Functional Analysis of the C.3705+5G>C Mutation in the SCN1A Gene:Cryptic Splicing Site Activation and Partial Exon Skipping

Several substitutions in Voltage-gated sodium channels (SCN1A) gene have been reported to cause aberrant splicing. Accordingly, this study aimed to investigate the potential effects of a transition in the fifth nucleotide at the donor splice site of intron 18 of the SCN1A gene previously described leading to SIGEI phenotype. Functional analyses using PCR mutagenesis, followed by an ex-vivo splicing assays, revealed that the c.3705+5Gu003eC mutation leads to the activation of a cryptic site into exon 18 leading to a partial exon skipping followed by a premature stop codon at position 1253 in the SCN1A protein. Bioinformatic tools showed an homology between cryptic and normal splicing consensus especially at position - 3, -2, -1, +1, +2 and +5; confirming the crucial role of these positions in the exon definition and explains the strength of the novel donor consensus. This analysis revealed also the enrichment of regions close to the new splice site in ESEs elements with high scores underlining the importance of ESEmediated SR protein function for accurate new splice site recognition. Our results demonstrate that splicing analysis of mRNA may help to understand both the functional consequences of mutations affected splicing consensus and the correlation between genotype and phenotype.