Candidate causal variants from three independent genetic signals at the 5q11.2 breast cancer risk locus regulate MAP3K1. D. M. Glubb1, K. A. Pooley2, K. Michailidou3, M. J. Maranian3, K. B. Meyer4, J. A. Betts1, K. M. Hillman1, S. Kaufmann1, G. Chenevix-Trench1, D. F. Easton2,3, A. M. Dunning2, S. L. Edwards1,5, J. D. French1,5, Breast Cancer Association Consortium 1) Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia; 2) Centre for Cancer Genetic Epidemiology, Department of Oncology, University of Cambridge, Cambridge, UK; 3) Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK; 4) Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK; 5) School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia.

   A genome wide association study has previously found a variant, rs889312, at 5q11.2 to be associated with breast cancer risk. To identify the causal variant(s) underlying this association, we analysed 909 genetic variants across 5q11.2 in 103,991 breast cancer cases and controls from 52 studies. Three sets of independent, correlated, highly trait-associated variants (iCHAVs), spanning a 183 kb region at 5q11.2, were identified in Europeans (46,451 cases and 42,599 controls). This region encompasses MAP3K1, a frequently mutated gene in breast cancer, encoding a stress-induced serine/threonine kinase which regulates apoptosis and induces cell proliferation through a RAS/RAF/MEK/ERK pathway. Using ENCODE data and chromatin conformation studies, we found that variants from the three iCHAVs coincide with five putative regulatory elements (PREs). We show that all five PREs physically interact with the MAP3K1 promoter through chromatin looping and have effects on MAP3K1 promoter activity in reporter gene assays. Analysis of iCHAV variants in the PREs revealed four variants that significantly alter MAP3K1 promoter activity: rs74345699 and rs62355900 (iCHAV1), rs16886397 (iCHAV2) and rs17432750 (iCHAV3). The risk (minor) alleles of iCHAV1 SNPs r74345699 and rs62355900 increase the effect of the PRE3 enhancer on MAP3K1 promoter activity in MCF7 breast cancer cells after estrogen stimulation. The risk (minor) allele of the iCHAV2 SNP rs16886397 confers enhancer activity on PRE5 and upregulates MAP3K1 promoter activity in MCF7 cells. The protective (minor) allele of the iCHAV3 SNP rs17432750 diminishes the effect of a PRE2 enhancer on MAP3K1 promoter activity in normal mammary epithelial cells. Furthermore, the protective allele of rs17432750 was shown by chromatin immunoprecipation to reduce GATA-3 binding and was associated with allele-specific chromatin looping between PRE2 and the MAP3K1 promoter. Notably, the risk associated alleles of all four functional iCHAV SNPs increased MAP3K1 transcriptional activity. Therefore, we propose that these risk alleles contribute to breast cancer risk by increasing MAP3K1 expression and promoting cancer cell survival and proliferation.

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