title: Functional analysis of splicing mutations in exon 7 of NF1 gene.
abstract: BACKGROUND
  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.
  
  
  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 NF1 exon 7 (NF1DeltaE7). Both the wild type and the mutated constructs shared NF1DeltaE7 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 NF1DeltaE7, while in the presence of N335N variant, the NF1DeltaE7 expression is abolished.
  
  
  CONCLUSIONS
  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.
authors.affiliation: IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Bottillo
authors.forename: Alessandro
authors.initials: A
authors.lastname: De Luca
authors.forename: Annalisa
authors.initials: A
authors.lastname: Schirinzi
authors.forename: Valentina
authors.initials: V
authors.lastname: Guida
authors.forename: Isabella
authors.initials: I
authors.lastname: Torrente
authors.forename: Stefano
authors.initials: S
authors.lastname: Calvieri
authors.forename: Cristina
authors.initials: C
authors.lastname: Gervasini
authors.forename: Lidia
authors.initials: L
authors.lastname: Larizza
authors.forename: Antonio
authors.initials: A
authors.lastname: Pizzuti
authors.forename: Bruno
authors.initials: B
authors.lastname: Dallapiccola
mesh_terms: D017398:Alternative Splicing; D004252:DNA Mutational Analysis; D005091:Exons; D016514:Genes, Neurofibromatosis 1; D006801:Humans; D009456:Neurofibromatosis 1 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000235:genetics
chemical_list: D012322:RNA Precursors