Fig 1.
BRCA1 variant carrier fibroblasts collected and reprogrammed into iPS cells.
a Schematic representation of the human BRCA1 mRNA, with pathogenic variants labelled in red, non-pathogenic labelled in green, and VUSs labelled in blue. BRCA1 exons are numbered from 2 to 24. b Table summarising the BRCA1 status of the study subjects, with schematic representations of the human BRCA1 protein variants. RING: Really Interesting New Gene; NLS: Nuclear Localisation Signal; DBD: DNA Binding Domain; SCD: Serine Containing Domain; BRCT: BRCA1 C-Terminus. Data shown are up to date as of January, 2020.
Fig 2.
Derivation of iPS cells from fibroblasts and characterisation (example of iC61G_2).
a Representative brightfield images of reprogramming cells at the indicated time points during iPS cells reprogramming process. Emergence of the first colonies was observed around day 7. White arrows show colonies. b Colonies adapted feeder free. Images were taken using an Olympus ix50 microscope at 4x and 10x magnifications. Scale bars represent 250 μm. c Representative staining for pluripotency markers. Nuclei were counterstained with DAPI (blue). Images were taken using an Eclipse 50i upright epifluorescence microscope at 50x magnification. Scale bars represent 100 μm. d Representative histological analysis of hematoxylin-eosin-stained images of sections of teratomas derived from iPS cells, showing all three germ-layers labelled (1 = endoderm, 2 = mesoderm, 3 = ectoderm). Images were taken using a Zeiss Axioscope Z plus at x10 magnification. e Representative aCGH graph.
Fig 3.
Assessment of BRCA1 protein expression level in iPS cells derived from fibroblasts.
a, b Expression levels demonstrated by Western blot of whole-cell lysates extracted from iPS cell lines. HSP90 was used as the loading control. c 293T cells were transfected with GFP-BRCA1-WT or variants. The truncated form of BRCA1 is indicated by an arrow. HSP90 was used as the loading control. d Immunofluorescence staining for BRCA1 nuclear foci formation following ionising radiation exposure. Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm.
Fig 4.
Whole exome sequencing analysis identifies a known pathogenic truncating variant in ATM in the Y856H patient.
a Screenshot of IGV. Track 1 shows mapped reads from iY856H. Track 2 shows mapped reads from matched fibroblasts. The green As in the grey alignments represent bases that did not match the reference sequence. b Validation by Sanger sequencing, in fibroblast and in iPS cells, of the heterozygous variant in the ATM gene identified by WES. c iPS cells were untreated, or treated by exposure to 0.5 Gy of ionising radiation (IR). Whole-cell lysates were prepared 1 hour post-IR, and analysed by Western blot for ATM phosphorylation at Serine 1981. HSP90 was used as the loading control. ATM autophosphorylation levels were measured using ImageJ quantification tool, and the HSP90 normalized intensity values are presented under the blots.
Fig 5.
Impact of BRCA1 variants on response to DNA damage repair.
a Cell viability assay of WT and indicated BRCA1 variant iPS cell lines following IR treatment. The viable fraction is expressed as a percentage of the viability values obtained for the respective untreated genotypes. (*) indicates p<0.05 using Student’s t-test. b The mask defining strategy for ImageStreamX. The upper panel shows raw data before the application of feature masks for the analysis. The lower panel shows feature masks in cyan. BF = brightfield. Magnification = 40x. c Representative images of iPS cells following IR exposure for the time points indicated. d All lines have been analysed prior to, and at 1, 12, and 24 hours post-irradiation. The percentage of cells with >5 foci has been plotted. (*) indicates significant difference (p = 0.02) between iWT1_2 and iC61G_2, (§§) indicates significant difference (p = 0.003) between iWT1_2 and iK381X_1, (##) indicates significant difference (p = 0.005) between iWT2_2 and iC61G_2, and (§§) indicates significant difference (p = 0.048) between iWT2_2 and iK381X_1 using Student’s t-test. No significant difference was observed between iY856H_2 and the two WT lines analysed. Error bars represent standard error of the mean. N = 3 for iWT1_2, iWT2_2, iK381X_1 and iY856H_2, N = 4 for iC61G_2.
Fig 6.
Effect of BRCA1 variants on HR pathway efficiency.
a The efficiency of repair was measured by quantification of GFP fluorescence expression, which can only occur when the linearized plasmid is accurately repaired. Ratios of GFP+ to DsRed+ were normalised to iWT1_2 iPS cells. Student’s t-test was used to measure the significance; p-values are shown on the graph. N≤3. b Number of gaps and breaks per metaphase spread of each iPS cell line, with or without ionising radiation treatment. Error bars represent standard error of the mean. Statistical significance was determined using a Mann-Whitney U test. p-values are shown on the graph. N = 30 metaphase cells for each data point. Representative images (inset) of a chromatid gap and a chromatid break.
Fig 7.
Differentiation of induced pluripotent stem cells into mammospheres.
a Representative images of iWT1_2, iWT2_2, iC61G_2, iK381X_1 and iY856H_2 iPS cells differentiated into primary mammospheres using MammoCult medium. b Representative images of iPS cells-derived mammospheres stained for two stem markers: Nestin (green) and OCT-4 (red). Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm. c Immunofluorescence staining for basal (CK14 and alpha-SMA) and luminal (CK18) markers on differentiated mammospheres. Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm.