Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

Abstract

The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.

Fig. 1. Characterisation of SULT1A1.

a Genomic viewer (UCSC) of the SULT1A1 gene locus with copy number variants; deletion (red) and duplication (blue). b Dosage effect in breast tumours with SULT1A1 copy number variants. CNVs copy number variants.

Fig. 2. Characterisation of MCF7–WT, and isogenic MCF7–C1, and MCF7–BRCA1^+/− cell lines.

a Sequence of heterozygous pathogenic BRCA1 c.2432_2433del variant introduced by CRISPR-Cas9. b Relative proliferation of MCF7–WT and clonally expanded CRISPR-Cas9 MCF7–C1 and MCF7–BRCA1^+/− cells for 72 h post seeding. Relative expression of c BRCA1, d ESR1, e CYP1A1, and f SULT1A1 for MCF7–WT, MCF7–C1 and MCF7–BRCA1^+/− cells. 4-OHE2 4-hydroxyestradiol, MMC Mitomycin C, Error bars = standard error of the mean; ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; n = 3 independent biological replicates; unpaired two-sided t-test.

Fig. 3. siRNA knockdown of SULT1A1 reduces proliferation and DNA damage of MCF7–BRCA1^+/− cells but not MCF7–C1 cells.

Expression of (a) SULT1A1 and (b) BRCA1 72 h post transfection. Relative DNA content of transfected (c) MCF7–C1 and (d) MCF7–BRCA1^+/− cells 72 h post transfection. Quantification of transfected DNA damage using the comet assay (e, f) and ϒ-H2AX/53BP1 foci quantification (g, h) for MCF7–C1 and MCF7–BRCA1^+/− cells, with and without siSULT1A1 transfection, 21 h post treatment with 1 μm 4-OHE2 (e and g) or 10 μm MMC (f and h). Significance of differences in relative expression and DNA content was determined by unpaired two-sided t-test. Gene expression and DNA content of siSULT1A1 transfected cells normalised to siControl transfected cells. Differences in DNA damage were determined by two-way analysis of variance. Example images of comet and ϒ-H2AX/53BP1 foci shown in Supplementary Figs. 3–6. 4-OHE2 = 4-hydroxyestradiol; MMC = Mitomycin C; Error bars = standard error of the mean; ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; n = 3 independent biological replicates.