Replication and functional genomic analyses of the breast cancer susceptibility locus at 6q25.1 generalize its importance in women of chinese, Japanese, and European ancestry

Abstract

We evaluated the generalizability of a single nucleotide polymorphism (SNP), rs2046210 (A/G allele), associated with breast cancer risk that was initially identified at 6q25.1 in a genome-wide association study conducted among Chinese women. In a pooled analysis of more than 31,000 women of East-Asian, European, and African ancestry, we found a positive association for rs2046210 and breast cancer risk in Chinese women [ORs (95% CI) = 1.30 (1.22-1.38) and 1.64 (1.50-1.80) for the AG and AA genotypes, respectively, P for trend = 1.54 × 10⁻³⁰], Japanese women [ORs (95% CI) = 1.31 (1.13-1.52) and 1.37 (1.06-1.76), P for trend = 2.51 × 10⁻⁴], and European-ancestry American women [ORs (95% CI) = 1.07 (0.99-1.16) and 1.18 (1.04-1.34), P for trend = 0.0069]. No association with this SNP, however, was observed in African American women [ORs (95% CI) = 0.81 (0.63-1.06) and 0.85 (0.65-1.11) for the AG and AA genotypes, respectively, P for trend = 0.4027]. In vitro functional genomic studies identified a putative functional variant, rs6913578. This SNP is 1,440 bp downstream of rs2046210 and is in high linkage disequilibrium with rs2046210 in Chinese (r(2) = 0.91) and European-ancestry (r² = 0.83) populations, but not in Africans (r² = 0.57). SNP rs6913578 was found to be associated with breast cancer risk in Chinese and European-ancestry American women. After adjusting for rs2046210, the association of rs6913578 with breast cancer risk in African Americans approached borderline significance. Results from this large consortium study confirmed the association of rs2046210 with breast cancer risk among women of Chinese, Japanese, and European ancestry. This association may be explained in part by a putatively functional variant (rs6913578) identified in the region.

Figure 1

ORs (95%) per risk allele for breast cancer by study site and ethnicity. The size of the boxes is proportional to the sample size of each study. The width of the diamonds represents the range of confident intervals of combined ORs derived from meta-analyses.

Figure 2

In vitro functional characterization of SNP rs2046210 and other potential functional SNPs in 6q25.1. A. Diagram of cloning strategy. A 36 kb region (chromosome 6:151,983,304–152,019,420) between the C6orf97 and ESR1 genes was divided into 4 DNA fragments (a–d), which were separately cloned into pGL3 basic and pGL3 promoter vectors. The 8.6 kb “a” fragment was further divided to three DNA fragments (e, f, and g) and subcloned into a pGL3 promoter vector. The “g” fragment harbored 4 SNPs (rs7740686, rs2046210, rs7763637, and rs6913578). B. Luciferase reporter activity assays: HEK293 cells were transiently transfected with pGL3 promoter/luciferase reporter constructs containing the 2.3 Kb “g” fragment. 1. Minor Alleles construct: contained the minor alleles for all four SNPs (rs7740686-T, rs2046210-A, rs7763637-A, and rs6913578 C); 2. Major Alleles construct: contained the major alleles for all four SNPs (rs7740686-A, rs2046210-G, rs7763637-G, and rs6913578-A); 3. rs7740686-A construct: containsed the rs7740686 major allele A and the minor alleles for the other three SNPs; 4. rs2046210-G construct: contained the rs2046210 major allele G and the minor alleles for the other three SNPs; 5. rs7763637-G construct: contained rs7763637 major allele G and the minor alleles for the other three SNPs; 6. rs6913578-A construct: contained rs6913578 major allele A and the minor alleles for the other three SNPs. Relative luciferase activity is shown as the mean ± SD of three experiments conducted in triplicate (relative to the Minor Allele construct). Statistical analysis was conducted by using Student's t test to compare the minor and major alleles (*P<0.01 when compared with the minor alleles, n=9). Relative luciferase activity is shown as the mean ± SD of three experiments conducted in triplicate (relative to the Minor Alleles construct). Statistical analysis was conducted using Student's t test to compare the minor and major alleles (*P<0.01 when compared with the minor alleles, n=9). C. Electrophoretic mobility shift assays. Nuclear protein extracts from MCF-7 (top panel) and HEK293 (bottom panel) cells were incubated with biotin-labeled probes corresponding to reference allele (lanes 1–5) or the risk allele (lanes 6–10) of rs6913578 in the absence or presence of competitors. Lanes 1 and 6, no nuclear extracts; lanes 2 and 7, unlabeled competitor in 200-fold molar excess; lanes 3 and 8 (5 mM MgCl2), lanes 4 and 9 (2.5 mM MgCl2), and lanes 5 and 10 (1.25 mM MgCl2), no competitor. I: free biotin-labeled probes. II: specific DNA-protein complex bands.