Open Access Research article

Functional analysis of splicing mutations in exon 7 of NF1 gene

Irene Bottillo12, Alessandro De Luca12, Annalisa Schirinzi12, Valentina Guida1, Isabella Torrente1, Stefano Calvieri3, Cristina Gervasini4, Lidia Larizza4, Antonio Pizzuti12 and Bruno Dallapiccola12*

Author Affiliations

1 IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy

2 Department of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy

3 Department of Dermatology-Venereology and Plastic and Reconstructive Surgery, University of Rome "La Sapienza", Rome, Italy

4 Division of Medical Genetics, San Paolo School of Medicine, University of Milan, Milan, Italy

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BMC Medical Genetics 2007, 8:4  doi:10.1186/1471-2350-8-4

Published: 12 February 2007



Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals. NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to missplicing.


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.


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 NF1 exon 7 (NF1ΔE7). Both the wild type and the mutated constructs shared NF1ΔE7 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 NF1ΔE7, while in the presence of N335N variant, the NF1ΔE7 expression is abolished.


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.