Spontaneous seizure and memory loss in mice expressing an epileptic encephalopathy variant in the calmodulin-binding domain of Kv7.2

Epileptic encephalopathy (EE) is characterized by seizures that respond poorly to antiseizure drugs, psychomotor delay, and cognitive and behavioral impairments. One of the frequently mutated genes in EE is KCNQ2, which encodes the Kv7.2 subunit of voltage-gated Kv7 potassium channels. Kv7 channels composed of Kv7.2 and Kv7.3 are enriched at the axonal surface, where they potently suppress neuronal excitability. Previously, we reported that the de novo dominant EE mutation M546V in human Kv7.2 blocks calmodulin binding to Kv7.2 and axonal surface expression of Kv7 channels via their intracellular retention. However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral comorbidities remains unknown. Here, we report conditional transgenic cKcnq2+/M547V mice, in which expression of mouse Kv7.2-M547V (equivalent to human Kv7.2-M546V) is induced in forebrain excitatory pyramidal neurons and astrocytes. These mice display early mortality, spontaneous seizures, enhanced seizure susceptibility, memory impairment, and repetitive behaviors. Furthermore, hippocampal pathology shows widespread neurodegeneration and reactive astrocytes. This study demonstrates that the impairment in axonal surface expression of Kv7 channels is associated with epileptic seizures, cognitive and behavioral deficits, and neuronal loss in KCNQ2-related EE.

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Loss of the glial glutamate transporter eaat2a leads to a combined developmental and epileptic encephalopathy in zebrafish

10.1101/2021.03.16.435577 ◽

2021 ◽

Author(s):

Adriana L. Hotz ◽

Ahmed Jamali ◽

Nicolas N. Rieser ◽

Stephanie Niklaus ◽

Ecem Aydin ◽

...

Keyword(s):

Neuronal Excitability ◽

Excitatory Amino Acid ◽

Brain Activity ◽

Glutamate Transporter ◽

Epileptic Seizures ◽

Synaptic Cleft ◽

Epileptic Encephalopathy ◽

Network Activity ◽

Excitatory Amino Acid Transporter ◽

The Impact

ABSTRACTAstroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Human patients with altered EAAT2 function exhibit epileptic seizures, suggesting an important role for astroglial glutamate transporters in balancing neuronal excitability. To study the impact of EAAT2 function at the neural network levels, we generated eaat2a mutant zebrafish. We observed that eaat2a-/- mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal brain activity was surprisingly reduced in eaat2a-/- mutant animals, which manifested in decreased locomotion, neuronal and astroglial calcium signals. Our results reveal an unexpected key role of the astroglial EAAT2a in balancing brain excitability, affecting both neuronal and astroglial network activity.

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Loss–of–function variants in the KCNQ5 gene are associated with genetic generalized epilepsies

10.1101/2021.04.20.21255696 ◽

2021 ◽

Author(s):

Johanna Krueger ◽

Julian Schubert ◽

Josua Kegele ◽

Audrey Labalme ◽

Miaomiao Mao ◽

...

Keyword(s):

Mammalian Cells ◽

De Novo ◽

Surface Expression ◽

Epileptic Encephalopathy ◽

Dominant Negative ◽

Dominant Negative Effect ◽

K Channel ◽

Loss Of Function ◽

Normal Surface ◽

Missense Variants

Objective: De novo missense variants in KCNQ5, encoding the voltage–gated K+ channel KV7.5, have been described as a cause of developmental and epileptic encephalopathy (DEE) or intellectual disability (ID). We set out to identify disease–related KCNQ5 variants in genetic generalized epilepsy (GGE) and their underlying mechanisms. Methods: 1292 families with GGE were studied by next-generation sequencing. Whole–cell patch–clamp recordings, biotinylation and phospholipid overlay assays were performed in mammalian cells combined with docking and homology modeling. Results: We identified three deleterious heterozygous missense variants, one truncation and one splice site alteration in five independent families with GGE with predominant absence seizures, two variants were also associated with mild to moderate ID. All three missense variants displayed a strongly decreased current density indicating a loss–of–function (LOF). When mutant channels were co–expressed with wild–type (WT) KV7.5 or KV7.5 and KV7.3 channels, three variants also revealed a significant dominant–negative effect on WT channels. Other gating parameters were unchanged. Biotinylation assays indicated a normal surface expression of the variants. The p.Arg359Cys variant altered PI(4,5)P2–interaction, presumably in the non–conducting preopen–closed state. Interpretation: Our study indicates that specific deleterious KCNQ5 variants are associated with GGE, partially combined with mild to moderate ID. The disease mechanism is a LOF partially with dominant–negative effects through functional, rather than trafficking deficits. LOF of KV7.5 channels will reduce the M–current, likely resulting in increased excitability of KV7.5–expressing neurons. Further studies on a network level are necessary to understand which circuits are affected and how the variants induce generalized seizures.

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PRRT2 gene variant in a child with dysmorphic features, congenital microcephaly, and severe epileptic seizures: genotype-phenotype correlation?

Italian Journal of Pediatrics ◽

10.1186/s13052-019-0755-2 ◽

2019 ◽

Vol 45 (1) ◽

Cited By ~ 3

Author(s):

Piero Pavone ◽

Giovanni. Corsello ◽

Sung Yoon Cho ◽

Xena Giada Pappalardo ◽

Martino Ruggieri ◽

...

Keyword(s):

Neuronal Excitability ◽

Transmembrane Protein ◽

Clinical Manifestations ◽

Gene Mutations ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Dysmorphic Features ◽

Severe Intellectual Disability ◽

Infantile Epilepsy ◽

Congenital Microcephaly

Abstract Background Mutations in Proline-rich Transmembrane Protein 2 (PRRT2) have been primarily associated with individuals presenting with infantile epilepsy, including benign familial infantile epilepsy, benign infantile epilepsy, and benign myoclonus of early infancy, and/or with dyskinetic paroxysms such as paroxysmal kinesigenic dyskinesia, paroxysmal non-kinesigenic dyskinesia, and exercise-induced dyskinesia. However, the clinical manifestations of this disorder vary widely. PRRT2 encodes a protein expressed in the central nervous system that is mainly localized in the pre-synaptic neurons and is involved in the modulation of synaptic neurotransmitter release. The anomalous function of this gene has been proposed to cause dysregulation of neuronal excitability and cerebral disorders. Case presentation We hereby report on a young child followed-up for three years who presents with a spectrum of clinical manifestations such as congenital microcephaly, dysmorphic features, severe intellectual disability, and drug-resistant epileptic encephalopathy in association with a synonymous variant in PRRT2 gene (c.501C > T; p.Thr167Ile) of unknown clinical significance variant (VUS) revealed by diagnostic exome sequencing. Conclusion Several hypotheses have been advanced on the specific role that PRRT2 gene mutations play to cause the clinical features of affected patients. To our knowledge, the severe phenotype seen in this case has never been reported in association with any clinically actionable variant, as the missense substitution detected in PRRT2 gene. Intriguingly, the same mutation was reported in the healthy father: the action of modifying factors in the affected child may be hypothesized. The report of similar observations could extend the spectrum of clinical manifestations linked to this mutation.

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Recurrent NUS1 canonical splice donor site mutation in two unrelated individuals with epilepsy, myoclonus, ataxia and scoliosis - a case report

BMC Neurology ◽

10.1186/s12883-019-1489-x ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Kouhei Den ◽

Yosuke Kudo ◽

Mitsuhiro Kato ◽

Kosuke Watanabe ◽

Hiroshi Doi ◽

...

Keyword(s):

Rna Splicing ◽

De Novo ◽

Involuntary Movement ◽

Donor Site ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Protein Glycosylation ◽

Splice Donor Site ◽

Splice Donor ◽

Site Mutation

Abstract Background We encountered two unrelated individuals suffering from neurological disorders, including epilepsy and scoliosis. Case presentation Whole-exome sequencing identified the same recurrent, de novo, pathogenic variant in NUS1 [NM_138459.4:c.691 + 1C > A] in both individuals. This variant is located in the conserved cis-prenyltransferase domain of the nuclear undecaprenyl pyrophosphate synthase 1 gene (NUS1), which encodes the Nogo-B receptor, an essential catalyst for protein glycosylation. This variant was confirmed to create a new splice donor site, resulting in aberrant RNA splicing resulting in a 91-bp deletion in exon 3 in both individuals. The mutant mRNA was partially degraded by nonsense mediated mRNA decay. To date, only four de novo variants and one homozygous variant have been reported in NUS1, which cause developmental and epileptic encephalopathy, early onset Parkinson’s disease, and a congenital disorder of glycosylation. Seven patients, including our two patients, have presented with epileptic seizures and intellectual disabilities. Conclusions Our study strongly supports the finding that this recurrent, de novo, variant in NUS1 causes developmental and epileptic encephalopathy with involuntary movement, ataxia and scoliosis.

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Biallelic Mutations in SLC1A2; an Additional Mode of Inheritance for SLC1A2-Related Epilepsy

Neuropediatrics ◽

10.1055/s-0037-1606370 ◽

2017 ◽

Vol 49 (01) ◽

pp. 059-062 ◽

Cited By ~ 6

Author(s):

Mirjana Gusic ◽

Roman Günthner ◽

Bader Alhaddad ◽

Reka Kovacs-Nagy ◽

Christine Makowski ◽

...

Keyword(s):

De Novo ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Genetic Mechanism ◽

Loss Of Function ◽

De Novo Mutations ◽

Functional Studies ◽

Additional Mode ◽

Biallelic Mutations ◽

Mode Of Inheritance

AbstractRecently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.

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Case Report: Clinical Features of a Chinese Boy With Epileptic Seizures and Intellectual Disabilities Who Carries a Truncated NUS1 Variant

Frontiers in Pediatrics ◽

10.3389/fped.2021.725231 ◽

2021 ◽

Vol 9 ◽

Author(s):

Pingli Zhang ◽

Di Cui ◽

Peiyuan Liao ◽

Xiang Yuan ◽

Nuan Yang ◽

...

Keyword(s):

Intellectual Disability ◽

Clinical Features ◽

De Novo ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Chinese Patient ◽

Pathogenic Variant ◽

Phenotype Correlation ◽

Congenital Disorder Of Glycosylation ◽

Pathogenic Variants

The mental retardation-55 with seizures (MRD55) is a rare genetic disease characterized by developmental delay, intellectual disability, language delay and multiple types of epileptic seizures. It is caused by pathogenic variants of the NUS1 gene, which encodes Nogo-B receptor (NgBR), a necessary subunit for the glycosylation reactions in mammals. To date, 25 disease-causing mutations of NUS1 have been reported, which are responsible for various diseases, including dystonia, Parkinson's disease, developmental and epileptic encephalopathy as well as congenital disorder of glycosylation. In addition, only 9 of these mutations were reported with detailed clinical features. There are no reports about Chinese cases with MRD55. In this study, a novel, de novo pathogenic variant of NUS1 (c.51_54delTCTG, p.L18Tfs*31) was identified in a Chinese patient with intellectual disability and epileptic seizures. This pathogenic variant resulted in truncated NgBR proteins, which might be the cause of the clinical features of the patient. Oxcarbazepine was an effective treatment for improving speech and movement of the patient, who consequently presented with no seizure. With this novel pathogenic variant found in NUS1, we expand the genotype spectrum of MRD55 and provide valuable insights into the potential genotype-phenotype correlation.

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Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate

10.1101/678219 ◽

2019 ◽

Author(s):

Alba Sanchis-Juan ◽

Marcia A Hasenahuer ◽

James A Baker ◽

Amy McTague ◽

Katy Barwick ◽

...

Keyword(s):

Structural Analysis ◽

Neuronal Excitability ◽

Transmembrane Domain ◽

De Novo ◽

Structural Information ◽

Missense Variant ◽

Epileptic Encephalopathy ◽

Missense Mutations ◽

Pathogenic Variants ◽

Activation Gate

AbstractCys-loop receptors are vital for controlling neuronal excitability in the brain and their dysfunction results in numerous neurological disorders. Recently, six de novo missense variants in GABRA2 gene, a member of this family, have been associated with early infantile epileptic encephalopathy (EIEE) and intellectual disability with seizures. Here, using whole-genome sequencing we identified a de novo missense variant in GABRA2 gene in a patient with EIEE and developmental delay. We perform protein structural analysis of the seven variants and show that all the mutations are in the transmembrane domain, either close to the desensitization gate, the activation gate or in inter-subunit interfaces. Further investigations demonstrated that the majority of pathogenic variants reported are at equivalent positions in other Cys-loop receptors, emphasizing the importance of these residues for the adequate function of the receptor. Also, a comparison of the distribution of the mutations in all the Cys-loop receptors showed that pathogenic variants are more common in the transmembrane helices, more specifically in the M2 helix, highlighting the importance of this segment. Our study expands the clinical spectrum of individuals with pathogenic missense mutations in GABRA2, defines the regions where pathogenic mutations are in the protein structure, and highlights the value of considering sequence, evolutionary, and structural information from other Cys-loop receptors as a strategy for variant interpretation of novel missense mutations in GABRA2.

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In vitro effects of S-Licarbazepine as a potential precision therapy on SCN8A variants causing neuropsychiatric disorders

10.1101/2021.04.24.441205 ◽

2021 ◽

Author(s):

Erva Bayraktar ◽

Yuanyuan Liu ◽

Ulrike B.S. Hedrich ◽

Yildirim Sara ◽

Holger Lerche ◽

...

Keyword(s):

Channel Coding ◽

Neuroblastoma Cells ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Fast Inactivation ◽

Psychomotor Delay ◽

Specific Effects ◽

Voltage Gated Sodium Channels ◽

Precision Therapy

Background and Purpose: Among genetic epilepsies, variants in sodium channel coding genes constitute a major subgroup. Variants in SCN8A, the coding gene for NaV1.6 channels, are characterized by a variety of symptoms including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, and others such as autistic features, ataxia or dystonia. Standard anticonvulsant treatment has only limited impact on the course of disease. Experimental Approach: Personalized therapeutic regimens tailored to disease-causing pathophysiological mechanisms may offer the specificity required to overcome intractability. Toward this aim, we investigated in vitro in neuroblastoma cells the effects of S-Licarbazepine, a third-generation dibenzazepine and enhancer of slow inactivation of voltage gated sodium channels, on three gain-of-function NaV1.6 variants linked to representative phenotypes of mild epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). Key Results: S-Licarbazepine strongly enhances the slow and — less pronounced — the fast inactivation of NaV1.6 wildtype channels. It acts similarly on all tested variants and irrespective of their particular biophysical dysfunction mechanism. Beyond that S–Licarbazepine has variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D, M1760I) and a trend to reduce the enhanced persistent Na+ current by A1622D variant channels. Conclusion and Implications: These data bring out that S-Licarbazepine not only owns substance-specific effects, but also holds variant-specific effects, which can variably contribute to functional compensation of distinct channel-specific biophysical properties and thereby highlighting the role of personalized approaches, which likely will be key to improved and successful treatment not only of SCN8A-related disease.

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