SCN1A intronic variants impact on Nav1.1 protein expression and sodium channel function, and associated with epilepsy phenotypic severity.

High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas deep intronic variants leading to haploinsufficiency could potentially cause milder phenotypes.

IFIH1 variants are associated with generalised epilepsy preceded by febrile seizures.

BACKGROUND: IFIH1 variants have been reported to be associated with immune-related disorders with/without seizures. It is unknown whether IFIH1 variants are associated with common epilepsy without acquired causes and the mechanism underlying phenotypic variation remains elusive. METHODS: Trio-based whole-exome sequencing was performed on patients with febrile seizures or epilepsy with antecedent febrile seizures. Previously reported variants were systematically reviewed to investigate genotype-phenotype associations. RESULTS: Two de novo heterozygous and three biallelic missense variants were identified in five patients with generalised epilepsy with antecedent febrile seizures. The variants were predicted to be damaging by in silico tools and were associated with hydrogen bonding changes to neighbouring amino acids or decreased protein stability. Patients exhibited an early onset age and became seizure-free with favourable outcome. Further analysis revealed that de novo missense variants located in the Hel region resulted in seizures with multiple neurological abnormalities, while those in the pincer domain or C-terminal domain led to seizures with normal neurodevelopment, suggesting a sub-molecular effect. Biallelic missense variants, which were inherited from unaffected parents and presented low allele frequencies in general populations, were associated with seizures without neurological abnormalities. Truncation variants were related to refractory epilepsy and severe developmental delay, suggesting a genotype-phenotype correlation. IFIH1 is predominantly expressed in the neonatal stage and decreases dramatically in the adulthood, which is consistent with the early onset age and favourable outcome of the patients. CONCLUSIONS: IFIH1 variants are potentially associated with generalised epilepsy with antecedent febrile seizures. The sub-molecular implication and genotype-phenotype association help explain phenotype variations of IFIH1 variants.

Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

NUS1 Variants Cause Lennox-Gastaut Syndrome Related to Unfolded Protein Reaction Activation.

NUS1 encodes the Nogo-B receptor, a critical regulator for unfolded protein reaction (UPR) signaling. Although several loss-of-function variants of NUS1 have been identified in patients with developmental and epileptic encephalopathy (DEE), the role of the NUS1 variant in Lennox-Gastaut syndrome (LGS), a severe child-onset DEE, remains unknown. In this study, we identified two de novo variants of NUS1, a missense variant (c.868 C > T/p.R290C) and a splice site variant (c.792-2 A > G), in two unrelated LGS patients using trio-based whole-exome sequencing performed in a cohort of 165 LGS patients. Both variants were absent in the gnomAD population and showed a significantly higher observed number of variants than expected genome-wide. The R290C variant was predicted to damage NUS1 and decrease its protein stability. The c.792-2 A > G variant caused premature termination of the protein. Knockdown of NUS1 activated the UPR pathway, resulting in apoptosis of HEK293T cells. Supplementing cells with expression of wild-type NUS1, but not the mutant (R290C), rescued UPR activation and apoptosis in NUS1 knockdown cells. Compared to wild-type Drosophila, seizure-like behaviors and excitability in projection neurons were significantly increased in Tango14 (homolog of human NUS1) knockdown and Tango14R290C/+ knock-in Drosophila. Additionally, abnormal development and a small body size were observed in both mutants. Activated UPR signaling was also detected in both mutants. Thus, NUS1 is a causative gene for LGS with dominant inheritance. The pathogenicity of these variants is related to the UPR signaling activation, which may be a common pathogenic mechanism of DEE.

Corrigendum: ATP6V0C is associated with febrile seizures and epilepsy with febrile seizures plus.

[This corrects the article DOI: 10.3389/fnmol.2022.889534.].

ZFHX3 variants cause childhood partial epilepsy and infantile spasms with favourable outcomes.

BACKGROUND: The ZFHX3 gene plays vital roles in embryonic development, cell proliferation, neuronal differentiation and neuronal death. This study aims to explore the relationship between ZFHX3 variants and epilepsy. METHODS: Whole-exome sequencing was performed in a cohort of 378 patients with partial (focal) epilepsy. A Drosophila Zfh2 knockdown model was used to validate the association between ZFHX3 and epilepsy. RESULTS: Compound heterozygous ZFHX3 variants were identified in eight unrelated cases. The burden of ZFHX3 variants was significantly higher in the case cohort, shown by multiple/specific statistical analyses. In Zfh2 knockdown flies, the incidence and duration of seizure-like behaviour were significantly greater than those in the controls. The Zfh2 knockdown flies exhibited more firing in excitatory neurons. All patients presented partial seizures. The five patients with variants in the C-terminus/N-terminus presented mild partial epilepsy. The other three patients included one who experienced frequent non-convulsive status epilepticus and two who had early spasms. These three patients had also neurodevelopmental abnormalities and were diagnosed as developmental epileptic encephalopathy (DEE), but achieved seizure-free after antiepileptic-drug treatment without adrenocorticotropic-hormone/steroids. The analyses of temporal expression (genetic dependent stages) indicated that ZFHX3 orthologous were highly expressed in the embryonic stage and decreased dramatically after birth. CONCLUSION: ZFHX3 is a novel causative gene of childhood partial epilepsy and DEE. The patients of infantile spasms achieved seizure-free after treatment without adrenocorticotropic-hormone/steroids implies a significance of genetic diagnosis in precise treatment. The genetic dependent stage provided an insight into the underlying mechanism of the evolutional course of illness.

CCDC88C variants are associated with focal epilepsy and genotype-phenotype correlation.

CCDC88C gene, which encodes coiled-coil domain containing 88C, is essential for cell communication during neural development. Variants in the CCDC88C caused congenital hydrocephalus, some accompanied by seizures. In patients with epilepsy without acquired etiologies, we performed whole-exome sequencing (trio-based). Two de novo and two biallelic CCDC88C variants were identified in four cases with focal (partial) epilepsy. These variants did not present or had low frequencies in the gnomAD populations and were predicted to be damaging by multiple computational algorithms. Patients with de novo variants presented with adult-onset epilepsy, whereas patients with biallelic variants displayed infant-onset epilepsy. They all responded well to anti-seizure medications and were seizure-free. Further analysis showed that de novo variants were located at crucial domains, whereas one paired biallelic variants were located outside the crucial domains, and the other paired variant had a non-classical splicing and a variant located at crucial domain, suggesting a sub-molecular effect. CCDC88C variants associated with congenital hydrocephalus were all truncated, whereas epilepsy-associated variants were mainly missense, the proportion of which was significantly higher than that of congenital hydrocephalus-associated variants. CCDC88C is potentially associated with focal epilepsy with favorable outcome. The underlying mechanisms of phenotypic variation may correlation between genotype and phenotype.

Novel indocyanine green-loaded photothermal nanoparticles targeting TRPV1 for thermal ablation treatment of severe murine asthma induced by ovalbumin and lipopolysaccharide.

To identify a replacement strategy for bronchial thermoplasty (BT) with non-invasive and free-of-severe side effect is urgently needed in the clinic for severe asthma treatment. In this study, PLGA-PEG@ICG@TRPV1 pAb (PIT) photothermal nanoparticles targeting bronchial TRPV1 were designed for photothermal therapy (PTT) against severe murine asthma induced by ovalbumin and lipopolysaccharide. PIT was formulated with a polyethylene glycol (PEG)-grafted poly (lactic-co-glycolic) acid (PLGA) coating as a skeleton structure to encapsulate indocyanine green (ICG) and was conjugated to the polyclonal antibody against transient receptor potential vanilloid 1 (TRPV1 pAb). The results revealed that PIT held good druggability due to its electronegativity and small diameter. PIT demonstrated great photothermal effects both in vivo and in vitro and exhibited good ability to target TRPV1 in vitro because of its selective cell uptake and specific cell toxicity toward TRPV1-overexpressing cells. The PIT treatment effectively reduced asthma symptoms in mice. This is evident from improvements in expiratory airflow limitation, significant decreases in inflammatory cell infiltration in the airways, and increases in goblet cell and columnar epithelial cell proliferation. In conclusion, PIT alleviates severe murine asthma symptoms through a combination of TRPV1 targeting and photothermal effects.

DLG3 variants caused X-linked epilepsy with/without neurodevelopmental disorders and the genotype-phenotype correlation.

Background: The DLG3 gene encodes disks large membrane-associated guanylate kinase scaffold protein 3, which plays essential roles in the clustering of N-methyl-D-aspartate receptors (NMDARs) at excitatory synapses. Previously, DLG3 has been identified as the causative gene of X-linked intellectual developmental disorder-90 (XLID-90; OMIM# 300850). This study aims to explore the phenotypic spectrum of DLG3 and the genotype-phenotype correlation. Methods: Trios-based whole-exome sequencing was performed in patients with epilepsy of unknown causes. To analyze the genotype-phenotype correlations, previously reported DLG3 variants were systematically reviewed. Results: DLG3 variants were identified in seven unrelated cases with epilepsy. These variants had no hemizygous frequencies in controls. All variants were predicted to be damaging by silico tools and alter the hydrogen bonds with surrounding residues and/or protein stability. Four cases mainly presented with generalized seizures, including generalized tonic-clonic and myoclonic seizures, and the other three cases exhibited secondary generalized tonic-clonic seizures and focal seizures. Multifocal discharges were recorded in all cases during electroencephalography monitoring, including the four cases with generalized discharges initially but multifocal discharges after drug treating. Protein-protein interaction network analysis revealed that DLG3 interacts with 52 genes with high confidence, in which the majority of disease-causing genes were associated with a wide spectrum of neurodevelopmental disorder (NDD) and epilepsy. Three patients with variants locating outside functional domains all achieved seizure-free, while the four patients with variants locating in functional domains presented poor control of seizures. Analysis of previously reported cases revealed that patients with non-null variants presented higher percentages of epilepsy than those with null variants, suggesting a genotype-phenotype correlation. Significance: This study suggested that DLG3 variants were associated with epilepsy with/without NDD, expanding the phenotypic spectrum of DLG3. The observed genotype-phenotype correlation potentially contributes to the understanding of the underlying mechanisms driving phenotypic variation.