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

Breast cancer research and treatment publish this investigation article

May 15th, 2018

Purpose
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.
Methods
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.
Results
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.
Conclusions
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




Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Infante M., Duran M., Marcos G., Lastra E., Gomez S. and Velasco EA.




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

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


Participants:

Consejo Superior de Investigaciones CientíficasGrupo de Splicing y Cáncer. Cancer Genetics. Instituto de Biología y Genética Molecular (IBGM). Consejo Superior de Investigaciones Científicas (CSIC).

Servicio de Oncología. Hospital Río Hortega.

Servicio de Oncología. Hospital Universitario de Burgos.

Universidad ComplutenseCentro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).







FACTOR YEAR Q
3.471 2018

NLMID: 8111104

PMID: 29766361

ISSN: 0167-6806



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


JOURNAL: Breast Cancer Res Treat


NUMERACIÓN: 171:53-63


AÑO: 2018


PUBLISHER: Dordrecht : Kluwer Academic


AUTHORS: Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Infante M., Duran M., Marcos G., Lastra E., Gomez S. and Velasco EA.


VISAVET PARTICIPANTS


Susana Gómez Barrero

DOI: https://doi.org/ 10.1007/s10549-018-4826-7


CITE THIS PUBLICATION:

Fraile-Bethencourt E., Valenzuela-Palomo A., Diez-Gomez B., Infante M., Duran M., Marcos G., Lastra E., Gomez S. and Velasco EA. Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants. Breast cancer research and treatment. 171:53-63 . 2018. (A). ISSN: 0167-6806. DOI: 10.1007/s10549-018-4826-7