Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.

OBJECTIVE: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. METHODS: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H/F+ mice. Spontaneous epileptiform events in Fhf1R52H/+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein. RESULTS: All Fhf1R52H/+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H/+ mice prior to seizure. SIGNIFICANCE: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.

A patient with juvenile-onset refractory status epilepticus caused by two novel compound heterozygous mutations in FARS2 gene.

FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase and is implicated in autosomal recessive combined oxidative phosphorylation deficiency 14. The clinical manifestation can be divided into early onset epileptic phenotype and spastic paraplegia phenotype. The purpose of this study was to report a case of juvenile manifesting refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. Microscopic and histochemical examination as well as next-generation sequencing and reconstruction of the three-dimensional structure of FARS2 protein were performed. A 17-year-old man with no developmental delays suffered from generalized tonic-clonic convulsion since 12 years of age and developed refractory status epilepticus 5 years later. No specific etiology was found following brain imaging, muscle biopsy and metabolic studies. DNA sequencing identified two novel compound heterozygous mutations in FARS2, (p.V197M and p.F402S), derived from each parents, respectively. These mutations affected the structure or thermodynamic stability of the protein. This is a case report of juvenile-onset refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. This case confirms and expands the clinicalphenotype and the genotypic spectrum of the FARS2 gene.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus.

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.

Expressional analysis of inwardly rectifying Kir4.1 channels in Noda epileptic rat (NER).

The inwardly rectifying potassium channel subunit Kir4.1 is expressed in brain astrocytes and involved in spatial K(+) buffering, regulating neural activity. To explore the pathophysiological alterations of Kir4.1 channels in epileptic disorders, we analyzed interictal expressional levels of Kir4.1 in the Noda epileptic rat (NER), a hereditary animal model for generalized tonic-clonic (GTC) seizures. Western blot analysis showed that Kir4.1 expression in NERs was significantly reduced in the occipito-temporal cortical region and thalamus. However, the expression of Kir5.1, another Kir subunit mediating spatial K(+) buffering, remained unaltered in any brain regions examined. Immunohistochemical analysis revealed that Kir4.1 was primarily expressed in glial fibrillary acidic protein (GFAP)-positive astrocytes (somata) and foot processes clustered around neurons proved with anti-neuronal nuclear antigen (NeuN) antibody. In NERs, Kir4.1 expression in astrocytic processes was region-selectively diminished in the amygdaloid nuclei (i.e., medial amygdaloid nucleus and basomedial amygdaloid nucleus) while Kir4.1 expression in astrocytic somata was unchanged. Furthermore, the amygdala regions with reduced Kir4.1 expression showed a marked elevation of Fos protein expression following GTC seizures. The present results suggest that reduced activity of astrocytic Kir4.1 channels in the amygdala is involved in limbic hyperexcitability in NERs.

Hyperinsulinemic hypoglycemia evolving to gestational diabetes and diabetes mellitus in a family carrying the inactivating ABCC8 E1506K mutation.

Congenital hyperinsulinism of infancy (CHI) is the most common cause of hypoglycemia in newborns and infants. Several molecular mechanisms are involved in the development of CHI, but the most common genetic defects are inactivating mutations of the ABCC8 or KCNJ11 genes. The classical treatment for CHI has been pancreatectomy that eventually leads to diabetes. More recently, conservative treatment has been attempted in some cases, with encouraging results. Whether or not the patients with heterozygous ABCC8 mutations submitted to conservative treatment may spontaneously develop type 2 diabetes in the long run, is a controversial issue. Here, we report a family carrying the dominant heterozygous germ line E1506K mutation in ABCC8 associated with persistent hypoglycemia in the newborn period and diabetes in adulthood. The mutation occurred as a de novo germ line mutation in the mother of the index patient. Her hypoglycemic symptoms as a child occurred after the fourth year of life and were very mild, but she developed glucose metabolism impairment in adulthood. On the other hand, in her daughter, the clinical manifestations of the disease occurred in the neonatal period and were more severe, leading to episodes of tonic-clonic seizures that were well controlled with octreotide or diazoxide. Our data corroborate the hypothesis that the dominant E1506K ABCC8 mutation, responsible for CHI, predisposes to the development of glucose intolerance and diabetes later in life.

Regional expression of Fos-like immunoreactivity following seizures in Noda epileptic rat (NER).

Noda epileptic rat (NER) is a genetic rat model of epilepsy that exhibit spontaneous generalized tonic-clonic (GTC) seizures with paroxysmal discharges. We analyzed the regional expression of Fos-like immunoreactivity (Fos-IR) following GTC seizures in NER to clarify the brain regions involved in the seizure generation. GTC seizures in NER elicited a marked increase in Fos expression in the piriform cortex, perirhinal-entorhinal cortex, insular cortex and other cortices including the motor cortex. In the limbic regions, Fos-IR was highest in the amygdalar nuclei (e.g., basomedial amygdaloid nucleus), followed by the cingulate cortex and hippocampus (i.e., dentate gyrus and CA3). As compared to the above forebrain regions, NER either with or without GTC seizures exhibited only marginal Fos expression in the basal ganglia (e.g., accumbens, striatum and globus pallidus), diencephalon (e.g., thalamus and hypothalamus) and lower brain stem structures (e.g., pons-medulla oblongata). These results suggest that GTC seizures in NER are of forebrain origin and are evoked primarily by activation of the limbic and/or cortical seizure circuits.

A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy.

The lde/lde rat is characterized by dwarfism, postnatal lethality, male hypogonadism, a high incidence of epilepsy and many vacuoles in the hippocampus and amygdala. We used a candidate approach to identify the gene responsible for the lde phenotype and assessed the susceptibility of lde/lde rats for audiogenic seizures. Following backcross breeding of lethal dwarfism with epilepsy (LDE) to Brown Norway rats, the lde/lde rats with an altered genetic background showed all pleiotropic phenotypes. The lde locus was mapped to a 1.5-Mbp region on rat chromosome 19 that included the latter half of the Wwox gene. Sequencing of the full-length Wwox transcript identified a 13-bp deletion in exon 9 in lde/lde rats. This mutation causes a frame shift, resulting in aberrant amino acid sequences at the C-terminal. Western blotting showed that both the full-length products of the Wwox gene and its isoform were present in normal testes and hippocampi, whereas both products were undetectable in the testes and hippocampi of lde/lde rats. Sound stimulation induced epileptic seizures in 95% of lde/lde rats, with starting as wild running (WR), sometimes progressing to tonic-clonic convulsions. Electroencephalogram (EEG) analysis showed interictal spikes, fast waves during WR and burst of spikes during clonic phases. The Wwox protein is expressed in the central nervous system (CNS), indicating that abnormal neuronal excitability in lde/lde rats may be because of a lack of Wwox function. The lde/lde rat is not only useful for understanding the multiple functions of Wwox but is also a unique model for studying the physiological function of Wwox in CNS.

Severe attacks of familial hemiplegic migraine, childhood epilepsy and ATP1A2 mutation.

We studied four members of a family suffering from typical attacks of familial hemiplegic migraine (FHM) caused by a new mutation, R548C, of ATP1A2 gene in exon 12. One individual had also childhood absence epilepsy and generalized tonic-clonic seizures (GTCS). GTCS were followed by a severe attack of hemiplegic migraine at four times. Sodium valproate enabled control of both the epileptic seizures and the most severe FHM attacks. This association of FHM and epileptic seizures and their control with the same treatment suggest similar pathophysiological mechanisms.

Griscelli syndrome type 2: a rare and lethal disorder.

Griscelli syndrome is a rare autosomal recessive disorder. It is characterized by pigment dilution and variable immune deficiency leading to increased susceptibility to certain infections and a tendency to develop a life-threatening hemophagocytic syndrome known as the accelerated phase. Griscelli syndrome is now classified into 3 types based on the genetic and molecular features. Primary neurological presentation without the accelerated phase is rare in type 2. In this article, the authors report a boy who was presented with seizures and diffuse white matter involvement unaccompanied by the other features of the accelerated phase. Mutation analysis in family members revealed the presence of a missense mutation in Rab27a gene. In addition to the rare presentation, this is the first case of Griscelli syndrome to be reported from Jordan.

A novel mitochondrial DNA mutation in COX1 leads to strokes, seizures, and lactic acidosis.

Cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain, with subunits originating both from the mitochondrial and nuclear genome. An eleven-year-old female presented initially with a seizure followed two months later with tonic-clonic seizures, weakness and aphasia. MRI of the cerebral hemispheres showed multiple infarcts. Previous history suggested gross and fine motor control deficits with learning difficulties. A muscle biopsy showed a specific decrease of COX staining in all fibres and pleomorphic mitochondria. Respiratory chain studies confirmed an isolated complex IV defect in muscle, whilst fibroblasts showed an initial COX activity below normal which rapidly came up to the normal range on culture. Sequencing of mtDNA revealed an heteroplasmic m.7023G>A mutation in the COX1 gene, with levels of 96% in muscle, 70% in blood and 50% in the initial skin fibroblast culture dropping to 10% in later passages. The mutation was present in a critical region of the COX1 gene, the V374M change being close to the two histidine residues His376 and His378 co-ordinating with the heme a and a (3), and His367 which co-ordinates a magnesium ion. This case highlights that a MELAS-like syndrome can occur with isolated COX deficiency.

Genetic polymorphism and idiopathic generalized epilepsy. Evidence of interaction between haptoglobin and ACP1 systems.

We have investigated possible interactions between ACP1 and Hp concerning their effects on the susceptibility to convulsive disorders. 129 children with idiopathic generalized epilepsy with tonic clonic seizures (IGE) and 127 controls were studied in the population of Rome. There is a significant interaction between Hp and ACP1 concerning their effects on epilepsy. The association of Hp with epilepsy depends on the ACP1 genotypes. In carriers of the *B/*B genotype of ACP1 the risk of epilepsy is much lower in Hp *1/*1 children than in other Hp types. This is not observed in carriers of other ACP1 types.The present data suggest an epistatic action of ACP1 concerning the effect of Hp on the susceptibility to convulsive disorders.

Novel de novo mutation of a conserved SCN1A amino-acid residue (R1596).

We report on the case of a 6-year-old boy with epilepsy involving febrile seizures and unprovoked generalized tonic clonic seizures. Genetic testing revealed a novel de novo mutation in the SCN1A gene (C>T 4786, R1596C). The epilepsy phenotype is within the spectrum of generalized epilepsy with febrile seizures plus. However, de novo mutations are more commonly reported in cases of severe myoclonic epilepsy of infancy, and are less often reported in generalized epilepsy with febrile seizures plus. The clinical utility of specific genetic testing in this case is discussed, as are criteria for determining the pathologic significance of novel DNA variants. In this case, the wild type of residue (R1596) is well-conserved across evolution from bacteria to humans, providing support for the hypothesis that this mutation causes epilepsy.

A new form of childhood onset, autosomal recessive spinocerebellar ataxia and epilepsy is localized at 16q21-q23.

Childhood ataxias are a complex set of inherited disorders. Ataxias associated with generalized tonic-clonic epilepsy are usually included with the progressive myoclonus epilepsies (PME). Five disease entities, Unverricht-Lundborg disease, Lafora's disease, neuronal ceroid lipofuscinoses, myoclonic epilepsy with ragged red fibres and sialidoses, account for the majority of PME cases. Two rare forms of ataxia plus epilepsy, sensory ataxic neuropathy, dysarthria and ophthalmoparesis, and infantile onset spinocerebellar ataxia were described recently and found to be caused by defective mitochondrial proteins. We report here a large consanguineous family from Saudi Arabia with four affected children presenting with generalized tonic-clonic epilepsy, ataxia and mental retardation, but neither myoclonus nor mental deterioration. MRI and muscle biopsy of one patient revealed, respectively, posterior white matter hyperintensities and vacuolization of the sarcotubular system. We localized the defective gene by homozygosity mapping to a 19 Mb interval in 16q21-q23 between markers D16S3091 and D16S3050. Linkage studies in this region will allow testing for homogeneity of this novel ataxia-epilepsy entity.

Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats.

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat. In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER. Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed. After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER. The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector. Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration. No inhibitory effects were observed 10 and 14 days after AxASPA administration. In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER. Survival rates did not differ between AxASPA- and AxLacZ-treated SERs. Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.

[Familial myoclonus-renal failure syndrome]. / Familiäres Myoklonus-Niereninsuffizienz-Syndrom.

In 1986 Andermann et al. described a syndrome presenting with renal failure, myoclonus, cerebellar symptoms, and epilepsy. They presumed a hereditary cause. We describe the first appearance of this syndrome in Europe, affecting three family members with comparable symptoms. Two of these patients were treated by us, and the third, already decreased, is described according to the available reports. The first clinical symptoms were manifested between the ages of 14 and 20. A female patient suffered from compensated kidney insufficiency and her two brothers aged 18 and 26 required dialysis. Biopsy of kidney tissue revealed nonspecific nephritis. All cases showed a cerebellar syndrome and action myoclonus. Two of them were diagnosed with epilepsy and grand mal seizures, and all suffered from demyelinizing or mixed polyneuropathy. Anamnesis of the family seems to indicate autosomal recessive inheritance.

A case of Adams-Oliver syndrome associated with acrania, microcephaly, hemiplegia, epilepsy, and mental retardation.

Adams-Oliver syndrome (AOS) is a rare congenital disorder, characterized by aplasia cutis congenita (ACC) of the scalp and variable degrees of terminal transverse limb defects. In this article, a newborn infant diagnosed as AOS for a large scalp defect, acrania, and finger malformations is presented. The patient was hospitalized and the scalp defect was successfully repaired with several surgical operations. During the hospitalization septicemia, meningitis, and convulsions developed, but they were successfully treated with appropriate antibiotics, antifungal, and anticonvulsive agents, He was discharged five months after admission to the hospital. Now, he is 3 years old, and has microcephaly, moderate mental retardation, left spastic hemiplegia, and epilepsy.

Effect of precollicular transection on audiogenic seizures in genetically epilepsy-prone rats.

Previous studies have demonstrated that generalized tonic-clonic seizures (GTCS) consisting of running/bouncing clonic and tonic extension can still be elicited in rats after brain transections which separate forebrain from brain stem, showing that forebrain circuitry is not required for GTCS. Inasmuch as sound-induced generalized tonic-clonic seizures in rodents are characterized by running-bouncing clonic and tonic convulsions, we have hypothesized that these are brain stem seizures that can occur independently of the forebrain. To test this hypothesis, we examined the response of two strains of genetically epilepsy-prone rats (GEPR-3s and GEPR-9s) to seizure-evoking auditory stimuli 3 h after a precollicular transection or sham surgery performed under ether anesthesia. In addition, the effect of a precollicular transection on audiogenic seizures was evaluated in normal rats made susceptible to such seizures by infusing NMDA into the inferior colliculus. Following the transection 58% of GEPR-9s displayed a sound-induced tonic-clonic convulsion and the remaining 42% exhibited a sound-induced seizure when subjected to stimulation 5 min after a subconvulsant dose of pentylenetetrazol (PTZ). While sham surgery and the precollicular transection both reduced sound-induced seizure severity in GEPR-3s, the full seizure response could be elicited by sound stimulation following a subconvulsant dose of PTZ. Moreover, the audiogenic seizures in normal rats rendered susceptible by NMDA were unaltered by the precollicular transection. These findings show that the anatomical circuitry required for generalized tonic-clonic seizures evoked by sound stimulation in rodents resides within the brain stem.