Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants
Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease.
The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [− 938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR.
Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): −59_−10del/µdel3 (16% of activity of the wild-type construct), −104_−55del/µdel4 (62%), −239_−190del/µdel7 (39%), −464_−415/µdel12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/µdel1 (356%), −14_+36del/µdel2 (180%), −194_−145del/µdel6 (154%), −284_−235del/µdel8 (168%), −329_−280del/µdel9 (111%), and −509_−460del/µdel13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113–163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72–83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS − 185 to + 105) where most active transcriptional motifs are known to be placed.
BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRCA2 promoter that may constitute potential targets for regulatory variants. Three SNPs moderately decreased luciferase activity, but confirmation of its potential pathogenicity requires further analysis. These data reinforce the need to screen the promoter regions of breast cancer genes with a view to discovering novel deleterious mutations.
KeywordsBreast cancer Ovarian cancer Susceptibility genes BRCA2 Transcription Promoter Regulatory mutations Luciferase assays
We are grateful to the breast/ovarian cancer patients and clinicians who participated in this study. EAV’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I + D + I 2013–2016, ISCIII (Fis: PI13/01749) co-funded by FEDER from Regional Development European Funds (European Union), and Grant CSI090U14 from the Consejería de Educación (ORDEN EDU/122/2014), Junta de Castilla y León. EFB was supported by a predoctoral fellowship from the University of Valladolid and Banco de Santander (2015–2019).
The datasets generated and/or analyzed during the current study are available in the Figshare repository, https://figshare.com/s/e7c982e6afb9907d56a0.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
- 12.Fraile-Bethencourt E, Díez-Gómez B, Velásquez-Zapata V et al (2017) Functional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet 13:e1006691. https://doi.org/10.1371/journal.pgen.1006691 CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Karimi M, Goldie LC, Cruickshank MN et al (2009) A critical assessment of the factors affecting reporter gene assays for promoter SNP function: a reassessment of -308 TNF polymorphism function using a novel integrated reporter system. Eur J Hum Genet 17:1454–1462. https://doi.org/10.1038/ejhg.2009.80 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.de la Hoya M, Soukarieh O, López-Perolio I et al (2016) Combined genetic and splicing analysis of BRCA1 c.[594-2A> C; 641A> G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms. Hum Mol Genet 25:2256–2268. https://doi.org/10.1093/hmg/ddw094 CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Gochhait S, Bukhari SIA, Bairwa N et al (2007) Implication of BRCA2 -26G> A 5′ untranslated region polymorphism in susceptibility to sporadic breast cancer and its modulation by p53 codon 72 Arg> Pro polymorphism. Breast Cancer Res 9:R71. https://doi.org/10.1186/bcr1780 CrossRefPubMedPubMedCentralGoogle Scholar