DNA repair capacity is impaired in healthy BRCA1 heterozygous mutation carriers
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BRCA1 germline mutations increase the lifetime risk of developing breast and ovarian cancers. However, taking into account the differences in disease manifestation among mutation carriers, it is probable that different BRCA1 mutations have distinct haploinsufficiency effects and lead to the formation of different phenotypes. Using lymphoblastoid cell lines derived from heterozygous BRCA1 mutation carriers and non-carriers, we investigated the haploinsufficiency effects of various mutation types using qPCR, immunofluorescence, and microarray technology. Lymphoblastoid cell lines carrying a truncating mutation showed significantly lower BRCA1 mRNA and protein levels and higher levels of gamma-H2AX than control cells or those harboring a missense mutation, indicating greater spontaneous DNA damage. Cells carrying either BRCA1 mutation type showed impaired RAD51 foci formation, suggesting defective repair in mutated cells. Moreover, compared to controls, cell lines carrying missense mutations displayed a more distinct expression profile than cells with truncating mutations, which is consistent with different mutations giving rise to distinct phenotypes. Alterations in the immune response pathway in cells harboring missense mutations point to possible mechanisms of breast cancer initiation in carriers of these mutations. Our findings offer insight into how various heterozygous mutations in BRCA1 could lead to impairment of BRCA1 function and provide strong evidence of haploinsufficiency in BRCA1 mutation carriers.
KeywordsBRCA1 Haploinsufficiency DNA repair Gene expression profiling Breast cancer
We thank the Confocal Microscopy Core Unit at CNIO for their help with high-throughput microscopy, and all the families who volunteered to participate in this study. The study was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) SAF2010-20493 and the Spanish Network on Rare Diseases (CIBERER).
Conflict of interest
The authors declare that they have no conflict of interest.
- 2.Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, Loman N, Olsson H, Johannsson O, Borg A, Pasini B, Radice P et al (2003) Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 72:1117–1130PubMedCentralPubMedCrossRefGoogle Scholar
- 4.Milne RL, Osorio A, Cajal TR, Vega A, Llort G, de la Hoya M, Diez O, Alonso MC, Lazaro C, Blanco I, Sanchez-de-Abajo A, Caldes T et al (2008) The average cumulative risks of breast and ovarian cancer for carriers of mutations in BRCA1 and BRCA2 attending genetic counseling units in Spain. Clin Cancer Res 14:2861–2869PubMedCrossRefGoogle Scholar
- 6.Gayther SA, Warren W, Mazoyer S, Russell PA, Harrington PA, Chiano M, Seal S, Hamoudi R, van Rensburg EJ, Dunning AM, Love R, Evans G et al (1995) Germline mutations of the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation. Nat Genet 11:428–433PubMedCrossRefGoogle Scholar
- 8.Drost R, Bouwman P, Rottenberg S, Boon U, Schut E, Klarenbeek S, Klijn C, van der Heijden I, van der Gulden H, Wientjens E, Pieterse M, Catteau A et al (2011) BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance. Cancer Cell 20:797–809PubMedCrossRefGoogle Scholar
- 9.Waddell N, Ten Haaf A, Marsh A, Johnson J, Walker LC, kConfab Investigators, Gongora M, Brown M, Grover P, Girolami M, Grimmond S, Chenevix-Trench G et al (2008) BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression. PLoS Gen 4:e1000080CrossRefGoogle Scholar
- 12.Kote-Jarai Z, Williams RD, Cattini N, Copeland M, Giddings I, Wooster R, tePoele RH, Workman P, Gusterson B, Peacock J, Gui G, Campbell C et al (2004) Gene expression profiling after radiation-induced DNA damage is strongly predictive of BRCA1 mutation carrier status. Clin Cancer Res 10:958–963PubMedCrossRefGoogle Scholar
- 13.Kote-Jarai Z, Matthews L, Osorio A, Shanley S, Giddings I, Moreews F, Locke I, Evans DG, Eccles D, Carrier Clinic C, Williams RD, Girolami M et al (2006) Accurate prediction of BRCA1 and BRCA2 heterozygous genotype using expression profiling after induced DNA damage. Clin Cancer Res 12:3896–3901PubMedCrossRefGoogle Scholar
- 14.Bellacosa A, Godwin AK, Peri S, Devarajan K, Caretti E, Vanderveer L, Bove B, Slater C, Zhou Y, Daly M, Howard S, Campbell KS et al (2010) Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations. Cancer Prev Res 3:48–61CrossRefGoogle Scholar
- 26.Bouwman P, van der Gulden H, van der Heijden I, Drost R, Klijn CN, Prasetyanti P, Pieterse M, Wientjens E, Seibler J, Hogervorst FB, Jonkers J (2013) A high-throughput functional complementation assay for classification of BRCA1 missense variants. Cancer Discov 3:1142–1155PubMedCrossRefGoogle Scholar