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 Kathleen Claes 1 , Jo Vandesompele 1 , Bruce Poppe 1 , Karine Dahan 2 , Ilse Coene 1 , Anne De Paepe 1 and Ludwine Messiaen* ,1 1 Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, Ghent, Belgium; 2 Centre Ge ÂneÂtique Humaine, Cliniques Universitaires de Saint-Luc, Avenue E Mounier 52 UCL 5220, Brussels, Belgium We report two novel mutations in the splice sites of BRCA1 exon 5: IVS5+3A4G, a Belgian founder mutation, and IVS376T4G, identi®ed in one family with a strong family history of breast cancer. Real-time RT ± PCR showed that IVS376T4G leads to a ®vefold increase of the BRCA1-Dex5 (isoform with an in frame skip of exon 5) ratio to the total BRCA1 expression level. IVS5+3A4G results in a 10-fold increase of the BRCA1-D22ntex5 ratio (isoform with an out of frame skip of the last 22 nucleotides of exon 5) and a twofold increase of the BRCA1-Dex5 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 signi®cance of the isoforms, we evaluated their prevalence in normal tissues and cancer cell lines. The BRCA1-D22ntex5 ratio was signi®cantly higher in an ovarian cancer cell line compared to normal ovarian tissue. Our ®ndings 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 aecting predisposition to hereditary breast and/or ovarian cancer. Oncogene (2002) 21, 4171 ± 4175. doi:10.1038/sj.onc. 1205520 Keywords: BRCA1; splice mutation; real time RT ± PCR; breast cancer By extensive linkage studies on families with a strong predisposition for breast and/or ovarian cancer and by positional cloning BRCA1 was mapped to 17q21 and BRCA2 to 13q12.3 (Easton et al., 1993; Miki et al., 1994; Wooster et al., 1995). A large number of dierent mutations in BRCA1 and BRCA2 implicated in the familial occurrence of breast and/or ovarian cancer, have been described in many pedigrees. In the BIC (Breast Information Core) database (http:// www. nhgri.nih.gov/Intramural_research / Lab_transfer/ Bic/Member/) splicing mutations only account for 7% of the germline mutations reported in BRCA1 and for 5% in BRCA2. In this study we report two novel splice site mutations outside the highly conserved 3' AG-acceptor (IVS376T4G) and 5' GT-donor (IVS5+3A4G) of exon 5 of the BRCA1 gene and showed how both mutations exert their pathological eect. Both mutations were identi®ed by DGGE (de- naturing gradient gel electrophoresis) on gDNA. IVS5+3A4G was identi®ed in nine apparently unrelated families and is a Belgian founder mutation (Claes et al., 1999, Claes et al., manuscript in preparation). IVS376T4G was detected in a proband diagnosed with breast cancer at age 43, who developed a second cancer in the same breast at age 46 years. Her sister developed a brain tumor at age 40 and is also carrying the mutation. Both inherited the mutation from their father. Several other family members in the paternal branch of the family have been diagnosed with breast cancer. Unfortunately, these patients were not available for further study. In all patients the complete coding region of both BRCA1 and BRCA2 was analysed by DGGE and no other mutation was found. In none of 100 unrelated healthy control persons IVS5+3A4G or IVS376T4G was detected, underscoring that these variants are very unlikely to represent polymorphic alterations in our population. In order to interprete the pathogenic eect of these variants, BRCA1 mRNA expression studies, including the search for alternative transcripts, were initiated. Fragment analysis of RT ± PCR products spanning exons 2 ± 7 showed that two alternative transcripts were more prevalent in patients carrying IVS5+3A4G than in controls (Figure 1): a transcript 22 nucleotides shorter than the full length fragment, containing an out of frame skip of the last 22 nucleotides of exon 5 (BRCA1-D22ntex5) and a transcript 78 basepairs Oncogene (2002) 21, 4171 ± 4175 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc *Correspondence: L Messiaen, Department Medical Genetics, Ghent University Hospital (0K5), De Pintelaan 185, B-9000 Ghent, Belgium; E-mail: Ludwine.Messiaen@rug.ac.be Received 29 January 2002; revised 7 March 2002; accepted 26 March 2002