Novel Carboxypeptidase A6 (CPA6) Mutations Identified in Patients with Juvenile Myoclonic and Generalized Epilepsy

Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy.     

Heterozygous Reelin Mutations Cause Autosomal-Dominant Lateral Temporal Epilepsy

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.     

Haploinsufficiency of glutamine synthetase increases susceptibility to experimental febrile seizures

Glutamine synthetase (GS) is a pivotal glial enzyme in the glutamate–glutamine cycle. GS is important in maintaining low extracellular glutamate concentrations and is downregulated in the hippocampus of temporal lobe epilepsy patients with mesial–temporal sclerosis, an epilepsy syndrome that is frequently associated with early life febrile seizures (FS). Human congenital loss of GS activity has been shown to result in brain malformations, seizures and death within days after birth. Recently, we showed that GS knockout mice die during embryonic development and that haploinsufficient GS mice have no obvious abnormalities or behavioral seizures. In the present study, we investigated whether reduced expression/activity of GS in haploinsufficient GS mice increased the susceptibility to experimentally induced FS. FS were elicited by warm-air-induced hyperthermia in 14-day-old mice and resulted in seizures in most animals. FS susceptibility was measured as latencies to four behavioral FS characteristics. Our phenotypic data show that haploinsufficient mice are more susceptible to experimentally induced FS ( P  < 0.005) than littermate controls. Haploinsufficient animals did not differ from controls in hippocampal amino acid content, structure (Nissl and calbindin), glial properties ( glial fibrillary acidic protein and vimentin) or expression of other components of the glutamate–glutamine cycle (excitatory amino acid transporter-2 and vesicular glutamate transporter-1). Thus, we identified GS as a FS susceptibility gene. GS activity-disrupting mutations have been described in the human population, but heterozygote mutations were not clearly associated with seizures or epilepsy. Our results indicate that individuals with reduced GS activity may have reduced FS seizure thresholds. Genetic association studies will be required to test this hypothesis.     

Combined effect between two functional polymorphisms of SLC6A12 gene is associated with temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common epilepsy subtype with complex genetic structure. A recent study in four populations (Ireland, UK, Australia and Finland) reported an allelic association between betaine/GABA transporter-1 (BGT-1 or SLC6A12) and mesial temporal lobe epilepsy with hippocampal sclerosis. To demonstrate the association between SLC6A12 gene polymorphisms and TLE, TaqMan method was used to genotype five single-nucleotide polymorphisms of SLC6A12 gene in 358 TLE patients and 596 nonepileptic control subjects of Chinese Han origin. Real-time PCR was used to detect the effects of variations on gene expression associated with TLE. Though, the single-marker analysis did not demonstrate allelic association with TLE, rs542736–rs557881 interaction showed significant association. The SLC6A12 expression levels in peripheral blood mononuclear cells were significantly higher in TLE patients than in control subjects and were correlated to rs542736 G–rs557881 A haplotypes. Our preliminary results suggested combined effect of two common polymorphisms on SLC6A12 gene may be associated with TLE, but the precise mechanism needs further investigation.     

A Point Mutation in <Emphasis Type="Italic">SCN1A</Emphasis> 5′ Genomic Region Decreases the Promoter Activity and Is Associated with Mild Epilepsy and Seizure Aggravation Induced by Antiepileptic Drug

The SCN1A gene with 1274 point mutations in the coding regions or genomic rearrangements is the most clinically relevant epilepsy gene. Recent studies have demonstrated that variations in the noncoding regions are potentially associated with epilepsies, but no distinct mutation has been reported. We sequenced the 5′ upstream region of SCN1A in 166 patients with epilepsy and febrile seizures who were negative for point mutations in the coding regions or genomic rearrangements. A heterozygous mutation h1u-1962 T?>?G was identified in a patient with partial epilepsy and febrile seizures, which was aggravated by oxcarbazepine. This mutation was transmitted from the patient’s asymptomatic mother and not found in the 110 normal controls. h1u-1962 T?>?G was located upstream the most frequently used noncoding exon and within the promoter sequences. Further experiments showed that this mutation decreased the promoter activity by 42.1 % compared with that of the paired haplotype (P?<?0.001). In contrast to the null expression that results in haploinsufficiency and severe phenotype, this mutation caused relatively less impairment, explaining the mild epilepsy with incomplete penetrance. The antiepileptic drug-induced seizure aggravation in this patient suggests clinical attention for mutations or variations in noncoding regions that may affect SCN1A expression.     

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.     

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.     

Developing a new animal model of temporal lobe epilepsy

Epilepsy affects 1-2 % of the population. For 30 % of these patients, their syndrome will be refractory to medical treatment. To improve our understanding and treatment of the epilepsies, we need to develop clinically relevant animal models. As temporal lobe epilepsy is often preceded by prolonged febrile seizures and in our population associated with a focal cortical dysplasia, we hypothesised that an underlying predisposing anatomical lesion would predispose individuals to develop prolonged febrile seizures and that temporal lobe epilepsy would later develop. As predicted, all the lesioned animals developed prolonged febrile seizures, while all other control groups only showed simple febrile seizures. After a latent period, 86 % of the animals who had experienced a prolonged seizure developed spontaneously recurrent limbic seizures. We now need to understand the anatomical and electrophysiological changes underlying this new epilepsy model to try and develop more effective treatments for the condition.     

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.     

Neocortex- and hippocampus-specific deletion of Gabrg2 causes temperature-dependent seizures in mice

Mutations in the GABRG2 gene encoding the γ-aminobutyric acid (GABA) A receptor gamma 2 subunit are associated with genetic epilepsy with febrile seizures plus, febrile seizures plus, febrile seizures, and other symptoms of epilepsy. However, the mechanisms underlying Gabrg2-mediated febrile seizures are poorly understood. Here, we used the Cre/loxP system to generate conditional knockout (CKO) mice with deficient Gabrg2 in the hippocampus and neocortex. Heterozygous CKO mice (Gabrg2^fl/wtCre⁺) exhibited temperature-dependent myoclonic jerks, generalised tonic-clonic seizures, increased anxiety-like symptoms, and a predisposition to induce seizures. Cortical electroencephalography showed the hyperexcitability in response to temperature elevation in Gabrg2^fl/wtCre⁺ mice, but not in wild-type mice. Gabrg2^fl/wtCre⁺ mice exhibited spontaneous seizures and susceptibility to temperature-induced seizures. Loss of neurons were observed in cortical layers V–VI and hippocampus of Gabrg2^fl/wtCre⁺ mice. Furthermore, the latency of temperature- or pentylenetetrazol-induced seizures were significantly decreased in Gabrg2^fl/wtCre⁺ mice compared with wild-type mice. In summary, Gabrg2^fl/wtCre⁺ mice with Gabrg2 deletion in the neocortex and hippocampus reproduce many features of febrile seizures and therefore provide a novel model to further understand this syndrome at the cellular and molecular level.Subject terms: Epilepsy, Genetics of the nervous system     

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system^1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds⁴. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish^5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.     

Absence of a general association between ABCB1 genetic variants and response to antiepileptic drugs in epilepsy patients

Over-expression of efflux transporter P-glycoprotein (PgP) encoded by ABCB1 gene has been implicated in poor responsive epilepsy. Several genetic variants have been shown to influence the expression levels of P-glycoprotein. The aim of the present study was to investigate the role of ABCB1 polymorphisms: C1236T, G2677T/A and C3435T in determining drug response to first line antiepileptic drugs (AEDs) namely phenobarbitone, phenytoin, carbamazepine and valproate in North Indian cohort of epilepsy patients. DNA samples were obtained from 392 consecutive epilepsy patients, out of which 228 had completed follow-up evaluation at 12 months. After attaining steady state of the AEDs in the first two months of study, 133 patients showed complete freedom from seizures (no-seizure group) and 95 patients continued to have seizures (recurrent-seizures group) in the remaining period of study. Comparison of “no-seizure” and “recurrent-seizures” groups revealed no significant differences in allelic, genotypic and haplotypic frequencies for all the studied variants. In conclusion, our finding disproves a general association between ABCB1 polymorphisms and drug response in epilepsy patients.     

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。     

Deletion of <Emphasis Type="Italic">mTOR</Emphasis> in Reactive Astrocytes Suppresses Chronic Seizures in a Mouse Model of Temporal Lobe Epilepsy

Germline and somatic mutations in key genes of the mammalian target of rapamycin (mTOR) pathway have been identified in seizure-associated disorders. mTOR mutations lead to aberrant activation of mTOR signaling, and, although affected neurons are critical for epileptogenesis, the role of mTOR activation in glial cells remains poorly understood. We previously reported a consistent activation of the mTOR pathway in astrocytes in the epileptic foci of temporal lobe epilepsy. In this study, it was demonstrated that mTOR deletion from reactive astrocytes prevents increases in seizure frequency over the disease course. By using a tamoxifen-inducible mTOR conditional knockout system and kainic acid, a model was developed that allowed astrocyte-specific mTOR gene deletion in mice with chronic epilepsy. Animals in which mTOR was deleted from 44 % of the astrocyte population exhibited a lower seizure frequency compared with controls. Down-regulation of mTOR significantly ameliorated astrogliosis in the sclerotic hippocampus but did not rescue mossy fiber sprouting. In cultured astrocytes, the mTOR pathway modulated the stability of the astroglial glutamate transporter 1 (Glt1) and influenced the ability of astrocytes to remove extracellular glutamate. Taken together, these data indicate that astrocytes with activated mTOR signaling may provide conditions that are favorable for spontaneous recurrent seizures.     

复杂性热性惊厥脑电图特征与癫痫发生的相关性

目的：探讨复杂性热性惊厥脑电图特征与癫痫发生的相关性。方法：2012年3 月到2014 年5 月选择在我院诊治为复杂性热 性惊厥的呼吸道感染患儿86 例作为观察组,同期选择在我院诊治的非热性惊厥的呼吸道感染患儿86 例作为对照组,两组都进 行脑电图监测与认知功能判定,对癫痫发生情况进行判定与分析。结果：观察组的言语智商、行为智商与总智商评分都明显低于 对照组(P<0.05)。观察组的癫痫发生率为9.3 %,脑电图异常率为8.1 %;而对照组的癫痫发生率为1.2 %,脑电图异常率为2.3 %, 对比差异都有统计学意义(P<0.05)。在观察组患儿中,Spearman 相关性分析显示脑电图异常与癫痫发生存在明显正向相关性(r=0. 349,P<0.05)。结论：复杂性热性惊厥伴随有脑电图异常,与癫痫发生存在明显正向相关性,损害患儿的认知功能。     

Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures

GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ∼4% of unsolved MAE cases.     

De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy

Severe myoclonic epilepsy of infancy (SMEI) is a rare disorder that occurs in isolated patients. The disease is characterized by generalized tonic, clonic, and tonic-clonic seizures that are initially induced by fever and begin during the first year of life. Later, patients also manifest other seizure types, including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life. Missense mutations in the gene that codes for a neuronal voltage-gated sodium-channel alpha-subunit (SCN1A) were identified in families with generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ is a mild type of epilepsy associated with febrile and afebrile seizures. Because both GEFS+ and SMEI involve fever-associated seizures, we screened seven unrelated patients with SMEI for mutations in SCN1A. We identified a mutation in each patient: four had frameshift mutations, one had a nonsense mutation, one had a splice-donor mutation, and one had a missense mutation. All mutations are de novo mutations and were not observed in 184 control chromosomes.     

Gain-of-function mutation in Gnao1: A murine model of epileptiform encephalopathy (EIEE17)?

G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gα_o, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1 ^+/G184S) that prevents G_o turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41–70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.