Novel enhancers including high LD SNPs were identified in breast epithelial cells.

<p>(A) Eleven enhancer regions, which included FunciSNP identified BCa high LD SNPs in epigenetically defined enhancers, were cloned and analyzed using the dual luciferase assays in MCF7 (blue), MDAMB231 (red), MCF10A (orange) and HMEC (blue). Each luciferase activity was divided by average luciferase activity of two negative controls, CT1 and 2. The average value of two negative controls was shown as a horizontal line across the breast cancer enhancers (BCEs) (gray). (B) The location of three SNPs (blue: rs4871782, green: rs28759353, black: rs10087810) at BCE5 and breast cancer risk tagSNP (red: rs13281615) in 8q24.21 region. (C) Allele-specific FAIRE assays were performed near three candidate SNPs for breast cancer risk at BCE5. Sequence results of Input DNA and FAIRE DNA are shown. The colors of the nucleotides from DNA sequencing: blue is C, green is A, black is G and red is T. Sequences near SNP were shown in a double-strand DNA (bottom). (D) Linkage Disequilibrium (LD) plot (r²) and haplotypes of three SNPs (in EUR) with breast cancer risk tagSNP, rs13281615 were shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063925#pone.0063925-Easton1" target="_blank">[5]</a>. Allele-specific in vitro dual luciferase assays were performed in HMEC (E) and MDAMB231 cells (F). The Analysis of variance statistical test (ANOVA) was used to confirm the difference and two-side p-values between alleles were calculated using the student t-test.