Journal: Nucleic Acids Research

Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

doi: 10.1093/nar/gku870

Figure Lengend Snippet: Scheme of consecutive experimental steps from selective BRCA1 gene isolation in yeast S. cerevisiae to its expression in the UWB1.289 BRCA1-deficient human cells. ( a ) Step 1: A direct TAR isolation of the BRCA1 gene from human genomic DNA. TAR vector contains two gene targeting hooks (green and blue boxes), a yeast centromeric locus ( CEN ) and a yeast selectable marker HIS3. Recombination between targeting sequences in the TAR vector and the targeted sequences of the genomic DNA fragment leads to the rescue of the BRCA1 -containing loci as a circular TAR/YAC molecule. Step 2: Retrofitting of the circular TAR/YAC isolate containing the full-length BRCA1 gene by pJBRV1 vector containing a 3′ HPRT-loxP-eGFP cassette. Recombination of the Bam HI-linearized pJBRV1 vector with a TAR/YAC in yeast leads to replacement of the ColE1 origin of replication by the F’ ′ factor origin of replication that allows enable subsequent propagation in a BAC form. Step 3: BRCA1 gene loading into a unique loxP site of the alphoid tetO -HAC (tetO-HAC) by Cre-loxP recombination system in hamster CHO cells. Step 4: MMCT of alphoid tetO -HAC/BRCA1 from CHO into the human BRCA1-deficient UWB1.289 cells for complementation analyses. Step 5: Elimination of the alphoid tetO -HAC/BRCA1 from UWB1.289 cells by expression of the tTS fusion construct. ( b ) PCR analysis of the TAR clones containing the full-length BRCA1 gene for the presence of exons before and after retrofitting in yeast and after BRCA1 insertion into a loxP site of the alphoid tetO -HAC in CHO cells. The numbers above correspond to the exon number (from 1 to 24). M, ladder. ( c ) FISH analysis of the alphoid tetO -HAC/BRCA1 in CHO cells using specific probes for HAC vector (in red) and for cDNA BRCA1 gene sequences (in green). ( d ) Transcriptional analysis of the human BRCA1 gene in CHO cells. Lane 1 corresponds to a positive control: RT-PCR of RNA purified from human MCF7 cells. Lane 2 corresponds to a negative control: RT-PCR of RNA purified from CHO cells. Lanes from 3 to 6 correspond to RNA purified from five independently obtained alphoid tetO -HAC/BRCA1-containing CHO clones. Lane 7 corresponds to the RT-PCR product of the control ERCCA2 gene. Clones 3, 4, 5 and 6 are BRCA1-positive. All primers designed are presented in Supplementary Table S1. The bands have a predicted size of RT-PCR products. All amplified fragments were gel-purified and sequenced and proved the identity of products to the human BRCA1 transcripts. M, ladder. ( e ) FISH analysis of the alphoid tetO -HAC/BRCA1 in UWB1.289 cells using specific probes for HAC vector (in red). ( f ) Western blot analysis of BRCA1-deficient UWB1.289 cells, alphoid tetO -HAC/BRCA1-containing UWB1.289 cells (five independently obtained clones after MMCT transfer from the clone #4 of CHO cells) using human-specific Abs against BRCA1. BRCA1 inserted into the alphoid tetO -HAC produces a protein of the predicted size. The human breast adenocarcinoma MCF7 cell line (hemizygous for the BRCA1 wild type with a reduced BRCA1 expression) was used as a positive control for expression of BRCA1.

Article Snippet: The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele ( ) was obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Techniques: Isolation, Expressing, Plasmid Preparation, Marker, Construct, Clone Assay, Positive Control, Reverse Transcription Polymerase Chain Reaction, Purification, Negative Control, Control, Amplification, Western Blot

Journal: Nucleic Acids Research

Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

doi: 10.1093/nar/gku870

Figure Lengend Snippet: Complementation tests for functionality of BRCA1 in UWB1.289 cells. ( a ) Accumulation of γ-H2AX in BRCA1-deficient cells (UWB1.289) carrying alphoid tetO -HAC/BRCA1 and in the cells that have lost the HAC after exposure to 2 Gy of γ-rays. Cells were stained 15 h after irradiation with anti-γ-H2AX antibodies (red) and with DAPI (blue). Representative microscopic fluorescence images of γ-H2AX foci accumulation are shown. ( b ) Quantitative data for γ-H2AX foci processing. Cells were collected after 15 h and the average number of γ-H2AX foci was quantified in BRCA1-positive and BRCA1-negative cells. NR cells have been included as a control. ( c ) Radiation sensitivity clonogenic survival assay. BRCA1-deficient UWB1.289 cells are radiation sensitive. Cells carrying alphoid tetO -HAC/BRCA1 and after HAC loss were plated in triplicate and irradiated with the indicated doses (from 0 to 8 Gy). Cells were stained with crystal violet and counted visually. Clonogenic survival was calculated as a number of colonies present in each plate normalized to the number of colonies in the NR control plates. Cells carrying alphoid tetO -HAC/BRCA1 (red) are less sensitive than the cells that lost the HAC (blue). Bars, mean of triplicate samples. ( d ) Co-immunoprecipitation analysis of BRCA1 and pol β proteins interaction. Cell extracts from the cells carrying alphoid tetO -HAC/BRCA1 and from the cells that have lost the HAC were immunoprecipitated with anti-pol β antibody or non-immune IgG. Immunoprecipitated proteins were detected by SDS-polyacrylamide gel electrophoresis and immunoblotting. ( e ) Viability of UWB1.289 cells carrying alphoid tetO -HAC/BRCA1 (red) and after HAC loss (blue) after treatment with paclitaxel. At 24 h after treatment the cell viability was measured. All experiments were repeated three times with each sample prepared in triplicate.

Article Snippet: The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele ( ) was obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Techniques: Staining, Irradiation, Fluorescence, Control, Clonogenic Cell Survival Assay, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Nucleic Acids Research

Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

doi: 10.1093/nar/gku870

Figure Lengend Snippet: Micronuclei (MNi) formation in the HAC/BRCA1-containing UWB1.289 cells and in the same cells that have lost the HAC

Article Snippet: The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele ( ) was obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Techniques:

Journal: Nucleic Acids Research

Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

doi: 10.1093/nar/gku870

Figure Lengend Snippet: De-repression of alpha-satellite DNA transcription in BRCA1-deficient UWB1.289 cells. ( a , c and d ) Quantitative RT-PCR experiments showing that the satellite DNA transcripts of HORs regions of the chromosome 21 (chr21a and chr21b), chromosome X (chrX) ( a ), chromosome 5 (D5Z1 and D5Z2) ( d ) and DNA transcripts of pericentromeric Sat2 repeats located at 10q21 ( c ) are significantly repressed in the cells carrying alphoid tetO -HAC/BRCA1 compared to that of the cells that have lost the HAC. C t values were normalized relative to each other. Error bars indicate SD. ( b ) Timing of transcription of HORs regions of chromosome X and chromosome 21. ( e ) ChIP analysis of different centromeric regions using antibodies against CENP-B (centromeric protein B) in BRCA1-deficient UWB1.289 cells containing alphoid tetO -HAC /BRCA1 and in the same cells that have lost the HAC. ( f ) Quantitative RT-PCR experiments showing the transcripts of the double homeobox 4 ( DUX4 ) gene located in the heterochromatic subtelomeric 4q35 region, the cancer-testis SPANX-B gene located at the Xq27 region and, as a control, a housekeeping gene PPIH, peptidyprolyl isomerase H (cyclophilin H) was used. Asterisk denotes significant difference.

Article Snippet: The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele ( ) was obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Techniques: Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

doi: 10.1093/nar/gku870

Figure Lengend Snippet: Expression of the human BRCA1 gene in porcine ST cells. ( a ) From hamster CHO cells exhibiting a high efficiency of microcell formation, the alphoid tetO -HAC/BRCA1 was transferred into the pig's testis fibroblast ST cells using MMCT technique. (b) FISH analysis of porcine cells containing the alphoid tetO -HAC/BRCA1. Chromosomal DNA was counterstained with DAPI (blue). Arrow indicates to the HAC. ( c ) Fluorescence image of cells carrying the alphoid tetO -HAC/BRCA1 containing the expressed eGFP transgene. ( d ) Expression of the human BRCA1 gene in porcine cells. RT-PCR analysis of genomic DNA isolated from the original ST cells (lane 4), from two porcine clones (pl4 and pl5) containing the alphoid tetO -HAC/BRCA1 (lanes 2 and 3) and from the human HeLa cells as a positive control (lane 1) using the human BRCA1-specific primers (Supplementary Table S1). The size of the predicted product is 839 bp. M-ladder containing alphoid tetO -HAC/BRCA1

Article Snippet: The BRCA1-deficient human ovarian cancer cell line UWB1.289 carrying a germline BRCA1 mutation within exon 11 and having a deletion of the wild-type allele ( ) was obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Techniques: Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Isolation, Clone Assay, Positive Control

Journal: Archives of pathology & laboratory medicine

Article Title: Familial Pancreatic Cancer

doi: 10.5858/133.3.365

Figure Lengend Snippet: Pancreatic Cancer-Associated Genetic Syndromes

Article Snippet: Goggins et al demonstrated that 7% of the patients with apparently sporadic pancreatic cancer at the Johns Hopkins Hospital had germline BRCA2 gene mutations 40 .

Techniques: Histopathology

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Donut plots showing the distribution of the a clinical classification (“ClinVar Class”) and b sequence ontology variant consequence (“Consequence”) for the 1717 CDS ± 5 bp variants with filtering allele frequency (FAF) > 0.001 (i.e., variants not meeting the BRCA1 and BRCA2 VCEP “BA1” benign stand-alone criterion), present in at least three individuals in the combined dataset, and with evidence from at least two datasets for BRCA2 . The clinical classification status (“ClinVar Class”) of variants was retrieved from the ClinVar database (last accessed on January 7, 2024). c Sankey plot depicting “suggested case-control likelihood ratio (ccLR) ACMG/AMP evidence” (excluding variants with a ccLR of “No evidence”) provided for unclassified variants (i.e., variants not reported in ClinVar or listed in ClinVar as VUS, variants of conflicting classifications of pathogenicity or variants with classification “not provided”), per sequence ontology variant consequence (“Consequence”).

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Sequencing, Variant Assay, Control

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Overlay of the case-control likelihood ratios (LRs) and the logistic regression odds ratio (OR) estimates is represented within each exon (middle panel). Exons are sequentially numbered from 1 to 23 and annotated from right to left to match the MANE Select transcripts. Although BRCA1 was initially described with 24 exons (GenBank Accession ID U14680.1 ), exon 4 is missing following further assessment of the gene. We implement the most updated version of exon numbering (excluding legacy exon numbering). Case-control LRs (top panel) are represented on a continuous log2-transformed y axis with axis breaks. For the case-control LR analysis, the red color gradient represents LR reaching suggested ACMG/AMP evidence in favor of pathogenicity with strength levels ranging from very strong (dark red) to supporting (yellow). The green color gradient represents LR reaching ACMG/AMP evidence against pathogenicity with strength levels ranging from very strong (dark green) to supporting (light green). Variants with LR of “No evidence” are not plotted. For the logistic regression analysis (bottom panel), orange color represents OR estimates reaching the strong PS4 criterion (OR ≥ 4.0, P value < 0.05, and confidence interval (CI) not including 2.0). Variants with OR estimates not reaching the PS4 criterion are not plotted. Associations were adjusted for age and study country (BCAC dataset), age and ethnic group (CARRIERS dataset), and age and genetic ancestry (UKB dataset). Only variants present in both cases and controls were analyzed. ORs with 95% CIs were estimated for each dataset and combined using a fixed-effects, inverse-variance meta-analysis in the ‘metafor’ R package to derive an overall test of association. A two-sided likelihood ratio test (LRT) was used to calculate P-values. No adjustments were made for multiple comparisons. For visualization purposes, the y-axis for logistic regression is represented in reverse order. LCI, lower confidence interval. Sequence ontology variant consequence (“Consequence”) is represented with different symbols.

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Control, Transformation Assay, Sequencing, Variant Assay

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Sankey plots for a BRCA1 and b BRCA2 . The variants assigned case-control likelihood ratio (LR) evidence in favor of or against pathogenicity (with suggested supporting, moderate, strong, or very strong evidence strength) are simplistically annotated as “Pathogenic”, “Benign”, “Suggested case-control LR (ccLR) ACMG/AMP Evidence”, respectively. Variants with LRs between 0.48 and 2.08 are defined as “No evidence” in the “Suggested ccLR ACMG/AMP Evidence” panel. The clinical classification status (“ClinVar Class”) of variants was retrieved from the ClinVar database (last accessed on January 7, 2024).

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Control

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Histograms showing the distribution of case-control likelihood ratios (LRs) categorized by a sequence ontology variant consequence for BRCA1 , b ClinVar classification for BRCA1 , c sequence ontology variant consequence for BRCA2 , d ClinVar classification for BRCA2 . Histograms categorized by ClinVar classification are divided into two panels; the top panel represents variants curated in ClinVar as (likely) benign or (likely) pathogenic, while the bottom panel represents unclassified variants (variants not reported in ClinVar or listed in ClinVar as variants of uncertain significance or with conflicting classifications of pathogenicity). For visualization purposes the x axis represents log10(LR) values. Dashed lines represent LRs between 0.48 and 2.08 considered as of “No evidence”.

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Control, Sequencing, Variant Assay

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Stacked bar plots of the suggested case-control likelihood ratio (LR) ACMG/AMP evidence per exon and sequence ontology variant consequence for a BRCA1 and b BRCA2 . Exons are sequentially numbered to match the MANE Select transcripts. Although BRCA1 was initially described with 24 exons (GenBank Accession ID U14680.1 ), exon 4 is missing following further assessment of the gene; legacy exon numbering for BRCA1 is represented in brackets. Variants assigned case-control LR evidence in favor of or against pathogenicity (with suggested supporting, moderate, strong or very strong evidence strength) are simplistically annotated as “Pathogenic”, and “Benign” in the key, respectively. Variants with LRs between 0.48 and 2.08 are defined as “No evidence”.

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Control, Sequencing, Variant Assay

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Concordance is shown separately for a BRCA1 and b BRCA2 . The top panels for each gene represent case-control likelihood ratios (LRs) compared to variants predicted as benign (“BP4 criterion”, “predicted benign” or “functional”) or pathogenic (“PP3 criterion”, “predicted pathogenic” or “loss-of-function”) by in silico prediction methods (AlphaMissense, BayesDel, MutPred2, VEST4 and REVEL) or through high-throughput functional assays (Findlay et al., 2018, Huang et al., 2025, Sahu et al., 2025, Hu et al., 2024, Mesman et al., 2019). Yellow and green colors represent variants predicted as pathogenic or benign by functional predictors, respectively. For visualization purposes the x axis represents log10(LR) values. Box plots with individual data points display the median value, with whiskers extending to a maximum of 1.5 × interquartile range (IQR) beyond the box. The notch in the box approximates the 95% confidence interval (CI) for the median. Bottom panels for each gene represent sequence-pathogenicity heatmaps demonstrating the concordance between the case-control LR (ccLR) method and functional predictors. For the case-control LR (ccLR) evidence, red color gradient represents LR reaching suggested ACMG/AMP evidence in favor of pathogenicity with strength levels ranging from very strong (dark red) to supporting (yellow). The green color gradient represents LR reaching suggested ACMG/AMP evidence against pathogenicity with strength levels ranging from very strong (dark green) to supporting (light green). Variants with ccLR of “No evidence” are not plotted. For the functional predictors, yellow and green colors represent evidence in favor and against pathogenicity, respectively (expressed as “pathogenic supporting”). The total number of variants included in the concordance analyses is depicted in Supplementary Data .

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Control, Functional Assay, In Silico, High Throughput Screening Assay, Sequencing

Journal: Nature Communications

Article Title: Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification

doi: 10.1038/s41467-025-59979-6

Figure Lengend Snippet: Using sequencing data of 96,691 female breast cancer cases and 302,116 unaffected controls from the Breast Cancer Association Consortium (BCAC), the Cancer Risk Estimates Related to Susceptibility (CARRIERS) consortium and the UK Biobank (UKB) we calculated case-control likelihood ratios (LRs) and odds ratios (ORs) for 11,207 BRCA1 and BRCA2 variants, of which 6909 are coding (coding sequence, CDS ± 5 bp). Derived LRs and ORs were further aligned to ACMG/AMP evidence strengths to provide evidence in favor of or against pathogenicity following sensitivity analyses-derived variant exclusion criteria.

Article Snippet: Specific guidelines for BRCA1 and BRCA2 , based on this framework, have been published by the ClinGen Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) BRCA1 and BRCA2 Variant Curation Expert Panel (VCEP) .

Techniques: Sequencing, Control, Derivative Assay, Variant Assay

Journal: Future Oncology

Article Title: A randomized Phase II study of veliparib with temozolomide or carboplatin/paclitaxel versus placebo with carboplatin/paclitaxel in BRCA1 / 2 metastatic breast cancer: design and rationale

doi: 10.2217/fon-2016-0412

Figure Lengend Snippet: Poly(ADP-ribose) polymerase inhibitors in clinical development for breast cancer.

Article Snippet: Talazoparib (BMN 673) , Medivation , Phase III: – Advanced setting monotherapy in germline BRCA1 / 2 -mutated breast cancer Phase II: – Advanced setting BRCA1 / 2 wild-type, triple-negative breast cancer and homologous recombination deficiency – Advanced setting BRCA1 / 2 -mutated breast cancer – Advanced setting in germline BRCA -intact breast cancer – Neoadjuvant setting in BRCA1 / 2 -mutated breast cancer.

Techniques: Adjuvant, Homologous Recombination