Breast cancer risk and survival at the 2q35 locus are mediated through chromatin looping and regulation of IGFBP5 expression. M. Ghoussaini1, S. L. Edwards2, J. D. French2, K. Michailidou3, S. Nord4, J. Dennis3, Q. Guo3, M. K. Schmidt5, K. Hillman2, S. Kaufmann2, E. Dicks3, S. Ahmed1, M. Maranian1, C. S. Healey1, C. Baynes1, C. Luccarini1, M. Bolla1, J. Wang1, V. N. Kristensen4, P. D. P. Pharoah1,3, G. Chenevix-Trench2, D. F. Easton1,3, A. M. Dunning1, The Breast Cancer Association Consortium 1) Centre for Cancer Genetic Epidemiology, Department of Oncology, University of Cambridge, Cambridge, UK; 2) Department of Genetics, Queensland Institute of Medical Research, Brisbane, Australia; 3) Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK; 4) Department of Genetics, University of Oslo, Oslo, Norway; 5) The Netherlands Cancer Institute, Division of Molecular Pathology, Amsterdam, the Netherlands.

   Genome wide association studies (GWAS) have previously identified a breast cancer susceptibility locus on 2q35 (rs13387042). In an effort to identify the likely causal variant(s), we performed fine-scale mapping of this region by genotyping 276 SNPs across a 210 Kb region in 89,050 subjects of European ancestry and 12,893 subjects of Asian ancestry from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). Genotypes for 1,284 SNPs were imputed using the 1000 genomes project as a reference. The most strongly associated variant in Europeans was rs4442975 (OR =0.86; 95% CI [0.84-0.88]; p=2.3x10-40); the association was confined to estrogen receptor positive disease. All but two perfectly correlated SNPs (rs4442975 and rs6721996) could be excluded as the likely causal variant (likelihood ratio more than 100:1). Both variants were much rarer in Asians than in Europeans (13% versus 49%); the association in Asians was in the same direction as in Europeans but not statistically significant. Follow-up experiments found no evidence for functional activity for rs6721996. SNP rs4442975 lies just outside an enhancer mark in breast cancer cell lines; using chromatin conformation studies, we identified a long-range physical interaction between the enhancer/flanking sequence encompassing rs4442975 and the IGFBP5 promoter located more than 350 Kb away. Expression analysis show that the rare allele of rs4442975 is associated with increased IGFBP5 levels in normal breast tissue (n=123, p=0.04). Consistent with this, we found that the rare allele of rs4442975 is associated with increased chromatin looping and interaction with the IGFBP5 promoter. The rare allele was also associated with poorer breast cancer survival in a meta-analysis including the BCAC dataset and nine GWAS (p=7x10-3). IGFBP5 plays an important role in breast cancer cell growth both in vivo and in vitro. These results suggest strongly that the 2q35 susceptibility locus is mediated through regulation of IGFBP5.

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