nach oben

Erschienen in:

15.05.2018 | Preclinical study

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

verfasst von: Eugenia Fraile-Bethencourt, Alberto Valenzuela-Palomo, Beatriz Díez-Gómez, Mar Infante, Mercedes Durán, Germán Marcos, Enrique Lastra, Susana Gómez-Barrero, Eladio A. Velasco

Erschienen in: Breast Cancer Research and Treatment | Ausgabe 1/2018

Einloggen, um Zugang zu erhalten

Abstract

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.

Nur mit Berechtigung zugänglich

Roy R, Chun J, Powell SN (2012) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer 12:68–78. https://doi.org/10.1038/nrc3181 CrossRef

Nielsen FC, van Overeem Hansen T, Sørensen CS (2016) Hereditary breast and ovarian cancer: new genes in confined pathways. Nat Rev Cancer 16:599–612. https://doi.org/10.1038/nrc.2016.72 CrossRefPubMed

Miki Y, Swensen J, Shattuck-Eidens D et al (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66–71CrossRefPubMed

Wooster R, Bignell G, Lancaster J et al (1995) Identification of the breast-cancer susceptibility gene Brca2. Nature 378:789–792CrossRefPubMed

Foulkes WD (2008) Inherited susceptibility to common cancers. N Engl J Med 359:2143–2153. https://doi.org/10.1056/NEJMra0802968 CrossRefPubMed

Stratton MR, Rahman N (2008) The emerging landscape of breast cancer susceptibility. Nat Genet 40:17–22. https://doi.org/10.1038/ng.2007.53 CrossRefPubMed

Balmaña J, Domchek SM, Tutt A, Garber JE (2011) Stumbling blocks on the path to personalized medicine in breast cancer: the case of PARP inhibitors for BRCA1/2-associated cancers. Cancer Discov 1:29–34. https://doi.org/10.1158/2159-8274.CD-11-0048 CrossRefPubMed

Turner NC, Ashworth A (2011) Biomarkers of PARP inhibitor sensitivity. Breast Cancer Res Treat 127:283–286. https://doi.org/10.1007/s10549-011-1375-8 CrossRefPubMed

De Vooght KMK, Wijk R, Van, Van Solinge WW (2009) Management of gene promoter mutations in molecular diagnostics. Clin Chem 55:698–708. https://doi.org/10.1373/clinchem.2008.120931 CrossRefPubMed

10.

Halvorsen M, Martin JS, Broadaway S, Laederach A (2010) Disease-associated mutations that alter the RNA structural ensemble. PLoS Genet 6:1–11. https://doi.org/10.1371/journal.pgen.1001074 CrossRef

11.

Brewster BL, Rossiello F, French JD et al (2012) Identification of fifteen novel germline variants in the BRCA1 3′UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site. Hum Mutat 33:1665–1675. https://doi.org/10.1002/humu.22159 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

13.

Sanz DJ, Acedo A, Infante M et al (2010) A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients. Clin Cancer Res 16:1957–1967. https://doi.org/10.1158/1078-0432.CCR-09-2564 CrossRefPubMed

14.

Quiles F, Menéndez M, Tornero E et al (2016) Investigating the effect of 28 BRCA1 and BRCA2 mutations on their related transcribed mRNA. Breast Cancer Res Treat. https://doi.org/10.1007/s10549-015-3676-9 PubMedCrossRef

15.

Théry JC, Krieger S, Gaildrat P et al (2011) Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes. Eur J Hum Genet 19:1052–1058. https://doi.org/10.1038/ejhg.2011.100 CrossRefPubMedPubMedCentral

16.

Lheureux S, Lambert B, Krieger S et al (2011) Two novel variants in the 3′UTR of the BRCA1 gene in familial breast and/or ovarian cancer. Breast Cancer Res Treat 125:885–891. https://doi.org/10.1007/s10549-010-1165-8 CrossRefPubMed

17.

Hindorff LA, Sethupathy P, Junkins HA et al (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci USA 106:9362–9367. https://doi.org/10.1073/pnas.0903103106 CrossRefPubMed

18.

Shim S-Y, Jeong HJ, Park HJ et al (2017) Functional variation of SHP-2 promoter is associated with preterm birth and delayed myelination and motor development in preterm infants. Sci Rep 7:6052. https://doi.org/10.1038/s41598-017-06401-x CrossRefPubMedPubMedCentral

19.

Haberle V, Lenhard B (2015) Promoter architectures and developmental gene regulation. Semin Cell Dev Biol. https://doi.org/10.1016/j.semcdb.2016.01.014 CrossRef

20.

Cooper DN (2002) Human gene mutation in pathology and evolution. J Inherit Metab Dis 25:157–182. https://doi.org/10.1023/A:1015621710660 CrossRefPubMed

21.

Poncz M, Ballantine M, Solowiejczyk D et al (1982) β-Thalassemia in a Kurdish Jew: single base changes in the T-A-T-A box. J Biol Chem 257:5994–5996PubMed

22.

Davis PL, Miron A, Andersen LM et al (1999) Isolation and initial characterization of the BRCA2 promoter. Oncogene 18:6000–6012CrossRefPubMed

23.

Velasco E, Infante M, Durán M et al (2007) Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes. Nat Protoc 2:237–246. https://doi.org/10.1038/nprot.2006.482 CrossRefPubMed

24.

de la Hoya M, Gutierrez-Enriquez S, Velasco E et al (2006) Genomic rearrangements at the BRCA1 locus in Spanish families with breast/ovarian cancer. Clin Chem 52:1480–1485. https://doi.org/10.1373/clinchem.2006.070110 CrossRefPubMed

25.

Li W, Cowley A, Uludag M et al (2015) The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res 43:W580–W584. https://doi.org/10.1093/nar/gkv279 CrossRefPubMedPubMedCentral

26.

Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463–465. https://doi.org/10.2144/000113418 CrossRefPubMedPubMedCentral

27.

Dos Santos ES, Caputo SM, Castera L et al (2017) Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition. Breast Cancer Res Treat. https://doi.org/10.1007/s10549-017-4602-0 PubMedCrossRef

28.

Dunham I, Kundaje A, Aldred SF et al (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74. https://doi.org/10.1038/nature11247 CrossRef

29.

Farré D, Roset R, Huerta M et al (2003) Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res 31:3651–3653CrossRefPubMedPubMedCentral

30.

Sandelin A, Alkema W, Engström P et al (2004) JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res 32:D91–D94. https://doi.org/10.1093/nar/gkh012 CrossRefPubMedPubMedCentral

31.

Acedo A, Sanz DJ, Durán M et al (2012) Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes. Breast Cancer Res 14:R87. https://doi.org/10.1186/bcr3202 CrossRefPubMedPubMedCentral

32.

Acedo A, Hernández-Moro C, Curiel-García Á et al (2015) Functional classification of BRCA2 DNA variants by splicing assays in a large minigene with 9 exons. Hum Mutat 36:210–221. https://doi.org/10.1002/humu.22725 CrossRefPubMedPubMedCentral

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 CrossRefPubMedPubMedCentral

34.

Liu Q, Thompson BA, Ward RL et al (2016) Understanding the pathogenicity of noncoding mismatch repair gene promoter variants in Lynch syndrome. Hum Mutat 37:417–426. https://doi.org/10.1002/humu.22971 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

36.

Michailidou K, Lindström S, Dennis J et al (2017) Association analysis identifies 65 new breast cancer risk loci. Nature 551:92–94. https://doi.org/10.1038/nature24284 CrossRefPubMedPubMedCentral

37.

GTEx Consortium (2013) The genotype-tissue expression (GTEx) project. Nat Genet 45:580–585. https://doi.org/10.1038/ng.2653 CrossRef

38.

Harper AR, Nayee S, Topol EJ (2015) Protective alleles and modifier variants in human health and disease. Nat Rev Genet 16:689–701. https://doi.org/10.1038/nrg4017 CrossRefPubMed

39.

Capasso M, Diskin S, Cimmino F et al (2014) Common genetic variants in NEFL influence gene expression and neuroblastoma risk. Cancer Res 74:6913–6924. https://doi.org/10.1158/0008-5472.CAN-14-0431 CrossRefPubMedPubMedCentral

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 CrossRefPubMedPubMedCentral

41.

Maia A-T, Antoniou AC, O’Reilly M et al (2012) Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers. Breast Cancer Res 14:R63. https://doi.org/10.1186/bcr3169 CrossRefPubMedPubMedCentral

42.

Kauffmann A, Rosselli F, Lazar V et al (2007) High expression of DNA repair pathways is associated with metastasis in melanoma patients. Oncogene 27:565–573. https://doi.org/10.1038/sj.onc.1210700 CrossRefPubMed

43.

Sarasin A, Dessen P (2010) DNA repair pathways and human metastatic malignant melanoma. Curr Mol Med 10:413–418CrossRefPubMed

44.

Klein HL (2008) The consequences of Rad51 overexpression for normal and tumor cells. DNA Repair 7:686–693. https://doi.org/10.1016/j.dnarep.2007.12.008 CrossRefPubMedPubMedCentral

45.

Bièche I, Noguès C, Lidereau R (1999) Overexpression of BRCA2 gene in sporadic breast tumours. Oncogene 18:5232–5238. https://doi.org/10.1038/sj.onc.1202903 CrossRefPubMed

Titel: Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants
verfasst von: Eugenia Fraile-Bethencourt
Alberto Valenzuela-Palomo
Beatriz Díez-Gómez
Mar Infante
Mercedes Durán
Germán Marcos
Enrique Lastra
Susana Gómez-Barrero
Eladio A. Velasco
Publikationsdatum: 15.05.2018
Verlag: Springer US
Erschienen in: Breast Cancer Research and Treatment / Ausgabe 1/2018
Print ISSN: 0167-6806
Elektronische ISSN: 1573-7217
DOI: https://doi.org/10.1007/s10549-018-4826-7

Klimawandel und Gesundheit: Was Sie in der Hausarztpraxis wissen müssen und tun können

Springer Medizin

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