Intronic variants inBRCA1andBRCA2that affect RNA splicing can be reliably selected by splice-site prediction programs

Abstract

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 (UVs) 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 programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T4A and c.498615G4T; BRCA2, c.76171 2T4G and c.875415G4A) 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.68-7T4A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six 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). No falsenegative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis.

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

“…15,16 Among the variants with type 1 predictions, but with no effect in the minigene assay, BRCA2 c.68-7T4A (Table 2) deserves particular attention, because, in patient blood RNA, it enhanced the exclusion of exon 3, which is also observed at low levels in control samples. These observations are in keeping with two recent reports, 8,20 but the relevance to cancer predisposition of this enhanced skipping of exon 3, which is in frame, is still questionable, and thus BRCA2 c.68-7T4A should still be considered as a VUS. The discrepancy observed in this case between the results of our minigene assay and those of RT-PCR analyses on patient blood RNA reflects the nature of the minigene assay used, which targets single exons.…”

“…However, in this series of BRCA variants, this stratification of assays would have provided only a moderate gain in time and costs, because only 15 of the 53 VUSs would have been excluded from functional assays, that is, the 15 type 2 intronic VUSs (10 and 5 for BRCA1 and BRCA2, respectively, see Supplementary Table S1). Indeed, no splicing alteration was induced by these intronic variants, in keeping with the previous suggestion 8 that intronic variants for which no effect on splicing is predicted, using multiple algorithms, could be excluded from further analyses. We support that suggestion, at least at the stage of the initial interpretation, in the molecular diagnostic setting, but one cannot exclude the possibility that intronic variants, distant from the exon boundaries, may induce more subtle splicing alterations.…”

“…Here we evaluated the bioinformatics predictions primarily against results of minigene assays because these assays do not depend on the availability of patient RNA. At variance with previous publications, 7,8 we studied all consecutive VUSs found in our molecular diagnostic activities without selection, except for VUS of the large exon 11 of BRCA1 and the large exon 11 of BRCA2, which require special minigene constructs. 14 We have used rather permissive criteria for the interpretation of the predictions of five algorithms (significant threshold of variation set at as low as 10% and concordance between two algorithms considered sufficient).…”

“…Several recent studies indicate that, for routine applications in molecular diagnostic laboratories, bioinformatics predictions are useful, at this time, only for variants that affect exon-intron junctions or generate new splice sites, or induce the use of cryptic sites by decreasing the strength of natural splice sites. [6][7][8][9][10] The main purpose of this study was to evaluate, in the molecular diagnostic context, the performance of bioinformatics predictions for the selection of variants that potentially induce splicing alterations. To this end, we studied 53 VUSs of BRCA1 or BRCA2 (30 in exons and 23 in introns) found in consecutive molecular screenings in the laboratories of the French Northwest Canceropole (Caen, Lille and Rouen) in the years 2008 and 2009.…”

Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients’ phenotypes

“…Although the predictions obtained are usually not enough to establish with sufficiently high accuracy the clinical impact of genetic variations on splicing, it has been proposed that SPPs could be used to perform a first selection of those variants that may have an effect on the pre-mRNA splicing before starting with time-consuming and labor-intensive mRNA analysis. 14 In this study, we have comprehensively reviewed the available information on splicing mutations of the GAA gene and we have evaluated the possible impact of these genetic variations on pre-mRNA-splicing process using different in silico approaches. In addition, using a minigene system assay, we have performed the functional characterization of three sequence variants previously found in Italian patients affected with LO GSDII.…”