Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells

Figures

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  Figure 1.. BRCA2:c.681+5G>C variant. (A) Pedigree of LP’s family. The proband is indicated with an arrow. Circles represent female family members, and squares represent male family members; a slash indicates deceased individuals. Colors denote cancer diagnoses, with the age at death (for those deceased) and age at cancer diagnosis specified. The current age of the proband is also provided. (B) Schematic representation of the variant’s location within intron 8 of the BRCA2 gene. Donor (5′) and acceptor (3′) splice sites (ss) are indicated.

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  Figure 2.. Splicing isoforms expressed in PBMCs from a heterozygous carrier of the BRCA2:c.681+5G>C variant. (A, B, E, F) PBMCs from a control individual and from LP were either mock-treated or treated with cycloheximide (CHX). Total RNA was isolated and analyzed by ddRT–PCR. Probes and primers are indicated. Graphs show the percentage of the indicated isoform relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. Error bars represent the SD. Significant differences: *P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant. (C, D) Sanger sequencing of the two aberrant BRCA2 cDNA fragments isolated from LP PBMCs. The mRNA positions (Human Genome Variation Society nomenclature) and protein consequences are indicated. Schematic representation of the detected isoforms. Source data are available for this figure.

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  Figure 3.. Splicing isoforms expressed in cells from homozygous carriers of the BRCA2:c.681+5G>C variant. (A) Pedigree of CC and DC’s family, two sisters who are homozygous carriers of the BRCA2:c.681+5G>C variant. The probands are indicated with arrows. Circles represent female family members, and squares represent male family members; a slash indicates deceased individuals. Colors denote cancer diagnoses, with the age at cancer diagnosis specified. The current age of the probands is also provided. (B, C, D) Quantification by ddRT–PCR of splicing isoforms expressed in PBMCs (B) and lymphoblastoid cell lines (C, D) from control individuals and DC and CC patients. Probes and primers are indicated. Cells were either mock-treated or treated with cycloheximide (CHX). Graphs on panel B show the percentage of the indicated isoform relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. (C, D) For lymphoblastoid cells, total (C) or nuclear (D) RNA was isolated and analyzed. Given that nuclei are enriched for unspliced pre-mRNA, on panels C and D, the percentage is relative to the sum of spliced and unspliced BRCA2 (18–19 exon junction and exon…

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  Figure S1.. Splicing isoforms expressed in lymphoblastoid cell lines from homozygous carriers of the BRCA2:c.681+5G>C variant. Quantification by ddRT–PCR of splicing isoforms expressed in lymphoblastoid cell lines derived from control individuals and patients DC and CC. Probes and primers are indicated. Cells were either mock-treated or treated with cycloheximide (CHX), as indicated. The graph shows the percentage of the isoform detected by the indicated probe relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. Error bars represent the SD. Significant differences: *P < 0.05; **P < 0.01; ns, non-significant. Source data are available for this figure.

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  Figure 4.. Deficient recruitment of RAD51 to DNA lesions in homozygous cells. (A) Immunoblot detection of the BRCA2 protein in extracts from control and CC lymphoblastoid cell lines. Cyclin A was used as a loading control. Serial dilutions of the control cell line are shown. (B, C) Control and patient CC-derived lymphoblastoid cell lines were treated with 5 μM etoposide for 15 min. (B) At 30 min after etoposide treatment, cells were analyzed by immunofluorescence with anti-gamma-H2AX antibody; nuclei were stained with DAPI. The graph on the right quantifies cells with ≥10 nuclear γ-H2AX foci relative to the total number of cells. (C) At 6 h after etoposide treatment, cells were analyzed by immunofluorescence with anti-gamma-H2AX and anti-RAD51 antibodies; nuclei were stained with DAPI. The graph on the right quantifies cells with ≥10 nuclear RAD51 foci relative to the total number of cells. Dot plots in (B, C) depict results from multiple assays, including two to three replicates using three control cell lines, and CC and DC patient cell lines. Each dot represents an individual replicate. Over 200 cells were counted per assay. Error bars represent the SD. Significant differences: *P < 0.05;…

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  Figure S2.. Repair of DNA lesions induced by etoposide. Control and patient CC-derived lymphoblastoid cell lines were treated with 5 μm etoposide for 15 min and analyzed 24 h later. Immunofluorescence with anti-gamma-H2AX antibody. Nuclei stained with DAPI. The number of γ-H2AX foci was quantified in over 200 cells per assay. Dot plots depict results from multiple assays, including two to three replicates using three control cell lines, and CC and DC patient cell lines. Each dot represents an individual replicate. Error bars represent the SD. Significant differences: ns, non-significant. Source data are available for this figure.

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  Figure 5.. Engineered exclusion of exon 7 in homozygous mutant cells partially restores DNA repair. (A) Schematic representation of the strategy used to disrupt exon 7 splice sites. (B) Quantification by ddRT–PCR of the Delta7,8 splicing isoform expressed in non-transfected CC cell line (red), and CC cell line transfected with the indicated guide RNAs (blue). Probe and primers are indicated. The graph shows the percentage of isoform detected by the indicated probe relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. (C, D) Indicated cells were treated with 10 μM etoposide for 15 min and analyzed 3 h later. Immunofluorescence with anti-gamma-H2AX and anti-RAD51 antibodies. Nuclei stained with DAPI. Confocal microscopy images after maximum intensity projection of Z-stacks of representative examples are shown. A control cell line was GM20172. The patient refers to CC-derived cell line. Edited clones were isolated from CC-derived cells transfected with gRNA 7. RAD51 foci were quantified in over 200 cells per assay. A dot plot shows results from multiple assays, with each dot representing an individual replicate. Significant differences: *P <…

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  Figure S3.. Splicing isoforms expressed in gene-edited clones. (A) Quantification by ddRT–PCR of the indicated splicing isoforms expressed in non-transfected CC cell line (red), and CC cell line transfected with the indicated guide RNAs (blue). Probes and primers are indicated. The graph shows the percentage of isoform detected by the indicated probe relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. (B) Quantification by ddRT–PCR of splicing isoforms expressed in gene-edited clones isolated from CC-derived cells transfected with gRNA 7. Probes and primers are indicated. The graph shows the percentage of isoform detected by the indicated probe relative to total BRCA2 transcripts detected by a probe spanning the junction between exons 13 and 14. (C) Sanger sequencing of the most abundant cDNA fragment amplified by RT–PCR using the BRCA2_e5e10 primer set; mRNA positions are annotated according to the Human Genome Variation Society (HGVS) nomenclature, along with the predicted protein consequences. A schematic representation of the detected isoform (Δ7,8 in-frame isoform) is also provided. (D) Immunoblot analysis of BRCA2 protein expression in…