Dataset Information

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. 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.

SUBMITTER: Bottillo I

PROVIDER: S-EPMC1802069 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Functional analysis of splicing mutations in exon 7 of NF1 gene.

Bottillo Irene I De Luca Alessandro A Schirinzi Annalisa A Guida Valentina V Torrente Isabella I Calvieri Stefano S Gervasini Cristina C Larizza Lidia L Pizzuti Antonio A Dallapiccola Bruno B

BMC medical genetics 20070212

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 17295913

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Project description:BackgroundAbnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay.ResultsWe analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion.ConclusionOur results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers.

Dataset Information

Functional analysis of splicing mutations in exon 7 of NF1 gene.

Publications

Functional analysis of splicing mutations in exon 7 of NF1 gene.

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