Barriers to generic antiseizure medication use: Results of a global survey by the International League Against Epilepsy Generic Substitution Task Force.

The objective of this study was to identify and quantify barriers to generic substitution of antiseizure medications (ASM). A questionnaire on generic ASM substitution was developed by the International League Against Epilepsy (ILAE) Task Force on Generic Substitution. Questions addressed understanding of bioequivalence, standards for generic products, experiences with substitution, and demographic data. The survey was web-based and distributed to ILAE chapters, their membership, and professional colleagues of task force members. Comparisons in responses were between ILAE regions and country income classification. A total of 800 individuals responded, with 44.2% being from the Asia-Oceania ILAE Region and 38.6% from European Region. The majority of respondents had little or no education in generic substitution or bioequivalence. Many respondents indicated lack of understanding aspects of generic substitution. Common barriers to generic substitution included limited access, poor or inconsistent quality, too expensive, or lack of regulatory control. Increase in seizures was the most common reported adverse outcome of substitution. Of medications on the World Health Organization Essential Medication list, problems with generic products were most frequent with carbamazepine, lamotrigine, and valproic acid. Several barriers with generic substitution of ASM revolved around mistrust of regulatory control and quality of generic ASM. Lack of education on generic substitution is also a concern. Generic ASM products may be the only option in some parts of the world and efforts should address these issues. Efforts to address these barriers should improve access to medications in all parts of the world.

A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels.

BACKGROUND: Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. METHODS: Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. RESULTS: Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. CONCLUSIONS: We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

The odyssey of complex neurogenetic disorders: From undetermined to positive.

The genetic and phenotypic heterogeneity of neurogenetic diseases forces patients and their families into a "diagnostic odyssey." An increase in the variability of genetic disorders and the corresponding gene-disease associations suggest the need to periodically re-evaluate the significance of variants of undetermined pathogenicity. Here, we report the diagnostic and clinical utility of Targeted Gene Panel Sequencing (TGPS) and Whole Exome Sequencing (WES) in 341 patients with suspected neurogenetic disorders from centers in Buenos Aires and Cincinnati over the last 4 years, focusing on the usefulness of reinterpreting variants previously classified as of uncertain significance. After a mean of ±2years (IC 95:0.73-3.27), approximately 30% of the variants of uncertain significance were reclassified as pathogenic. The use of next generation sequencing methods has facilitated the identification of both germline and mosaic pathogenic variants, expanding the diagnostic yield. These results demonstrate the high clinical impact of periodic reanalysis of undetermined variants in clinical neurology.

Identification of a somatic mutation in the RHEB gene through high depth and ultra-high depth next generation sequencing in a patient with Hemimegalencephaly and drug resistant Epilepsy.

Malformations of cortical development are a frequent cause of drug-resistant Epilepsy and developmental delay. Hemimegalencephaly is a Malformation of cortical development characterized by enlargement of all or a part of one cerebral hemisphere. Germline and somatic mutation in genes belonging to the Mammalian Target of Rapamycin (mTOR) pathway has been identified in patients suffering from epilepsy secondary to Hemimegalencephaly and focal cortical dysplasia. We present here a patient suffering from severe neonatal Epilepsy since 3 h of life secondary to Hemimegalencephaly, requiring an anatomic hemispherectomy surgical procedure for seizure control, where by means of next-generation sequencing at an ultra-high depth coverage, we were able to identify a novel somatic mutation in the RHEB gene (NM_005614: c.119A > T: p. Glu40Val). The histopathological diagnosis was Cortical Dysplasia type IIB determined by the presence of dysmorphic neurons of variable size with nuclear alteration and balloon cells in the context of Hemimegalencephaly, which are similar to that have been demonstrated in hyperactivating RHEB models. This is the first report of a somatic mutation in RHEB gene in a patient suffering from Epilepsy secondary to Hemimegalencephaly. It highlights different current topics in the fields of genetics of Malformations of cortical development: a-somatic mosaicism is not uncommon in these neurodevelopmental disorders; b-the molecular diagnostic approach should involve the use of state-of-the-art methods and the sampling of different tissues; c-new findings might facilitate therapeutics discoveries while providing an improved understanding of normal brain development.

Germline and somatic mutations in cortical malformations: Molecular defects in Argentinean patients with neuronal migration disorders.

Neuronal migration disorders are a clinically and genetically heterogeneous group of malformations of cortical development, frequently responsible for severe disability. Despite the increasing knowledge of the molecular mechanisms underlying this group of diseases, their genetic diagnosis remains unattainable in a high proportion of cases. Here, we present the results of 38 patients with lissencephaly, periventricular heterotopia and subcortical band heterotopia from Argentina. We performed Sanger and Next Generation Sequencing (NGS) of DCX, FLNA and ARX and searched for copy number variations by MLPA in PAFAH1B1, DCX, POMT1, and POMGNT1. Additionally, somatic mosaicism at 5% or higher was investigated by means of targeted high coverage NGS of DCX, ARX, and PAFAH1B1. Our approach had a diagnostic yield of 36%. Pathogenic or likely pathogenic variants were identified in 14 patients, including 10 germline (five novel) and 4 somatic mutations in FLNA, DCX, ARX and PAFAH1B1 genes. This study represents the largest series of patients comprehensively characterized in our population. Our findings reinforce the importance of somatic mutations in the pathophysiology and diagnosis of neuronal migration disorders and contribute to expand their phenotype-genotype correlations.

De novo mutations in epileptic encephalopathies.

Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox-Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 × 10(-3)). Among these are GABRB3, with de novo mutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10(-10) and P = 7.8 × 10(-12), respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P < 10(-8)), as has been reported previously for autism spectrum disorders.

The epilepsy phenome/genome project.

BACKGROUND: Epilepsy is a common neurological disorder that affects approximately 50 million people worldwide. Both risk of epilepsy and response to treatment partly depend on genetic factors, and gene identification is a promising approach to target new prediction, treatment, and prevention strategies. However, despite significant progress in the identification of genes causing epilepsy in families with a Mendelian inheritance pattern, there is relatively little known about the genetic factors responsible for common forms of epilepsy and so-called epileptic encephalopathies. Study design The Epilepsy Phenome/Genome Project (EPGP) is a multi-institutional, retrospective phenotype-genotype study designed to gather and analyze detailed phenotypic information and DNA samples on 5250 participants, including probands with specific forms of epilepsy and, in a subset, parents of probands who do not have epilepsy. RESULTS: EPGP is being executed in four phases: study initiation, pilot, study expansion/establishment, and close-out. This article discusses a number of key challenges and solutions encountered during the first three phases of the project, including those related to (1) study initiation and management, (2) recruitment and phenotyping, and (3) data validation. The study has now enrolled 4223 participants. CONCLUSIONS: EPGP has demonstrated the value of organizing a large network into cores with specific roles, managed by a strong Administrative Core that utilizes frequent communication and a collaborative model with tools such as study timelines and performance-payment models. The study also highlights the critical importance of an effective informatics system, highly structured recruitment methods, and expert data review.

SLC6A4 gene variants and temporal lobe epilepsy susceptibility: a meta-analysis.

There seems to be a role for serotoninergic neuro-transmission in the pathophysiology of the epilepsies. Different groups have studied the role of regulatory variants in the SLC6A4 gene, which code for the central serotonin transporter, in the complex genetics of temporal lobe epilepsy (TLE) obtaining contradictory findings. Therefore, a systematic review and critical analysis of this topic seem to be timely. Published studies up to October 2011 of TLE and the SLC6A4 promoter and intron 2 variant number repeat polymorphisms (VNTR) were identified by searches of Medline, Scopus and ISI-Web of Sciences databases. Meta-analysis of TLE case-control data were performed to assess the association of SLC6A4 VNTRs with TLE susceptibility. Pooled odds ratios were estimated by means of a genetic-model-free approach. The quality of the included studies was assessed by a score. The studies included compared a total of 991 TLE cases and 1,202 controls. We did not find synthetic evidence of association between SLC6A4 promoter and intron 2 variants and the risk of TLE. However, the intron 2 VNTR seems to have opposite effects in different populations. In this meta-analysis our findings were inconclusive in order to associate any of the 5-HT receptor gene variants with the risk of TLE.

Postoperative neuropsychological outcome in patients with mesial temporal lobe epilepsy in Argentina.

The aim of the present study is to compare pre- and postsurgical neuropsychological outcome in individuals suffering from mesial temporal lobe epilepsy (mTLE), in order to evaluate prognosis. The selected thirty-five patients had medically mTLE and had undergone an anterior temporal lobectomy (ATL). Neuropsychological evaluation was performed in three different stages: before ATL, 6 months after resection, and a year afterwards. Neuropsychological protocol evaluated attention, verbal memory, visual memory, executive function, language, intelligence, and handedness. There was a significant improvement (P = 0.030) in the group with visual memory deficit after surgery, whereas no changes were observed across patients with verbal memory deficit. No changes were observed in language after surgery. Executive function showed significant improvement 6 months after surgery (P = 0.035). Postoperative outcome of cognitive impairments depends on baseline neuropsychological status of the patients with TLE. In our case series, deficits found in patients with mTLE after ATL did not result in a subjective complaint.

Noninvasive approach to focal cortical dysplasias: clinical, EEG, and neuroimaging features.

Purpose. The main purpose is to define more accurately the epileptogenic zone (EZ) with noninvasive methods in those patients with MRI diagnosis of focal cortical dysplasia (FCD) and epilepsy who are candidates of epilepsy surgery. Methods. Twenty patients were evaluated prospectively between 2007 and 2010 with comprehensive clinical evaluation, video-electroencephalography, diffusion tensor imaging (DTI), and high-resolution EEG to localize the equivalent current dipole (ECD). Key Findings. In 11 cases with white matter asymmetries in DTI the ECDs were located next to lesion on MRI with mean distance of 14.63 millimeters with topographical correlation with the EZ. Significance. We could establish a hypothesis of EZ based on Video-EEG, high-resolution EEG, ECD method, MRI, and DTI. These results are consistent with the hypothesis that the EZ in the FCD is complex and is often larger than visible lesion in MRI.

Contribución de las imágenes de resonancia magnética por tensor de difusión al diagnóstico de displasias corticales focales / Contribution of diffusion tensor magnetic resonance imaging to the diagnosis of focal cortical dysplasias

Introducción. Las displasias corticales focales (DCF) son malformaciones corticales y, aunque evidencian característicastípicas en la resonancia magnética (RM) convencional, la determinación precisa de la zona epileptógena es controvertida. La evolución postoperatoria menos favorable con respecto a otras epilepsias sintomáticas se explicaría mediante la existencia de áreas epileptógenas no visibles en la RM convencional. Las imágenes por tensor de difusión (DTI) son sensibles a anomalías microestructurales sutiles, y la anisotropía fraccional, un indicador indirecto, demuestra áreas de reducción en la sustancia blanca subyacente a las DCF, excediendo las alteraciones detectadas con la RM convencional en casosaislados de trabajos previos.Objetivo. Analizar las características de la anisotropía fraccional en una serie de pacientes con DCF, para evaluar la contribución diagnóstica de la RM por DTI. Sujetos y métodos. Se escanearon 21 controles y 11 pacientes con DCF visible en la RM, con registro de variables clínicas eimaginológicas. Se realizó un análisis visual de los mapas de anisotropía fraccional en busca de asimetrías entre hemisferios,sesgado a los datos clínicos o de RM estructural. Resultados. Dos mujeres y nueve varones, de 30,0 ± 9,7 años de edad; tiempo de evolución de la epilepsia: 22,0 ± 9,3años; frecuencia promedio de las crisis: 3/mes (rango: 0,16-8/mes). Todos evidenciaron asimetrías interhemisféricas, en 10 pacientes (90%) excediendo los límites estructurales de la DCF. Ninguno de los controles demostró asimetrías en la anisotropía fraccional. No se encontró relación significativa con las variables confrontadas.Conclusiones. Se necesita estudiar un mayor número de pacientes para valorar la utilidad de las DTI para definir la localizacióny extensión de la zona epileptógena en esta población (AU)

Introduction. Focal cortical dysplasias (FCD) are cortical malformations and, although they display typical characteristicsin conventional magnetic resonance imaging (MRI), the precise determination of the epileptogenic zone remains a controversial issue. The less favourable progress during the post-operative period with respect to other symptomaticepilepsies could be explained by the existence of epileptogenic areas that do not show up in conventional MRI. Diffusion tensor imaging (DTI) is sensitive to subtle icrostructural abnormalities, and fractional anisotropy, which is an indirectindicator, shows areas with reductions in the underlying white matter that go beyond the alterations detected withconventional MRI in isolated cases in previous works. Aim. In this study we analyse the characteristics of fractional anisotropy in a series of patients with FCD in order to evaluatethe contribution made to diagnosis by MRI by DTI. Subjects and methods. Twenty-one controls and eleven patients with FCD that was visible in MRI were scanned, andclinical and imaging variables were both recorded. A visual analysis of the fractional anisotropy maps was conducted to search for asymmetries between hemispheres and biases in the clinical or structural MRI data. Results. Two females and nine males, aged 30 ± 9.7 years took part in the study; time to progression of epilepsy: 22 ± 9.3 years; average frequency of the seizures: 3/month (range: 0.16-8/month). All of them showed inter-hemispheric asymmetries, which went beyond the structural limits of FCD in the case of 10 of the patients (90%). None of the controlsdisplayed asymmetries in the fractional anisotropy. No significant relation was found with the variables that were compared.Conclusions. Further studies need to be conducted with larger numbers of patients in order to evaluate the usefulness of DTI in defining the location and extension of the epileptogenic zone in this population (AU)

Diagnosis of mitochondrial disorders applying massive pyrosequencing.

Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.

Association between equivalent current dipole source localization and focal cortical dysplasia in epilepsy patients.

We analysed the association between focal cortical dysplasia (FCD) visible in MRI and the location of equivalent current dipole (ECD) of single interictal scalp EEG spikes (IIS) in 11 epilepsy patients. We calculated several indicators of distance of ECDs to the FCD border. The results confirm some previous studies suggesting that the epileptogenic zone associated to the location of ECDs extends beyond the FCD visible in MRI. The analysis suggests the ECDs to be in a shell parallel to part of the FCD surface.

Cerebrotendinous xanthomatosis revealed in drug-resistant epilepsy diagnostic workup.

Cerebrotendinous xanthomatosis (CTX) is a treatable disorder of bile acid production caused by mutations in the mitochondrial enzyme sterol 27-hydroxilase. This inborn error of bile acid metabolism results in lipid pathologic accumulation in multiple tissues. Progressive neuropsychiatric disturbances are a frequent manifestation of this disease. Although seizures have been frequently noticed as part of CTX manifestations, there have not been reports of CTX being diagnosed in drug-resistant epilepsy diagnostic workup nor of seizure response to chenodeoxycholic acid treatment. Here, the authors present a case of a drug-resistant epilepsy patient with a complex phenotype where a diagnosis of CTX was done and showed a significant reduction in seizure frequency after chenodeoxycholic acid supplementation. This report illustrates the importance of considering treatable neurometabolic disorders in epileptic patients showing complex phenotypes.

Doublecortin (DCX) immunoreactivity in hippocampus of chronic refractory temporal lobe epilepsy patients with hippocampal sclerosis.

INTRODUCTION: Status epilepticus increases the production of new neurons (hippocampal neurogenesis) and promotes aberrant migration. However chronic experimental models of epilepsy and studies performed in human epilepsy showed controversial results suggesting a reduction in hippocampal neurogenesis in late stages of the disease. Doublecortin (DCX) has been validated to determine alterations in the production of new neurons in the human hippocampus. OBJECTIVES: Determine DCX expression in human hippocampal sclerosis (HS) from patients who underwent epilepsy surgery for refractory temporal lobe epilepsy (TLE). METHODS: Hippocampal sections of 9 patients with HS and TLE who underwent surgery, were processed using immunoperoxidase for DCX. Archival material from 5 normal post-mortem hippocampus were simultaneously processed. RESULTS: Significantly lower staining intensity was observed in DCX-positive neurons localized in dentate gyrus (DG) and in CA1 of epileptic hippocampus; lower DCX reactive area was observed in pyramidal layers of CA1; and a reduced in the mean number of DCX-positive neurons were determined in DG compared to normal hippocampus (p<0.05). CONCLUSIONS: This study found a decrease in DCX expression in hippocampus of patients with HS and chronic and refractory TLE suggesting alterations in NG and hippocampal synaptogenesis with potential cognitive and emotional repercussion.

ApoE epsilon4 genotype and the age at onset of temporal lobe epilepsy: a case-control study and meta-analysis.

In order to investigate the role of ApoE epsilon4 as a modifier of the age at onset of temporal lobe epilepsy (TLE), we performed a molecular epidemiology study in 78 patients with mesial temporal lobe epilepsy and hippocampal sclerosis. Genotyping was done by a PCR-RFLP assay. In order to better estimate the role of this variant as a modifier of the age at onset, we also performed a systematic review of the literature. We included our results into a meta-analysis along with data available from seven published studies with 728 patients that looked into the role of ApoE epsilon4 in TLE. We found that ApoE epsilon4 carriers in our population had a non-significant earlier age of epilepsy onset than non-carriers. The meta-analysis confirmed this finding, showing that ApoE epsilon4 carriers had epilepsy onset almost 4 years earlier than non-carriers (mean difference 5.15 years; CI 95% 2.08-6.22; p=0.001). In conclusion, the ApoE epsilon4 isoform is a genetic factor that might influence the age at onset of TLE.

Psychogenic nonepileptic seizures after head injury: a case report.

Psychogenic nonepileptic seizures (PNESs) are diagnosed when disruptive changes in behaviour, thinking, or emotion resemble epileptic seizures (ESs), but no paroxysmal discharges are seen on electroencephalogram (EEG) and do not originate from another medical illness. The gold standard for PNES diagnosis is video electroencephalogram (Video-EEG). PNESs are defined by modern psychiatry as conversion and dissociative disorders but these disorders may coexist with many others psychiatric disorders, including depression, posttraumatic stress disorder, and personality disorders. It is well known that epileptic seizures are a frequent and well-studied complication of traumatic head injury (THI). However, THI may also generate psychic symptoms including PNES. In this paper we describe a patient who developed PNES after THI in a bus accident and received a diagnosis of refractory epilepsy for 24 years until she underwent Video-EEG.

ApoE epsilon4 is not associated with posictal confusion in patients with mesial temporal lobe epilepsy with hippocampal sclerosis.

A previous report found an association between ApoE isoforms and postictal confusion in medically intractable temporal lobe epilepsy (TLE). We performed a molecular epidemiology study in an independent sample of 77 TLE patients. We failed to replicate the original allelic association between ApoE epsilon4 allele and postictal confusion in our population (chi(2)=1.67; d.f.=1; p=0.2). Thus, the association between ApoE epsilon4 allele and postictal confusion still needs to be fully investigated in different and independent populations.

Serotonin transporter gene variation and refractory mesial temporal epilepsy with hippocampal sclerosis.

We performed a molecular epidemiology study in a population of 105 mesial temporal lobe epilepsy with hippocampal sclerosis (MTE-HS) patients in order to investigate the role of a polymorphism in the serotonin transporter gene (SLC6A4) in the prediction of antiepileptic drug (AED) treatment response. Homozygous carriers of the 12-repeat allele had an almost fourfold increase in risk for a MTE-HS not responding to medical treatment (OR 3.88; CI 95% 1.40-10.7; p=0.006) compared to carriers of the 10-repeat allele. Therefore, a polymorphism of SLC6A4 might be a genetic marker of pharmacoresistance in MTE-HS patients.

Psychotic disorders in Argentine patients with refractory temporal lobe epilepsy: a case-control study.

The issue of psychotic disorders in epilepsy has given rise to great controversy among professionals; however, there are not many studies in this area and the physiopathological mechanisms remain unknown. The aim of this study was to describe the spectrum of psychotic disorders in an Argentine population with refractory temporal lobe epilepsy (RTLE) and to determine the risk factors associated with psychotic disorders. Clinical variables of the epileptic syndrome were compared among a selected population with RTLE with and without psychotic disorders (DSM-IV/Ictal Classification of psychoses). Logistic regression was performed. Sixty-three patients with psychotic disorders (Psychotic Group, PG) and 60 controls (Control Group, CG) were included. The most frequent psychotic disorders were brief psychotic episodes (35%) (DSM-IV) and interictal psychosis (50%) (Ictal Classification). Risk factors for psychotic disorders were bilateral hippocampal sclerosis, history of status epilepticus, and duration of epilepsy greater than 20 years.