all other epilepsy specimens and controls. Significance We identified a cohort of young children with drug resistance from seizure onset, bad EEG with posterior emphasis, lack of any focal neurological deficits but severe cognitive impairment, subtle hypoplasia of the epileptogenic area on MRI, and histopathologically defined and molecularly confirmed by DNA methylation analysis as FCD ILAE Type 1A.

During the past decade, there have been considerable advances in understanding of the genetic and morphogenic processes underlying cortical malformations and developmental brain tumours. Focal malformations are caused by somatic (postzygotic) variants in genes related to cell growth (ie, in the mTOR pathway in focal cortical dysplasia type 2), which are acquired in neuronal progenitors during neurodevelopment. In comparison, developmental brain tumours result from somatic variants in genes related to cell proliferation (eg, in the MAP-kinase pathway in ganglioglioma), which affect proliferating glioneuronal precursors. The timing of the genetic event and the specific gene involved during neurodevelopment will drive the nature and size of the lesion, whether it is a developmental malformation or a brain tumour. There is also emerging evidence that epigenetic processes underlie a molecular memory in epileptogenesis. This knowledge will together facilitate understanding of why and how patients with these lesions have epilepsy, and could form a basis for a move towards precision medicine for this challenging cohort of patients.

ThePTPN11gene was recently described as a novel lesional epilepsy gene by extensive exome-wide sequencing studies. However, germline mutations ofPTPN11and otherRAS-/MAP-Kinase signaling pathwaygenescause Noonan syndrome, a multisystem disorder characterized by abnormal facial features, developmental delay, and sporadically, also brain tumors. Herein, we performed a deep phenotype-genotype analysis of a comprehensive series of ganglioglioma (GG) with brain somatic alterations of thePTPN11gene compared to GG with other common MAP-Kinase signaling pathway alterations. Seventy-two GG were submitted to whole exome sequencing and genotyping and 86 low grade epilepsy associated tumors (LEAT) to DNA-methylation analysis. Clinical data were retrieved from hospital files including postsurgical disease onset, age at surgery, brain localization, and seizure outcome. A comprehensive histopathology staining panel was available in all cases. We identified eight GG withPTPN11alterations, copy number variant (CNV) gains of chromosome 12, and the commonality of additional CNV gains inFGFR4, RHEB, NF1, KRASas well asBRAFV600Ealterations. Histopathology revealed an atypical and complex glio-neuronal phenotype with subpial tumor spread and large, pleomorphic, and multinuclear cellular features. Only three out of eight patients with GG andPTPN11alterations were free of disabling-seizures two years after surgery (38% Engel I). This was remarkably different from our series of GG withBRAFV600Emutations (85% Engel I). Our data point to a subgroup of GG with cellular atypia in glial and neuronal cell components, adverse postsurgical outcome, and genetically characterized byPTPN11and other Noonan syndrome-related alterations of theRAS-/MAP-Kinase signaling pathway. These findings need prospective validation in clinical practice as they argue for an adapted WHO grading system in developmental, glio-neuronal tumors associated with early-onset focal epilepsy. These findings also open avenues for targeted medical treatment.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a causal factor in regeneration failure. We demonstrate that the SLRPs Chondroadherin, Fibromodulin, Lumican, and Prolargin are enriched in human, but not zebrafish, CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer structural and mechanical properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as previously unknown inhibitory ECM factors in the human CNS that impair axon regeneration by modifying tissue mechanics and structure.ONE SENTENCE SUMMARYComposition, structural organization, and mechanical properties of the injury ECM direct central nervous system regeneration.

OBJECTIVE: Memory impairment is common in patients with temporal lobe epilepsy and seriously affects life quality. Chronic stress is a recognized cofactor in epilepsy and can also impair memory function. Furthermore, increased cortisol levels have been reported in epilepsy patients. Animal models have suggested that aggravating effects of stress on memory and synaptic plasticity were mediated via glucocorticoids. The aim of this study was, therefore, to investigate the effect of glucocorticoid receptor (GR) modulation on synaptic plasticity in the human cortex of epilepsy patients. METHODS: We performed field potential recordings in acute slices from the temporal neocortex of patients who underwent surgery for drug-resistant temporal lobe epilepsy. Synaptic plasticity was investigated by a theta-burst stimulation (TBS) protocol for induction of long-term potentiation (LTP) in the presence of GR modulators. RESULTS: LTP was impaired in temporal cortex from epilepsy patients. Pretreatment of the slices with the GR antagonist mifepristone (RU486) improved LTP induction, suggesting that LTP impairment was due to baseline GR activation in the human cortex. The highly potent GR agonist dexamethasone additionally weakened synaptic strength in an activity-dependent manner when applied after TBS. SIGNIFICANCE: Our results show a direct negative glucocorticoid effect on synaptic potentiation in the human cortex and imply chronic activation of GRs. Chronic stress may therefore contribute to memory impairment in patients with temporal lobe epilepsy. Furthermore, the activity-dependent acute inhibitory effect of dexamethasone suggests a mechanism of synaptic downscaling by which postictally increased cortisol levels may prevent pathologic plasticity upon seizures.

Epigenetics refers broadly to processes that influence medium to long-term gene expression by changing the readability and accessibility of the genetic code. The Neurobiology Commission of the International League Against Epilepsy (ILAE) recently convened a Task Force to explore and disseminate advances in epigenetics to better understand their role and intersection with genetics and the neurobiology of epilepsies and their co-morbidities, and to accelerate translation of these findings into the development of better therapies. Here, we provide a topic primer on epigenetics, explaining the key processes and findings to date in experimental and human epilepsy. We review the growing list of genes with epigenetic functions that have been linked with epilepsy in humans. We consider potential practical applications, including using epigenetic signals as biomarkers for tissue- and biofluid-based diagnostics and the prospects for developing epigenetic-based treatments for epilepsy. We include a glossary of terms, FAQs and other supports to facilitate a broad understanding of the topic for the non-expert. Last, we review the limitations, research gaps and the next challenges. In summary, epigenetic processes represent important mechanisms controlling the activity of genes, providing opportunities for insight into disease mechanisms, biomarkers and novel therapies for epilepsy.

Understanding the exact molecular mechanisms involved in the aetiology of epileptogenic pathologies with or without tumour activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants in low-grade epilepsy-associated tumours (7.92 ± 5.65 single-nucleotide variants) than in brain tissue from malformations of cortical development (6.11 ± 4 single-nucleotide variants) or hippocampal sclerosis (5.1 ± 3.04 single-nucleotide variants). Tumour tissues also had the largest number of likely pathogenic variant carrying cells. low-grade epilepsy-associated tumours had the highest proportion of samples with one or more somatic copy-number variants (24.7%), followed by malformations of cortical development (5.4%) and hippocampal sclerosis (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among low-grade epilepsy-associated tumours. For germline variant-associated malformations of cortical development genes such as TSC2, DEPDC5 and PTEN, germline single-nucleotide variants were frequently identified within large loss of heterozygosity regions, supporting the recently proposed ‘second hit’ disease mechanism in these genes. We detect somatic variants in 12 established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the aetiology of low-grade epilepsy-associated tumours (e.g. BRAF) and malformations of cortical development (e.g. SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with low-grade epilepsy-associated tumours and NRAS Q61 mutated protein with a complex malformation of cortical development characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21–q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical-genetic analyses showed that the numbers of somatic single-nucleotide variant across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with low-grade epilepsy-associated tumours. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening and guides future directions for clinical implementation of epilepsy surgery genetics.

Focal malformations of cortical development (MCD) are linked to somatic brain mutations occurring during neurodevelopment. Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is a newly recognized clinico-pathological entity associated with pediatric drug-resistant focal epilepsy, and amenable to neurosurgical treatment. MOGHE is histopathologically characterized by clusters of increased oligodendroglial cell densities, patchy zones of hypomyelination, and heterotopic neurons in the white matter. The molecular etiology of MOGHE remained unknown so far. We hypothesized a contribution of mosaic brain variants and performed deep targeted gene sequencing on 20 surgical MOGHE brain samples from a single-center cohort of pediatric patients. We identified somatic pathogenic SLC35A2 variants in 9/20 (45%) patients with mosaic rates ranging from 7 to 52%. SLC35A2 encodes a UDP-galactose transporter, previously implicated in other malformations of cortical development (MCD) and a rare type of congenital disorder of glycosylation. To further clarify the histological features of SLC35A2-brain tissues, we then collected 17 samples with pathogenic SLC35A2 variants from a multicenter cohort of MCD cases. Histopathological reassessment including anti-Olig2 staining confirmed a MOGHE diagnosis in all cases. Analysis by droplet digital PCR of pools of microdissected cells from one MOGHE tissue revealed a variant enrichment in clustered oligodendroglial cells and heterotopic neurons. Through an international consortium, we assembled an unprecedented series of 26 SLC35A2-MOGHE cases providing evidence that mosaic SLC35A2 variants, likely occurred in a neuroglial progenitor cell during brain development, are a genetic marker for MOGHE.

It is challenging to estimate genetic variant burden across different subtypes of epilepsy. Herein, we used a comparative approach to assess the genetic variant burden and genotype–phenotype correlations in four most common brain lesions in patients with drug-resistant focal epilepsy. Targeted sequencing analysis was performed for a panel of 161 genes with a mean coverage of >400×. Lesional tissue was histopathologically reviewed and dissected from hippocampal sclerosis (n = 15), ganglioglioma (n = 16), dysembryoplastic neuroepithelial tumors (n = 8), and focal cortical dysplasia type II (n = 15). Peripheral blood (n = 12) or surgical tissue samples histopathologically classified as lesion-free (n = 42) were available for comparison. Variants were classified as pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics guidelines. Overall, we identified pathogenic and likely pathogenic variants in 25.9% of patients with a mean coverage of 383×. The highest number of pathogenic/likely pathogenic variants was observed in patients with ganglioglioma (43.75%; all somatic) and dysembryoplastic neuroepithelial tumors (37.5%; all somatic), and in 20% of cases with focal cortical dysplasia type II (13.33% somatic, 6.67% germline). Pathogenic/likely pathogenic positive genes were disorder specific and BRAF V600E the only recurrent pathogenic variant. This study represents a reference for the genetic variant burden across the four most common lesion entities in patients with drug-resistant focal epilepsy. The observed large variability in variant burden by epileptic lesion type calls for whole exome sequencing of histopathologically well-characterized tissue in a diagnostic setting and in research to discover novel disease-associated genes.

Structural brain lesions, including the broad range of malformations of cortical development (MCD) and glioneuronal tumors, are among the most common causes of drug‐resistant focal epilepsy. Epilepsy surgery can provide a curative treatment option in respective patients. The currently available pre‐surgical multi‐modal diagnostic armamentarium includes high‐ and ultra‐high resolution magnetic resonance imaging (MRI) and intracerebral EEG to identify a focal structural brain lesion as epilepsy underlying etiology. However, specificity and accuracy in diagnosing the type of lesion have proven to be limited. Moreover, the diagnostic process does not stop with the decision for surgery. The neuropathological diagnosis remains the gold standard for disease classification and patient stratification, but is particularly complex with high inter‐observer variability. Here, the identification of lesion‐specific mosaic variants together with epigenetic profiling of lesional brain tissue became new tools to more reliably identify disease entities. In this review, we will discuss how the paradigm shifts from histopathology toward an integrated diagnostic approach in cancer and the more recent development of the DNA methylation‐based brain tumor classifier have started to influence epilepsy diagnostics. Some examples will be highlighted showing how the diagnosis and our mechanistic understanding of difficult to classify structural brain lesions associated with focal epilepsy has improved with molecular genetic data being considered in decision making., The identification of lesion‐specific somatic variants, gene fusion, and epigenetic profiles has helped to rationalize disease classification in cancer and more recently also in epilepsy. Many structural brain lesions which are commonly associated with epilepsy including low‐grade epilepsy‐associated tumors (LEAT) and the broad spectrum of malformations of cortical development (MCD) have proven difficult to diagnose based on their histopathology alone. Here, molecular diagnostic approaches can be a game‐changer and help provide more accurate diagnoses as well as identify new clinically meaningful disease entities.

© 2021 The Authors., The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., Hyun-Dong Chang is supported by the Dr. Rolf M. Schwiete Foundation. Susanne Melzer and Attila Tarnok thank De Novo Software for providing FCS Express. Enrico Lugli is supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC IG20676). Gabriele De Simone and Giovanni Galletti were supported by Fellowships from the Fondazione Italiana per la Ricerca sul Cancro-Associazione Italiana per la Ricerca sul Cancro (FIRC-AIRC). Jun Dong is supported by Deutsche Forschungsgemeinschft (DFG, German Research Foundation) Projektnummer 389687267 and Chinesisch-Deutsches Zentrum für Wissenschaftsförderung [Sino-German Center for Research Promotion (SGC)] grant C-0072. Nicola Gagliani, Samuel Huber and Franziska Muscate are supported by DFG fundings: SFB841,GA 2441/3-1, HU 1714/10-1. The tetramer APC-conjugated H-2K (d) HIV-1 gag197-205 AMQMLKETI used in TDS assay for mouse blood T cells was obtained through the NIH Tetramer Facility. Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14-1. Supported by the following grants: AIRC 5X1000 2018 id. 21147 (Lorenzo Moretta); AIRC IG 2017 id. 19920 (Lorenzo Moretta); RC-2020 OPBG (Lorenzo Moretta); AIRC and European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 800924 (Linda Quatrini). Dirk Baumjohann was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Emmy Noether Programme BA 5132/1-2 (252623821) and Germany's Excellence Strategy EXC2151 (390873048).

Polymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here, we analyzed 26 PMG patients employing array-based DNA methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases, about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q. Electronic supplementary material The online version of this article (10.1007/s00401-020-02228-5) contains supplementary material, which is available to authorized users.

Objectives Focal cortical dysplasia (FCD) is a major cause of drug‐resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation–based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes. Methods DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls. Results Differential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway–related genes. Significance Our studies extend the evidence for disease‐specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Sechs Jahre nach Veroffentlichung soll der Nutzen der ILAE-Konsensus-Klassifikation Fokaler Kortikaler Dysplasien (FCD) bewertet werden. FCDs sind haufige Ursachen fruhkindlicher und medikamentos schwierig zu behandelnder fokaler Epilepsien. Die neurochirurgische Therapie bietet oftmals eine grose Chance auf dauerhafte Heilung. Dies ist aber wesentlich vom histopathologischen Subtyp abhangig, und das Resektionsfeld muss durch umfassende prachirurgische Diagnostik definiert werden, z. B. durch die vorlaufige Abschatzung der Art der FCD anhand des MRT und ihre Nachbarschaftsbeziehung zu eloquenten Arealen, sowie durch iktale und interiktale EEG-Befunde. Die ILAE-Klassifikation grenzt 3 histomorphologisch unterschiedliche FCD-Subtypen ab, welche nach aktuellem Kenntnisstand auch unterschiedlichen Pathomechanismen zugrunde liegen. FCD Typ 1 sind seltene Erkrankungen bei weit uberwiegend jungen Kindern mit radiologisch, elektro-klinisch als auch pathologisch schwierig zu diagnostizierenden Epilepsien – mit deutlich geringeren Chancen auf eine postoperative Anfallsfreiheit als dies bei einer FCD Typ 2 der Fall ist. Die FCD Typ 2 stellt eine klinisch-pathologisch gut charakterisierte und chirurgisch oftmals gut behandelbare Gruppe dar. Molekulargenetisch finden sich Mutationen im mTOR-Signalweg, was daran denken lasst, dass zukunftig evtl. eine zielgerichtete medikamentose Therapie moglich wird. Wahrenddessen sind assoziierte FCD Typ 3 in der wissenschaftlichen Literatur zwar haufig beschrieben, ihre Entstehung und Eigenstandigkeit jedoch umstritten. Klinischer Verlauf und neurochirurgischer Therapieerfolg sind meist von der Hauptlasion abhangig, welche mit Hippocampussklerosen (HS), epilepsieassoziierten Tumoren (LEAT), vaskularen Fehlbildungen, Enzephalitiden und anderen fruhkindlichen erworbenen Lasionen ein breites Spektrum abdecken. Eine Revision der bestehenden ILAE-Klassifikation wird bereits diskutiert, v. a. um dem Einzug molekulardiagnostischer Methoden wie in vielen anderen Bereichen der Medizin gerecht zu werden. Wesentliche Anderungen im bestehenden Prinzip in der Einteilung der drei FCD-Typen sind allerding nicht zu erwarten.

Objective The microscopic review of hematoxylin-eosin-stained images of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex remains challenging. Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. We trained a convolutional neural network (CNN) to classify both entities and visualize the results. Additionally, we propose a new Web-based deep learning application as proof of concept of how deep learning could enter the pathologic routine. Methods A digital processing pipeline was developed for a series of 56 cases of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex to obtain 4000 regions of interest and 200 000 subsamples with different zoom and rotation angles to train a neural network. Guided gradient-weighted class activation maps (Guided Grad-CAMs) were generated to visualize morphological features used by the CNN to distinguish both entities. Results Our best-performing network achieved 91% accuracy and 0.88 area under the receiver operating characteristic curve at the tile level for an unseen test set. Novel histopathologic patterns were found through the visualized Guided Grad-CAMs. These patterns were assembled into a classification score to augment decision-making in routine histopathology workup. This score was successfully validated by 11 expert neuropathologists and 12 nonexperts, boosting nonexperts to expert level performance. Significance Our newly developed Web application combines the visualization of whole slide images with the possibility of deep learning-aided classification between focal cortical dysplasia IIb and tuberous sclerosis complex. This approach will help to introduce deep learning applications and visualization for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Epilepsy is a heterogeneous condition with disparate etiologies and phenotypic and genotypic characteristics. Clinical and research aspects are accordingly varied, ranging from epidemiological to molecular, spanning clinical trials and outcomes, gene and drug discovery, imaging, electroencephalography, pathology, epilepsy surgery, digital technologies, and numerous others. Epilepsy data are collected in the terabytes and petabytes, pushing the limits of current capabilities. Modern computing firepower and advances in machine and deep learning, pioneered in other diseases, open up exciting possibilities for epilepsy too. However, without carefully designed approaches to acquiring, standardizing, curating, and making available such data, there is a risk of failure. Thus, careful construction of relevant ontologies, with intimate stakeholder inputs, provides the requisite scaffolding for more ambitious big data undertakings, such as an epilepsy data commons. In this review, we assess the clinical and research epilepsy landscapes in the big data arena, current challenges, and future directions, and make the case for a systematic approach to epilepsy big data.

We trained a convolutional neural network (CNN) to classify H.E. stained microscopic images of focal cortical dysplasia type IIb (FCD IIb) and cortical tuber of tuberous sclerosis complex (TSC). Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. The microscopic review of routine stainings of such surgical specimens remains challenging. A digital processing pipeline was developed for a series of 56 FCD IIb and TSC cases to obtain 4000 regions of interest and 200.000 sub-samples with different zoom and rotation angles to train a CNN. Our best performing network achieved 91% accuracy and 0.88 AUCROC (area under the receiver operating characteristic curve) on a hold-out test-set. Guided gradient-weighted class activation maps visualized morphological features used by the CNN to distinguish both entities. We then developed a web application, which combined the visualization of whole slide images (WSI) with the possibility for classification between FCD IIb and TSC on demand by our pretrained and build-in CNN classifier. This approach might help to introduce deep learning applications for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

The revolution in high-throughput omics technologies has dramatically expanded our understanding of the epilepsies as complex diseases. It is now clear that further progress in treating the full spectrum of seizure disorders requires a systems-level framework for analyzing and integrating data from multiple omics technologies that moves beyond the search for single molecular alterations to an understanding of dysregulated pathways in epilepsy. Taking such a pathway-centered view requires further integrating the tools of systems biology into epilepsy research. In this appraisal, we highlight and summarize systems biology approaches in basic epilepsy studies as they were discussed during the 2017 Workshop on the Neurobiology of Epilepsy (WONOEP). During the 3-day event, participants exchanged emerging results and thoughts on developing the systems biology of epilepsy, and the promise and limitations of these approaches for the near term.

We comprehensively studied the clinical presentation, stereo-EEG and MRI findings, histopathological diagnosis, and brain somatic mutations in a retrospective series of drug-resistant patients with difficult-to-localize epilepsy due to focal cortical dysplasia at the bottom of a sulcus (BOS-FCD). We identified 10 patients with BOS-FCD from the Cleveland Clinic epilepsy surgery database submitted for intracranial video-EEG monitoring. Brain MRI, including voxel-based morphometric analysis and surgical tissue submitted for histopathology, was reviewed. Paraffin tissue samples from five patients were made available for targeted next-generation sequencing. Postsurgical follow-up was available in nine patients. BOS-FCD was identified in the superior frontal sulcus in six patients, inferior frontal sulcus in one patient, central sulcus in one patient, and intraparietal sulcus in two patients. All patients had stereotyped seizures. Intracranial EEG recordings identified ictal onset at the BOS-FCD in all 10 patients, whereas ictal scalp EEG had a localizing value in only six patients. Complete resection was achieved by lesionectomy or focal corticectomy in nine patients. Histopathologically, six patients had FCD type IIb and three had FCD type IIa. Next-generation sequencing analysis of DNA extracted from lesion-enriched (micro-dissected) tissue from five patients with FCD type II led to the identification of a germline frameshift insertion in DEPDC5, introducing a premature stop in one patient. Eight out of nine patients with available follow-up were completely seizure-free (Engel Class IA) after a mean follow-up period of six years. Our results confirm previous studies classifying difficult-to-localize BOS-FCD into the emerging spectrum of FCD ILAE type II mTORopathies. Further studies with large patient numbers and ultra-deep genetic testing may help to bridge the current knowledge gap in genetic aetiologies of FCD.

The brain is a complex system composed of networks of interacting elements, from genes to circuits, whose function (and dysfunction) is not derivable from the superposition of individual components. Epilepsy is frequently described as a network disease, but to date, there is no standardized framework within which network concepts applicable to all levels from genes to whole brain can be used to generate deeper insights into the pathogenesis of seizures or the associated morbidities. To address this shortcoming, the Neurobiology Commission of the International League Against Epilepsy dedicated a Workshop on Neurobiology of Epilepsy (XIV WONOEP 2017) with the aim of formalizing network concepts as they apply to epilepsy and to critically discuss whether and how such concepts could augment current research endeavors. Here, we review concepts and strategies derived by considering epilepsy as a disease of different network hierarchies that range from genes to clinical phenotypes. We propose that the concept of networks is important for understanding epilepsy and is critical for developing new study designs. These approaches could ultimately facilitate the development of novel diagnostic and therapeutic strategies.

SummaryObjective Current medications for patients with epilepsy work in only two of three patients. For those medications that do work, they only suppress seizures. They treat the symptoms, but do not modify the underlying disease, forcing patients to take these drugs with significant side effects, often for the rest of their lives. A major limitation in our ability to advance new therapeutics that permanently prevent, reduce the frequency of, or cure epilepsy comes from a lack of understanding of the disease coupled with a lack of reliable biomarkers that can predict who has or who will get epilepsy. Methods The main goal of this report is to present a number of approaches for identifying reliable biomarkers from observing patients with brain disorders that have a high probability of producing epilepsy. Results A given biomarker, or more likely a profile of biomarkers, will have both a quantity and a time course during epileptogenesis that can be used to predict who will get the disease, to confirm epilepsy as a diagnosis, to identify coexisting pathologies, and to monitor the course of treatments. Significance Additional studies in patients and animal models could identify common and clinically valuable biomarkers to successfully translate animal studies into new and effective clinical trials.

Highlights • Hippocampal MTR in TLE does not add to volume or T2 signal regarding lateralization. • Extracellular CSPG are the most relevant correlate to MTR in hippocampal sclerosis. • Increased CSPG counterbalances neuron loss over the reduction in hippocampal MTR., Objective Temporal lobe epilepsy patients (TLE) often present with hippocampal atrophy, increased T2 relaxation, and reduced magnetization transfer ratio (MTR) in magnetic resonance images (MRI). The histological correlates of the reduced hippocampal MTR are so far unknown. Since MTR is dependent on the tissue’s macromolecules, our aim was to evaluate the correlations between cellular populations, extracellular matrix molecules and the MTR in TLE patients. Methods Patients with TLE (n = 26) and voluntaries (=20) were scanned in a 3 Tesla MRI scanner, and MTR images were calculated from 3DT1 sequences with magnetization pulse on resonance. Immunohistochemistry for neurons, reactive astrocytes, activated microglia, and extracellular matrix chondroitin sulfate were performed in formalin fixed, paraffin embedded tissues of TLE and autopsy controls (n = 10). Results were considered significant with adjusted p

Objective The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb. Methods Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein. Results All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations. Interpretation Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb. Ann Neurol 2015;77:312–319

Epilepsy is a frequent neurological disorder, although onset and progression of seizures remain difficult to predict in affected patients, irrespective of their epileptogenic condition. Previous studies in animal models as well as human epileptic brain tissue revealed a remarkably diverse pattern of gene expression implicating epigenetic changes to contribute to disease progression. Here we mapped for the first time global DNA methylation patterns in chronic epileptic rats and controls. Using methyl-CpG capture associated with massive parallel sequencing (Methyl-Seq) we report the genomic methylation signature of the chronic epileptic state. We observed a predominant increase, rather than loss of DNA methylation in chronic rat epilepsy. Aberrant methylation patterns were inversely correlated with gene expression changes using mRNA sequencing from same animals and tissue specimens. Administration of a ketogenic, high-fat, low-carbohydrate diet attenuated seizure progression and ameliorated DNA methylation mediated changes in gene expression. This is the first report of unsupervised clustering of an epigenetic mark being used in epilepsy research to separate epileptic from non-epileptic animals as well as from animals receiving anti-convulsive dietary treatment. We further discuss the potential impact of epigenetic changes as a pathogenic mechanism of epileptogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1168-8) contains supplementary material, which is available to authorized users.

Summary Seizures cannot be medically controlled in approximately 40% of people with epilepsy. Although we are beginning to understand how to better treat certain seizure types, we still do not know the regulatory events that determine antiepileptic drug resistance. Proposed pathoetiologic mechanisms include altered expression of drug targets (i.e., receptor or ion channel modifications), endothelial drug transporter activation (i.e., increasing drug clearance), or intrinsic severity factors. The latter hypothesis results from an often confirmed clinical observation, that seizure severity is a reliable predictor for the development of pharmacoresistance (PR) in epilepsy. Herein, we propose, that genome modifications that do not involve changes to the DNA sequence per se (i.e., epigenetic changes) could confer PR in patients with epilepsy. Seizures cause excessive neuronal membrane depolarization, which can influence the cellular nucleus; we thus hypothesize that seizures can mediate epigenetic modifications that result in persistent genomic methylation, histone density, and posttranslational modifications, as well as noncoding RNA-based changes. Although experimental evidence is lacking in epilepsy, such mechanisms are well characterized in cancer, either as a result of anticancer drugs themselves or cancer-related intrinsic signals (i.e., noncoding RNAs). We suggest that similar mechanisms also play a role in PR epilepsies. Addressing such epigenetic mechanisms may be a successful strategy to increase the brain's sensitivity to antiepileptic drugs and may even act as disease-modifying treatment.

Rapid developments in molecular genetic technology and research have swiftly advanced our understanding of neuro-oncology. As a consequence, the WHO invited their expert panels to revise the current classification system of brain tumours and to introduce, for the first time, a molecular genetic approach for selected tumour entities, thus setting a new gold standard in histopathology. The revised 5th edition of the 'blue book' was released in May 2016 and will have a major impact in stratifying diagnosis and treatment. However, low-grade neuroepithelial tumours that present with early-onset focal epilepsy and are mostly seen in children and young adults (previously designated as long-term epilepsy-associated neuroepithelial tumours, LEAT) lack such innovative clinicopathological and molecular genetic tools. The Neuropathology Task Force of the International League against Epilepsy will critically discuss this issue, and will offer perspectives on how to decipher and validate clinically meaningful LEAT entities using the current WHO approach that integrates clinicopathological and genetic classification systems.

The ubiquitous metabolic intermediary and nucleoside adenosine is a “master regulator” in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of the Adk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influence Adk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to the Adk promoter influences the regulation of Adk expression.

This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns.

DNA methylation is a covalent chromatin modification, characterized by the biochemical addition of a methyl group (-CH3) to cytosine nucleotides via a DNA methyltransferase enzyme. 5'-Methylcytosine (5-mC), frequently called the fifth base, has been implicated in genome stability, silencing of transposable elements, and repression of gene expression. Through the latter, DNA methylation dynamics broadly influence brain development, function, and aging. Aberrant DNA methylation patterns, either localized to specific gene regions or scattered throughout the genome, are associated with many neurologic disorders. Herein, we discuss the emerging role of DNA methylation in epileptogenesis and the perspectives arising from epigenetic medicine as new therapeutic strategy in difficult-to-treat epilepsies.

Focal cortical dysplasias (FCD) which represent a composite group of cortical malformations are increasingly recognized as morphological substrate for severe therapy-refractory epilepsy in children and young adults. However, presurgical evaluation remains challenging as not all FCD variants can be reliably detected by high-resolution magnetic resonance imaging (MRI). Here, we studied a cohort of 52 epilepsy patients with neuropathological evidence for FCD using the 2011 classification of the International League against Epilepsy (ILAE) and systematically analysed those histopathologic features applicable also for MRI diagnostics. Histopathologic parameters included quantitative measurements of cellular profiles, cortical thickness, heterotopic neurons in white matter, and myelination that were compared between FCD subtypes and age-/localization-matched controls (n = 36) using multivariate analysis. Dysmorphic neurons in both FCD Type II variants showed significantly increased diameter of their cell bodies and nuclei. Cortical thickness was also increased with a distinct loss of myelin content specifying FCD Type IIb from IIa. The data further suggested that myelination deficits in FCD Type IIb result from compromised oligodendroglial lineage differentiation and we concluded that the "transmantle sign" is a unique finding in FCD Type IIb. In contrast, FCD Type Ia was characterized by a smaller cortical ribbon and higher neuronal densities, but these parameters failed to reach statistical significance (considering age- and location-dependent variability in controls). All FCD variants showed abnormal grey-white matter boundaries with increased numbers of heterotopic neurons. Similar results were obtained also at deep white matter location. Thus, many FCD variants may indeed escape visual MRI inspection, but suspicious areas with increased or decreased cortical thickness as well as grey-white matter blurring may be uncovered using post-processing protocols of neuroimaging data. The systematic analysis of well-specified histopathological features could be helpful to improve sensitivity and specificity in MRI detection during pre-surgical work-up of patients with drug-resistant focal epilepsies.

Mesial temporal sclerosis (MTS) is the most common lesion in chronic, intractable temporal lobe epilepsies (TLE) and characterized by segmental neuronal cell loss in major hippocampal segments. Another histopathological hallmark includes granule cell dispersion (GCD), an architectural disturbance of the dentate gyrus encountered in approximately 50% of patients with mesial temporal sclerosis. Reelin, which plays a key role during hippocampal development and maintenance of laminar organization, is synthesized and released by Cajal-Retzius cells of the dentate molecular layer, and previous studies have shown that Reelin transcript levels are downregulated in human temporal lobe epilepsies specimens. To investigate whether epigenetic silencing by Reelin promoter methylation may be an underlying pathogenetic mechanism of GCD, DNA was harvested from 3 microdissected hippocampal subregions (i.e. molecular and granule cell layers of the dentate gyrus and presubiculum) from 8 MTS specimens with GCD, 5 TLE samples without GCD, and 3 autopsy controls. Promoter methylation was analyzed after bisulfite treatment, cloning, and direct sequencing; immunohistochemistry was performed to identify Cajal-Retzius cells. Reelin promoter methylation was found to be greater in TLE specimens than in controls; promoter methylation correlated with GCD among TLE specimens (p < 0.0002). No other clinical or histopathological parameter (i.e. sex, age, seizure duration, medication or extent, of MTS) correlated with promoter methylation. These data support a compromised Reelin-signaling pathway and identify promoter methylation as an epigenetic mechanism in the pathogenesis of TLE.

In humans, genomic DNA is organized in 23 chromosome pairs coding for roughly 25,000 genes. Not all of them are active at all times. During development, a broad range of different cell types needs to be generated in a highly ordered and reproducible manner, requiring selective gene expression programs. Epigenetics can be regarded as the information management system that is able to index or bookmark distinct regions in our genome to regulate the readout of DNA. It further comprises the molecular memory of any given cell, allowing it to store information of previously experienced external (e.g., environmental) or internal (e.g., developmental) stimuli, to learn from this experience and to respond. The underlying epigenetic mechanisms can be synergistic, antagonistic, or mutually exclusive and their large variety combined with the variability and interdependence is thought to provide the molecular basis for any phenotypic variation in physiological and pathological conditions. Thus, widespread reconfiguration of the epigenome is not only a key feature of neurodevelopment, brain maturation, and adult brain function but also disease.

The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb.Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein.All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations.Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb.