Pathological splice mutations outside the invariant AG/GT splice sites of BRCA1 exon 5 increase alternative transcript levels in the 5' end of the BRCA1 gene.

We report two novel mutations in the splice sites of BRCA1 exon 5: IVS5+3A>G, a Belgian founder mutation, and IVS3-6T>G, identified in one family with a strong family history of breast cancer. Real-time RT-PCR showed that IVS3-6T>G leads to a fivefold increase of the BRCA1-Deltaex5 (isoform with an in frame skip of exon 5) ratio to the total BRCA1 expression level. IVS5+3A>G results in a 10-fold increase of the BRCA1-Delta22ntex5 ratio (isoform with an out of frame skip of the last 22 nucleotides of exon 5) and a twofold increase of the BRCA1-Deltaex5 ratio. These altered ratios are most likely to result from increased expression of the alternative transcripts, although we cannot completely rule out a small decrease of the total BRCA1 expression level due to highly variable BRCA1 levels in cultured cell lines. In order to explore the functional significance of the isoforms, we evaluated their prevalence in normal tissues and cancer cell lines. The BRCA1-Delta22ntex5 ratio was significantly higher in an ovarian cancer cell line compared to normal ovarian tissue. Our findings suggest that revealing the defects caused by some splice mutations requires accurate quantitative methods. We hypothesize that disruption of alternative transcript ratios of BRCA1 may be a dominant mechanism affecting predisposition to hereditary breast and/or ovarian cancer.