Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

Abstract

TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOGs, we analyzed ∼480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 × 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 × 10(-8)) and BRCA1 mutation carrier (P = 1.1 × 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 × 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 × 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 × 10(-12)) and BRCA1 mutation carrier (P = 1.6 × 10(-14)) breast and invasive ovarian (P = 1.3 × 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.

Figure 1

Association results for all SNPs for seven phenotypes including: (a) telomere length, (b) overall breast cancer, (c) breast cancer risk in BRCA1 mutation carriers, (d) ER-negative breast cancer, (e) ER-positive breast cancer, (f) serous low malignant potential (LMP) ovarian cancer and (g) serous invasive ovarian cancer. Directly-genotyped SNPs are represented by solid black circles and imputed SNPs (r²>0.3, minor allele frequency (MAF)>0.02) as open red circles, plotted as the negative log of the P-value against relative position across the locus. Schematics of the gene structures are shown above panel a. Association peaks, as described in the text are labeled at the top of the figure, and are shown as grey regions, when appropriate.

Figure 2

Associated signals within the TERT gene. Peak regions described in the text are labeled: Peaks 2 and 3 overlap around introns 2–4, and Peak 1 encompasses the promoter. The positions of associated SNPs are shown as black and red ticks representing genotyped and imputed SNPs, respectively. The TERT gene structure is depicted with exons (boxes) joined by introns (lines). The positions of all analyzed iCOGS SNPs are marked. Data from the UCSC genome browser including epigenetic marks for H3K4Me1 and H3K4Me3, ESPERR regulatory potential, and vertebrate conservation tracks are shown. Regions cloned into reporter constructs are depicted as the green bar (TERT promoter) or blue bars (PRE-A and PRE-B). The pattern of linkage disequilibrium based on the BCAC population is shown where white represents r²=0 and black r²=1.

Figure 3

Open chromatin signatures around rs10069690. (A) Map of the PCR amplicon sites A-D used to annotate a 1 kilobase region surrounding rs10069690 and rs2442652. Primer sequences are listed in Supplementary Table 11. (B) PCR analysis of FAIRE-processed chromatin from FACS-sorted myoepithelial/stem, luminal progenitor, mature luminal and stromal cell enriched fractions derived from breast tissues of four subjects Q695, Q723, Q706, and Q674. Error bars represent standard errors of triplicate PCR reactions.

Figure 4

TERT promoter and putative regulatory element (PRE) activity. Luciferase reporter assays following transient transfection of ER-negative breast cancer cell line (MDA-MB-468), ER-positive breast cancer cell line (MCF7), and ovarian cancer cell line (A2780). Error bars represent standard error between at least three separate experiments. (a-c) Luciferase reporter assays following transient transfection with pGL2-control (SV40 promoter and enhancer), pGL2-basic (lacks promoter and enhancer), and the TERT reporter vectors TERT wildtype (wt) (3.9kb of TERT promoter), the minor (T) alleles of rs2736107, rs2736108, rs2736109, and rs2736107/8/9 (T-alleles at all sites). Comparisons with TERT wt performed using one-way ANOVA with post hoc Dunnett's tests are represented by ** (P-value <0.001) and * (P-value 0.005). (d–f) PRE-A or PRE-B were cloned downstream of either the TERT wt or TERT rs2736107/8/9 (TERTh) promoter-driven reporters with and without SNPs rs10069690, rs2242652 and rs7705526. Comparisons with TERT wt or TERTh performed using one way ANOVA with post hoc Dunnett's tests are represented by *** (P-value <0.0001) and ** (P-value <0.001).