A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy

We recently described mutations of the neuronal sodium-channel alpha-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.     

Neuronal sodium-channel alpha1-subunit mutations in generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by the presence of febrile and afebrile seizures. The first gene, GEFS1, was mapped to chromosome 19q and was identified as the sodium-channel beta1-subunit, SCN1B. A second locus on chromosome 2q, GEFS2, was recently identified as the sodium-channel alpha1-subunit, SCN1A. Single-stranded conformation analysis (SSCA) of SCN1A was performed in 53 unrelated index cases to estimate the frequency of mutations in patients with GEFS+. No mutations were found in 17 isolated cases of GEFS+. Three novel SCN1A mutations-D188V, V1353L, and I1656M-were found in 36 familial cases; of the remaining 33 families, 3 had mutations in SCN1B. On the basis of SSCA, the combined frequency of SCN1A and SCN1B mutations in familial cases of GEFS+ was found to be 17%.     

Altered Function of the SCN1A Voltage-gated Sodium Channel Leads to ��-Aminobutyric Acid-ergic (GABAergic) Interneuron Abnormalities

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),⁷ severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a^RH/RH mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a^RH/+ heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a^RH/+ and Scn1a^RH/RH mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.     

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high-performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ~25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ~12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice-site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.     

Genome-wide linkage of febrile seizures and epilepsy to the FEB4 locus at 5q14.3-q23.1 and no MASS1 mutation

Febrile seizures (FS) represent the most common seizure disorder in childhood and contribution of a genetic predisposition has been clearly proven. In some families FS is associated with a wide variety of afebrile seizures. Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome with a spectrum of phenotypes including FS, atypical febrile seizures (FS+) and afebrile generalized and partial seizures. Mutations in the genes SCN1B, SCN1A and GABRG2 were identified in GEFS+ families. GEFS+ is genetically heterogeneous and mutations in these three genes were detected in only a minority of the families. We performed a 10 cM density genome-wide scan in a multigenerational family with febrile seizures and epilepsy and obtained a maximal multipoint LOD score of 3.12 with markers on chromosome 5q14.3-q23.1. Fine mapping and segregation analysis defined a genetic interval of ≈33 cM between D5S2103 and D5S1975. This candidate region overlapped with a previously reported locus for febrile seizures (FEB4) in the Japanese population, in which MASS1 was proposed as disease gene. Mutation analysis of the exons and exon–intron boundaries of MASS1 in our family did not reveal a disease causing mutation. Our linkage data confirm for the first time that a locus on chromosome 5q14-q23 plays a role in idiopathic epilepsies. However, our mutation data is negative and do not support a role for MASS1 suggesting that another gene within or near the FEB4 locus might exist.     

Single-channel properties of human NaV1.1 and mechanism of channel dysfunction in SCN1A-associated epilepsy

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel alpha-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is approximately 0.3 ms in the -30 to -10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.     

Febrile temperatures unmask biophysical defects in Nav1.1 epilepsy mutations supportive of seizure initiation

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Na_v1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Na_v1.1 mutants—R859H (GEFS+) and R865G (DS)—at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Na_v1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.     

Truncation of the GABAA-Receptor γ2 Subunit in a Family with Generalized Epilepsy with Febrile Seizures Plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.     

13例SCN2A 基因突变相关儿童神经系统疾病表型分析

摘要 目的：总结并分析SCN2A基因突变引起的儿童神经系统疾病相关表型谱特点。方法：采用回顾性研究,收集2018年6月至2021年6月在上海交通大学医学院附属上海儿童医学中心神经内科诊治的患儿,并经二代基因测序检测,纳入SCN2A基因突变者,研究并总结患儿神经系统临床表型特点。结果：共纳入13例SCN2A突变患儿,包括新生突变9例和遗传性突变4例。其中11例患儿伴有癫痫发作,发作年龄为1日龄~1岁11月龄,4例在新生儿期起病 （36%）,1~3 月龄起病2例（18%）,4~12月龄起病2例（18%）,1岁后起病3例（27%）;发作类型中强直阵挛发作、痉挛发作、局灶性发作均各有4例（36%）,阵挛发作1例（9%）。另有2例无癫痫发作的患儿,1例表现为全面性发育迟缓,另一例表现为发育迟缓合并孤独症谱系疾病。11例癫痫患儿中,丛集性发作患儿10例。遗传性突变4例患儿中2例智力、运动发育正常;9例新生突变的患儿中8例伴有运动、智力发育落后,1例发育正常。11例癫痫患儿表型中良性家族性新生儿癫痫1例,新生儿惊厥2例,婴儿痉挛症2例,不能分类的早发性癫痫性脑病3例,儿童期起病的癫痫性脑病2例,热厥附加症1例。结论：SCN2A基因突变引起的儿童神经系统疾病以癫痫表现居多、癫痫表型谱广,少数表现为不伴癫痫发作的发育迟缓和孤独症谱系疾病。     

Scn1a dysfunction alters behavior but not the effect of stress on seizure response

Mutations in the voltage‐gated sodium channel gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition, dysfunction in SCN1A is increasingly being linked to neuropsychiatric abnormalities, social deficits and cognitive disabilities. We have previously reported that mice heterozygous for the SCN1A R1648H mutation identified in a GEFS+ family have infrequent spontaneous seizures, increased susceptibility to chemically and hyperthermia‐induced generalized seizures and sleep abnormalities. In this study, we characterized the behavior of heterozygous mice expressing the SCN1A R1648H mutation (Scn1a^RH/+) and the effect of stress on spontaneous and induced seizures. We also examined the effect of the R1648H mutation on the hypothalamic–pituitary–adrenal (HPA) axis response. We confirmed our previous finding that Scn1a^RH/+ mutants are hyperactive, and also identified deficits in social behavior, spatial memory, cued fear conditioning, pre‐pulse inhibition and risk assessment. Furthermore, while exposure to a stressor did increase seizure susceptibility, the effect seen in the Scn1a^RH/+ mutants was similar to that seen in wild‐type littermates. In addition, Scn1a dysfunction does not appear to alter HPA axis function in adult animals. Our results suggest that the behavioral abnormalities associated with Scn1a dysfunction encompass a wider range of phenotypes than previously reported and factors such as stress exposure may alter disease severity in patients with SCN1A mutations.     

Identification of a New Locus for Generalized Epilepsy with Febrile Seizures Plus (GEFS+) on Chromosome 2q24-q33

We report the identification of a new locus for generalized epilepsy with febrile seizures plus (GEFS+). Six family members manifested isolated typical febrile seizures (FS), and five had typical FS associated with generalized epilepsy (FS+, generalized tonic/clonic seizures). Afebrile seizures occurred from childhood until the teenage years. The maximum two-point LOD score was 3.99 for markers D2S294 and D2S2314. Flanking markers place the GEFS+ locus between D2S141 and D2S116, with multipoint analysis favoring the 13-cM interval spanned by D2S294 and D2S364. This locus is the second GEFS+ locus to be reported, which suggests that this syndrome is genetically heterogeneous.     

A Second Locus for Familial Generalized Epilepsy with Febrile Seizures Plus Maps to Chromosome 2q21-q33

We report a clinical and genetic study of a family with a phenotype resembling generalized epilepsy with febrile seizures plus (GEFS+), described by Berkovic and colleagues. Patients express a very variable phenotype combining febrile seizures, generalized seizures often precipitated by fever at age >6 years, and partial seizures, with a variable degree of severity. Linkage analysis has excluded both the beta 1 subunit gene (SCN1B) of a voltage-gated sodium (Na+) channel responsible for GEFS+ and the two loci, FEB1 and FEB2, previously implicated in febrile seizures. A genomewide search, under the assumption of incomplete penetrance at 85% and a phenocopy rate of 5%, permitted identification of a new locus on chromosome 2q21-q33. The maximum pairwise LOD score was 3.00 at recombination fraction 0 for marker D2S2330. Haplotype reconstruction defined a large (22-cM) candidate interval flanked by markers D2S156 and D2S2314. Four genes coding for different isoforms of the alpha-subunit voltage-gated sodium channels (SCN1A, SCN2A1, SCN2A2, and SCN3A) located in this region are strong candidates for the disease gene.     

Evaluation of candidate genes from orphan FEB and GEFS+ loci by analysis of human brain gene expression atlases

Febrile seizures, or febrile convulsions (FEB), represent the most common form of childhood seizures and are believed to be influenced by variations in several susceptibility genes. Most of the associated loci, however, remain 'orphan', i.e. the susceptibility genes they contain still remain to be identified. Further orphan loci have been mapped for a related disorder, genetic (generalized) epilepsy with febrile seizures plus (GEFS+).We show that both spatially mapped and 'traditional' gene expression data from the human brain can be successfully employed to predict the most promising candidate genes for FEB and GEFS+, apply our prediction method to the remaining orphan loci and discuss the validity of the predictions. For several of the orphan FEB/GEFS+ loci we propose excellent, and not always obvious, candidates for mutation screening in order to aid in gaining a better understanding of the genetic origin of the susceptibility to seizures.     

Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy

Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun et al. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABA_A receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.     

De Novo Loss-of-Function Mutations in CHD2 Cause a Fever-Sensitive Myoclonic Epileptic Encephalopathy Sharing Features with Dravet Syndrome

Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures.     

Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy

Severe Myoclonic Epilepsy in Infancy (SMEI) is an intractable epileptic syndrome with onset in the first year of life and is commonly caused by de novo mutations in the SCN1A gene, encoding the α1-subunit of the neuronal voltage-gated sodium channel. We report two unrelated families in which probands were affected by SMEI and their parents showed a single febrile seizure during early childhood or no neurological symptoms. Semiquantitative analysis of SCN1A mutations allowed the detection of a somatic and germline mosaicism in one of the parents. The study provides the first example of parental mosaicisms in SMEI and opens a new insight into the phenotypic variability and complex inheritance of this condition. The identification of germline mosaicisms has important consequences in genetic counseling of SMEI when SCN1A mutations appear to occur de novo with standard screening methods.     

Molecular basis of an inherited epilepsy

Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.     

A new locus for generalized epilepsy with febrile seizures plus maps to chromosome 2

Generalized epilepsy with febrile seizures plus (GEFS+) is a recently recognized but relatively common form of inherited childhood-onset epilepsy with heterogeneous epilepsy phenotypes. We genotyped 41 family members, including 21 affected individuals, to localize the gene causing epilepsy in a large family segregating an autosomal dominant form of GEFS+. A genomewide search examining 197 markers identified linkage of GEFS+ to chromosome 2, on the basis of an initial positive LOD score for marker D2S294 (Z=4.4, recombination fraction [straight theta] = 0). A total of 24 markers were tested on chromosome 2q, to define the smallest candidate region for GEFS+. The highest two-point LOD score (Zmax=5.29; straight theta=0) was obtained with marker D2S324. Critical recombination events mapped the GEFS+ gene to a 29-cM region flanked by markers D2S156 and D2S311, with the idiopathic generalized epilepsy locus thereby assigned to chromosome 2q23-q31. The existence of the heterogeneous epilepsy phenotypes in this kindred suggests that seizure predisposition determined by the GEFS+ gene on chromosome 2q could be modified by other genes and/or by environmental factors, to produce the different seizure types observed.     

Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation

The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.