Yue-Sheng Long

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.

Altered expression of voltage-gated potassium channel 4.2 and voltage-gated potassium channel 4-interacting protein, and changes in intracellular calcium levels following lithium-pilocarpine-induced status epilepticus

The A-type voltage-gated potassium channels (Kv4) have been proved to play a major role as modulators of somatodendritic excitability. Recent studies indicate that neuronal hyperactivity in epilepsy is associated with changes in Kv4. However, the precise regulation of Kv4 in the development of epilepsy and its underlying mechanism remain unclear. In this study, we investigated whether the expression of the Kv4.2 channel and of its major modulator, voltage-dependent potassium channel-interacting protein (KChIP1), is altered following lithium-pilocarpine induced status epilepticus (SE) and the chronic-epilepsy phase in the rat model. We found that Kv4.2 and KChIP1 expression was transiently up-regulated following SE, whereas it was down-regulated during the chronic phase: this was most prominent in the CA1 and CA3 regions. The time-course analysis of the protein expression level showed that the peak Kv4.2 up-regulation was between 6 and 24 h after SE, whereas KChIP1 expression was increased earlier and for a shorter period. The temporospatial changes in Kv4.2 were very similar to those of its major modulator KChIP1. We compared the difference in 4-aminopyridine (4-AP)-induced intracellular calcium ([Ca(2+)]i) elevation between model and control brain slices. The results showed that the [Ca(2+)]i elevation induced by the Kv4 channel blocker 4-AP was aggravated and prolonged in the model slice after SE. The functional relevance of these changes in Ca(2+) homeostasis and Kv4.2 and KChIP1 expression may be associated with intrinsic neuronal excitability regulation and epileptogenesis.

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high‐performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ∼25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ∼12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice‐site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.

Association of HLA‐B*1502 allele with lamotrigine‐induced Stevens–Johnson syndrome and toxic epidermal necrolysis in Han Chinese subjects: a meta‐analysis

Despite several studies investigating the association between the human leukocyte antigen HLA‐B*1502 allele and lamotrigine‐induced Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in Han Chinese subjects, the relationship remains unclear.

Milder phenotype with SCN1A truncation mutation other than SMEI.

Till now truncation mutations of voltage-gated sodium channel alpha subunit type I (SCN1A) gene were mostly found in severe myoclonic epilepsy of infancy (SMEI) patients. In this research we first identified two novel de novo truncation mutations (S662X and M145fx148) in two patients whose phenotypes were quite milder compared with SMEI patients. One patient was diagnosed as generalized epilepsy with febrile seizures plus (GEFS+); the other had focal seizures. Both patients had good response to anti-epileptic therapy (valproate or the combination of valproate and topiramate). Our findings extended the utility of the SCN1A gene testing and further confirmed the complex relationship between genotype and phenotype of SCN1A mutations. Further work is needed to optimize the protocol for specific genetic testing in children with epilepsy.

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 MBD2s 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 MBD2s 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.

A novel role of fragile X mental retardation protein in pre-mRNA alternative splicing through RNA-binding protein 14

Fragile X mental retardation protein (FMRP), an important RNA-binding protein responsible for fragile X syndrome, is involved in posttranscriptional control of gene expression that links with brain development and synaptic functions. Here, we reveal a novel role of FMRP in pre-mRNA alternative splicing, a general event of posttranscriptional regulation. Using co-immunoprecipitation and immunofluorescence assays, we identified that FMRP interacts with an alternative-splicing-associated protein RNA-binding protein 14 (RBM14) in a RNA-dependent fashion, and the two proteins partially colocalize in the nuclei of hippocampal neurons. We show that the relative skipping/inclusion ratio of the micro-exon L in the Protrudin gene and exon 10 in the Tau gene decreased in the hippocampus of Fmr1 knockout (KO) mice. Knockdown of either FMRP or RBM14 alters the relative skipping/inclusion ratio of Protrudin and Tau in cultured Neuro-2a cells, similar to that in the Fmr1 KO mice. Furthermore, overexpression of FMRP leads to an opposite pattern of the splicing, which can be offset by RBM14 knockdown. RNA immunoprecipitation assays indicate that FMRP promotes RBM14s binding to the mRNA targets. In addition, overexpression of the long form of Protrudin or the short form of Tau promotes protrusion growth of the retinoic acid-treated, neuronal-differentiated Neuro-2a cells. Together, these data suggest a novel function of FMRP in the regulation of pre-mRNA alternative splicing through RBM14 that may be associated with normal brain function and FMRP-related neurological disorders.

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.