Abstract 2565: Functional characterization of genetic variants at 16q12 associated with breast cancer in women of African ancestry.

Introduction: Women in the African Diaspora experience a disproportionate burden of pre-menopausal breast cancer in comparison to other races for reasons that remain unknown and understudied. To understand the etiologic heterogeneity in breast cancer and identify risk factors particular to women of African ancestry, we conducted a fine-mapping study of breast cancer susceptibility loci using a large cohort of 1502 cases and 1378 controls, all of African ancestry. We identified significant single nucleotide polymorphisms (SNPs) at chromosome 16q12. Because our association signal within a linkage disequilibrium (LD) block at 16q12 is close to the TOX3 and LOC643714 genes and some of these SNPs were reported to be associated with FOXA-1 binding sites, we performed functional analysis of 16q12 SNPs to investigate biological importance of the genetic variants in the development of breast cancer. Methods: To provide functional annotation of SNPs in relation to gene expression, we isolated DNA and RNA from 19 breast cell lines and genotyped 27 SNPs of 16q12 by sequencing. Expression levels of TOX3 and LOC643714 were quantified using qRT-PCR. Modulation of promoter activity by allelic difference was determined by a luciferase reporter gene assay. Modified histone signature was assessed by chromatin immunoprecipitation (ChIP) assays using antibodies against methylated histones. Local chromosome abnormalities of 16p12 will be validated through fluorescence in situ hybridization (FISH). Results: Based on our fine mapping data and LD block analysis, we selected 27 SNPs located in the promoter and intron 1 of LOC643714. To determine the potential of risk-associated SNPs serving as cis-acting regulatory elements for LOC643714 or TOX3, we performed ChIP assays and found an active chromatin conformation of the region with strong enhancer signature. Difference in the promoter activity was also observed between G and A alleles of rs3104793. Quantification of gene expression revealed greater expression of LOC643714 and TOX3 in cancer cells than in normal breast cells. Notably, breast cancer cell lines from black women showed higher expression of TOX3 compared to cells from white women. After assessing copy number variation (CNV) of the loci, we will integrate all the data (genotyping and gene expression) to examine their correlations. Finally, we will perform eQTL analysis in lymphoblast and breast cancer cells. Conclusion: Our data suggest a potential biological role for 16q12 SNPs in regulating gene expression in the development of breast cancer. This underscores the need for more functional studies on population-specific common variants to understand their contribution to breast cancer risks and etiologic heterogeneity in diverse populations. Citation Format: Yoo-Jeong Han, Jing Zhang, Yonglan Zheng, DeZheng Huo, Olufunmilayo I. Olopade. Functional characterization of genetic variants at 16q12 associated with breast cancer in women of African ancestry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2565. doi:10.1158/1538-7445.AM2013-2565