Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170

Abstract

We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression.

Figure 1

Association results for all SNPs with six phenotypes. (a–f) The phenotypes analyzed include risk of ER⁺ breast cancer in BCAC (a), risk of ER⁻ breast cancer in BCAC (b), risk of triple-negative breast cancer, derived from the CIMBA meta-analysis of BRCA1 mutation carriers with ER⁻ tumors (c), risk of HER2⁺ breast cancer in BCAC (d), mammographic dense area in MODE (e) and tumor grade after adjustment for ER status in BCAC (f). P values for each SNP (from unconditional logistic regression) are shown plotted as the negative log-transformed P value against relative position across the locus. A schematic of the gene structures is shown above a and d. The physical positions of signals 1–5 are shown as colored, numbered stripes. Dotted horizontal lines indicate the genome-wide significance level.

Figure 2

ER expression and allelic imbalance correlate with signal 1 SNPs. (a) Negative correlation between the signal 1 SNP rs2046210 and ER protein expression. Black dots represent ER expression from individual samples measured by immunohistochemistry (H score). Horizontal lines represent the mean H score for each genotype. The P value was calculated using a Spearman rank correlation test. (b) Box plots of ESR1 gene expression (log₂ transformed) in breast tumor and adjacent normal samples. Boxes extend from the 25th to the 75th percentile, horizontal bars represent the median, whiskers indicate the full range of ESR1 expression and outliers are represented as circles. (c) Allelic imbalance in ESR1 expression by genotypic status at breast cancer risk variants. Data are classified according to the genotypes at risk SNPs (heterozygous versus homozygous). Black dots represent the average major allele fraction of the marker SNPs across ESR1 for an individual from TCGA with breast cancer. Red lines and whiskers correspond to means ± 1 s.d. For rs7740686 (signal 1) and rs9397437 (signal 2), Levene’s test (equality of variances) was used to calculate the P values; for rs851985 (signal 3), a two-tailed t test (equality of means) was used to calculate the P value.

Figure 3

Chromatin interactions across the 6q25.1 risk region. (a) Signals 1–5 are numbered and shown as colored stripes. RMND1, ARMT1, CCDC170 and ESR1 gene structures are depicted with exons (vertical bars) joined by introns (lines). Gene-enhancer predictions from PreSTIGE, ChIP-seq binding profiles for H3K27ac and Encyclopedia of DNA Elements (ENCODE) RNA polymerase II ChIA-PET interactions in MCF-7 cells are shown. (b–d) 3C anchor points (3C baits) and interrogated sequences (3C regions) are depicted as black boxes and gray shading, respectively. 3C interaction profiles in ER⁺ MCF-7 and ER⁻ Bre-80 breast cell lines are shown for signals 1 and 2 (b), signals 3 and 4 (c) and signal 5 (d). 3C libraries were generated with EcoRI, with the anchor point set at the ESR1, RMND1-ARMT1 or CCDC170 promoter region. Graphs present the results from three biological replicates; error bars, s.d.

Figure 4

Risk alleles reduce ESR1 and RMND1 promoter activity. Luciferase reporter assays were performed following transient transfection of ER⁺ MCF-7 breast cancer cells. PREs containing the major SNP alleles were cloned downstream of target gene promoter-driven luciferase constructs (prom) for the creation of reference (Ref-PRE) constructs. Minor SNP alleles were engineered into the constructs and are designated by the rsID of the corresponding SNP. “Haplotype” denotes a construct that contains the minor alleles of both candidate SNPs within signal 1 or 3. Error bars, 95% confidence intervals from three independent experiments. P values were determined by two-way ANOVA followed by Dunnett’s multiple-comparisons test: **P < 0.01, ***P < 0.001.

Figure 5

GATA3 and CTCF binding in vivo. (a) ChIP and quantitative PCR (qPCR) assays using antibody against GATA3 or CTCF in ER⁺ BT-474 breast cancer cells. A region within the second intron of ESR1 served as a negative control (NC). Normal rabbit IgG was used as a non-specific antibody control. Graphs present the results of two biological replicates; error bars, s.d. (b) 3C followed by sequencing for the signal 4 PRE containing rs1361024 in heterozygous ER⁺ MCF-7 breast cancer cells shows allele-specific chromatin looping. The chromatograms represent one of three independent 3C libraries generated and sequenced.