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.

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.

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.

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.

HCFC1 variants in the proteolysis domain are associated with X-linked idiopathic partial epilepsy: Exploring the underlying mechanism.

BACKGROUND: HCFC1 encodes transcriptional co-regulator HCF-1, which undergoes an unusual proteolytic maturation at a centrally located proteolysis domain. HCFC1 variants were associated with X-linked cobalamin metabolism disorders and mental retardation-3. This study aimed to explore the role of HCFC1 variants in common epilepsy and the mechanism underlying phenotype heterogeneity. METHODS: Whole-exome sequencing was performed in a cohort of 313 patients with idiopathic partial (focal) epilepsy. Functional studies determined the effects of the variants on the proteolytic maturation of HCF-1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. RESULTS: We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability. CONCLUSION: HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF-1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub-molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.

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.

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.

BCOR variants are associated with X-linked recessive partial epilepsy.

OBJECTIVE: BCOR gene, encoding a corepressor of BCL6, plays an important role in fetal development. BCOR mutations were previously associated with oculofaciocardiodental syndrome (OFCD or MCOPS2, OMIM# 300166). The BCOR protein is ubiquitously expressed in multiple areas, including the brain. However, the role of BCOR in neurological disorder remains elusive. METHODS: Trios-based whole-exome sequencing was performed in a cohort of 323 cases with partial epilepsy without acquired causes. RESULTS: Seven hemizygous missense BCOR variants, including c 0.103 G>C/p.Asp35His, c.1079 A>G/p.His360Arg, c 0.1097 C>T/p.Thr366Ile, c 0.3301 C>T/p.Pro1101Ser, c 0.3391 C>T/p.Arg1131Trp, c 0.4199 G>A/p.Arg1400Gln, and c 0.5254 G>A/p.Asp1752Asn, were identified in seven cases with partial epilepsy. Two patients presented partial seizures with generalized seizures and/or generalized discharges. One case showed cortical dysplasia in the right temporal-occipital area on MRI. Two cases presented mild developmental delay. However, all patients achieved seizure-free. The frequency of BCOR variants in the present cohort was significantly higher than that in the controls of healthy Chinese volunteers and all populations of Genome Aggregation Database (gnomAD). Computational modeling, including hydrogen bond and prediction of protein stability, implied that the variants lead to structural impairment. Previously, OFCD associated BCOR mutations were mostly destructive mutations in an X-linked dominant (XLD) pattern; in contrast, the BCOR variants identified in this study were all missense variants, which were associated with partial epilepsy in an X-linked recessive (XLR) pattern. The proportion of missense mutations in epilepsy was significantly higher than that in OFCD. CONCLUSIONS: BCOR was potentially a candidate pathogenic gene of partial epilepsy with or without developmental delay. The genotype-phenotype correlation helps understanding the mechanism underlying phenotypic variation.

PRRT2 gene mutations associated with infantile convulsions induced by sucking and the genotype-phenotype correlation.

Introduction: PRRT2 is a major causative gene for self-limited familial neonatal-infantile epilepsy, paroxysmal kinesigenic dyskinesia, and paroxysmal kinesigenic dyskinesia with infantile convulsions. Voluntary movement trigger is prominent in adolescence and adulthood, but the triggers are unknown in infants. Methods: A gene panel designed for targeted next-generation sequencing (NGS) was used to screen genetic abnormalities in a cohort of 45 cases with infantile convulsions. The copy number variation was detected by a computational method based on the normalized depth of coverage and validated by a quantitative real-time polymerase chain reaction (RT-qPCR) method. The genotype-phenotype correlation of the PRRT2 mutation gene was analyzed. Results: A de novo heterozygous PRRT2 deletion was identified in a child who had infantile convulsions induced by vigorous sucking. Seizures happened during the change of feeding behavior from breast to formula, which led to hungry and vigorous sucking. Ictal electroencephalograms recorded seizures with focal origination, which provided direct evidence of epileptic seizures in infants with PRRT2 mutations. Seizures stopped soon after the feeding behavior was changed by reducing feeding interval time and extending feeding duration. Data reanalysis on our previously reported cases with PRRT2 mutations showed that six of 18 (33.3%) patients had infantile convulsions or infantile non-convulsion seizures during feeding. The mutations included two truncating mutations (c.579dupA/p.Glu194Argfs*6, and c.649dupC/p.Arg217Profs*8) that were identified in each of the three affected individuals. Conclusions: This study suggests that feeding, especially vigorous sucking, is potentially a trigger and highlights the significance of feeding behavior in preventing seizures in infants with PRRT2 mutations. Identification of PRRT2 haploinsufficiency mutations in the patients with infantile convulsions induced by sucking suggested a potential genotype-phenotype correlation.

SHROOM4 Variants Are Associated With X-Linked Epilepsy With Features of Generalized Seizures or Generalized Discharges.

Objective: SHROOM4 gene encodes an actin-binding proteins, which plays an important role in cytoskeletal architecture, synaptogenesis, and maintaining gamma-aminobutyric acid receptors-mediated inhibition. SHROOM4 mutations were reported in patients with the Stocco dos Santos type of X-linked syndromic intellectual developmental disorder (SDSX; OMIM# 300434). In this study, we investigated the association between SHROOM4 and epilepsy. Methods: Trios-based whole-exome sequencing was performed in a cohort of 320 cases with idiopathic generalized epilepsy or idiopathic partial epilepsy. Protein modeling was used to assess the damaging effects of variations. Results: Six hemizygous missense SHROOM4 variants, including c.13C > A/p. Pro5Thr, c.3236C > T/p.Glu1079Ala, c.3581C > T/p.Ser1194Leu, c.4288C > T/p.Arg1430Cys, c.4303G > A/p.Val1435Met, c.4331C > T/p.Pro1444Leu, were identified in six cases with idiopathic epilepsy without intellectual disability. All patients presented with features of generalized seizures or generalized discharges. These hemizygous variants had no or extremely low allele frequencies in controls and showed statistically higher frequency in the case cohort than controls. All variants were predicted to alter hydrogen bond with surrounding amino acids or decreased protein stability. The SHROOM4 variants reported in patients with SDSX were mostly destructive or duplicative variants; in contrast, the SHROOM4 variants were all missense variants, suggesting a potential genotype-phenotype correlation. The two missense variants associated with SDSX were located in the middle of SHROOM4 protein, whereas variants associated with idiopathic epilepsy were located around the N-terminal PDZ domain and the C-terminal ASD2 domain. Significance: SHROOM4 was potentially a candidate pathogenic gene of idiopathic epilepsy without intellectual disability. The genotype-phenotype correlation and sub-regional effect helps understanding the mechanism underlying phenotypic variation.

Recessive LAMA5 Variants Associated With Partial Epilepsy and Spasms in Infancy.

Objective: The LAMA5 gene encodes the laminin subunit α5, the most abundant laminin α subunit in the human brain. It forms heterotrimers with the subunit ß1/ß2 and γ1/γ3 and regulates neurodevelopmental processes. Genes encoding subunits of the laminin heterotrimers containing subunit α5 have been reported to be associated with human diseases. Among LAMAs encoding the laminin α subunit, LAMA1-4 have also been reported to be associated with human disease. In this study, we investigated the association between LAMA5 and epilepsy. Methods: Trios-based whole-exome sequencing was performed in a cohort of 118 infants suffering from focal seizures with or without spasms. Protein modeling was used to assess the damaging effects of variations. The LAMAs expression was analyzed with data from the GTEX and VarCards databases. Results: Six pairs of compound heterozygous missense variants in LAMA5 were identified in six unrelated patients. All affected individuals suffered from focal seizures with mild developmental delay, and three patients presented also spasms. These variants had no or low allele frequencies in controls and presented statistically higher frequency in the case cohort than in controls. The recessive burden analysis showed that recessive LAMA5 variants identified in this cohort were significantly more than the expected number in the East Asian population. Protein modeling showed that at least one variant in each pair of biallelic variants affected hydrogen bonds with surrounding amino acids. Among the biallelic variants in cases with only focal seizures, two variants of each pair were located in different structural domains or domains/links, whereas in the cases with spasms, the biallelic variants were constituted by two variants in the identical functional domains or both with hydrogen bond changes. Conclusion: Recessive LAMA5 variants were potentially associated with infant epilepsy. The establishment of the association between LAMA5 and epilepsy will facilitate the genetic diagnosis and management in patients with infant epilepsy.

CACNA1A Mutations Associated With Epilepsies and Their Molecular Sub-Regional Implications.

Purpose: Previously, mutations in the voltage-gated calcium channel subunit alpha1 A (CACNA1A) gene have been reported to be associated with paroxysmal disorders, typically as episodic ataxia type 2. To determine the relationship between CACNA1A and epilepsies and the role of molecular sub-regional on the phenotypic heterogeneity. Methods: Trio-based whole-exome sequencing was performed in 318 cases with partial epilepsy and 150 cases with generalized epilepsy. We then reviewed all previously reported CACNA1A mutations and analyzed the genotype-phenotype correlations with molecular sub-regional implications. Results: We identified 12 CACNA1A mutations in ten unrelated cases of epilepsy, including four de novo null mutations (c.2963_2964insG/p.Gly989Argfs*78, c.3089 + 1G > A, c.4755 + 1G > T, and c.6340-1G > A), four de novo missense mutations (c.203G > T/p.Arg68Leu, c.3965G > A/p.Gly1322Glu, c.5032C > T/p.Arg1678Cys, and c.5393C > T/p.Ser1798Leu), and two pairs of compound heterozygous missense mutations (c.4891A > G/p.Ile1631Val& c.5978C > T/p.Pro1993Leu and c.3233C > T/p.Ser1078Leu&c.6061G > A/p.Glu2021Lys). The eight de novo mutations were evaluated as pathogenic or likely pathogenic mutations according to the criteria of American College of Medical Genetics and Genomics (ACMG). The frequencies of the compound heterozygous CACNA1A mutations identified in this cohort were significantly higher than that in the controls of East Asian and all populations (P = 7.30 × 10-4, P = 2.53 × 10-4). All of the ten cases were ultimately seizure-free after antiepileptic treatment, although frequent epileptic seizures were observed in four cases. Further analysis revealed that episodic ataxia type 2 (EA2) had a tendency of higher frequency of null mutations than epilepsies. The missense mutations in severe epileptic phenotypes were more frequently located in the pore region than those in milder epileptic phenotypes (P = 1.67 × 10-4); de novo mutations in the epilepsy with intellectual disability (ID) had a higher percentage than those in the epilepsy without ID (P = 1.92 × 10-3). Conclusion: This study suggested that CACNA1A mutations were potentially associated with pure epilepsy and the spectrum of epileptic phenotypes potentially ranged from the mild form of epilepsies such as absence epilepsy or partial epilepsy, to the severe form of developmental epileptic encephalopathy. The clinical phenotypes variability is potentially associated with the molecular sub-regional of the mutations.

CELSR3 variants are associated with febrile seizures and epilepsy with antecedent febrile seizures.

AIMS: To identify novel pathogenic gene of febrile seizures (FS)/epilepsy with antecedent FS (EFS+). METHODS: The trio-based whole-exome sequencing was performed in a cohort of 462 cases with FS/EFS+. Silico programs, sequence alignment, and protein modeling were used to predict the damaging of variants. Statistical testing was performed to analyze gene-based burden of variants. RESULTS: Five heterozygous missense variants in CELSR3 were detected in five cases (families) with eight individuals (five females, three males) affected. Two variants were de novo, and three were identified in families with more than one individual affected. All the variants were predicted to be damaging in silico tools. Protein modeling showed that the variants resulted in disappearance of multiple hydrogen bonds and one disulfide bond, which potentially caused functional impairments of protein. The frequency of CELSR3 variants identified in this study was significantly higher than that in controls. All affected individuals were diagnosed with FS/EFS+, including six patients with FS and two patients with EFS+. All cases presented favorable outcomes without neurodevelopmental disorders. CONCLUSIONS: CELSR3 variants are potentially associated with FS/EFS+.

GRIN2A Variants Associated With Idiopathic Generalized Epilepsies.

Objective: The objective of this study is to explore the role of GRIN2A gene in idiopathic generalized epilepsies and the potential underlying mechanism for phenotypic variation. Methods: Whole-exome sequencing was performed in a cohort of 88 patients with idiopathic generalized epilepsies. Electro-physiological alterations of the recombinant N-methyl-D-aspartate receptors (NMDARs) containing GluN2A mutants were examined using two-electrode voltage-clamp recordings. The alterations of protein expression were detected by immunofluorescence staining and biotinylation. Previous studies reported that epilepsy related GRIN2A missense mutations were reviewed. The correlation among phenotypes, functional alterations, and molecular locations was analyzed. Results: Three novel heterozygous missense GRIN2A mutations (c.1770A > C/p.K590N, c.2636A > G/p.K879R, and c.3199C > T/p.R1067W) were identified in three unrelated cases. Electrophysiological analysis demonstrated R1067W significantly increased the current density of GluN1/GluN2A NMDARs. Immunofluorescence staining indicated GluN2A mutants had abundant distribution in the membrane and cytoplasm. Western blotting showed the ratios of surface and total expression of the three GluN2A-mutants were significantly increased comparing to the wild type. Further analysis on the reported missense mutations demonstrated that mutations with severe gain-of-function were associated with epileptic encephalopathy, while mutations with mild gain of function were associated with mild phenotypes, suggesting a quantitative correlation between gain-of-function and phenotypic severity. The mutations located around transmembrane domains were more frequently associated with severe phenotypes and absence seizure-related mutations were mostly located in carboxyl-terminal domain, suggesting molecular sub-regional effects. Significance: This study revealed GRIN2A gene was potentially a candidate pathogenic gene of idiopathic generalized epilepsies. The functional quantitative correlation and the molecular sub-regional implication of mutations helped in explaining the relatively mild clinical phenotypes and incomplete penetrance associated with GRIN2A variants.

HLA Risk Alleles in Aromatic Antiepileptic Drug-Induced Maculopapular Exanthema.

To characterize human leukocyte antigen (HLA) loci as risk factors in aromatic antiepileptic drug-induced maculopapular exanthema (AED-MPE). A case-control study was performed to investigate HLA loci involved in AED-MPE in a southern Han Chinese population. Between January 2007 and June 2019, 267 patients with carbamazepine (CBZ), oxcarbazepine (OXC), or lamotrigine (LTG) associated MPE and 387 matched drug-tolerant controls from six centers were enrolled. HLA-A/B/C/DRB1 genotypes were determined using sequence-based typing. Potential risk alleles were validated by meta-analysis using data from different populations and in silico analysis of protein-drug interactions. HLA-DRB1*04:06 was significantly associated with OXC-MPE (p = 0.002, p c = 0.04). HLA-B*38:02 was associated with CBZ-MPE (p = 0.03). When pooled, HLA-A*24:02, HLA-A*30:01, and HLA-B*35:01 additionally revealed significant association with AED-MPE. Logistic regression analysis showed a multiplicative interaction between HLA-A*24:02 and HLA-B*38:02 in CBZ-MPE. Meta-analysis of data from different populations revealed that HLA-24*:02 and HLA-A*30:01 were associated with AED-MPE (p = 0.02 and p = 0.04, respectively). In silico analysis of protein-drug interaction demonstrated that HLA-A*24:02 and HLA-A*30:01 had higher affinities with the three aromatic AEDs than the risk-free HLA-A allele. HLA-DRB1*04:06 showed relatively specific high affinity with S-monohydroxy derivative of OXC. HLA-DRB1*04:06 is a specific risk allele for OXC-induced MPE in the Southern Han Chinese. HLA-A*24:02, possibly HLA-A*30:01, are common risk factors for AED-MPE. The multiplicative risk potential between HLA-A*24:02 and HLA-B*38:02 suggests that patients with two risk alleles are at greater risk than those with one risk allele. Inclusion of these HLA alleles in pre-treatment screening would help estimating the risk of AED-MPE.

CHD4 variants are associated with childhood idiopathic epilepsy with sinus arrhythmia.

AIMS: CHD4 gene, encoding chromodomain helicase DNA-binding protein 4, is a vital gene for fetal development. In this study, we aimed to explore the association between CHD4 variants and idiopathic epilepsy. METHODS: Trios-based whole-exome sequencing was performed in a cohort of 482 patients with childhood idiopathic epilepsy. The Clinical Validity Framework of ClinGen and an evaluating method from five clinical-genetic aspects were used to determine the association between CHD4 variants and epilepsy. RESULTS: Four novel heterozygous missense mutations in CHD4, including two de novo mutations (c.1597A>G/p.K533E and c.4936G>A/p.E1646K) and two inherited mutations with co-segregation (c.856C>G/p.P286A and c.4977C>G/p.D1659E), were identified in four unrelated families with eight individuals affected. Seven affected individuals had sinus arrhythmia. From the molecular sub-regional point of view, the missense mutations located in the central regions from SNF2-like region to DUF1087 domain were associated with multisystem developmental disorders, while idiopathic epilepsy-related mutations were outside this region. Strong evidence from ClinGen Clinical Validity Framework and evidences from four of the five clinical-genetic aspects suggested an association between CHD4 variants and epilepsy. CONCLUSIONS: CHD4 was potentially a candidate pathogenic gene of childhood idiopathic epilepsy with arrhythmia. The molecular sub-regional effect of CHD4 mutations helped explaining the mechanisms underlying phenotypic variations.

RYR2 Mutations Are Associated With Benign Epilepsy of Childhood With Centrotemporal Spikes With or Without Arrhythmia.

RYR2 encodes ryanodine receptor 2 protein (RYR-2) that is mainly located on endoplasmic reticulum membrane and regulates intracellular calcium concentration. The RYR-2 protein is ubiquitously distributed and highly expressed in the heart and brain. Previous studies have identified the RYR2 mutations in the etiology of arrhythmogenic right ventricular dysplasia 2 and catecholaminergic polymorphic ventricular tachycardia. However, the relationship between RYR2 gene and epilepsy is not determined. In this study, we screened for novel genetic variants in a group of 292 cases (families) with benign epilepsy of childhood with centrotemporal spikes (BECTS) by trio-based whole-exome sequencing. RYR2 mutations were identified in five cases with BECTS, including one heterozygous frameshift mutation (c.14361dup/p.Arg4790Pro fs∗6), two heterozygous missense mutations (c.2353G > A/p.Asp785Asn and c.8574G > A/p.Met2858Ile), and two pairs of compound heterozygous mutations (c.4652A > G/p.Asn1551Ser and c.11693T > C/p.Ile3898Thr, c.7469T > C/p.Val2490Ala and c.12770G > A/p.Arg4257Gln, respectively). Asp785Asn was a de novo missense mutation. All the missense mutations were suggested to be damaging by at least three web-based prediction tools. These mutations do not present or at low minor allele frequency in gnomAD database and present statistically higher frequency in the cohort of BECTS than in the control populations of gnomAD. Asp785Asn, Asn1551Ser, and Ile3898Thr were predicted to affect hydrogen bonds with surrounding amino acids. Three affected individuals had arrhythmia (sinus arrhythmia and occasional atrial premature). The two probands with compound heterozygous missense mutations presented mild cardiac structural abnormalities. Strong evidence from ClinGen Clinical Validity Framework suggested an association between RYR2 variants and epilepsy. This study suggests that RYR2 gene is potentially a candidate pathogenic gene of BECTS. More attention should be paid to epilepsy patients with RYR2 mutations, which were associated with arrhythmia and sudden unexpected death in previous reports.

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.