Breast Cancer Research and Treatment

, Volume 171, Issue 1, pp 53–63 | Cite as

Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants

  • Eugenia Fraile-Bethencourt
  • Alberto Valenzuela-Palomo
  • Beatriz Díez-Gómez
  • Mar Infante
  • Mercedes Durán
  • Germán Marcos
  • Enrique Lastra
  • Susana Gómez-Barrero
  • Eladio A. VelascoEmail author
Preclinical study



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.


Breast 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).

Data availability

The datasets generated and/or analyzed during the current study are available in the Figshare repository,

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

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

Informed consent was obtained from all individual participants included in the study.

Supplementary material

10549_2018_4826_MOESM1_ESM.pptx (157 kb)
Supplementary material 1 (PPTX 156 KB)
10549_2018_4826_MOESM2_ESM.pptx (172 kb)
Supplementary material 2 (PPTX 171 KB)
10549_2018_4826_MOESM3_ESM.docx (20 kb)
Supplementary material 3 (DOCX 19 KB)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Eugenia Fraile-Bethencourt
    • 1
  • Alberto Valenzuela-Palomo
    • 1
  • Beatriz Díez-Gómez
    • 1
  • Mar Infante
    • 2
  • Mercedes Durán
    • 2
  • Germán Marcos
    • 3
  • Enrique Lastra
    • 4
  • Susana Gómez-Barrero
    • 5
  • Eladio A. Velasco
    • 1
    Email author
  1. 1.Grupo de Splicing y CáncerInstituto de Biología y Genética Molecular (IBGM), Consejo Superior de Investigaciones Científicas (CSIC-UVa)ValladolidSpain
  2. 2.Cancer GeneticsInstituto de Biología y Genética Molecular (UVa-CSIC)ValladolidSpain
  3. 3.Servicio de OncologíaHospital Río HortegaValladolidSpain
  4. 4.Servicio de OncologíaHospital Universitario de BurgosBurgosSpain
  5. 5.VISAVET-Universidad Complutense de MadridMadridSpain

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