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.

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.

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.

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.

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.

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.

Association study between interleukin 1 beta gene and epileptic disorders: a HuGe review and meta-analysis.

Previous studies have examined the association of a single nucleotide polymorphism at the promoter region of interleukin 1B (IL-1 beta-511T) with temporal lobe epilepsy and febrile seizures susceptibility, but those studies have been inconclusive. Published studies up to March 2007 of temporal lobe epilepsy, febrile seizures and the IL-1 beta-511T single nucleotide polymorphism were identified by searches of Medline and Embase databases. Meta-analysis of temporal lobe epilepsy and febrile seizures case-control data were performed to assess the association of IL-1 beta-511T with temporal lobe epilepsy, temporal lobe epilepsy with hippocampal sclerosis, febrile seizures, and other epileptic disorders. Pooled odds ratios (OR) were estimated by means of a genetic-model-free approach. The quality of the included studies was assessed by a score. The results show a modest association (OR, 1.48; 95% confidence interval, 1.09-2.00; P = 0.01) between the IL-1 beta-511T polymorphism and temporal lobe epilepsy with hippocampal sclerosis.

Transcriptionally less active prodynorphin promoter alleles are associated with temporal lobe epilepsy: a case-control study and meta-analysis.

We performed an association study in a population of patients with Mesial Temporal Lobe Epilepsy (TLE) with Hippocampal Sclerosis (MTEHS) together with a systematic revision of the literature to investigate the role of transcriptionally less active polymorphic alleles of Prodynorphin (PDYN) gene in this pathology. We included 102 patients with a diagnosis of MTEHS and 86 healthy controls. The positive antecedent of family history for epileptic events defined a TLE subgroup with familial predisposition for epileptic disorders. The PDYN promoter polymorphism was genotyped by means of a PCR assay. For meta-analysis, we identified case-control association studies between TLE and PDYN by searching PUBMED. The pooled OR was estimated using a fixed effects model under dominant and co-dominant heredity models. No differences in genotypic and allelic frequencies were found between cases and controls (p=0.61) in our population, neither in the whole cohort nor in the analysis limited to TLE with familial predisposition (p=0.71). The Meta-Analysis included 591 TLE patients and 1117 healthy controls. We found an association between L allele (p=0.003; OR=1.40; IC 95=1.12-1.74) and a modestly higher risk to develop TLE in the group of patients with familial predisposition. Therefore, functional allelic variants in the PDYN promoter might modify the risk to develop TLE in subjects with familial predisposition.

GABABR1 (G1465A) gene variation and temporal lobe epilepsy controversy: new evidence.

The G1465A polymorphism in the gene of the GABA type B receptor subunit 1 (GABABR1) has been linked to the risk for temporal lobe epilepsy (TLE). However, six replication studies did not show significant association between the G1465A GABABR1 gene variant and TLE. The authors examined this association in a sample of 102 patients with mesial TLE with hippocampal sclerosis (MTLE-HS) and 71 controls. The genotype distribution varied significantly between patients and controls. Heterozygous carriers of the A-allele had a 10-fold increase in risk for MTLE-HS (OR 10.01; 95% CI 3.98-25.18, p=3.788E-08).

NADPH diaphorase reactive neurons in temporal lobe cortex of patients with intractable epilepsy and hippocampal sclerosis.

Several studies have demonstrated a controversial involvement of NO in epileptogenesis. The aim of this study is to compare the NADPH diaphorase (NADPH-d) reactivity in the temporal cortex between surgical specimens of patients with intractable epilepsy and hippocampal sclerosis and autopsy controls. Brain samples of patients and postmortem controls were stained with the NADPH-d technique. Sprouting and larger areas of NADPH-d reactive neurons were found in the temporal cortex of epileptic patients.

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.

Psychiatric disorders in patients with psychogenic non-epileptic seizures, with and without comorbid epilepsy.

PURPOSE: The aim of this study is to describe similarities and differences in epidemiological, psychiatric and semiologic variables between patients with psychogenic none epileptic seizures (PNES) and comorbid epilepsy (mixed PNES), and patients with PNES without comorbid epilepsy (pure PNES). RESULTS: Forty-three patients with PNES diagnosed by Video-EEG were included. Twenty-four had pure PNES, and ninteen mixed PNES. Female population, age, duration of PNES, psychiatric institutionalization, psychopharmacotherapy, dissociative disorders and posttraumatic stress disorder (PTSD), were significantly higher in the pure PNES patients. Suicide attempts, antiepileptic therapy, conversive, affective and personality disorders were frequent in both groups. In the analysis of seizure semiology, the total lack of responsiveness was significantly higher in the mixed PNES group. CONCLUSIONS: Pure PNES patients showed similarities and differences in the psychiatric profile, with a greater prevalence of dissociative disorders and PTSD, factors related to psychiatric severity.

Periventricular nodular and subcortical neuronal heterotopia in adult epileptic patients.

Developmental malformations are brain abnormalities that occur during embryogenesis. Neuronal migration disorders, including heterotopic lesions, constitute one type of such abnormalities. The aim of the study was to compare the epileptic clinical patterns of patients with periventricular nodular heterotopia (PNH) (G1) with those affected by subcortical heterotopia (SCH) (G2) looking for differences between both groups which, eventually, might suggest the type of the underlying malformation. The variables studied in both groups were: type of the heterotopia depicted on MRI studies, sex, age, age at seizure onset, annual seizure frequency, localization of the ictal symptomatogenic zone, characteristics of the EEG, other associated anomalies on the magnetic resonance images (MRI) besides the heterotopia, and response to treatment. The only difference found between both groups was the type of heterotopia as shown by MRI studies. The other assessed variables did not significantly (p>0.05) differ between groups. No differences in the clinical features characterizing epilepsy could be found in patients with PNH or SCH, being the images the only tool able to differentiate them.

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.

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.

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.