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.

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.

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.

DEPDC5 Variants Associated Malformations of Cortical Development and Focal Epilepsy With Febrile Seizure Plus/Febrile Seizures: The Role of Molecular Sub-Regional Effect.

To explore the phenotype spectrum of DEPDC5 variants and the possible mechanisms underlying phenotypical variation, we performed targeted next-generation sequencing in 305 patients with focal epilepsies and 91 patients with generalized epilepsies. Protein modeling was performed to predict the effects of missense mutations. All previously reported epilepsy-related DEPDC5 variants were reviewed. The genotype-phenotype correlations with molecular sub-regional implications were analyzed. We identified a homozygous DEPDC5 mutation (p.Pro1031His) in a case with focal cortical dysplasia and eight heterozygous mutations in 11 families with mild focal epilepsies, including 13 patients in eight families with focal epilepsy with febrile seizures plus/febrile seizures (FEFS + /FS). The mutations included one termination codon mutation (p.Ser1601_Ter1604del_ext133), three truncating mutations (p.Val151Serfs∗27, p.Arg239∗, and p.Arg838∗), and four missense mutations (p.Tyr7Cys, p.Tyr836Cys, p.Pro1031His, and p.Gly1545Ser) that were predicted to affect hydrogen bonds and protein stability. Analysis on epilepsy-related DEPDC5 variants revealed that malformations of cortical development (MCDs) had a tendency of higher frequency of null mutations than those without MCD. MCD-associated heterozygous missense mutations were clustered in structural axis for binding arrangement (SABA) domain and close to the binding sites to NPRL2/NPRL3 complex, whereas those associated with FEFS + /FS were a distance away from the binding sites. Evidence from four aspects and one possible evidence from sub-regional implication suggested MCD and FEFS + /FS as phenotypes of DEPDC5 variants. This study suggested that the phenotypes of DEPDC5 variants vary from mild FEFS + /FS to severe MCD. Heterozygous DEPDC5 mutations are generally less pathogenic and commonly associated with mild phenotypes. Bi-allelic mutations and second hit of somatic mutations, together with the genotype-phenotype correlation and sub-regional implication of DEPDC5 variants, explain severe phenotypes.

A Species-Correlated Transitional Residue D132 on Human FMRP Plays a Role in Nuclear Localization via an RNA-Dependent Interaction With PABP1.

Fragile X mental retardation protein (FMRP), a key determinant of normal brain development and neuronal plasticity, plays critical roles in nucleocytoplasmic shuttling of mRNAs. However, the factors involved in FMRP nuclear localization remain to be determined. Using cross-species sequence comparison, we show that an aspartate in position 132 (D132), located within the conserved nuclear localization signal (NLS) of FMRP, appears in human and other mammals, while glutamate 132 (E132) appears in rodents and birds. Human FMRP-D132E alters the secondary structure of the protein and reduces its nuclear localization, while the reciprocal substitution in mouse FMRP-E132D promotes its nuclear localization. Human FMRP could interact with poly(A)-binding protein 1 (PABP1) which is impeded by the D132E mutation. Reversely, mouse FMRP could not interact with PABP1, but the E132D mutation leads to the FMRP-PABP1 interaction. We further show that overexpression of human FMRP-D132E mutant promotes the formation of cytoplasmic aggregates of PABP1 in human cells, but not of mouse FMRP-E132D in mouse cells. PABP1 knockdown reduces the nuclear localization of human FMRP, but not mouse FMRP. Furthermore, RNase A treatment decreases the PABP1 levels in the anti-V5-immunoprecipitates using the V5-hFMRP-transfected cells, suggesting an interaction between human FMRP and PABP1 in an RNA-dependent fashion. Thus, our data suggest that the FMRP protein with the human-used D132 accommodates a novel protein-RNA-protein interaction which may implicate a connection between FMRP residue transition and neural evolution.

Development-related aberrations in Kv1.1 α-subunit exert disruptive effects on bioelectrical activities of neurons in a mouse model of fragile X syndrome.

Kv1.1, a Shaker homologue potassium channel, plays a critical role in homeostatic regulation of neuronal excitability. Aberrations in the functional properties of Kv1.1 have been implicated in several neurological disorders featured by neuronal hyperexcitability. Fragile X syndrome (FXS), the most common form of inherited mental retardation, is characterized by hyperexcitability in neural network and intrinsic membrane properties. The Kv1.1 channel provides an intriguing mechanistic candidate for FXS. We investigated the development-related expression pattern of the Kv1.1 α-subunit by using a Fmr1 knockout (KO) mouse model of FXS. Markedly decreased protein expression of Kv1.1 was found in neonatal and adult stages when compared to age-matched wild-type (WT) mice. Immunohistochemical investigations supported the delayed development-related increases in Kv1.1 expression, especially in CA3 pyramidal neurons. By applying a Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), we isolated the Kv1.1-mediated currents in the CA3 pyramidal neurons. The isolated DTX-κ-sensitive current of neurons from KO mice exhibited decreased amplitude, lower threshold of activation, and faster recovery from inactivation. The equivalent reduction in potassium current in the WT neurons following application of the appropriate amount of DTX-κ reproduced the enhanced firing abilities of KO neurons, suggesting the Kv1.1 channel as a critical contributor to the hyperexcitability of KO neurons. The role of Kv1.1 in controlling neuronal discharges was further supported by the parallel developmental trajectories of Kv1.1 expression, current amplitude, and discharge impacts, with a significant correlation between the amplitude of Kv1.1-mediated currents and Kv1.1-blocking-induced firing enhancement. These data suggest that the expression of the Kv1.1 α-subunit has a profound pathological relevance to hyperexcitability in FXS, as well as implications for normal development, maintenance, and control of neuronal activities.

[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.

Epigenetic Downregulation of Scn3a Expression by Valproate: a Possible Role in Its Anticonvulsant Activity.

Upregulation of sodium channel SCN3A expression in epileptic tissues is known to contribute to enhancing neuronal excitability and the development of epilepsy. Therefore, certain strategies to reduce SCN3A expression may be helpful for seizure control. Here, we reveal a novel role of valproate (VPA) in the epigenetic downregulation of Scn3a expression. We found that VPA, instead of carbamazepine (CBZ) and lamotrigine (LTG), could significantly downregulate Scn3a expression in mouse Neuro-2a cells. Luciferase assays and CpG methylation analyses showed that VPA induced the methylation at the -39C site in Scn3a promoter which decreased the promoter activity. We further showed that VPA downregulated the expression of methyl-CpG-binding domain protein 2 (MBD2) at the posttranscriptional level and knockdown of MBD2 increased Scn3a expression. In addition, we found that VPA induced the expression of fat mass and obesity-associated (FTO) protein and FTO knockdown abolished the repressive effects of VPA on MBD2 and Nav1.3 expressions. Furthermore, VPA, instead of other two anticonvulsant drugs, induced the expressions of Scn3a and Mbd2 and reduced Fto expression in the hippocampus of VPA-treated seizure mice. Taken together, this study suggests an epigenetic pathway for the VPA-induced downregulation of Scn3a expression, which provides a possible role of this pathway in the anticonvulsant action of VPA.

Involvement of FMRP in Primary MicroRNA Processing via Enhancing Drosha Translation.

Fragile X mental retardation protein (FMRP), associated with fragile X syndrome, is known as an RNA-binding protein to regulate gene expression at post-transcriptional level in the brain. FMRP is also involved in microRNA (miRNA) biogenesis during the process of precursor miRNA (pre-miRNA) into mature miRNA. However, there is no description of the effect of FMRP on primary miRNA (pri-miRNA) processing. Here, we uncover a novel role of FMRP in pri-miRNA processing via controlling Drosha translation. We show that the expression of DROSHA protein, instead of its messenger RNA (mRNA) transcripts, is downregulated in both the hippocampus of Fmr1-knockout mice and the FMRP-knockdown Neuro-2a cells. Overexpression or knockdown FMRP does not alter Drosha mRNA stability. Immunoprecipitation and polysome analyses demonstrate that FMRP binds to the Drosha mRNA and enhances its translation. Additionally, we show that loss of FMRP in Fmr1-deficient mice results in the accumulation of three in six analyzed pri-miRNAs and the reduction of the corresponding pre-miRNAs and mature miRNAs. Thus, our data suggest that FMRP is involved in pri-miRNA processing via enhancing DROSHA expression that may play an important role in fragile X syndrome.

GAPDH-mediated posttranscriptional regulations of sodium channel Scn1a and Scn3a genes under seizure and ketogenic diet conditions.

Abnormal expressions of sodium channel SCN1A and SCN3A genes alter neural excitability that are believed to contribute to the pathogenesis of epilepsy, a long-term risk of recurrent seizures. Ketogenic diet (KD), a high-fat and low-carbohydrate treatment for difficult-to-control (refractory) epilepsy in children, has been suggested to reverse gene expression patterns. Here, we reveal a novel role of GAPDH on the posttranscriptional regulation of mouse Scn1a and Scn3a expressions under seizure and KD conditions. We show that GAPDH binds to a conserved region in the 3' UTRs of human and mouse SCN1A and SCN3A genes, which decreases and increases genes' expressions by affecting mRNA stability through SCN1A 3' UTR and SCN3A 3' UTR, respectively. In seizure mice, the upregulation and phosphorylation of GAPDH enhance its binding to the 3' UTR, which lead to downregulation of Scn1a and upregulation of Scn3a. Furthermore, administration of KD generates ß-hydroxybutyric acid which rescues the abnormal expressions of Scn1a and Scn3a by weakening the GAPDH's binding to the element. Taken together, these data suggest that GAPDH-mediated expression regulation of sodium channel genes may be associated with epilepsy and the anticonvulsant action of KD.

A MicroRNA Profile in Fmr1 Knockout Mice Reveals MicroRNA Expression Alterations with Possible Roles in Fragile X Syndrome.

Fragile X syndrome (FXS), a common form of inherited mental retardation, is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is involved in brain functions by interacting with mRNAs and microRNAs (miRNAs) that selectively control gene expression at translational level. However, little is known about the role of FMRP in regulating miRNA expression. Here, we found a development-dependant dynamic expression of Fmr1 gene (encoding FMRP) in mouse hippocampus with a small peak at postnatal day 7 (P7). MiRNA microarray analysis showed that the levels of 38 miRNAs showed a significant increase with about 15 ~ 250-folds and the levels of 26 miRNAs showed a significant decrease with only about 2 ~ 4-folds in the hippocampus of P7 Fmr1 knockout (KO) mice. The qRT-PCR assay showed that nine of the most increased miRNAs (>100-folds in microarrays) increased about 40 ~ 70-folds and their pre-miRNAs increased about 5 ~ 10-folds, but no significant difference in their pri-miRNA levels was observed, suggesting that the alterations of partial miRNAs are an indirect consequence of FMRP lacking. We further demonstrated that a set of protein-coding mRNAs, potentially targeted by the nine miRNAs, were down-regulated in the hippocampus of Fmr1 KO mice. Finally, luciferase assays demonstrated that miR-34b, miR-340, and miR-148a could down-regulate the reporter gene expression by interacting with the Met 3' UTR. Taken together, these findings suggest that the miRNA expression alterations resulted from the absence of FMRP might contribute to molecular pathology of FXS.

Transcription of the human sodium channel SCN1A gene is repressed by a scaffolding protein RACK1.

Voltage-gated sodium channel α subunit type I (Nav1.1, encoded by SCN1A gene) plays a critical role in the initiation of action potential in the central nervous system. Downregulated expression of SCN1A is believed to be associated with epilepsy. Here, we found that the SCN1A promoter (P1c), located at the 5' untranslated exon 1c, drove the reporter gene expression in human NT2 cells, and a region between nt +53 and +62 downstream of the P1c promoter repressed the promoter activity. Further analyses showed that a scaffolding protein RACK1 (receptor for activated C kinase 1) was involved in binding to this silencer. Knockdown of RACK1 expression in NT2 cells deprived the repressive role of the silencer on the P1c promoter and increased SCN1A transcription, suggesting the potential involvement of RACK1 in negatively regulating SCN1A transcription via interaction with the silencer. Furthermore, we demonstrated that the binding of the protein complex including RACK1 to the SCN1A promoter motif was decreased in neuron-like differentiation of the NT2 cells induced by retinoic acid and resulted in the upregulation of SCN1A transcription. Taken together, this study reports a novel role of RACK1 in regulating SCN1A expression that participates in retinoic acid-induced neuronal differentiation of NT2 cells.

Human transcription factor genes involved in neuronal development tend to have high GC content and CpG elements in the proximal promoter region.

Transcription factors (TFs) play critical roles in the development of the nervous system, but the transcriptional regulatory mechanisms of these genes are poorly understood. Here we analyzed 5-kb of the 5' flanking genomic DNA sequences of 41 TF genes involved in neuronal development. The results showed that the TF genes tend to have higher GC contents in the proximal region and most of the TF genes have at least one proximal GC-rich (GC content > 60%) promoter with a CpG island. The promoter distribution analysis showed that the GC-poor promoters were sporadically distributed within the 5-kb flanking genomic sequence (FGS); however, more than half (37 of 70) of the GC-rich promoters were located in the proximal region between nucleotides -1 and -500. Luciferase assays showed that partial GC-rich promoters increased gene expression in SH-SY5Y cells and that CpG methylation repressed the promoter activity. This study suggests a potential general mechanism for regulation of TF expression.

Promoter analysis of mouse Scn3a gene and regulation of the promoter activity by GC box and CpG methylation.

Voltage-gated sodium channel α-subunit type III (Na(v)1.3) is mainly expressed in the central nervous system and is associated with neurological disorders. The expression of mouse Scn3a product (Na(v)1.3) mainly occurs in embryonic and early postnatal brain but not in adult brain. Here, we report for the first time the identification and characterization of the mouse Scn3a gene promoter region and regulation of the promoter activity by GC box and CpG methylation. Luciferase assay showed that the promoter region F1.2 (nt -1,049 to +157) had significantly higher activity in PC12 cells, comparing with that in SH-SY5Y cells and HEK293 cells. A stepwise 5' truncation of the promoter region found that the minimal functional promoter located within the region nt -168 to +157. Deletion of a GC box (nt -254 to -258) in the mouse Scn3a promoter decreased the promoter activity. CpG methylation of the F1.2 without the GC box completely repressed the promoter activity, suggesting that the GC box is a critical element in the CpG-methylated Scn3a promoter. These results suggest that the GC box and CpG methylation might play important roles in regulating mouse Scn3a gene expression.

Identification of the promoter region and the 5'-untranslated exons of the human voltage-gated sodium channel Nav1.1 gene (SCN1A) and enhancement of gene expression by the 5'-untranslated exons.

Voltage-gated sodium channels play critical roles in the excitability of the brain. A decreased level of Na(v)1.1 has been identified as the cause of severe myoclonic epilepsy in infancy. In the present study, we identified the transcription start site and three 5'-untranslated exons of SCN1A by using 5'-full RACE. The 2.5-kb region upstream of the transcription start site was targeted as a potential location of the promoter. The 2.5-kb genomic fragment (P(2.5), from +26 to -2,500) and the 2.7-kb fragment (P(2.7), P(2.5) combined with the 227-bp 5'-untranslated exons) were cloned to produce luciferase constructs. The P(2.5) and the P(2.7) drove luciferase gene expression in the human neuroblastoma cell line SH-SY5Y but not in the human embryonic kidney cell line HEK-293. The 5'-untranslated exons could greatly enhance gene expression in SH-SY5Y cells. The P(2.7) could be used as a functional unit to study the role of SCN1A noncoding sequences in gene expression. These findings will also help in exploring the possibility of promoter mutant-induced diseases and revealing the mechanism underlying the regulation of SCN1A expression in the normal brain.