Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6

Michela Raponi (1, 2) , Jana Kralovicova (2) , Ellen Copson (2) , Petr Divina (3) , Diana M. Eccles (2, 4) , Peter Johnson (2) , Diana Baralle (2, 5) , Igor Vorechovsky (6)

1 Human Genetics Division [Southampton]
2 Genetics
3 Bioinformatics
4 Wessex Clinical Genetics Service
5 Medical Genetics
6 Human Genetics

Missense, nonsense and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency amongst disease-causing exonic substitutions is unknown. Here, we have tested missense and silent mutations deposited in the BRCA1 mutation databases of unclassified variants for their effects on exon inclusion. Analysis of 21 BRCA1 variants using minigene assays revealed a single exon-skipping mutation c.231G>T. Comprehensive mutagenesis of an adjacent 12-nt segment showed that this silent mutation resulted in a higher level of exon skipping than 35 other single-nucleotide substitutions. Exon inclusion levels of mutant constructs correlated significantly with predicted splicing enhancers/silencers, prompting the development of two online utilities freely available at http://www.dbass.org.uk. EX-SKIP quickly estimates which allele is more susceptible to exon skipping whereas HOT-SKIP examines all possible mutations at each exon position and identifies candidate exon-skipping positions/substitutions. We demonstrate that the distribution of exon-skipping and disease-associated substitutions previously identified in coding regions was biased toward top-ranking HOT-SKIP mutations. Finally, we show that proteins 9G8, SC35, SF2/ASF, Tra2 and hnRNP A1 were associated with significant alterations of BRCA1 exon 6 inclusion in the mRNA. Together, these results facilitate prediction of exonic substitutions that reduce exon inclusion in mature transcripts.

Mots clés: Life Sciences

Michela Raponi, Jana Kralovicova, Ellen Copson, Petr Divina, Diana M. Eccles, et al.. Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6. Human Mutation, 2011, 32 (4), pp.436. ⟨10.1002/humu.21458⟩. ⟨hal-00620579⟩