Investigating the genetic basis of fever-associated syndromic epilepsies using copy number variation analysis.
ABSTRACT: Fever-associated syndromic epilepsies ranging from febrile seizures plus (FS+) to Dravet syndrome have a significant genetic component. However, apart from SCN1A mutations in >80% of patients with Dravet syndrome, the genetic underpinnings of these epilepsies remain largely unknown. Therefore, we performed a genome-wide screening for copy number variations (CNVs) in 36 patients with SCN1A-negative fever-associated syndromic epilepsies. Phenotypes included Dravet syndrome (n = 23; 64%), genetic epilepsy with febrile seizures plus (GEFS+) and febrile seizures plus (FS+) (n = 11; 31%) and unclassified fever-associated epilepsies (n = 2; 6%). Array comparative genomic hybridization (CGH) was performed using Agilent 4 × 180K arrays. We identified 13 rare CNVs in 8 (22%) of 36 individuals. These included known pathogenic CNVs in 4 (11%) of 36 patients: a 1q21.1 duplication in a proband with Dravet syndrome, a 14q23.3 deletion in a proband with FS+, and two deletions at 16p11.2 and 1q44 in two individuals with fever-associated epilepsy with concomitant autism and/or intellectual disability. In addition, a 3q13.11 duplication in a patient with FS+ and two de novo duplications at 7p14.2 and 18q12.2 in a patient with atypical Dravet syndrome were classified as likely pathogenic. Six CNVs were of unknown significance. The identified genomic aberrations overlap with known neurodevelopmental disorders, suggesting that fever-associated epilepsy syndromes may be a recurrent clinical presentation of known microdeletion syndromes.
Project description:It has been established that febrile seizures and its extended syndromes like generalized epilepsy with febrile seizures (FS) plus (GEFS+) and Dravet syndrome have been associated with mutations especially in SCN1A and GABRG2 genes. In patients, the onset of FS is likely due to the combined effect of temperature and inflammation in genetically vulnerable individuals because fever is often associated with infection. Much effort has been spent to understand the mechanisms underlying fever induction of seizures. In addition to the role of cytokines in FS, previous studies in Scn1a+/- knockout mice, a model of Dravet syndrome, indicated that temperature elevation alone could result in seizure generation, and the effect of elevated temperature inducing seizures was age-dependent. Here, we report the thermal effect in a different mouse model of Dravet syndrome, the Gabrg2+/Q390X knockin mouse. We demonstrated age-dependent dysregulated temperature control and that temperature elevation produced myoclonic jerks, generalized tonic clonic seizures (GTCSs) and heightened anxiety-like symptoms in Gabrg2+/Q390X mice. The study indicated that regardless of other inflammatory factors, brief heat alone increased brain excitability and induced multiple types of seizures in Gabrg2+/Q390X mice, suggesting that mutations like GABRG2(Q390X) may alter brain thermal regulation and precipitate seizures during temperature elevations.
Project description:A follow-up study of a large Utah family with significant linkage to chromosome 2q24 led us to identify a new febrile seizure (FS) gene, SCN9A encoding Na(v)1.7. In 21 affected members, we uncovered a potential mutation in a highly conserved amino acid, p.N641Y, in the large cytoplasmic loop between transmembrane domains I and II that was absent from 586 ethnically matched population control chromosomes. To establish a functional role for this mutation in seizure susceptibility, we introduced the orthologous mutation into the murine Scn9a ortholog using targeted homologous recombination. Compared to wild-type mice, homozygous Scn9a(N641Y/N641Y) knockin mice exhibit significantly reduced thresholds to electrically induced clonic and tonic-clonic seizures, and increased corneal kindling acquisition rates. Together, these data strongly support the SCN9A p.N641Y mutation as disease-causing in this family. To confirm the role of SCN9A in FS, we analyzed a collection of 92 unrelated FS patients and identified additional highly conserved Na(v)1.7 missense variants in 5% of the patients. After one of these children with FS later developed Dravet syndrome (severe myoclonic epilepsy of infancy), we sequenced the SCN1A gene, a gene known to be associated with Dravet syndrome, and identified a heterozygous frameshift mutation. Subsequent analysis of 109 Dravet syndrome patients yielded nine Na(v)1.7 missense variants (8% of the patients), all in highly conserved amino acids. Six of these Dravet syndrome patients with SCN9A missense variants also harbored either missense or splice site SCN1A mutations and three had no SCN1A mutations. This study provides evidence for a role of SCN9A in human epilepsies, both as a cause of FS and as a partner with SCN1A mutations.
Project description:Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage-gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage-gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss-of-function mutations in SCN1A result in Dravet syndrome, a severe infant-onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a(+/-) ) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a(+/-) mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a(+/-) mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a(+/-) mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a(+/-) mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA-seq analysis of strain-dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a(+/-) mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.
Project description:Epilepsy is a common neurological disorder that affects 1% of the population. Approximately, 30% of individuals with epilepsy are refractory to treatment, highlighting the need for novel therapies. Conventional anticonvulsant screening relies predominantly on induced seizure models. However, these models may not be etiologically relevant for genetic epilepsies. Mutations in SCN1A are a common cause of Dravet Syndrome, a severe epileptic encephalopathy. Dravet syndrome typically begins in infancy with seizures provoked by fever and then progresses to include afebrile pleomorphic seizure types. Affected children respond poorly to available anticonvulsants. Scn1a+/- heterozygous knockout mice recapitulate features of Dravet syndrome and provide a potential screening platform to investigate novel therapeutics. In this study, we conducted a screening of conventional anticonvulsants in Scn1a+/- mice to establish assays that most closely correlate with human response data.On the basis of clinical response data from a large, single center, retrospective survey of Dravet syndrome case records, we selected nine drugs for screening in Scn1a+/- mice to determine which phenotypic measures correlate best with human therapeutic response. We evaluated several screening paradigms and incorporated pharmacokinetic monitoring to establish drug exposure levels.Scn1a+/- mice exhibited responses to anticonvulsant treatment similar to those observed clinically. Sodium channel blockers were not effective or exacerbated seizures in Scn1a+/- mice. Overall, clobazam was the most effective anticonvulsant in Scn1a+/- mice, consistent with its effect in Dravet syndrome.Genetic models of spontaneous epilepsy provide alternative screening platforms and may augment the AED development process. In this study, we established an effective screening platform that pharmacologically validated Scn1a+/- mice for preclinical screening of potential Dravet syndrome therapeutics.
Project description:UNLABELLED:Classic Dravet syndrome is also termed severe myoclonic epilepsy of infancy (SMEI). There are subtle phenotypic variants of Dravet which may have all the features of the syndrome except one, such as without myoclonic seizures, onset in the second year or without generalized spike and wave on EEG. These have been termed borderline variants of SMEI. Rather than ascribing multiple different names to marginally different phenotypes, the term Dravet syndrome is now preferred to describe the group of severe infantile onset epilepsies (OMIM #607208, #182389, #604403) associated with mutations in SCN1A (OMIM *182389). SCN1A-related seizure disorders can be inherited in an autosomal dominant manner but most are due to de novo mutations. SCN1A testing can be done through bi-directional DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) for: 1) individuals with electroclinical phenotype of Dravet Syndrome or clinical sub-types - several seizure types in one individual with onset in infancy, refractory to medication and with generalised spike and wave on EEG, or 2) infants less than 1 year old with 2 or more prolonged hemiclonic febrile seizures in early infancy. DISCLAIMER:This summary is based on a UK Genetic Testing Network (UKGTN) approved Gene Dossier application.
Project description:Mutations in the neuronal voltage-gated sodium channel genes SCN1A and SCN2A are associated with inherited epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (severe myoclonic epilepsy of infancy). The clinical presentation and severity of these epilepsies vary widely, even in people with the same mutation, suggesting the action of environmental or genetic modifiers. To gain support for the hypothesis that genetic modifiers can influence clinical presentation in patients with SCN1A-derived GEFS+, we used mouse models to study the effect of combining the human GEFS+ mutation SCN1A-R1648H with SCN2A, KCNQ2, and SCN8A mutations. Knock-in mice heterozygous for the R1648H mutation (Scn1a(RH/+)) have decreased thresholds to induced seizures and infrequent spontaneous seizures, whereas homozygotes display spontaneous seizures and premature lethality. Scn2a(Q54) transgenic mice have a mutation in Scn2a that results in spontaneous, adult-onset partial motor seizures, and mice carrying the Kcnq2-V182M mutation exhibit increased susceptibility to induced seizures, and rare spontaneous seizures as adults. Combining the Scn1a-R1648H allele with either Scn2a(Q54) or Kcnq2(V182M/+) results in early-onset, generalized tonic-clonic seizures and juvenile lethality in double heterozygous mice. In contrast, Scn8a mutants exhibit increased resistance to induced seizures. Combining the Scn1a-R1648H and Scn8a-med-jo alleles restores normal thresholds to flurothyl-induced seizures in Scn1a(RH/+) heterozygotes and improved survival of Scn1a(RH/RH) homozygotes. Our results demonstrate that variants in Scn2a, Kcnq2, and Scn8a can dramatically influence the phenotype of mice carrying the Scn1a-R1648H mutation and suggest that ion channel variants may contribute to the clinical variation seen in patients with monogenic epilepsy.
Project description:To explore the prognostic value of initial clinical and mutational findings in infants with SCN1A mutations.Combining sex, age/fever at first seizure, family history of epilepsy, EEG, and mutation type, we analyzed the accuracy of significant associations in predicting Dravet syndrome vs milder outcomes in 182 mutation carriers ascertained after seizure onset. To assess the diagnostic accuracy of all parameters, we calculated sensitivity, specificity, receiver operating characteristic (ROC) curves, diagnostic odds ratios, and positive and negative predictive values and the accuracy of combined information. We also included in the study demographic and mutational data of the healthy relatives of mutation carrier patients.Ninety-seven individuals (48.5%) had Dravet syndrome, 49 (23.8%) had generalized/genetic epilepsy with febrile seizures plus, 30 (14.8%) had febrile seizures, 6 (3.5%) had focal epilepsy, and 18 (8.9%) were healthy relatives. The association study indicated that age at first seizure and frameshift mutations were associated with Dravet syndrome. The risk of Dravet syndrome was 85% in the 0- to 6-month group, 51% in the 6- to 12-month range, and 0% after the 12th month. ROC analysis identified onset within the sixth month as the diagnostic cutoff for progression to Dravet syndrome (sensitivity = 83.3%, specificity = 76.6%).In individuals with SCN1A mutations, age at seizure onset appears to predict outcome better than mutation type. Because outcome is not predetermined by genetic factors only, early recognition and treatment that mitigates prolonged/repeated seizures in the first year of life might also limit the progression to epileptic encephalopathy.
Project description:A substantial number of mutations have been identified in voltage-gated sodium channel genes that result in various forms of human epilepsy. SCN1A mutations result in a spectrum of severity ranging from mild febrile seizures to Dravet syndrome, an infant-onset epileptic encephalopathy. Dravet syndrome patients experience multiple seizures types that are often refractory to treatment, developmental delays, and elevated risk for SUDEP. The same sodium channel mutation can produce epilepsy phenotypes of varying clinical severity. This suggests that other factors, including genetic, modify the primary mutation and change disease severity. Mouse models provide a useful tool in studying the genetic basis of epilepsy. The mouse strain background can alter phenotype severity, supporting a contribution of genetic modifiers in epilepsy. The Scn1a+/- mouse model has a strain-dependent epilepsy phenotype. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have a normal phenotype and lifespan, while [129xC57BL/6J]F1-Scn1a+/- mice experience spontaneous seizures, hyperthermia-induced seizures and high rates of premature death. We hypothesize the phenotypic differences are due to strain-specific genetic modifiers that influence expressivity of the Scn1a+/- phenotype. Low resolution mapping of Scn1a+/- identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival. One locus of interest, Dsm1 located on chromosome 5, was fine mapped to a 9 Mb region using interval specific congenics. RNA-Seq was then utilized to identify candidate modifier genes within this narrowed region. Three genes with significant total gene expression differences between 129S6/SvEvTac and [129xC57BL/6J]F1 were identified, including the GABAA receptor subunit, Gabra2. Further analysis of Gabra2 demonstrated allele-specific expression. Pharmological manipulation by clobazam, a common anticonvulsant with preferential affinity for the GABRA2 receptor, revealed dose-dependent protection against hyperthermia-induced seizures in Scn1a+/- mice. These findings support Gabra2 as a genetic modifier of the Scn1a+/- mouse model of Dravet syndrome.
Project description:OBJECTIVE: To determine the genetic etiology of the severe early infantile onset syndrome of malignant migrating partial seizures of infancy (MPSI). METHODS: Fifteen unrelated children with MPSI were screened for mutations in genes associated with infantile epileptic encephalopathies: SCN1A, CDKL5, STXBP1, PCDH19, and POLG. Microarray studies were performed to identify copy number variations. RESULTS: One patient had a de novo SCN1A missense mutation p.R862G that affects the voltage sensor segment of SCN1A. A second patient had a de novo 11.06 Mb deletion of chromosome 2q24.2q31.1 encompassing more than 40 genes that included SCN1A. Screening of CDKL5 (13/15 patients), STXBP1 (13/15), PCDH19 (9/11 females), and the 3 common European mutations of POLG (11/15) was negative. Pathogenic copy number variations were not detected in 11/12 cases. CONCLUSION: Epilepsies associated with SCN1A mutations range in severity from febrile seizures to severe epileptic encephalopathies including Dravet syndrome and severe infantile multifocal epilepsy. MPSI is now the most severe SCN1A phenotype described to date. While not a common cause of MPSI, SCN1A screening should now be considered in patients with this devastating epileptic encephalopathy.
Project description:Dravet syndrome is an infant-onset epileptic encephalopathy with multiple seizure types that are often refractory to conventional therapies. Treatment with standard benzodiazepines like clobazam, in combination with valproate and stiripentol, provides only modest seizure control. While benzodiazepines are a first-line therapy for Dravet syndrome, they are limited by their ability to only modulate synaptic receptors. Unlike benzodiazepines, neuroactive steroids potentiate a wider-range of GABAA receptors. The synthetic neuroactive steroid SGE-516 is a potent positive allosteric modulator of both synaptic and extrasynaptic GABAA receptors. Prior work demonstrated anticonvulsant activity of SGE-516 in acute seizure assays in rodents. In this study, we evaluated activity of SGE-516 on epilepsy phenotypes in the Scn1a +/- mouse model that recapitulates many features of Dravet syndrome, including spontaneous seizures, premature death and seizures triggered by hyperthermia. To evaluate SGE-516 in Scn1a +/- mice, we determined the effect of treatment on hyperthermia-induced seizures, spontaneous seizure frequency and survival. SGE-516 treatment protected against hyperthermia-induced seizures, reduced spontaneous seizure frequency and prolonged survival in the Scn1a +/- mice. This provides the first evidence of SGE-516 activity in a mouse model of Dravet syndrome, and supports further investigation of neuroactive steroids as potential anticonvulsant compounds for refractory epilepsies.