Abstracts

Rationale: Mutations involving the voltage-gated sodium channel ? ? gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. In this study, we investigated the upstream regions of the SCN1A 5' non-coding exons to elucidate the clinical importance of the regulatory region of the SCN1A gene in patients with epilepsy.Methods: To identify the 5 ends of the human and mouse SCN1A transcripts, we performed RACE-PCR. To investigate whether the human genomic DNA fragments upstream of the major exons exerted promoter activity, dual luciferase reporter assays were performed. We also performed Northern blotting with probes for human non-coding exons to analyze the expression of those non-coding exons in various regions of the human brain. We performed multiplex ligation-dependent probe amplification (MLPA) assays to detect genomic deletions/duplications, using probes covering the 26 SCN1A coding exons and SCN1A 5 region. Total of 130 patients comprising 71 Dravet syndrome, nine generalized epilepsy and 50 focal epilepsy were screened. None showed any SCN1A mutations in prior sequence analyses of all 26 coding exons and exon-intron boundaries.Results: We firstly investigated the upstream regions of the SCN1A 5' non-coding exons and found that both human and mouse SCN1A contain two different major 5 exons, distal exon A and proximal exon B, respectively. Next we performed dual luciferase reporter assays, and showed that genomic fragments upstream of human exons hA and hB contain potential SCN1A promoter regions in both neuronal SH-SY5Y neuroblastoma and in non-neuronal kidney cells. Northern blot analysis showed that human exons hA and hB were simultaneously detected in several adult human brain regions, suggesting that these two major promoters were simultaneously active in various brain regions. Because the upstream genomic DNA sequences of SCN1A non-coding exons hA and hB are putative promoter regions, we postulated that heterozygous deletions involving these regions could reduce SCN1A transcription and result in epileptic seizures. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5 non-coding exons and regions with promoter activity but not affecting the coding exons. We also identified five patients with deletions/duplication in the coding region. The deletions involving the regions with SCN1A promoter activity were subsequently confirmed with long-range PCR or FISH methods. Finally, no aberrant peak signals for MLPA probes in the SCN1A 5' region were observed among 312 ethnically matched healthy controls, indicating that the deletions observed in this SCN1A 5' region were likely to be clinically significant.Conclusions: This study provides the first cases of microdeletion limited to the SCN1A 5 promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome.