Yi-Wu Shi

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 Nav1.1 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 (P2.5, from +26 to –2,500) and the 2.7‐kb fragment (P2.7, P2.5 combined with the 227‐bp 5′‐untranslated exons) were cloned to produce luciferase constructs. The P2.5 and the P2.7 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 P2.7 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.

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

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

Electrophysiological Differences between the Same Pore Region Mutation in SCN1A and SCN3A

Mutations in the sodium channel gene, SCN1A (NaV1.1), have been linked to a spectrum of epilepsy syndromes, and many of these mutations occur in the pore region of the channel. Electrophysiological characterization has revealed that most SCN1A mutations in the pore region result in complete loss of function. SCN3A mutations have also been identified in patients with epilepsy; however, mutations in this pore region maintain some degree of electrophysiological function. It is thus speculated that compared to SCN3A disruptions, SCN1A mutations have a more pronounced effect on electrophysiological function. In this study, we identified a novel mutation, N302S, in the SCN3A pore region of a child with epilepsy. To investigate if mutations at the pore regions of SCN3A and SCN1A have different impacts on channel function, we studied the electrophysiological properties of N302S in NaV1.3 and its homologous mutation (with the same amino acid substitution) in NaV1.1 (N301S). Functional analysis demonstrated that SCN1A-N301S had no measurable sodium current, indicating a complete loss of function, while SCN3A-N302S slightly reduced channel activity. This observation indicates that the same pore region mutation affects SCN1A more than SCN3A. Our study further revealed a huge difference in electrophysiological function between SCN1A and SCN3A mutations in the pore region; this might explain the more common SCN1A mutations detected in patients with epilepsy and the more severe phenotypes associated with these mutations.

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 (Nav1.3) is mainly expressed in the central nervous system and is associated with neurological disorders. The expression of mouse Scn3a product (Nav1.3) 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.

Ion Channel Genes and Epilepsy: Functional Alteration, Pathogenic Potential, and Mechanism of Epilepsy

Ion channels are crucial in the generation and modulation of excitability in the nervous system and have been implicated in human epilepsy. Forty-one epilepsy-associated ion channel genes and their mutations are systematically reviewed. In this paper, we analyzed the genotypes, functional alterations (funotypes), and phenotypes of these mutations. Eleven genes featured loss-of-function mutations and six had gain-of-function mutations. Nine genes displayed diversified funotypes, among which a distinct funotype-phenotype correlation was found in SCN1A. These data suggest that the funotype is an essential consideration in evaluating the pathogenicity of mutations and a distinct funotype or funotype-phenotype correlation helps to define the pathogenic potential of a gene.

Novel mutations and phenotypes of epilepsy-associated genes in epileptic encephalopathies

Epileptic encephalopathies are severe epilepsy disorders with strong genetic bases. We performed targeted next‐generation sequencing (NGS) in 70 patients with epileptic encephalopathies. The likely pathogenicity of variants in candidate genes was evaluated by American College of Medical Genetics and Genomics (ACMG) scoring taken together with the accepted clinical presentation. Thirty‐three candidate variants were detected after population filtration and computational prediction. According to ACMG, 21 candidate variants, including 18 de novo variants, were assessed to be pathogenic/likely pathogenic with clinical concordance. Twelve variants were initially assessed as uncertain significance by ACMG, among which 3 were considered causative and 3 others were considered possibly causative after analysis of clinical concordance. In total, 24 variants were identified as putatively causative, among which 19 were novel findings. SCN1A mutations were identified in 50% of patients with Dravet syndrome. TSC1/TSC2 mutations were detected in 66.7% of patients with tuberous sclerosis. STXBP1 mutations were the main findings in patients with West syndrome. Mutations in SCN2A, KCNT1, KCNQ2 and CLCN4 were identified in patients with epileptic infantile with migrating focal seizures; among them, KCNQ2 and CLCN4 were first identified as potential causative genes. Only one CHD2 mutation was detected in patients with Lennox‐Gastaut syndrome. This study highlighted the utility of targeted NGS in genetic diagnoses of epileptic encephalopathies and a comprehensive evaluation of the pathogenicity of variants based on ACMG scoring and assessment of clinical concordance. Epileptic encephalopathies differ in genetic causes, and the genotype‐phenotype correlations would provide insights into the underlying pathogenic mechanisms.

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.868Cxa0>xa0T/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.

A Point Mutation in SCN1A 5′ Genomic Region Decreases the Promoter Activity and Is Associated with Mild Epilepsy and Seizure Aggravation Induced by Antiepileptic Drug

The SCN1A gene with 1274 point mutations in the coding regions or genomic rearrangements is the most clinically relevant epilepsy gene. Recent studies have demonstrated that variations in the noncoding regions are potentially associated with epilepsies, but no distinct mutation has been reported. We sequenced the 5′ upstream region of SCN1A in 166 patients with epilepsy and febrile seizures who were negative for point mutations in the coding regions or genomic rearrangements. A heterozygous mutation h1u-1962xa0Tu2009>u2009G was identified in a patient with partial epilepsy and febrile seizures, which was aggravated by oxcarbazepine. This mutation was transmitted from the patient’s asymptomatic mother and not found in the 110 normal controls. h1u-1962xa0Tu2009>u2009G was located upstream the most frequently used noncoding exon and within the promoter sequences. Further experiments showed that this mutation decreased the promoter activity by 42.1xa0% compared with that of the paired haplotype (Pu2009<u20090.001). In contrast to the null expression that results in haploinsufficiency and severe phenotype, this mutation caused relatively less impairment, explaining the mild epilepsy with incomplete penetrance. The antiepileptic drug-induced seizure aggravation in this patient suggests clinical attention for mutations or variations in noncoding regions that may affect SCN1A expression.

Few individuals with Lennox-Gastaut syndrome have autism spectrum disorder: a comparison with Dravet syndrome

BackgroundAutism spectrum disorder (ASD) in epilepsy has been a topic of increasing interest, which in general occurs in 15–35% of the patients with epilepsy, more frequently in those with intellectual disability (ID). Lennox-Gastaut syndrome (LGS) and Dravet syndrome (DS) are two typical forms of intractable epileptic encephalopathy associated with ID. We previously reported that ASD was diagnosed in 24.3% of patients with DS, higher in those with profound ID. Given the severe epilepsy and high frequency of ID in LGS, it is necessary to know whether ASD is a common psychomotor co-morbidity of LGS. This study evaluated the autistic behaviors and intelligence in patients with LGS and further compared that between LGS and DS, aiming to understand the complex pathogenesis of epilepsy-ASD-ID triad.MethodsA total of 50 patients with LGS and 45 patients with DS were enrolled and followed up for at least 3xa0years. The clinical characteristics were analyzed, and evaluations of ASD and ID were performed.ResultsNo patients with LGS fully met the diagnostic criteria for ASD, but three of them exhibited more or less autistic behaviors. Majority (86%) of LGS patients presented ID, among which moderate to severe ID was the most common. Early onset age and symptomatic etiology were risk predictors for ID. The prevalence of ASD in LGS was significantly lower than that in DS (0/50 vs. 10/45, pu2009<u20090.001), while the prevalence and severity of ID showed no significant difference between the two forms of epileptic encephalopathy.ConclusionsThis study demonstrated a significant difference in the co-morbidity of ASD between LGS and DS, although they had a similar prevalence and severity of ID, refuting the proposal that the prevalence of ASD in epilepsy is accounted for by ID. These findings suggest that the co-morbidity of ASD, ID, and epilepsy may result from multifaceted pathogenic mechanisms.