The BRCA1 Variant p.Ser36Tyr Abrogates BRCA1 Protein Function and Potentially Confers a Moderate Risk of Breast Cancer

<div><p>The identification of variants of unknown clinical significance (VUS) in the <i>BRCA1</i> gene complicates genetic counselling and causes additional anxiety to carriers. <i>In silico</i> approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.</p></div

Ribbon representation of the BARD1:BRCA1 complex.

<p>A. The RING domains of BRCA1 and BARD1 (PDB 1JM7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400-Brzovic3" target="_blank">[63]</a>) are represented in magenta and blue respectively and the residues isoleucine 26, serine 36 and C61G are indicated in yellow. Orange spheres represent the Zn²⁺ ions. This model demonstrates that serine 36 lies within the second β-sheet of the RING structure. B. and C. represent the complex in a different orientation revealing the position of serine 36 (B.) and its substitution by tyrosine (C.) may distort the structure. The model was modified using PyMOL software.</p

p.Ser36Tyr (S36Y) BRCA1:BARD1 complex is withheld within the cytoplasm following HU treatment.

<p>Lysates of transfected cells were fractionated into nuclear and cytosolic extracts following S-phase synchronization and treatment with HU. BARD1 was co-precipitated with the ectopically expressed BRCA1 in the A. Nuclear and B. Cytosolic extracts with the anti-DYKDDDDK tag antibody which recognizes only the exogenous BRCA1. A. Immunoblot analysis of the pull-downs demonstrated that unlike wild type BRCA1, both the p.Ser36Tyr and p.Cys61Gly (C61G) variants exhibit reduced ability to co-precipitate BARD1 in the nuclear extracts of stressed cells. B. However, in the cytosolic extracts the p.Ser36Tyr variant, but not p.Cys61Gly, co-precipitated a higher proportion of BARD1 in treated cells compared to wild type transfected cells. In untreated cells, both variants, including wild type BRCA1, co-precipitated larger amounts of BARD1 compared to treated cells. Bar charts showing that C. in the nuclei of treated cells, both the p.Ser36Tyr and p.Cys61Gly variants exhibit reduced ability to co-precipitate with BARD1, whereas D. in the cytosol the p.Ser36Tyr variant co-precipitates similar levels of BARD1 compared to wild type BRCA1, but it is not statistically significant. Immunoblot analysis of nuclear and cytoplasmic extracts demonstrating their purity is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093400#pone.0093400.s002" target="_blank">Fig. S2</a>. The results are representative of 2 experiments.</p

Expression of full-length, epitope-tagged BRCA1 in HEK293T cells.

<p>Protein expression analysis of wild type BRCA1, p.Ser36Tyr (S36Y) VUS and of the known pathogenic p.Cys61Gly (C61G) variant following transient transfection in HEK293T cells was confirmed with the anti-DYKDDDDK tag antibody that recognizes ectopically expressed BRCA1. Immunoblot analysis detected a 220 KDa band which corresponds to full-length BRCA1 protein. Both p.Ser36Tyr and p.Cys61Gly variants demonstrated reduced protein expression compared to wild type BRCA1. Co-transfection with wild type BRCA1 and BARD1 induced an increase in BARD1 protein expression (Supp Fig.1). EV corresponds to empty vector and WT to wild type protein. β-actin served as loading control. The results are representative of between 3 and 5 experiments.</p

p.Ser36Tyr (S36Y) BRCA1 variant demonstrates reduced ability to interact with BARD1 in S-phase synchronized cells.

<p>A. Exogenously expressed BRCA1 co-precipitates with BARD1 in cells that were co-transfected with BRCA1 and BARD1, S-phase synchronized and treated with HU. Immunoblot analysis of the pull-downs performed with the anti-DYKDDDDK tag antibody which recognizes only the epitope-tagged exogenous BRCA1 protein, demonstrated that BARD1 levels co-precipitated by wild type BRCA1 are higher than those co-precipitated by each of the p.Ser36Tyr and p.Cys61Gly (C61G) variants in both resting or HU treated cells. B. Immunoblot analysis of whole cell extracts detected decreased BARD1 and exogenous BRCA1 expression in p.Ser36Tyr and p.Cys61Gly BRCA1 variant transfected cells compared to the wild type transfected cells. β-actin served as loading control. C. Bar charts comparing the levels of BARD1 co-precipitated by p.Ser36Tyr and p.Cys61Gly variant with wild type BRCA1 in untreated cells or cells treated with HU, demonstrate that both variants exhibit reduced ability to interact with BARD1 compared to wild type BRCA1. D. Bar charts showing the expression levels of wild type BRCA1 and p.Ser36Tyr and p.Cys61Gly variants following normalization with β-actin in whole cell extracts, demonstrate that both variants are expressed at lower levels compared to wild type BRCA1. The results are representative of 3 experiments.</p

p.Ser36Tyr (S36Y) BRCA1 variant exhibits diminished co-localization with conjugated ubiquitin foci in HU treated cells.

<p>Immunofluorescence analysis of transfected, S-phase synchronized cells demonstrated that the p.Ser36Tyr BRCA1 variant fails to co-localize with conjugated ubiquitin detected by the FK2 antibody that recognizes only conjugated ubiquitin structures. A high proportion of p.Ser36Tyr BRCA1 and conjugated ubiquitin were detected outside the nucleus similar to wild type transfected cells. HU treatment induced mobilization and co-localization of wild type BRCA1 and conjugated ubiquitin in the nuclei of transfected cells. Contrary to the wild type transfected cells, HU treatment did not induce p.Ser36Tyr nuclear mobilization to the same levels and in many cells the p.Ser36Tyr protein did not co-localize with conjugated ubiquitin foci. Three different locations are shown for the p.Ser36Tyr variant following treatment with HU. Nuclei were stained with DAPI. The forth column is the merge of BRCA1 and conjugated ubiquitin and where green and red signals overlap a yellow signal is seen indicating co-localization. The fifth column is the merge of all stains. Where all signals overlap a white signal is seen, where red and blue signal overlap a pink signal is seen and where green and blue signals overlap a violet signal is seen indicating co-localization. p.Cys61Gly (C61G) and empty vector (EV) control are displayed in Supporting Fig. 3). Statistical analysis confirmed that the observed effects are significant (p<0.05). Insets show the arrow pointed cells following enlargement. The results are representative of 3 experiments. Scale bar: 40 μm.</p

Summary of the pathogenic mutations detected and family history of the probands.

+<p>Mutation nomenclature according to NM_000249.3 (<i>MLH1</i>), NM_000251.2 (<i>MSH2</i>) and NM_000179.2 (<i>MSH6</i>). For the nomenclature of mutations nucleotide 1 is the A of the ATG-translation initiation codon.</p>++<p>BrC – brain cancer.</p><p>CRC – colorectal cancer.</p><p>EC – endometrial cancer.</p><p>RC – renal cancer.</p

Summary of the missense mutations identified.

+<p>Mutation nomenclature according to NM_000249.3 (<i>MLH1</i>), NM_000251.2 (<i>MSH2</i>) and NM_000179.2 (<i>MSH6</i>). For the nomenclature of mutations nucleotide 1 is the A of the ATG-translation initiation codon.</p>++<p>Class 1: not pathogenic.</p><p>Class 3: uncertain.</p><p>Class 5: pathogenic.</p>+++<p>BC – breast cancer.</p><p>CRC – colorectal cancer.</p><p>GC – gastric cancer.</p><p>OC – ovarian cancer.</p