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.

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.

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.

Variants in BSN gene associated with epilepsy with favourable outcome.

BACKGROUND: BSN gene encodes Bassoon, an essential protein to assemble the cytomatrix at the active zone of neurotransmitter release. This study aims to explore the relationship between BSN variants and epilepsy. METHODS: Whole-exome sequencing was performed in a cohort of 313 cases (trios) with epilepsies of unknown causes. Additional cases with BSN variants were collected from China Epilepsy Gene V.1.0 Matching Platform. The Clinical Validity Framework of ClinGen was used to evaluate the relationship between BSN variants and epilepsy. RESULTS: Four pairs of compound heterozygous variants and one cosegregating heterozygous missense variant in BSN were identified in five unrelated families. These variants presented statistically higher frequency in the case cohort than in controls. Additional two de novo heterozygous nonsense variants and one cosegregating heterozygous missense variant were identified in three unrelated cases from the gene matching platform, which were not present in the Genome Aggregation Database. The missense variants tended to be located in C-terminus, including the two monoallelic missense variants. Protein modelling showed that at least one missense variant in each pair of compound heterozygous variants had hydrogen bond alterations. Clinically, two cases were diagnosed as idiopathic generalised epilepsy, two as focal epilepsy and the remaining four as epilepsy with febrile seizures plus. Seven out of eight probands showed infancy or childhood-onset epilepsy. Eight out of 10 affected individuals had a history of febrile convulsions. All the cases were seizure-free. The cases with monoallelic variants achieved seizure-free without treatment or under monotherapy, while cases with biallelic missense variants mostly required combined therapy. The evidence from ClinGen Framework suggested an association between BSN variants and epilepsy. CONCLUSION: The BSN gene was potentially a novel candidate gene for epilepsy. The phenotypical severity was associated with the genotypes and the molecular subregional effects of the variants.

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.

I-line photolithographic metalenses enabled by distributed optical proximity correction with a deep-learning model.

High pattern fidelity is paramount to the performance of metalenses and metasurfaces, but is difficult to achieve using economic photolithography technologies due to low resolutions and limited process windows of diverse subwavelength structures. These hurdles can be overcome by photomask sizing or reshaping, also known as optical proximity correction (OPC). However, the lithographic simulators critical to model-based OPC require precise calibration and have not yet been specifically developed for metasurface patterning. Here, we demonstrate an accurate lithographic model based on Hopkin's image formulation and fully convolutional networks (FCN) to control the critical dimension (CD) patterning of a near-infrared (NIR) metalens through a distributed OPC flow using i-line photolithography. The lithographic model achieves an average ΔCD/CD = 1.69% due to process variations. The model-based OPC successfully produces the 260 nm CD in a metalens layout, which corresponds to a lithographic constant k1 of 0.46 and is primarily limited by the resolution of the photoresist. Consequently, our fabricated NIR metalens with a diameter of 1.5 mm and numerical aperture (NA) of 0.45 achieves a measured focusing efficiency of 64%, which is close to the calculated value of 69% and among the highest reported values using i-line photolithography.

Potential role of regulatory DNA variants in modifying the risk of severe cutaneous reactions induced by aromatic anti-seizure medications.

OBJECTIVE: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions. Antiseizure medications (ASMs) with aromatic ring structure, including carbamazepine, are among the most common culprits. Screening for human leukocyte antigen (HLA) allele HLA-B*15:02 is recommended prior to initiating treatment with carbamazepine in Asians, but this allele has low positive predictive value. METHODS: We performed whole genome sequencing and analyzed 6 199 696 common variants among 113 aromatic ASM-induced SJS/TEN cases and 84 tolerant controls of Han Chinese ethnicity. RESULTS: In the primary analysis, nine variants reached genome-wide significance (p < 5e-08), one in the carbamazepine subanalysis (85 cases vs. 77 controls) and a further eight identified in HLA-B*15:02-negative subanalysis (35 cases and 53 controls). Interaction analysis between each novel variant from the primary analysis found that five increased risk irrespective of HLA-B*15:02 status or zygosity. HLA-B*15:02-positive individuals were found to have reduced risk if they also carried a chromosome 12 variant, chr12.9426934 (heterozygotes: relative risk = .71, p = .001; homozygotes: relative risk = .23, p < .001). All significant variants lie within intronic or intergenic regions with poorly understood functional consequence. In silico functional analysis of suggestive variants (p < 5e-6) identified through the primary and subanalyses (stratified by HLA-B*15:02 status and drug exposure) suggests that genetic variation within regulatory DNA may contribute to risk indirectly by disrupting the regulation of pathology-related genes. The genes implicated were specific either to the primary analysis (CD9), HLA-B*15:02 carriers (DOCK10), noncarriers (ABCA1), carbamazepine exposure (HLA-E), or phenytoin exposure (CD24). SIGNIFICANCE: We identified variants that could explain why some carriers of HLA-B*15:02 tolerate treatment, and why some noncarriers develop ASM-induced SJS/TEN. Additionally, this analysis suggests that the mixing of HLA-B*15:02 carrier status in previous studies might have masked variants contributing to susceptibility, and that inheritance of risk for ASM-induced SJS/TEN is complex, likely involving multiple risk variants.

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.

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

BACKGROUND: The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. METHODS: We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. RESULTS: Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. CONCLUSIONS: We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.

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.

[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.