Differences in SCN1A intronic variants result in diverse aberrant splicing patterns and are related to the phenotypes of epilepsy with febrile seizures.

Abstract Objective Intronic variants of the SCN1A gene are detected in patients with epilepsy with febrile seizures (EFS), which includes a series of phenotypes with different severities. However, the pathogenicity of intronic variants and their genotype-phenotype correlation remain under characterized. The purpose of this study was to determine the changes in mRNA splicing caused by SCN1A intronic variants associated with EFS and their association with phenotypes. Methods Five SCN1A intronic variants detected in patients with focal epilepsy with antecedent febrile seizures plus (FEFS+) and Dravet syndrome (DS) were molecularly cloned. Through an in vitro minigene splicing assay, their influence on mRNA splicing was qualitatively and quantitatively compared and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Q-PCR). Results The severe phenotype of DS-associated variants c.602 + 1G > A and c.4853-1G > C, which occurred in canonical splice sites of introns, caused exon skipping and little retention of full-length mRNA, while the milder phenotype of FEFS+-associated variants c.473 + 5G > A, c.473 + 5G > C and c.4853-25T > A, which occurred in potential splice sites or in deep intronic regions, presented partial exon skipping or intronic insertion and significantly higher retention of full-length mRNA at different levels. Full-length mRNA retention was negatively correlated with the location of intronic variants and phenotype severity. Conclusion The different aberrant splicing patterns resulting from SCN1A intronic variants with different positions represent a potential molecular mechanism for phenotypic differences in EFS. This research provides valuable clues for functional studies on the pathogenicity of intronic variants and for the evaluation of genotype-phenotype correlations.