Functional analysis of splicing mutations in exon 7 of NFI gene

Background: Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1: 3,500 individuals. NFI exon 7 displays weakly defined exon- intron boundaries, and is particularly prone to missplicing. Methods: In this study we investigated the expression of exon 7 transcripts using bioinformatic identification of splicing regulatory sequences, and functional minigene analysis of four sequence changes [ c. 910C > T ( R304X), c. 945G > A/ c. 946C > A ( Q315Q/ L316M), c. 1005T > C ( N335N)] identified in exon 7 of three different NF1 patients. Results: Our results detected the presence of three exonic splicing enhancers ( ESEs) and one putative exonic splicing silencer ( ESS) element. The wild type minigene assay resulted in three alternative isoforms, including a transcript lacking NFI exon 7 ( NFI Delta E7). Both the wild type and the mutated constructs shared NFI Delta E7 in addition to the complete messenger, but displayed a different ratio between the two transcripts. In the presence of R304X and Q315Q/ L316M mutations, the relative proportion between the different isoforms is shifted toward the expression of NFI Delta E7, while in the presence of N335N variant, the NFI Delta E7 expression is abolished. Conclusion: In conclusion, it appears mandatory to investigate the role of each nucleotide change within the NF1 coding sequence, since a significant proportion of NF1 exon 7 mutations affects pre-mRNA splicing, by disrupting exonic splicing motifs and modifying the delicate balance between aberrantly and correctly spliced transcripts.