Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

Abstract

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10(-20)), ER-negative BC (P=1.1 × 10(-13)), BRCA1-associated BC (P=7.7 × 10(-16)) and triple negative BC (P-diff=2 × 10(-5)). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10(-3)) and ABHD8 (P<2 × 10(-3)). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3'-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk.

Figure 1. Regional association plot disease-specific risk associations.

Results for ER negative breast cancer from BCAC, for ovarian cancer from OCAC and for BRCA1 mutation carriers with breast cancer from CIMBA are shown. Also shown are the results of a meta-analysis for BRCA1 and general population ER negative breast cancer cases. The grey bars indicate the boundaries of the two association peaks, and the dotted horizontal line indicates the cutoff for genome-wide significance (χ²-test P=5 × 10⁻⁸). Previously identified GWAS SNPs are indicated with italic font. Genes in the region are displayed beneath the association results.

Figure 2. Expression quantitative trait locus analyses.

Significant eQTL associations identified between rs4808616 and ABHD8 expression in (a) ovarian cancer tissues and (b) in normal breast tissues. (c) A significant association was also identified between rs4808616 and ANKLE1 expression in primary normal ovarian/fallopian tube epithelial cell cultures. The horizontal line indicates the median expression, the limits of the boxes denote the first and third quartiles, and the whiskers represent 1.5 times the interquartile range of the data. Outliers are indicated with circles.

Figure 3. Chromosome conformation capture analysis of long-range interactions at the 19p13 region.

3C interaction profiles in breast and ovarian cell lines. 3C libraries were generated with NcoI, with the anchor point set at the ABHD8 promoter region. (a) A physical map of the region interrogated by 3C is shown, with annotated genes shown in blue, the 13 risk-associated SNPs shown in red, the ABHD8 promoter fragment shown in green and the position of the interacting NcoI fragment represented by the purple bar (not to scale). (b) Relative interaction frequencies between the ABHD8 promoter and regions spanning risk associated SNPs in normal breast (Bre80) and ovarian (IOSE11) epithelial cells lines, and in breast (MCF7) and ovarian (A2780) cancer cell lines. A peak of interaction with the ABHD8 was observed for one region (purple bar) in all four cell lines. There were no interactions detected between the purple region and the BABAM or USHBP1 promoters. The interacting region contains four candidate causal SNPs (from left to right) rs4808075, rs10419397, rs56069439 and rs4808076. Error bars represent s.d. (N=3).

Figure 4. Epigenetic marks intersecting candidate causal SNPs in the 19p13 susceptibility region and analyses of UTR SNPs.

The thirteen candidate SNPs were aligned with open chromatin and enhancer marks (H3K27ac and H3K4me1) in high-grade serous ovarian cancer cells (UWB1.289 and CaOV3) and ovarian cancer precursor cells (ovarian epithelial cells, IOSE and fallopian epithelial cells, FT). Enhancer and insulator (CTCF) data for human mammary epithelial cells (HMECs) were obtained from ENCODE. Five SNPs coincide with biofeatures in breast and/or ovarian cells (indicated in red).

Figure 5. Allele specific analysis of susceptibility SNPs.

(a) Location of SNPs in putative regulatory elements (PREs) and 5′ untranslated regions. (b) RNA stability assays in primary ovarian epithelial cell lines for risk-associated UTR SNPs in ABHD8 and ANKLE1. Normal ovarian epithelial cell lines carrying different genotypes of the risk SNP rs4808616, located in the 3′ UTR of ABHD8. Rs4808616 is tightly correlated with rs111961716 (R²=0.98) located in the 3′ UTR of ANKLE1. The risk allele of rs111961716 was associated with decreased mRNA stability of ANKLE1 compared with the protective allele (P=0.006, ANOVA). Different genotypes of rs4808616 are not associated with the stability the ABHD8 transcript. (c–e) Luciferase assays to evaluate SNP-dependent promoter and enhancer activity. (c) The ANKLE1 promoter SNP did not affect ANKLE1 expression in ovarian cancer cells (A2780) and normal breast cells (Bre80). (d) Allele-specific activity of PRE-A, PRE-B and PRE-C on the ANKLE1 promoter. (e) Allele-specific activity of PRE-A, PRE-B and PRE-C on ABHD8 promoter activity. *P>0.05, **P>0.01, ***P>0.001, ****P>0.0001, two-way ANOVA. RLU, relative light units.

Figure 6. Effects of deletion of the putative enhancer containing the rs56069439 risk SNP in breast and ovarian epithelial cells.

(a) Illustration of the 57 bp region in an intron of ANKLE1 containing rs56069439; H3K4me1 marks overlapped rs56069439 in ovarian, fallopian and breast cells. Location of the two guide RNAs (gRNAs) used to create the stable Δrs56069439 deletion by CRISPR/Cas9 genome editing, cutting sites are indicated with the green arrow. PAM, protospacer adjacent motif. (b) PCR analysis of targeted region in representative MCF10A (breast) epithelial cell clones. Control clones were transfected with the vector backbone only. (c) Verification of deletions by Sanger sequencing, and alignment to the genome using BLAT. (d) Gene expression analysis using TaqMan probes showing downregulation of ANKLE1 was associated with deletion of a region containing rs56069439.

Figure 7. Phenotypic effects of overexpressing full length ABHD8GFP fusion transcript in normal breast and ovarian epithelial cells.

(a) ABHD8 overexpression induced a significant decrease in migration in both breast (MCF10A) and ovarian (IOSE19) cells; (b) ABHD8 overexpression induced a significant decrease in invasion in breast epithelial cells and a similar trend of decreased invasion in ovarian epithelial cells.