The use of splice site prediction programs as a reliable tool to select intronic variants in BRCA1 and BRCA2 that affect RNA splicing

M.P.G. Vreeswijk1, J.N. Kraan1, H. van der Klift1, G.R. Vink1, C.J. Cornelisse2, J. Wijnen1, E. Bakker1, C.J. van Asperen1, P. Devilee1,2
Center for Human and Clinical Genetics1 and Department of Pathology2, Leiden University Medical Center

A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants include variants that are located in the intronic sequences of BRCA1 and BRCA2.

The purpose of this study was to assess the use of splice site prediction analysis as a first tool to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed extensive analysis of six genetic variants in splice site regions of BRCA1 and BRCA2 by in vitro molecular characterization of RNA. In four cases (BRCA1 c.81-6T>A and c.4986+5G>T; BRCA2 c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance.

Six splice site prediction programs were used to predict whether an effect on RNA splicing was expected for these 6 variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e. the wild-type splice site was recognized). In four cases (2.5%) a false positive prediction was observed with one of the programs, i.e. the programs predicted an effect on RNA splicing which was not confirmed by molecular analysis. No false negative predictions were observed, indicating that an effect on RNA splicing was always predicted by the SSPP. We therefore recommend the use of these programs by the DNA diagnostic laboratories to select intronic variants that are likely to affect RNA splicing for further analysis.