Multimodal mapping of regional brain vulnerability to focal cortical dysplasia.

Focal cortical dysplasia (FCD) type II is a highly epileptogenic developmental malformation and a common cause of surgically treated drug-resistant epilepsy. While clinical observations suggest frequent occurrence in the frontal lobe, mechanisms for such propensity remain unexplored. Here, we hypothesized that cortex-wide spatial associations of FCD distribution with cortical cytoarchitecture, gene expression and organizational axes may offer complementary insights into processes that predispose given cortical regions to harbour FCD. We mapped the cortex-wide MRI distribution of FCDs in 337 patients collected from 13 sites worldwide. We then determined its associations with (i) cytoarchitectural features using histological atlases by Von Economo and Koskinas and BigBrain; (ii) whole-brain gene expression and spatiotemporal dynamics from prenatal to adulthood stages using the Allen Human Brain Atlas and PsychENCODE BrainSpan; and (iii) macroscale developmental axes of cortical organization. FCD lesions were preferentially located in the prefrontal and fronto-limbic cortices typified by low neuron density, large soma and thick grey matter. Transcriptomic associations with FCD distribution uncovered a prenatal component related to neuroglial proliferation and differentiation, likely accounting for the dysplastic makeup, and a postnatal component related to synaptogenesis and circuit organization, possibly contributing to circuit-level hyperexcitability. FCD distribution showed a strong association with the anterior region of the antero-posterior axis derived from heritability analysis of interregional structural covariance of cortical thickness, but not with structural and functional hierarchical axes. Reliability of all results was confirmed through resampling techniques. Multimodal associations with cytoarchitecture, gene expression and axes of cortical organization indicate that prenatal neurogenesis and postnatal synaptogenesis may be key points of developmental vulnerability of the frontal lobe to FCD. Concordant with a causal role of atypical neuroglial proliferation and growth, our results indicate that FCD-vulnerable cortices display properties indicative of earlier termination of neurogenesis and initiation of cell growth. They also suggest a potential contribution of aberrant postnatal synaptogenesis and circuit development to FCD epileptogenicity.

Predictors of genetic diagnosis in individuals with developmental and epileptic encephalopathies.

OBJECTIVE: To evaluate the clinical predictors of positive genetic investigation in developmental and epileptic encephalopathies, beyond the influence of Dravet Syndrome. METHODS: The study included 98 patients diagnosed with developmental and epileptic encephalopathies. The patients underwent Sanger sequencing of SCN1A, Chromosomal Microarray Analysis, and Whole Exome Sequencing. The association of clinical variables with a positive genetic test was investigated using univariate and multivariate analysis. RESULTS: Genetic diagnosis was identified in 47 (48 %) patients with developmental and epileptic encephalopathies. Beyond Dravet Syndrome influence, first seizure in the context of fever (p < 0.01), seizures precipitated by temperature (p = 0.04), cognitive regression (p = 0.04), hypotonia (p < 0.01), and focal seizures (p = 0.03) increased the chances of a positive genetic investigation. In contrast, atonic seizures (p = 0.01) and generalized discharges on electroencephalogram (p = 0.02) decreased the chances. Dravet Syndrome was positively associated with a genetic developmental and epileptic encephalopathies etiology (p < 0.01), whereas epilepsy with myoclonic-atonic seizures (p = 0.01), developmental and epileptic encephalopathies with spike-wave activation in sleep (p = 0.04), and Lennox-Gastaut syndrome (p = 0.03) were negatively associated. In multivariate analysis, the first seizure in the context of fever (p < 0.01) and hypotonia (p = 0.02) were positively, and atonic seizures (p = 0.01) were negatively and independently associated with a genetic etiology. CONCLUSION: The predictive variables of genetic investigation in developmental and epileptic encephalopathies are first seizure in the context of fever and hypotonia, whereas atonic seizures decrease the chances of finding a genetic cause for developmental and epileptic encephalopathies. Regarding epileptic syndromes, Dravet Syndrome is highly associated with a positive genetic test, whereas epilepsy with myoclonic-atonic seizures, developmental and epileptic encephalopathies with spike-wave activation in sleep, and Lennox-Gastaut syndrome are rarely associated with a positive genetic investigation.

Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Transcriptomic analysis of dorsal and ventral subiculum after induction of acute seizures by electric stimulation of the perforant pathway in rats.

Preconditioning is a mechanism in which injuries induced by non-lethal hypoxia or seizures trigger cellular resistance to subsequent events. Norwood et al., in a 2010 study, showed that an 8-h-long period of electrical stimulation of the perforant pathway in rats is required for the induction of hippocampal sclerosis. However, in order to avoid generalized seizures, status epilepticus (SE), and death, a state of resistance to seizures must be induced in the hippocampus by a preconditioning paradigm consisting of two daily 30-min stimulation periods. Due to the importance of the subiculum in the hippocampal formation, this study aims to investigate differential gene expression patterns in the dorsal and ventral subiculum using RNA-sequencing, after induction of a preconditioning protocol by electrical stimulation of the perforant pathway. The dorsal (dSub) and ventral (vSub) subiculum regions were collected by laser-microdissection 24 h after preconditioning protocol induction in rats. RNA sequencing was performed in a Hiseq 4000 platform, reads were aligned using the STAR and DESEq2 statistics package was used to estimate gene expression. We identified 1176 differentially expressed genes comparing control to preconditioned subiculum regions, 204 genes were differentially expressed in dSub and 972 in vSub. The gene ontology enrichment analysis showed that the most significant common enrichment pathway considering up-regulated genes in dSub and vSub was steroid metabolism. In contrast, the most significant enrichment pathway considering down-regulated genes in vSub was axon guidance. Our results indicate that preconditioning induces changes in the expression of genes related to synaptic reorganization, increased cholesterol metabolism, and astrogliosis in both dSub and vSub. Both regions also presented a decrease in the expression of genes related to glutamatergic transmission and an increase in expression of genes related to complement system activation and GABAergic transmission. The down-regulation of proapoptotic and axon guidance genes in the ventral subiculum suggests that preconditioning may induce a neuroprotective environment in this region.

The ILAE consensus classification of focal cortical dysplasia: An update proposed by an ad hoc task force of the ILAE diagnostic methods commission.

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.

Slowly progressive behavioral frontotemporal dementia syndrome in a family co-segregating the C9orf72 expansion and a Synaptophysin mutation.

INTRODUCTION: Synaptophysin, already related to X-linked intellectual disability, is expressed mainly in the central nervous system. Studies in humans indicate that the downregulation of synaptophysin could be involved in the development of dementia. Our study presents the first familial case of behavioral variant frontotemporal dementia associated with the co-occurrence of the repeat expansion in C9orf72 and a pathogenic variant in the SYP gene. METHODS: Exome sequencing and repeat-primed PCR for C9orf72 were performed for two siblings with clinical and imaging findings suggestive of slowly progressive behavioral frontotemporal dementia. RESULTS: We found that both siblings have the hexanucleotide expansion in C9orf72 and a null variant in the SYP gene. The most affected sibling presents the putative variant in a hemizygous state. With milder symptoms, his sister has the same pathogenic variant in heterozygosis, compatible with X-linked inheritance. DISCUSSION: Our results strengthened previous suggestive evidence that the phenotypes associated with C9orf72 repeat expansion are variable and probably influenced by additional genetic modifiers. We hypothesized that the pathogenic variant in the SYP gene might have modified the typical phenotype associated with the C9orf72 mutation.

Increased runs of homozygosity in the autosomal genome of Brazilian individuals with neurodevelopmental delay/intellectual disability and/or multiple congenital anomalies investigated by chromosomal microarray analysis.

Runs of homozygosity (ROH) in the human genome may be clinically relevant. The aim of this study was to report the frequency of increased ROH of the autosomal genome in individuals with neurodevelopmental delay/intellectual disability and/or multiple congenital anomalies, and to compare these data with a control group. Data consisted of calls of homozygosity from 265 patients and 289 controls. In total, 7.2% (19/265) of the patients showed multiple ROH exceeding 1% of autosomal genome, compared to 1.4% (4/289) in the control group (p=0.0006). Homozygosity ranged from 1.38% to 22.12% among patients, and from 1.53 to 2.40% in the control group. In turn, 1.9% (5/265) of patients presented ROH ≥10Mb in a single chromosome, compared to 0.3% (1/289) of individuals from the control group (p=0.0801). By excluding cases with reported consanguineous parents (15/24), the frequency of increased ROH was 3.4% (9/250) among patients and 1.7% (5/289) in the control group, considering multiple ROH exceeding 1% of the autosome genome and ROH ≥10Mb in a single chromosome together, although not statistically significant (p=0.1873). These results reinforce the importance of investigating ROH, which with complementary diagnostic tests can improve the diagnostic yield for patients with such conditions.

Multi-omics analysis suggests enhanced epileptogenesis in the Cornu Ammonis 3 of the pilocarpine model of mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder characterized by the occurrence of seizures, and histopathological abnormalities in the mesial temporal lobe structures, mainly hippocampal sclerosis (HS). We used a multi-omics approach to determine the profile of transcript and protein expression in the dorsal and ventral hippocampal dentate gyrus (DG) and Cornu Ammonis 3 (CA3) in an animal model of MTLE induced by pilocarpine. We performed label-free proteomics and RNAseq from laser-microdissected tissue isolated from pilocarpine-induced Wistar rats. We divided the DG and CA3 into dorsal and ventral areas and analyzed them separately. We performed a data integration analysis and evaluated enriched signaling pathways, as well as the integrated networks generated based on the gene ontology processes. Our results indicate differences in the transcriptomic and proteomic profiles among the DG and the CA3 subfields of the hippocampus. Moreover, our data suggest that epileptogenesis is enhanced in the CA3 region when compared to the DG, with most abnormalities in transcript and protein levels occurring in the CA3. Furthermore, our results show that the epileptogenesis in the pilocarpine model involves predominantly abnormal regulation of excitatory neuronal mechanisms mediated by N-methyl D-aspartate (NMDA) receptors, changes in the serotonin signaling, and neuronal activity controlled by calcium/calmodulin-dependent protein kinase (CaMK) regulation and leucine-rich repeat kinase 2 (LRRK2)/WNT signaling pathways.

Inflammatory and neurotrophic factor plasma levels are related to epilepsy independently of etiology.

OBJECTIVE: Inflammation plays an essential role in epilepsy. Studies indicate that cytokines and neurotrophic factors can act in neuroexcitability and epileptogenesis. We aimed to investigate the association between plasma inflammatory and neurotrophic markers, seizure frequency, and chronic epilepsy subtypes. METHODS: We studied 446 patients with epilepsy and 166 healthy controls. We classified patients according to etiology and seizure frequency. We measured plasma levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, IL-17, interferon-γ (IFNγ), tumor necrosis factor α (TNFα), soluble TNF receptor 1 (sTNFr1), sTNFr2, brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT3), NT4/5, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) by enzyme-linked immunosorbent assay or cytometric bead array. RESULTS: The plasma levels of BDNF, NT3, NGF, and sTNFr2 were higher, whereas IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, TNFα, CNTF, and sTNFr1 were lower in patients than controls. IL1, GDNF, and NT4/5 were similar between groups. These markers did not correlate with age, sex, and epilepsy duration. The molecule sTNFr2 was the best marker to discriminate patients from controls (area under the curve = .857), also differing between patients with frequent and infrequent seizures. SIGNIFICANCE: This large cohort confirmed that patients with epilepsy have abnormal levels of plasma inflammatory and neurotrophic markers independent of the underlying etiology. Plasma level of sTNFr2 was related to seizure frequency and discriminated people with or without epilepsy with good accuracy, making it a potential biomarker for epilepsy and seizure burden.

Toward a better definition of focal cortical dysplasia: An iterative histopathological and genetic agreement trial.

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme. METHODS: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients. RESULTS: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases. SIGNIFICANCE: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.

New avenues in molecular genetics for the diagnosis and application of therapeutics to the epilepsies.

Genetic epidemiology studies have shown that most epilepsies involve some genetic cause. In addition, twin studies have helped strengthen the hypothesis that in most patients with epilepsy, a complex inheritance is involved. More recently, with the development of high-density single-nucleotide polymorphism (SNP) microarrays and next-generation sequencing (NGS) technologies, the discovery of genes related to the epilepsies has accelerated tremendously. Especially, the use of whole exome sequencing (WES) has had a considerable impact on the identification of rare genetic variants with large effect sizes, including inherited or de novo mutations in severe forms of childhood epilepsies. The identification of pathogenic variants in patients with these childhood epilepsies provides many benefits for patients and families, such as the confirmation of the genetic nature of the diseases. This process will allow for better genetic counseling, more accurate therapy decisions, and a significant positive emotional impact. However, to study the genetic component of the more common forms of epilepsy, the use of high-density SNP arrays in genome-wide association studies (GWAS) seems to be the strategy of choice. As such, researchers can identify loci containing genetic variants associated with the common forms of epilepsy. The knowledge generated over the past two decades about the effects of the mutations that cause the monogenic epilepsy is tremendous; however, the scientific community is just starting to apply this information in order to generate better target treatments.

Placental transcriptome profile of women with sickle cell disease reveals differentially expressed genes involved in migration, trophoblast differentiation and inflammation.

Sickle cell disease (SCD) is a group of disorders whose common characteristic is the presence of hemoglobin (Hb) S in erythrocytes. The main consequence of this abnormality is vaso-occlusion, which can affect almost all organs including the placenta. This study aimed to evaluate the gene expression profile in placentas of women with SCD by means of total RNA sequencing. For this, we proposed a case-control study, with three groups of pregnant women: HbSS (n = 10), HbSC (n = 14) and HbAA (n = 21). The results showed differences in expression in a number of genes such as NOS2 (fold change, FC = 4.52), HLAG (FC = 5.56), ASCL2 (FC = 3.61), CXCL10 (FC = -3.66) and IL1R2 (FC = 3.92) for the HbSC group and S100A8 (FC = -3.82), CPXM2 (FC = 4.57), CXCL10 (FC = -4.59), CXCL11 (FC = -3.72) and CAMP (FC = -4.55) for the HbSS group. Differentially expressed genes are mainly associated with migration, trophoblast differentiation and inflammation. The causes leading to altered gene expression in placentas of sickle cell patients are not fully understood, but the presence of intravascular hemolysis and vaso-occlusion, with cycles of ischemia and reperfusion, may contribute to the emergence of an environment which can be very harmful for placental physiology, altering the nutrient supply and metabolic exchange for fetal growth.

The Machado-Joseph disease-associated form of ataxin-3 impacts dynamics of clathrin-coated pits.

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin-3 is the main cause of neurodegeneration in Machado-Joseph disease. Ataxin-3 contains an N-terminal catalytic domain, called Josephin domain, and a highly aggregation-prone C-terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin-mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin-3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin-3 could impact CME. Here, we report that both the wild-type and the expanded ataxin-3 reduce transferrin internalization and expanded ataxin-3 impacts dynamics of clathrin-coated pits (CCPs) by reducing CCP nucleation and increasing short-lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.

Revisiting the clinical impact of variants in EFHC1 in patients with different phenotypes of genetic generalized epilepsy.

The most common form of genetic generalized epilepsy (GGE) is juvenile myoclonic epilepsy (JME), which accounts for 5 to 10% of all epilepsy cases. The gene EFHC1 has been implicated as a putative cause of JME. However, it remains debatable whether testing for EFHC1 mutations should be included in the diagnostic epilepsy gene panels. To investigate the clinical utility of EFHC1 testing, we studied 125 individuals: 100 with JME and 25 with other GGEs. We amplified and sequenced all EFHC1 coding exons. Then, we predicted the pathogenicity or benign impact of the variants using the analyses proposed by the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP). Mutation screening revealed 11 missense variants in 44 probands with JME (44%) and one of the seven individuals with generalized tonic-clonic seizures on awakening (14%). Six of the 11 variants (54%) were classified as 'benign,' and the remaining variants were considered variants of uncertain significance (VUS). There is currently a limitation to test for genes that predispose an individual to complex, nonmonogenic phenotypes. Thus, we show suggestive evidence that EFHC1 testing lacks a scientific foundation based on the disputed nature of the gene-disease relationship and should be currently limited to research purposes.

Methodological differences can affect sequencing depth with a possible impact on the accuracy of genetic diagnosis.

For a better interpretation of variants, evidence-based databases, such as ClinVar, compile data on the presumed relationships between variants and phenotypes. In this study, we aimed to analyze the pattern of sequencing depth in variants from whole-exome sequencing data in the 1000 Genomes project phase 3, focusing on the variants present in the ClinVar database that were predicted to affect protein-coding regions. We demonstrate that the distribution of the sequencing depth varies across different sequencing centers (pair-wise comparison, p < 0.001). Most importantly, we found that the distribution pattern of sequencing depth is specific to each facility, making it possible to correctly assign 96.9% of the samples to their sequencing center. Thus, indicating the presence of a systematic bias, related to the methods used in the different facilities, which generates significant variations in breadth and depth in whole-exome sequencing data in clinically relevant regions. Our results show that methodological differences, leading to significant heterogeneity in sequencing depth, may potentially influence the accuracy of genetic diagnosis. Furthermore, our findings highlight how it is still challenging to integrate results from different sequencing centers, which may also have an impact on genomic research.

The impact of post-alignment processing procedures on whole-exome sequencing data.

The use of post-alignment procedures has been suggested to prevent the identification of false-positives in massive DNA sequencing data. Insertions and deletions are most likely to be misinterpreted by variant calling algorithms. Using known genetic variants as references for post-processing pipelines can minimize mismatches. They allow reads to be correctly realigned and recalibrated, resulting in more parsimonious variant calling. In this work, we aim to investigate the impact of using different sets of common variants as references to facilitate variant calling from whole-exome sequencing data. We selected reference variants from common insertions and deletions available within the 1K Genomes project data and from databases from the Latin American Database of Genetic Variation (LatinGen). We used the Genome Analysis Toolkit to perform post-processing procedures like local realignment, quality recalibration procedures, and variant calling in whole exome samples. We identified an increased number of variants from the call set for all groups when no post-processing procedure was performed. We found that there was a higher concordance rate between variants called using 1K Genomes and LatinGen. Therefore, we believe that the increased number of rare variants identified in the analysis without realignment or quality recalibration indicated that they were likely false-positives.

Structural signature of SCA3: From presymptomatic to late disease stages.

OBJECTIVE: Machado-Joseph disease (SCA3/MJD) is the most frequent spinocerebellar ataxia worldwide and characterized by brainstem, basal ganglia, and cerebellar damage. However, little is known about the natural history of the disease. This motivated us to determine the extension and progression of central nervous system involvement in SCA3/MJD using multimodal magnetic resonance imaging (MRI)-based analyses in a large cohort of patients (n = 79) and presymptomatic subjects (n = 12). METHODS: All subjects underwent MRI in a 3T device to assess gray and white matter. To evaluate the cerebral and cerebellar cortices, we used measures from FreeSurfer and SUIT. T1-multiatlas assessed deep gray matter. Diffusion tensor imaging multiatlas was used to investigate cerebral white matter (WM) and SpineSeg to assess the cervical spinal cord. RESULTS: There was widespread WM and cerebellar damage, in contrast to the restricted motor cortex involvement when all patients are compared to age- and sex-matched controls. Presymtomatic patients showed WM microstructural abnormalities mainly in the cerebellar and cerebral peduncles and volumetric reduction of midbrain, spinal cord, and substantia nigra. To assess the disease progression, we divided patients into four subgroups defined by time from ataxia onset. There was a clear pattern of evolving structural compromise, starting in infratentorial structures and progressing up to the cerebral cortex. CONCLUSION: Structural damage in SCA3/MJD begins in the spinal cord, cerebellar peduncles, as well as substantia nigra and progresses to cerebral areas in the long term. These structural differences reveal some insights into the pathogenesis of SCA3/MJD and suggest a staging scheme to map the progression of the disease. Ann Neurol 2018;84:401-408.

Dysregulation of NEUROG2 plays a key role in focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. METHODS: We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing for genes in the mTOR pathway were performed to validate and further explore our initial study. RESULTS: hsa-let-7f (p = 0.039), hsa-miR-31 (p = 0.0078), and hsa-miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p < 0.05), was upregulated. We also found that the RND2 gene, a NEUROG2-target, is upregulated (p < 0.001). In vitro experiments showed that hsa-miR-34a downregulates NEUROG2 by binding to its 5'-untranslated region. Moreover, we observed strong nuclear expression of NEUROG2 in balloon cells and dysmorphic neurons and found that 28.5% of our patients presented brain somatic mutations in genes of the mTOR pathway. INTERPRETATION: Our findings suggest a new molecular mechanism, in which NEUROG2 has a pivotal and central role in the pathogenesis of FCD type II. In this way, we found that the downregulation of hsa-miR-34a leads to upregulation of NEUROG2, and consequently to overexpression of the RND2 gene. These findings indicate that a faulty coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and complete dyslamination in FCD type II. Ann Neurol 2018;83:623-635.

ATP Synthase Subunit Beta Immunostaining is Reduced in the Sclerotic Hippocampus of Epilepsy Patients.

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKAα3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected "en bloc" for HS treatment (n = 15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n = 10). NKAα3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss.