Women at high risk of familial cancer were genotyped through NGS with a costum-made panel of high-risk genes, namely BRCA1, BRCA2, PTEN, TP53, BRCA1-interacting helicase 1, RAD51C, RAD51D, ATM, partner and localizer of BRCA2, checkpoint kinase 2 and cadherin-1, which are described to be essential in the clinical guidelines for breast cancer studies (21,23,24). All exons and exon-intron boundaries had 100% coverage. In addition, two healthy, non-carrier controls (NC) with no VUS identified after NGS for the same clinical panel were also included in this study.

The participants enrolled were selected from a collaboration with the company Ophiomics Precision Medicine. All participants were informed about the present study and the collection of blood samples by venous puncture were preceded by the signing of an informed consent form agreeing to the use of their blood samples for research. Detailed family history of oncological diseases for each patient/participant was also collected. All personal data was anonymized, and the samples were coded. The present study was approved by the National Commission of Data Protection (approval no. 10637/2016) for the use of samples for research and also by the Ethical Commission of NOVA Medical School/Faculdade Ciências Médicas (NMS/FCM; approval no. 54/2018/CEFCM).

Genomic DNA extraction from total peripheral blood cells was performed with the GeneJET Whole Blood Genomic DNA Purification Mini kit (Thermo Fisher Scientific, Inc.) according to manufacturer's instructions, with a minor change: the final elution volume was 75 µl. The quantification and the quality of the extracted DNA was evaluated with the electrophoresis system Agilent 2200 TapeStation System (Agilent Technologies, Inc.) using the Agilent Genomic DNA ScreenTape and Reagents kit (Agilent Technologies, Inc.) according to manufacturer's instructions.

Genomic DNA extracted from peripheral blood (200 µl) of patients was evaluated for DNA concentration and integrity; DNA isolated from each sample was quantified in 2200 TapeStation using the Genomic DNA ScreenTape (Agilent Technologies, Inc.). Genomic DNA libraries were prepared using the Ion Ampliseq Library kit (2.0) using the custom Ion Ampliseq panel described above and quantified by quantitative PCR with the Ion Library Quantification kit (Thermo Fisher Scientific, Inc.). The emulsion PCR of amplified libraries was performed using Ion Chef (Thermo Fisher Scientific, Inc.). Sequencing runs were performed with Ion personal machine using 316 Chips (Thermo Fisher Scientific, Inc.) aiming for a mean sequencing depth coverage of 100×. Variant annotation was performed in reference to Human Genome version GRCh38 and based on information contained in the databases ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), DGVa (https://www.ebi.ac.uk/dgva/), dbSNP (https://www.ncbi.nlm.nih.gov/snp/), HGMD-PUBLIC (https://www.hgmd.cf.ac.uk/ac/index.php), EBI Variation HomoSapiens (https://www.ebi.ac.uk/eva/). The bioinformatics algorithms used to predict the functional impact of variants were: PolyPhen (http://genetics.bwh.harvard.edu/pph2/), SIFT (https://sift.bii.a-star.edu.sg), LoF (http://aloft.gersteinlab.org), Condel (https://bbglab.irbbarcelona.org/fannsdb/help/condel.html), BLOSUM62 scoring matrix used in BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and CAROL (https://www.sanger.ac.uk/tool/carol/).

Detection of copy number variants for BRCA1 and BRCA2 genes was performed by MLPA with the panels MLPA® Salsa® P002-BRCA1 and MLPA® Salsa® P090-BRCA2 (MRC-Holland BV), respectively. The Portuguese founder mutation (c_156_157 inserção Alu) BRCA2 (OMIM:600185) was also screened by PCR. All reported variants classified as pathogenic or unknown significance, occurring in coding regions and at frequencies >10% were validated by Sanger sequencing (ABI3100 Avant; Thermo Fisher Scientific, Inc.).

PCR reactions to prepare samples for Sanger sequencing were performed according to Platinum® PCR SuperMix High Fidelity (Invitrogen; Thermo Fisher Scientific, Inc.) manufacturer's instructions. Briefly, a total volume of 20 µl was used, containing 18 µl of Platinum® PCR SuperMix High Fidelity (Invitrogen; Thermo Fisher Scientific, Inc.), 0.2 µM of forward (5′-AATGATAGGCGGACTCCCAG-3′) and reverse (5′-GAGGCTTGCCTTCTTCCGAT-3′) primers. High quality genomic DNA (5–50 ng) extracted from peripheral blood cells was added to the PCR mix and the PCR reactions were performed in a Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific, Inc.). The cycling conditions employed were initial denaturation at 94°C, 2 min, 30 cycles of amplification at 94°C, 15 sec, 55°C, 15 sec, 68°C, 1 min and held at 10°C. The size and quantity of each fragment analyzed was evaluated with the electrophoresis system Agilent 2200 TapeStation System (Agilent Technologies, Inc.) using the kit DNA ScreenTape and Reagents (Agilent Technologies, Inc.) according to manufacturer's instructions. Each sample was sequenced with both forward and reverse primers in an Applied Biosystems 3130 Genetic Analyzer (Thermo Fisher Scientific, Inc.) according to manufacturer's instructions.

Blood samples were irradiated in vitro using a ⁶⁰Co radiation source in a Precisa 22 irradiator at the Ionizing Radiation Installations at Center for Nuclear Sciences and Technologies-Instituto Superior Técnico (C2TN-IST) in Lisbon. Each donor sample was irradiated with a dose of 2 Gy and a non-irradiated control (0 Gy) was included. A total of ~4 ml whole blood was isolated from each donor and subject to irradiation after which each assay was performed as described below. To perform the comet assay, lymphocytes were isolated and then distributed into 4-ml glass tubes for irradiation.

After irradiation, blood samples were cultured in triplicates or quadruplicates for each donor. The experiments were performed as previously described (25–27) with minor modifications. Briefly, 500 µl irradiated and non-irradiated whole blood was added to 4.5 ml RPMI-1640 medium with L-Glutamine (MilliporeSigma), supplemented with 25% FBS (MilliporeSigma), 1.5% penicillin-streptomycin (Pen-Strep), 0.5% sodic heparin (5,000 UI/ml; B. Braun Medical Inc.) and 2.5% phytohemagglutinin (Gibco; Thermo Fisher Scientific, Inc.). Cultures were maintained in an incubator at 37°C and 5% CO₂ at an 40° angle for 48 h. After 24 h, colcemid (0.08 µg/ml; Gibco; Thermo Fisher Scientific, Inc.) was added to the culture. At the end of the 48 h, cultures were centrifuged at 400 × g for 5 min at RT (RT). The pellet was then resuspended with mild stirring before 10 ml KCl solution [0.56% (p/v)] previously warmed to 37°C was added and homogenization by inversion. The tubes were incubated at 37°C for 20 min to promote hypotonic shock and then centrifuged at 400 × g for 5 min at RT. The cells were fixed under stirring with 5 ml fixative mixture of methanol:acetic acid [3:1 (v/v)] previously cooled at −20°C, before being centrifuged at 400 × g for 5 min at RT. These two steps of fixation and subsequent centrifugation were repeated two or three times, until the supernatant became clear. Finally, 10 ml fixative mixture was added to each tube and the samples were stored at −20°C.

Samples held at −20°C were centrifuged at 400 × g for 5 min at RT following which the supernatant was removed and the suspension homogenized by gentle tapping. Glass slides were washed and immersed in distilled water at 4°C and a few drops of the cell suspension were spread onto each slide. Once well dried for 24 h at RT, the slides were stained with Giemsa's solution 4% (v/v) in 0.01 M phosphate buffer (pH 6.8) for 10 min. Excess dye was then washed off under running water. Once well dried again, permanent slides were prepared using a mounting medium [Entellan® (MilliporeSigma)]. Slides were then scored using an optical microscope at ×1,000 magnification. Scoring was performed in 200 complete metaphases (46 chromosomes) by two independent evaluators (100 each), according to the criteria described by Rueff et al (28) and following the recommendations of the International Atomic Energy Agency (IAEA) (29). Each metaphase was analyzed according to the following criteria: The presence of chromosomal aberrations, namely chromatid with gaps or breaks; chromosomes with gaps or breaks; excess of acentric fragments; dicentric chromosomes DIC; and rings. The metaphases containing ≥1 chromosome aberration except gaps were accounted for the frequency (%) of aberrant cells excluding gaps [chromosomal aberration excluding gaps (CAEG)].

For the CBMN assay, blood samples were cultured after irradiation in triplicate or quadruplicate for each donor (26,30,31). Briefly, 0.5 ml irradiated whole blood was added into each tube containing 4.5 ml RPMI-1640 medium with L-Glutamine, supplemented with 25% FBS, 1.5% of Pen-Strep, 0.5% of sodic heparin and 2.5% of phytohemagglutinin. Cultures were maintained at 37°C, 5% CO₂ and at an angle of 40° for ~72 h. After 44 h, cytochalasin-B (6 µg/ml; MilliporeSigma) was added. At the end of the 72 h, cultures were centrifuged at 110 × g for 10 min at RT. After discarding the supernatant, cells were washed twice with 5 ml washing solution [RPMI-1640 medium with L-Glutamine and NaHCO₃ (0.1 g/l), supplemented with 2% FBS] and centrifuged at 110 × g for 7 min at RT. Mild hypotonic treatment was then performed by adding 5 ml 4:1 distilled water: RPMI-1640 medium with L-Glutamine (pH 7.2) and NaHCO₃ (0.1 g/l), supplemented with 2% FBS, followed by centrifugation at 110 × g for 5 min at RT. After concentrating the pellet by discarding most of the supernatant, a drop of cell suspension was placed onto each glass slide and a smear was performed.

Once the glass slides were completely dry, they were fixed with pre-cooled 5 ml methanol:acetic acid solution [3:1 (v/v)] for 20 min at −20°C. The slides were then dried and stained with Giemsa's solution in 0.01 M phosphate buffer (pH 6.8) for 8 min at RT. The permanent slides were prepared as aforementioned with Entellan® mounting medium. Slides were imaged and scored using an optical microscope at ×400 magnification. For each donor and dose, 2,000 binucleated cells were scored by two independent scorers (1,000 each) according to the IAEA criteria (29). The number of micronucleated binucleated cells were recorded.

Peripheral blood mononuclear cells (PBMCs) were prepared from fresh blood samples and isolated through density gradient centrifugation using Histopaque-1077 (MilliporeSigma) according to the manufacturer's protocols. Briefly, blood was diluted with an equal volume of PBS before 5 ml of this diluted blood was carefully added to a canonical centrifuge tube, which contains 3.5 ml Histopaque-1077, before being centrifuged at 700 × g for 30 min at RT. PBMCs were then harvested from the interface and washed with PBS and centrifuged again at 200 × g for 10 min at RT. The pellet was suspended in RPMI-1640 medium supplemented with 25% FBS and 1.5% Pen-Strep. For irradiation, part of the cell suspension (~4 ml) was used, whereas the rest was used as control. Cell suspensions were held on ice until the single-cell gel electrophoresis assay was performed. For chemical exposure, 1×10⁶ PBMCs were cultured in 12-well plates and exposed for 2 h with doxorubicin (BioAustralis) at 37°C at 5% CO₂. The samples were then centrifuged at 200 × g for 5 min at RT, washed in 1 ml PBS and centrifuged again. The pellet was then resuspended in 100 µl 0.5% low-melting point agarose.

The comet assay (SCGE) was performed as previously described (32) with slight modifications. Briefly, the cell suspensions were spread on glass microscope slides previously coated with 1% normal-melting point agarose and kept at 4°C for 20 min. The slides were then left overnight in a cold lysis buffer (2.5 M NaCl, 10 mM Tris, 100 mM EDTA and 1% Triton, pH 10). After the overnight lysis, slides were washed with previously cooled double-distilled water and remained immersed for 10 min at 4°C. They were then immersed in cold electrophoresis buffer (10 M NaOH and 200 mM EDTA, pH >13) for 20 min at 4°C. Electrophoresis was conducted for 20 min at 25 V (400 mA) before the slides were neutralized three times with neutralization buffer (0.4 M Tris, pH 7.5) at 5 min each, dried with ethanol (50, 75 and 100%; 5 min each) and stained with 3X GelRed (Biotium, Inc.). Slides were scored using a fluorescent microscope (Zeiss Z2; Carl Zeiss AG) at ×200 magnification, before ~200 cells were selected and images captured. The cell images captured were then examined using the CometScore V1.5 Software, which calculated the % DNA in the tail.

The functional assays through chemical exposure were performed for all samples carrying the VUS and for NC controls. In total. three different concentrations of doxorubicin were chosen (0.1, 1.0 and 5.0 µM). The duration of chemical exposure varied according to the assay performed. Treated samples were incubated at 37°C for specific periods of time.

Following doxorubicin treatment for 2 h at 37°C with 5% CO₂, the samples were centrifuged at 200 × g for 5 min at RT. RPMI-1640 medium (1 ml) supplemented with 25% of FBS and 1.5% of Pen-Strep was then added to each sample and incubated for 30 min at 37°C with 5% CO₂. Samples were centrifuged at 200 × g at RT and 1 ml PBS and 1 µl violet fluorescent reactive dye (LIVE/DEAD™ Fixable Violet Dead Cell Stain kit; Thermo Fisher Scientific, Inc.) was added to the respective sample and incubated for 30 min at RT protected from light. The samples were then centrifuged again at 200 × g for 5 min at RT and the pellet was washed with 1 ml PBS, followed by centrifugation at 200 × g for 5 at RT min again. This was followed by fixation in 500 µl 2% formaldehyde for 15 min on ice. The samples were centrifuged again at the same speed and time at 4°C, and each pellet was resuspended in 500 µl 70% cold ethanol in PBS before being kept overnight at 4°C. The next day, the samples were centrifuged at 200 × g for 5 min at 4°C and each pellet was resuspended in 1 ml blocking buffer (containing 4% BSA in PBS, 4% goat serum and 0.25% Triton X-100) and were centrifuged further in the same conditions as before. In total, 1:500 antibody [Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (CR55T33), PE, eBioscience; Thermo Fisher Scientific, Inc.] was added to the respective pellet, followed by 2 h incubation at RT protected from light. Cells were washed with 1.5 ml 1% BSA and centrifuged at 200 × g for 5 min at RT. Each pellet was resuspended in 200 µl 0.1% BSA. The samples were analyzed by flow cytometry using a BD FACSCanto II Cytometer (BD Biosciences), where 20,000 events were counted. Image analysis were performed using the FlowJo v10 software (FlowJo LLC).

Caspase assays were performed using commercially available kits, specifically by CaspaTag™ Caspase-3/7 In Situ Assay kit and CaspaTag™ Caspase-9 In Situ Assay kit (Thermo Fisher Scientific, Inc.). The methodology was performed according to the manufacturer's protocols, with minor alterations. The methodology used for both assays was the same, with the main difference being the FLICA concentration specific for each one, according to the manufacturer's instructions. Briefly, after treatment for 2 h (or overnight for Caspase-9) at 37°C with 5% CO₂, samples were centrifuged at 200 × g for 5 min at RT before the pellet was resuspended in 200 µl PBS. In total, 10 µl 6X FLICA for Caspases 3/7 and 15X FLICA for Caspase 9 were added to the respective tubes. They were then incubated for 1 h at 37°C with 5% CO₂ protected from light. Tubes were gently swirled three times, before 1 ml 1X wash buffer (10X wash buffer provided in the kit) was added and centrifuged at 400 × g for 5 min at RT. The pellet was resuspended in 1 ml 1X wash buffer and centrifuged again at 400 × g for 5 min at RT. The pellet was resuspended in 400 µl 1X wash buffer and 2 µl propidium iodide (provided in kit) was added to the respective tubes. Samples were analyzed by flow cytometry using a BD FACSCanto II Cytometer (BD Biosciences) and 20,000 events were counted. Image analysis were performed using the FlowJo v10 software (FlowJo LLC).

TUNEL assay was performed using the APO-BrdU™ TUNEL Assay kit (Thermo Fisher Scientific, Inc.). The methodology was performed according to the manufacturer's protocols with minor alterations. Briefly, after treatment for 4 h at 37°C with 5% CO₂, samples were centrifuged at 200 × g for 5 min at RT. In total, 500 µl PBS was added to the samples before PBMCs were pelleted (300 × g for 5 min at RT) followed by fixation in 500 µl 2% formaldehyde for 15 min on ice. Samples were then centrifuged at 300 × g for 5 min at 4°C and each pellet was resuspended in 1 ml PBS and centrifuged again at 300 × g for 5 min at 4°C. The pellet was resuspended in 500 µl PBS and 1 ml 70% cold ethanol in PBS before being incubated for 30 min on ice. Samples were centrifuged (300 × g for 5 min at 4°C) and the pellet was resuspended and centrifuged twice with 1 ml wash buffer (provided in kit). Each pellet was then resuspended in 50 µl DNA-labeling solution, which contains reaction buffer, TdT enzyme, BrdUTP and ddH₂O, before being kept overnight at 22–24°C. The next day, samples were resuspended and centrifuged twice in same conditions as before (300 × g for 5 min at RT) with 1 ml rinse buffer (provided in kit), 10⁶ cells were added to 100 µl diluted solution, which contains the Alexa Fluor™ 488-conjugated anti-BrdU mouse monoclonal antibody PRB-1 (provided in kit) and rinse buffer. They were then incubated for 30 min at RT protected from light. Samples were analyzed by flow cytometry using a BD FACSCanto II Cytometer (BD Biosciences), where 20,000 events were counted. Image analysis were performed using FlowJo v10 software (FlowJo LLC).

All graphs were plotted using the GraphPad Prism 9 software (Dotmatics). Data were presented as the means ± standard deviation. All graphs were obtained for the grouped samples according to their genetic status: NC carriers or VUS carriers. Statistical analysis was performed using GraphPad Prism 9 taking into account the pooled samples. For the CA and MN assays, χ² or Fisher's exact tests was applied, where P<0.05 was considered to indicate a statistically significant association. For the SCGE or comet assays, Kolmogorov-Smirnov normality test was performed to examine if samples followed a Gaussian distribution. If this was not observed, then non-parametric tests were used to analyze the data. To compare controls (0 Gy) and irradiated samples (2 Gy), Wilcoxon signed rank test was applied. The non-parametric Mann-Whitney U test was performed to compare the different groups of samples. P<0.05 was considered to indicate a statistically significant difference.

The results obtained by NGS revealed two women with a VUS in the BRCA1 gene (NM_007294.3:c.1067A>G) ambiguously defined as probably pathogenic according to the PolyPhen2 database and as benign in the ClinVar database were identified (Table I). These VUS-carriers were female individuals with no identified tumors, belonging to two distinct families with high incidence of oncologic diseases and the same VUS (rs1799950). The familial history of each woman and their pedigrees were constructed and are shown in Fig. 1.

In order to validate the sensitivity of the functional assays to verify the pathogenicity of the gene variant, and since it was not possible to include a pathogenic BRCA1 variant selected by NGS in the present study, two women also with high-risk genealogy harboring variants in the ATM gene that are likely to be pathogenic were included in the study (Table II). These carriers of ATM mutations are first degree-relatives (mother and daughter). The two were diagnosed with breast cancer in a family with a relevant pedigree history of oncological diseases (Fig. 2). In addition, this variant was also identified in a third relative in this family (case II.3), who was also diagnosed with breast cancer along with her twin sister (case II.2; ATM2) and her niece (case III.1; ATM1), emphasizing the high probability of this being a pathogenic variant. Together with the BRCA1 gene, the ATM gene is also involved in the DDR pathway and is important in the cellular response to genotoxic agents.

DNA damage was induced by γ-radiation and chemical exposure to doxorubicin, a DNA damaging agent that is also used as a first line chemotherapeutic for several cancers. The present study was intended to be exploratory and a proof of concept. The genotoxic and functional studies performed between NC carriers and carriers of VUS and ATM mutation are described below and were chosen with the objective of measuring the extent of DNA damage and several apoptosis end-points.

Samples from two participants, VUS_BRCA1_1 and NC 2, were first used to establish a dose response curve for the present study. Dose-response curves at 0, 1, 2 and 5 Gy were performed for the MN and CA assays (Fig. 3). The radiation dose chosen (2 Gy) was previously described (33) and is used in biological dosimetry requirements.

Table III shows the results obtained after analysis, where it is possible to observe a global increase in the frequency of CAEG globally following radiation exposure. The most frequent CA present were acentric fragments and DIC which, apart from rings, are the main CAs identified following γ-radiation exposure. Fig. 4 shows representative images of metaphases showing these structures that were observed during analysis. The results obtained individually for each woman were then grouped to evaluate the effect attributed to the presence of each genetic variant. The frequency of CAEG observed is shown in Fig. 5. No significant differences could be observed among the NC carriers, VUS_BRCA1 carriers and ATM carriers.

Micronuclei slides were then analyzed. For each participant, 1,000 binucleated cells were counted and independently analyzed by two independent evaluators. The data obtained for the micronuclei distribution in each participant are shown in Table IV. Fig. 6 shows representative images captured during the data analysis, with the results observed. An overall analysis of the results showed an increase in the rates of binucleated cells with micronuclei (MNBN) and total micronuclei (TMN) following exposure to a dose of 2 Gy, compared with those in the control group of 0 Gy. Furthermore, the occurrence of ≥2 micronuclei after radiation exposure (Table III) was observed across all samples.

The main aim of the present study was to functionally characterize the genetic variant by assessing the cellular response to γ-radiation. Given the limited number of samples analyzed, the data were grouped according to the presence of each genetic variant: VUS carriers, ATM carriers and NC carriers. Fig. 7 represents the distribution of MNs in each group. Analysis of the MN frequency distribution revealed a significant decrease in the MNBN frequency in VUS and ATM carriers compared with NC carriers.

The % DNA in tail for all samples was measured to evaluate the effect of γ-radiation and chemical exposure to doxorubicin. A dose-dependent effect was observed in both experiments, with higher doses inducing more lesions (Fig. 8). Comparing the effects of 2 Gy on NC and VUS carriers, an increase in the number of DNA lesions was observed in the VUS carriers, with an even more significant increase in ATM carriers, compared with the NC carriers. However, this effect was not observed after doxorubicin exposure except for the basal level, where VUS carriers have significantly more basal DNA damage than NC carriers (Fig. 8B).

The γH2AX assay is one functional method that can be used to detect DSBs following chemical exposure. The data obtained was grouped into NC and VUS carriers. The results obtained demonstrated a dose-dependent effect in both NC and VUS carriers (Fig. 9). However, the differences between VUS carriers and NC carriers were non-significant.

The caspase signaling cascade is responsible for the activation of apoptosis and inflammatory processes, which allow cells to maintain their genomic stability whilst controlling programed cell death. In the present study, two caspases with two different functions in the apoptosis pathway were assessed; caspase 9 (initiator caspase) and caspases 3–7 (executioner caspases) (34). These caspases operate in the intrinsic pathway, which is triggered in response to death stimuli generated by DNA damage. Chemical exposure was found to slightly activate caspase 9, while almost no activation could be detected for caspase 3–7 (Fig. 10). Caspases 3–7 activity exhibited only slight variations at the different doxorubicin concentrations. However, the activity of caspase 9 increased sharply at higher doxorubicin concentrations, suggesting that activation of the initiator caspase cascade occurred due to DNA damage.

To the best of the authors' knowledge, cells undergoing apoptosis exhibit several changes in nuclear morphology, especially during the later stages of programmed cell death or apoptosis. These features include DNA fragmentation and DNA strand breaks, both of which are relevant when evaluating the biological role of DNA repair genes. The damage inflicted in PBMCs by doxorubicin exposure was also evaluated using the TUNEL assay as quantified by flow cytometry. This assay measures DNA fragments resulting from the apoptotic process. A significant difference at higher concentrations of doxorubicin between NC carriers and VUS carriers was observed (Fig. 11), with NC carriers being more sensitive to DNA fragmentation, consistent with the results obtained for MN (Fig. 4).