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Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 ( NF1 ) due to intronic sequence exonization
Author(s) -
Raponi M.,
Upadhyaya M.,
Baralle D.
Publication year - 2006
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.9412
Subject(s) - minigene , biology , rna splicing , intron , genetics , exon , transition (genetics) , mutation , splice site mutation , neurofibromatosis , exonic splicing enhancer , splice , microbiology and biotechnology , alternative splicing , gene , rna
Genomic variations with no apparent effect (“neutral polymorphisms”) may have a significant effect on splicing. The effect of this type of mutation is difficult to spot, unless a functional assay is undertaken. In our study, DNA sequencing of a patient with clinically defined neurofibromatosis type 1 (NF1) showed only a single polymorphism in intron 30 due to an A>G transition 279 nucleotides from the 3' splice site. Using a minigene splicing assay we conclusively show that this change produces a cryptic exon with a 3' SS defined by the nucleotide change and the unexpected activation of a very weak 5'SS. Further site directed mutagenesis studies aimed at identifying the signals involved in the cryptic exon inclusion were carried out. Interestingly we find that particular characteristics of the cryptic 5' SS are essential for its inclusion. Significantly an additional single nucleotide change disrupting the cryptic 5'ss consensus sequence rescues the effect of the pathogenetic mutation resulting in normal splicing. © 2006 Wiley‐Liss, Inc.