The human BRCA1 gene is under transcriptional control of two alternative promoters, α and β, that drive the transcription of exon 1a or exon 1b, respectively. Additionally, the 5'-UTR region, encompassing both exons 1, contains multiple putative and functional regulatory sequences. At the RNA level each exon 1 is linked, by splicing, with exon 2 that contains the translation start site. The aim of this study was to search for sequence alterations within the BRCA1 promoter and 5'-UTR in patients with breast and/or ovarian cancer, and to assess whether these sequence variants influence activity of the BRCA1 promoter region.

The 5' region of the BRCA1 gene, containing promoters α and β as well as exons 1a and 1b and the fragment of intron 1, was sequenced in 87 breast/ovarian cancer cases. All patients had a strong family history of breast and ovarian cancer, but were found mutation-negative in our previous search for founder mutations in BRCA1 (185delAG, 300T/G, 4153delA, 5382insC) and BRCA2 (6174delT, 9631delC). The frequency of the 2223delAAAAA deletion was assessed using allele-specific PCR amplification (ASA) in a larger group of breast/ovarian cancer patients fulfilling the aforementioned criteria. The functional significance of sequence variants within the 5'-UTR of BRCA1 was analyzed by luciferase assay. A 1.5 kb DNA region encompassing minimal BRCA1 promoter and 5'-UTR, both wild type and variant sequence, was cloned into the pGL3 vector containing the luciferase reporter gene. The luciferase activity reflected the influence of the sequence alterations on the transcriptional activity of the BRCA1 promoter and other gene regulatory regions.

We found several sequence variants within the examined non-coding region of BRCA1. The frequency of the largest sequence alteration found, deletion 2223delAAAAA (according to the Acc. U37574) within exon 1b, was determined in a group of 150 patients. Three families have been identified bearing the said deletion. We also found two linked nucleotide substitutions (2642A>T, 2743T>C) in BRCA1 intron 1. The functional impact of the most frequent sequence alterations was examined in lung cancer cell line NCI-H1299 and breast cancer cell line MCF7. In the MCF7 cell line all tested variants of BRCA1 promoter/5'-UTR showed lower activity than the control wild-type sequence, while in NCI-H1299 cells the variant promoter/5'-UTR activity was higher than the control. However, observed differences of luciferase activity were not statistically significant.

Our luciferase assay showed that sequence variants detected in our study within the BRCA1 promoter/5'-UTR do not change the functional activity of the BRCA1 promoter in the experimental system that we have used, and may not be associated with an increased risk of breast and ovarian cancer. The detailed results of the analyses will be presented.