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.

Optimization of in silico tools for predicting genetic variants: individualizing for genes with molecular sub-regional stratification.

Genes are unique in functional role and differ in their sensitivities to genetic defects, but with difficulties in pathogenicity prediction. This study attempted to improve the performance of existing in silico algorithms and find a common solution based on individualization strategy. We initiated the individualization with the epilepsy-related SCN1A variants by sub-regional stratification. SCN1A missense variants related to epilepsy were retrieved from mutation databases, and benign missense variants were collected from ExAC database. Predictions were performed by using 10 traditional tools with stepwise optimizations. Model predictive ability was evaluated using the five-fold cross-validations on variants of SCN1A, SCN2A, and KCNQ2. Additional validation was performed in SCN1A variants of damage-confirmed/familial epilepsy. The performance of commonly used predictors was less satisfactory for SCN1A with accuracy less than 80% and varied dramatically by functional domains of Nav1.1. Multistep individualized optimizations, including cutoff resetting, domain-based stratification, and combination of predicting algorithms, significantly increased predictive performance. Similar improvements were obtained for variants in SCN2A and KCNQ2. The predictive performance of the recently developed ensemble tools, such as Mendelian clinically applicable pathogenicity, combined annotation-dependent depletion and Eigen, was also improved dramatically by application of the strategy with molecular sub-regional stratification. The prediction scores of SCN1A variants showed linear correlations with the degree of functional defects and the severity of clinical phenotypes. This study highlights the need of individualized optimization with molecular sub-regional stratification for each gene in practice.

UNC13B variants associated with partial epilepsy with favourable outcome.

The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), which is highly expressed in the brain-predominantly in the cerebral cortex-and plays an essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of the UNC13B mutation in human disease is not known. In this study, we screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed a favourable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database (gnomAD). Computational modelling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a) knockdown Drosophila. Electrophysiological recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results indicate that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favourable outcome under anti-epileptic therapy in our patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition.

CELSR1 variants are associated with partial epilepsy of childhood.

CELSR1 gene, encoding cadherin EGF LAG seven-pass G-type receptor 1, is mainly expressed in neural stem cells during the embryonic period. It plays an important role in neurodevelopment. However, the relationship between CELSR1 and disease of the central nervous system has not been defined. In this study, we performed trios-based whole-exome sequencing in a cohort of 356 unrelated cases with partial epilepsy without acquired causes and identified CELSR1 variants in six unrelated cases. The variants included one de novo heterozygous nonsense variant, one de novo heterozygous missense variant, and four compound heterozygous missense variants that had one variant was located in the extracellular region and the other in the cytoplasm. The patients with biallelic variants presented severe epileptic phenotypes, whereas those with heterozygous variants were associated with a mild epileptic phenotype of benign epilepsy with centrotemporal spikes (BECTS). These variants had no or low allele frequency in the gnomAD database. The frequencies of the CELSR1 variants in this cohort were significantly higher than those in the control populations. The evidence from ClinGen Clinical-Validity Framework suggested a strong association between CELSR1 variants and epilepsy. These findings provide evidence that CELSR1 is potentially a candidate pathogenic gene of partial epilepsy of childhood.

Familial paroxysmal kinesigenic dyskinesia is associated with mutations in the KCNA1 gene.

Paroxysmal kinesigenic dyskinesia (PKD) is a heterogeneous movement disorder characterized by recurrent dyskinesia attacks triggered by sudden movement. PRRT2 has been identified as the first causative gene of PKD. However, it is only responsible for approximately half of affected individuals, indicating that other loci are most likely involved in the etiology of this disorder. To explore the underlying causative gene of PRRT2-negative PKD, we used a combination strategy including linkage analysis, whole-exome sequencing and copy number variations analysis to detect the genetic variants within a family with PKD. We identified a linkage locus on chromosome 12 (12p13.32-12p12.3) and detected a novel heterozygous mutation c.956 T>G (p.319 L>R) in the potassium voltage-gated channel subfamily A member 1, KCNA1. Whole-exome sequencing in another 58 Chinese patients with PKD who lacked mutations in PRRT2 revealed another novel mutation in the KCNA1 gene [c.765 C>A (p.255 N>K)] within another family. Biochemical analysis revealed that the L319R mutant accelerated protein degradation via the proteasome pathway and disrupted membrane expression of the Kv1.1 channel. Electrophysiological examinations in transfected HEK293 cells showed that both the L319R and N255K mutants resulted in reduced potassium currents and respective altered gating properties, with a dominant negative effect on the Kv1.1 wild-type channel. Our study suggests that these mutations in KCNA1 cause the Kv1.1 channel dysfunction, which leads to familial PKD. The current study further extended the genotypic spectrum of this disorder, indicating that Kv1.1 channel dysfunction maybe one of the underlying defects in PKD.

Synaptic clustering differences due to different GABRB3 mutations cause variable epilepsy syndromes.

GABRB3 is highly expressed early in the developing brain, and its encoded ß3 subunit is critical for GABAA receptor assembly and trafficking as well as stem cell differentiation in embryonic brain. To date, over 400 mutations or variants have been identified in GABRB3. Mutations in GABRB3 have been increasingly recognized as a major cause for severe paediatric epilepsy syndromes such as Lennox-Gastaut syndrome, Dravet syndrome and infantile spasms with intellectual disability as well as relatively mild epilepsy syndromes such as childhood absence epilepsy. There is no plausible molecular pathology for disease phenotypic heterogeneity. Here we used a very high-throughput flow cytometry assay to evaluate the impact of multiple human mutations in GABRB3 on receptor trafficking. In this study we found that surface expression of mutant ß3 subunits is variable. However, it was consistent that surface expression of partnering γ2 subunits was lower when co-expressed with mutant than with wild-type subunits. Because γ2 subunits are critical for synaptic GABAA receptor clustering, this provides an important clue for understanding the pathophysiology of GABRB3 mutations. To validate our findings further, we obtained an in-depth comparison of two novel mutations [GABRB3 (N328D) and GABRB3 (E357K)] associated with epilepsy with different severities of epilepsy phenotype. GABRB3 (N328D) is associated with the relatively severe Lennox-Gastaut syndrome, and GABRB3 (E357K) is associated with the relatively mild juvenile absence epilepsy syndrome. With functional characterizations in both heterologous cells and rodent cortical neurons by patch-clamp recordings, confocal microscopy and immunoblotting, we found that both the GABRB3 (N328D) and GABRB3 (E357K) mutations reduced total subunit expression in neurons but not in HEK293T cells. Both mutant subunits, however, were reduced on the cell surface and in synapses, but the Lennox-Gastaut syndrome mutant ß3 (N328D) subunit was more reduced than the juvenile absence epilepsy mutant ß3 (E357K) subunit. Interestingly, both mutant ß3 subunits impaired postsynaptic clustering of wild-type GABAA receptor γ2 subunits and prevented γ2 subunits from incorporating into GABAA receptors at synapses, although by different cellular mechanisms. Importantly, wild-type γ2 subunits were reduced and less clustered at inhibitory synapses in Gabrb3+/- knockout mice. This suggests that impaired receptor localization to synapses is a common pathophysiological mechanism for GABRB3 mutations, although the extent of impairment may be different among mutant subunits. The study thus identifies the novel mechanism of impaired targeting of receptors containing mutant ß3 subunits and provides critical insights into understanding how GABRB3 mutations produce severe epilepsy syndromes and epilepsy phenotypic heterogeneity.

Evaluating the pathogenic potential of genes with de novo variants in epileptic encephalopathies.

Epileptic encephalopathies comprise a group of catastrophic epilepsies with heterogeneous genetic etiology. Although next-generation sequencing techniques can reveal a number of de novo variants in epileptic encephalopathies, evaluating the pathogenicity of these variants can be challenging. Determining the pathogenic potential of genes in epileptic encephalopathies is critical before evaluating the pathogenicity of variants identified in an individual. We reviewed de novo variants in epileptic encephalopathies, including their genotypes and functional consequences. We then evaluated the pathogenic potential of genes, with the following additional considerations: (1) recurrence of variants in unrelated cases, (2) information of previously defined phenotypes, and (3) data from genetic experimental studies. Genes related to epileptic encephalopathy revealed pathogenicity with distinct functional alterations, i.e., either a gain of function or loss of function in the majority; however, several genes warranted further study to confirm their pathogenic potential. Whether a gene was associated with distinct phenotype, the genotype (or functional alteration)--phenotype correlation, and quantitative correlation between genetic impairment and phenotype severity were suggested to be specific evidence in determining the pathogenic role of genes. Data from epileptic encephalopathy-related genes would be helpful in outlining guidelines for evaluating the pathogenic potential of genes in other genetic disorders.

ARHGEF9 mutations in epileptic encephalopathy/intellectual disability: toward understanding the mechanism underlying phenotypic variation.

ARHGEF9 resides on Xq11.1 and encodes collybistin, which is crucial in gephyrin clustering and GABAA receptor localization. ARHGEF9 mutations have been identified in patients with heterogeneous phenotypes, including epilepsy of variable severity and intellectual disability. However, the mechanism underlying phenotype variation is unknown. Using next-generation sequencing, we identified a novel mutation, c.868C > T/p.R290C, which co-segregated with epileptic encephalopathy, and validated its association with epileptic encephalopathy. Further analysis revealed that all ARHGEF9 mutations were associated with intellectual disability, suggesting its critical role in psychomotor development. Three missense mutations in the PH domain were not associated with epilepsy, suggesting that the co-occurrence of epilepsy depends on the affected functional domains. Missense mutations with severe molecular alteration in the DH domain, or located in the DH-gephyrin binding region, or adjacent to the SH3-NL2 binding site were associated with severe epilepsy, implying that the clinical severity was potentially determined by alteration of molecular structure and location of mutations. Male patients with ARHGEF9 mutations presented more severe phenotypes than female patients, which suggests a gene-dose effect and supports the pathogenic role of ARHGEF9 mutations. This study highlights the role of molecular alteration in phenotype expression and facilitates evaluation of the pathogenicity of ARHGEF9 mutations in clinical practice.

MDH2 is an RNA binding protein involved in downregulation of sodium channel Scn1a expression under seizure condition.

Voltage-gated sodium channel α-subunit type I (NaV1.1, encoded by SCN1A gene) plays a critical role in the excitability of brain. Downregulation of SCN1A expression is associated with epilepsy, a common neurological disorder characterized by recurrent seizures. Here we reveal a novel role of malate dehydrogenase 2 (MDH2) in the posttranscriptional regulation of SCN1A expression under seizure condition. We identified that MDH2 was an RNA binding protein that could bind two of the four conserved regions in the 3' UTRs of SCN1A. We further showed that knockdown of MDH2 or inactivation of MDH2 activity in HEK-293 cells increased the reporter gene expression through the 3' UTR of SCN1A, and MDH2 overexpression decreased gene expression by affecting mRNA stability. In the hippocampus of seizure mice, the upregulation of MDH2 expression contributed to the decrease of the NaV1.1 levels at posttranscriptional level. In addition, we showed that the H2O2 levels increased in the hippocampus of the seizure mice, and H2O2 could promote the binding of MDH2 to the binding sites of Scn1a gene, whereas ß-mercaptoethanol decreased the binding capability, indicating an important effect of the seizure-induced oxidation on the MDH2-mediated downregulation of Scn1a expression. Taken together, these data suggest that MDH2, functioning as an RNA-binding protein, is involved in the posttranscriptional downregulation of SCN1A expression under seizure condition.

[Efficacy and safety of cyclophosphamide as a sequential immunotherapy drug for anti-N-methyl-D-aspartate receptor encephalitis in children].

OBJECTIVE: To evaluate the efficacy and safety of cyclophosphamide as a second-line drug in the treatment of children with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. METHODS: Six children with anti-NMDAR encephalitis, who showed poor response to steroids and intravenous immunoglobulin, were given cyclophosphamide as a second-line immunotherapy. Follow-up was performed to evaluate the efficacy and safety of cyclophosphamide. RESULTS: After first-line immunotherapy for 1-4 weeks, the six patients had reduced psychiatric symptoms, seizures, and involuntary movements; three patients had an improved level of consciousness and were able to make simple conversations. However, all the patients still showed slow response, as well as cortical dysfunction symptoms such as aphasia, alexia, agraphia, acalculia, apraxia, and movement disorders. The six patients continued to receive cyclophosphamide as a sequential therapy. They were able to answer simple questions 7 days after treatment. Three school-aged patients were able to make simple calculation, had greatly improved reading and writing ability, and almost recovered self-care ability 2-3 weeks later. The cognitive function of the six patients was almost restored to the level before the onset of disease, and their living ability returned to normal 2-3 months later. During the treatment period, there were no adverse reactions or abnormal results of routine blood test and liver and kidney function tests. CONCLUSIONS: Children with anti-NMDAR encephalitis should be given appropriate immunotherapy as soon as possible. Cyclophosphamide as a sequential therapy has good efficacy and safety.

Alteration of Scn3a expression is mediated via CpG methylation and MBD2 in mouse hippocampus during postnatal development and seizure condition.

Increased expression of sodium channel SCN3A, an embryonic-expressed gene, has been identified in epileptic tissues, which is believed to contribute to the development of epilepsy. However, the regulatory mechanism of SCN3A expression under epileptic condition is still unknown. Here we showed a high level of Scn3a mRNA expression in mouse embryonic hippocampus with gradually decreasing to a low level during the postnatal development and a methylation of a specific CpG site (-39C) in the Scn3a promoter was increased in hippocampus during postnatal development, corresponding to the downregulation of Scn3a expression. Furthermore, in vitro methylation and -39C>T mutation of the Scn3a promoter decreased the reporter gene expression, suggesting an important role of the -39C site in regulating gene expression. We then demonstrated that the sequence containing -39C was a MBD2-binding motif and the CpG methylation of the promoter region increased the capability of MBD2's binding to the motif. Knockdown of MBD2 in mouse N1E-115 cells led to the -39C methylation and the downregulation of Scn3a transcription by decreasing the Scn3a promoter activity. In the hippocampus of seizure mice, the expressions of Scn3a and Mbd2 were upregulated after 10-day KA treatment. At the same time point, the -39C site was demethylated and the capability of MBD2's binding to the Scn3a promoter motif was decreased. Taken together, these findings suggest that CpG methylation and MBD2 are involved in altering Scn3a expression during postnatal development and seizure condition.

The SCN1A mutation database: updating information and analysis of the relationships among genotype, functional alteration, and phenotype.

Mutations in the SCN1A gene have been identified in epilepsy patients with widely variable phenotypes and modes of inheritance and in asymptomatic carriers. This raises challenges in evaluating the pathogenicity of SCN1A mutations. We systematically reviewed all SCN1A mutations and established a database containing information on functional alterations. In total, 1,257 mutations have been identified, of which 81.8% were not recurrent. There was a negative correlation between phenotype severity and missense mutation frequency. Further analyses suggested close relationships among genotype, functional alteration, and phenotype. Missense mutations located in different sodium channel regions were associated with distinct functional changes. Missense mutations in the pore region were characterized by the complete loss of function, similar to haploinsufficiency. Mutations with severe phenotypes were more frequently located in the pore region, suggesting that functional alterations are critical in evaluating pathogenicity and can be applied to patient management. A negative correlation was found between phenotype severity and familial incidence, and incomplete penetrance was associated with missense and splice site mutations, but not truncations or genomic rearrangements, suggesting clinical genetic counseling applications. Mosaic mutations with a load of 12.5-25.0% were potentially pathogenic with low penetrance, suggesting the need for future studies on less pathogenic genomic variations.

A novel variant in the 3' UTR of human SCN1A gene from a patient with Dravet syndrome decreases mRNA stability mediated by GAPDH's binding.

Mutations in the SCN1A gene-encoding voltage-gated sodium channel α-I subunit (Nav1.1) cause various spectrum of epilepsies including Dravet syndrome (DS), a severe and intractable form. A large number of SCN1A mutations identified from the DS patients lead to the loss of function or truncation of Nav1.1 that result in a haploinsufficiency effects, indicating that the exact expression level of SCN1A should be essential to maintain normal brain function. In this study, we have identified five variants c.*1025T>C, c.*1031A>T, c.*1739C>T, c.*1794C>T and c.*1961C>T in the SCN1A 3' UTR in the patients with DS. The c.*1025T>C, c.*1031A>T and c.*1794C>T are conserved among different species. Of all the five variants, only c.*1794C>T is a novel variant and alters the predicted secondary structure of the 3' UTR. We also show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) only binds to the 3' UTR sequence containing the mutation allele 1794U but not the wild-type allele 1794C, indicating that the mutation allele forms a new GAPDH-binding site. Functional analyses show that the variant negatively regulates the reporter gene expression by affecting the mRNA stability that is mediated by GAPDH's binding, and this phenomenon could be reversed by shRNA-induced GAPDH knockdown. These findings suggest that GAPDH and the 3'-UTR variant are involved in regulating SCN1A expression at post-transcriptional level, which may provide an important clue for further investigating on the relationship between 3'-UTR variants and SCN1A-related diseases.

Epilepsy-associated genes: an update.

PURPOSE: To provide an updated list of epilepsy-associated genes based on clinical-genetic evidence. METHODS: Epilepsy-associated genes were systematically searched and cross-checked from the OMIM, HGMD, and PubMed databases up to July 2023. To facilitate the reference for the epilepsy-associated genes that are potentially common in clinical practice, the epilepsy-associated genes were ranked by the mutation number in the HGMD database and by case number in the China Epilepsy Gene 1.0 project, which targeted common epilepsy. RESULTS: Based on the OMIM database, 1506 genes were identified to be associated with epilepsy and were classified into three categories according to their potential association with epilepsy or other abnormal phenotypes, including 168 epilepsy genes that were associated with epilepsies as pure or core symptoms, 364 genes that were associated with neurodevelopmental disorders as the main symptom and epilepsy, and 974 epilepsy-related genes that were associated with gross physical/systemic abnormalities accompanied by epilepsy/seizures. Among the epilepsy genes, 115 genes (68.5%) were associated with epileptic encephalopathy. After cross-checking with the HGMD and PubMed databases, an additional 1440 genes were listed as potential epilepsy-associated genes, of which 278 genes have been repeatedly identified variants in patients with epilepsy. The top 100 frequently reported/identified epilepsy-associated genes from the HGMD database and the China Epilepsy Gene 1.0 project were listed, among which 40 genes were identical in both sources. SIGNIFICANCE: Recognition of epilepsy-associated genes will facilitate genetic screening strategies and be helpful for precise molecular diagnosis and treatment of epilepsy in clinical practice.

Reprint of: Recessive APC2 missense variants associated with epilepsies without neurodevelopmental disorders.

OBJECTIVES: The APC2 gene, encoding adenomatous polyposis coli protein-2, is involved in cytoskeletal regulation in neurons responding to endogenous extracellular signals and plays an important role in brain development. Previously, the APC2 variants have been reported to be associated with cortical dysplasia and intellectual disability. This study aims to explore the association between APC2 variants and epilepsy. METHODS: Whole-exome sequencing (WES) was performed in cases (trios) with epilepsies of unknown causes. The damaging effects of variants were predicted by protein modeling and in silico tools. Previously reported APC2 variants were reviewed to analyze the genotype-phenotype correlations. RESULTS: Four pairs of compound heterozygous missense variants were identified in four unrelated patients with epilepsy without brain malformation/intellectual disability. All variants presented no or low allele frequencies in the controls. The missense variants were predicted to be damaging by silico tools, and affect hydrogen bonding with surrounding amino acids or decreased protein stability. Patients with variants that resulted in significant changes in protein stability exhibited more severe and intractable epilepsy, whereas patients with variants that had minor effect on protein stability exhibited relatively mild phenotypes. The previously reported APC2 variants in patients with complex cortical dysplasia with other brain malformations-10 (CDCBM10; MIM: 618677) were all truncating variants; in contrast, the variants identified in epilepsy in this study were all missense variants, suggesting a potential genotype-phenotype correlation. SIGNIFICANCE: This study suggests that APC2 is potentially associated with epilepsy without brain malformation/intellectual disability. The genotype-phenotype correlation helps to understand the underlying mechanisms of phenotypic heterogeneity.

Recessive APC2 missense variants associated with epilepsies without neurodevelopmental disorders.

OBJECTIVES: The APC2 gene, encoding adenomatous polyposis coli protein-2, is involved in cytoskeletal regulation in neurons responding to endogenous extracellular signals and plays an important role in brain development. Previously, the APC2 variants have been reported to be associated with cortical dysplasia and intellectual disability. This study aims to explore the association between APC2 variants and epilepsy. METHODS: Whole-exome sequencing (WES) was performed in cases (trios) with epilepsies of unknown causes. The damaging effects of variants were predicted by protein modeling and in silico tools. Previously reported APC2 variants were reviewed to analyze the genotype-phenotype correlations. RESULTS: Four pairs of compound heterozygous missense variants were identified in four unrelated patients with epilepsy without brain malformation/intellectual disability. All variants presented no or low allele frequencies in the controls. The missense variants were predicted to be damaging by silico tools, and affect hydrogen bonding with surrounding amino acids or decreased protein stability. Patients with variants that resulted in significant changes in protein stability exhibited more severe and intractable epilepsy, whereas patients with variants that had minor effect on protein stability exhibited relatively mild phenotypes. The previously reported APC2 variants in patients with complex cortical dysplasia with other brain malformations-10 (CDCBM10; MIM: 618677) were all truncating variants; in contrast, the variants identified in epilepsy in this study were all missense variants, suggesting a potential genotype-phenotype correlation. SIGNIFICANCE: This study suggests that APC2 is potentially associated with epilepsy without brain malformation/intellectual disability. The genotype-phenotype correlation helps to understand the underlying mechanisms of phenotypic heterogeneity.

Variants in BRWD3 associated with X-linked partial epilepsy without intellectual disability.

AIMS: Etiology of the majority patients with idiopathic partial epilepsy (IPE) remains elusive. We thus screened the potential disease-associated variants in the patients with IPE. METHODS: Trios-based whole exome sequencing was performed in a cohort of 320 patients with IPE. Frequency and molecular effects of variants were predicted. RESULTS: Three novel BRWD3 variants were identified in five unrelated cases with IPE, which were four male cases and one female case. The variants included two recurrent missense variants (c.836C>T/p.Thr279Ile and c.4234A>C/p.Ile1412Leu) and one intronic variant close to splice site (c.2475 + 6A>G). The two missense variants were located in WD40 repeat domain and bromodomain, respectively. They were predicted to be damaging by silico tools and change hydrogen bonds with surrounding amino acids. The frequency of mutant alleles in this cohort was significantly higher than that in the controls of East Asian and all population of gnomAD. All these variants were inherited from the asymptomatic mothers. Four male cases presented frequent seizures at onset, while the female case only had two fever-triggered seizures. They showed good responses to valproate and lamotrigine, then finally became seizure free. All the cases had no intellectual disability. Further analysis demonstrated that all previously reported destructive variants of BRWD3 caused intellectual disability, while missense variants located in WD40 repeat domains and bromodomains of BRWD3 were associated with epilepsy. CONCLUSION: BRWD3 gene is potentially associated with X-linked partial epilepsy without intellectual disability. The genotypes and locations of BRWD3 variants may explain for their phenotypic variation.

De novo GABRA1 variants in childhood epilepsies and the molecular subregional effects.

Background: The GABRA1 gene, encoding the GABRAR subunit α1, plays vital roles in inhibitory neurons. Previously, the GABRA1 gene has been identified to be associated with developmental and epileptic encephalopathy (DEE) and idiopathic generalized epilepsy (IGE). This study aims to explore the phenotypic spectrum of GABRA1 and molecular subregional effect analysis. Methods: Trios-based whole-exome sequencing was performed in patients with epilepsy. Previously reported GABRA1 mutations were systematically reviewed to analyze the molecular subregional effects. Results: De novo GABRA1 mutations were identified in six unrelated patients with heterogeneous epilepsy, including three missense mutations (p.His83Asn, p.Val207Phe, and p.Arg214Cys) and one frameshift mutation (p.Thr453Hisfs*47). The two missense mutations, p.His83Asn and p.Val207Phe, were predicted to decrease the protein stability but no hydrogen bond alteration, with which the two patients also presented with mild genetic epilepsy with febrile seizures plus and achieved seizure-free status by monotherapy. The missense variant p.Arg214Cys was predicted to decrease protein stability and destroy hydrogen bonds with surrounding residues, which was recurrently identified in three cases with severe DEE. The frameshift variant p.Thr453Hisfs*47 was located in the last fifth residue of the C-terminus and caused an extension of 47 amino acids, with which the patients presented with moderated epilepsy with generalized tonic-clonic seizures alone (GTCA) but achieved seizure-free status by four drugs. The four variants were not presented in gnomAD and were evaluated as "pathogenic/likely pathogenic" according to ACMG criteria. Analysis of all reported cases indicated that patients with mutations in the N-terminal extracellular region presented a significantly higher percentage of FS and DEE, and the patients with variants in the transmembrane region presented earlier seizure onset ages. Significance: This study suggested that GABRA1 variants were potentially associated with a spectrum of epilepsies, including EFS+, DEE, and GTCA. Phenotypic severity may be associated with the damaging effect of variants. The molecular subregional effects help in understanding the underlying mechanism of phenotypic variation.