The first three authors contributed equally to this work.
Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6†
Article first published online: 8 MAR 2011
© 2011 Wiley-Liss, Inc.
Volume 32, Issue 4, pages 436–444, April 2011
How to Cite
Raponi, M., Kralovicova, J., Copson, E., Divina, P., Eccles, D., Johnson, P., Baralle, D. and Vorechovsky, I. (2011), Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6. Hum. Mutat., 32: 436–444. doi: 10.1002/humu.21458
Communicated by Mario Tosi
- Issue published online: 15 MAR 2011
- Article first published online: 8 MAR 2011
- Accepted manuscript online: 1 FEB 2011 02:33PM EST
- Manuscript Accepted: 7 JAN 2011
- Manuscript Received: 31 JUL 2010
- The EURASNET
- Action Medical Research
- The JDRF International
Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among 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 the 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. Hum Mutat 32:1–9, 2011. © 2011 Wiley-Liss, Inc.