Research ArticleCANCER

Aneuploidy and a deregulated DNA damage response suggest haploinsufficiency in breast tissues of BRCA2 mutation carriers

See allHide authors and affiliations

Science Advances  29 Jan 2020:
Vol. 6, no. 5, eaay2611
DOI: 10.1126/sciadv.aay2611
  • Fig. 1 Single-cell whole-genome analysis of BRCA2mut/+ human primary breast epithelial cells.

    (A) Workflow depicts dissociation and isolation of human breast epithelial cells from BRCA2 carrier (BRCA2mut/+) prophylactic mastectomy and control [wild-type (WT)] elective mammoplasty cases for subsequent analyses, as indicated. Dot plot at center shows representative flow cytometry sorting via CD49f and EpCAM of ML, LP, and basal epithelial cells. FACS, fluorescence-activated cell sorting. (B) Summary of single-cell whole-genome sequencing (WGS) analysis of flow-sorted, primary uncultured breast epithelial cells. Copy number variation (CNV) calls for individual cells (rows) across the genome (x axis; Chr, chromosome) are shown, with gains and losses boxed. Cell types and genotypes are indicated at the top left, and individual patient ID numbers are indicated at the right. In total, 252 sequenced breast epithelial cells from BRCA2mut/+ (n = 5) and control (n = 2) tissue specimens are depicted. (C) Bar chart depicting the prevalence of CNVs in LP (L) and basal (B) cells of BRCA2 carrier and control (WT) patients. Color code depicts the number of CNVs identified per cell. (D) LP cells from BRCA2 carriers are significantly more likely to harbor CNVs than basal cells. P value is determined by χ2 test.

  • Fig. 2 Polyclonal, subchromosomal aneuploidy is a hallmark of BRCA2mut/+ breast epithelial cells.

    (A) Representative segmentation plots of individual LP (n = 4) and basal (n = 2) cells harboring CNVs from four BRCA2 mutation carriers. Y axis depicts normalized WGS read counts across the genome (x axis). Red dots indicate region of gain, whereas blue dots indicate losses. Patient ID numbers are indicated at the right. (B) Segmentation plots of three LP cells that share a clonal loss (red box) in a BRCA2 carrier (patient 131). Zoomed-in images of the clonal loss are shown at the right. (C) Representative chromatograms from single-cell PCR-based Sanger sequencing of genomic DNA in a BRCA2mut/+ LP cell. The presence of a heterozygous single-nucleotide polymorphism (SNP) and the superimposition of sequences adjacent to the frameshift mutation suggest that LOH has not occurred.

  • Fig. 3 BRCA2mut/+ breast epithelial cells exhibit DNA damage and an impaired replication stress checkpoint response.

    (A) Representative images of comet assays performed on primary human breast epithelial cells isolated from control (WT) and BRCA2mut/+ tissues. Red lines highlight “tail” of broken DNA. Graph below summarizes data from n = 3 patients per genotype (50 cells per patient). Cells were either untreated (Unt) or treated with HU for 4 hours. Data are depicted as fold change in tail DNA intensity. P values are determined by unpaired t test. ***P < 0.001 and ****P < 0.0001. Error bars indicate SD. (B) Representative confocal immunofluorescence staining of primary breast epithelial cells for p-CHEK1 (Ser317) shows increased nuclear staining following HU treatment only in control (WT) but not in BRCA2mut/+ cells. Graph at the right summarizes nuclear fluorescence of individual cells (dots) (n = 4 patients for control and n = 3 patients for BRCA2mut/+; four fields counted per condition per patient). P values are determined by unpaired t test. ***P < 0.001 and ****P < 0.0001. Horizontal lines indicate means and SDs. Scale bars, 20 μm. (C) Chromatograms depicting Sanger DNA sequencing of a cytospin of primary breast cells assayed in (B) from a BRCA2mut/+ patient harboring BRCA2 5799_5802delCCAA (p.Asn1933Lysfs). The superimposition of sequences adjacent to the frameshift mutation suggests that LOH has not occurred. (D) Heat map of RNA sequencing (RNA-seq) data from freshly sorted cells shows differential expression of RSRD (replication stress response deficiency) genes (24) in BRCA2mut/+ LP cells (n = 7 patients) compared to control (WT) LP cells (n = 9 patients). Columns correspond to individual patients.

  • Fig. 4 BRCA2mut/+ LP cells display increased TP53 activity and suppressed NF-κB/SASP pathway expression.

    (A) Bar charts show the mean expression levels of canonical TP53 target genes in freshly sorted BRCA2 carrier LP cells (n = 7 patients) compared to controls (WT; n = 9 patients), assessed by RNA-seq. Error bars denote SEM. P values are determined by Mann-Whitney test. *P < 0.05 and **P < 0.01. XPC, Xeroderma pigmentosum group C-complementing protein; FPKM, Fragments Per Kilobase of transcript per Million mapped reads. (B) Heat map depicts down-regulation of NF-κB/SASP pathway genes in BRCA2 carrier LP cells compared to controls (WT), assessed by RNA-seq as in (A). Columns correspond to individual patients. Direct NF-κB target genes are highlighted in red. (C) Western blot analysis shows that NFKB1 (p50) and NFKB2 (p52) subunits are expressed at lower levels in BRCA2mut/+ breast tissues compared to control (WT) tissues (n = 3 patients per genotype). β-Tubulin serves as a loading control. (D) Negative enrichment of a SASP signature in GSEA of RNA-seq data from freshly sorted LP cells of BRCA2 carriers (n = 7 patients) and controls (WT; n = 9 patients). NES, normalized enrichment score; FDR, false discovery rate.

  • Fig. 5 Noncancerous breast tissues of BRCA2 mutation carriers demonstrate age-associated deregulation of epithelial cell proportions compared to controls.

    (A) Representative flow cytometry analysis showing distinct epithelial subpopulations (basal, LP, and ML) isolated from breast tissues of control (WT) and BRCA2 mutation carriers following sorting via CD49f and EpCAM staining. Numbers indicate percentages of each epithelial cell subpopulation. (B) Linear regression analysis of LP and basal cell proportions by age for controls (WT) (n = 26 patients) and BRCA2 carriers (n = 28 patients). The LP/basal ratio by patient provides additional validation as it accounts for technical factors that may have subtle effects on absolute cell numbers. (C) TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining of representative control (WT) and BRCA2 carrier tissues. Summary data obtained by counting four fields for five patients per genotype are shown. ****P < 0.0001 by Fisher’s exact test.

  • Fig. 6 Summary of findings in primary BRCA2mut/+ breast tissues.

    Epithelial progenitor cells of heterozygous germline BRCA2 carriers exhibit DNA damage, failed replication stress, and damage responses, together with attenuated apoptosis. LOH analyses suggest that these findings may reflect a haploinsufficient phenotype for BRCA2 in vivo.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/5/eaay2611/DC1

    Supplementary Materials and Methods

    Fig. S1. Analysis of single-cell WGS data from normal human skin and brain cells.

    Fig. S2. Identification and characterization of CNVs in freshly collected BRCA2mut/+ breast epithelial cells.

    Fig. S3. Characterization of replication stress response deficiency and haploinsufficiency in BRCA2mut/+ breast epithelial cells.

    Fig. S4. Suppression of NF-κB/SASP response associated with loss of BRCA2.

    Fig. S5. Proportions of mammary epithelial cell subsets in BRCA2 carrier and control tissues.

    Table S1. Characteristics of patients undergoing WGS of breast tissues.

    Table S2. Summary of single-cell site-specific PCR/sequencing data.

    Table S3. Characteristics of patients undergoing RNA-seq of breast tissues.

    Reference (43)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Analysis of single-cell WGS data from normal human skin and brain cells.
    • Fig. S2. Identification and characterization of CNVs in freshly collected BRCA2mut/+ breast epithelial cells.
    • Fig. S3. Characterization of replication stress response deficiency and haploinsufficiency in BRCA2mut/+ breast epithelial cells.
    • Fig. S4. Suppression of NF-κB/SASP response associated with loss of BRCA2.
    • Fig. S5. Proportions of mammary epithelial cell subsets in BRCA2 carrier and control tissues.
    • Table S1. Characteristics of patients undergoing WGS of breast tissues.
    • Table S2. Summary of single-cell site-specific PCR/sequencing data.
    • Table S3. Characteristics of patients undergoing RNA-seq of breast tissues.
    • Reference (43)

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article