BRCA1 frameshift variants leading to extended incorrect protein C termini.

Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus constitute loss-of-function variants and underscore the need for comprehensive assessment of individual variants.

Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay.

BRCA1 (Breast Cancer 1, early onset) is linked to breast and ovarian cancer predisposition. Still, the risks conferred by a significant portion of BRCA1 variants identified in the population remains unknown. Most of these variants of uncertain significance are missense alterations. However, the functional implications of small in-frame deletions and/or insertions (indels) are also difficult to predict. Our group has previously evaluated the functional impact of 347 missense variants using an extensively validated transcriptional activity assay. Here we show a systematic assessment of 30 naturally occurring in-frame indels located at the C-terminal region of BRCA1. We identified positions sensitive and tolerant to alterations, expanding the knowledge of structural determinants of BRCA1 function. We further designed and assessed the impact of four single codon deletions in the tBRCT linker region and six nonsense variants at the C-terminus end of BRCA1. Amino acid substitutions, deletions or insertions in the disordered region do not significantly impact activity and are not likely to constitute pathogenic alleles. On the other hand, a sizeable fraction of in-frame indels at the BRCT domain significantly impact function. We then use a Bayesian integrative statistical model to derive the probability of pathogenicity for each variant. Our data highlights the importance of assessing the impact of small in-frame indels in BRCA1 to improve risk assessment and clinical decisions for carriers.

Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

Two distinct mechanisms underlie estrogen-receptor-negative breast cancer susceptibility at the 2p23.2 locus.

Genome wide-association studies (GWAS) have established over 400 breast cancer risk loci defined by common single nucleotide polymorphisms (SNPs), including several associated with estrogen-receptor (ER)-negative disease. Most of these loci have not been studied systematically and the mechanistic underpinnings of risk are largely unknown. Here we explored the landscape of genomic features at an ER-negative breast cancer susceptibility locus at chromosome 2p23.2 and assessed the functionality of 81 SNPs with strong evidence of association from previous fine mapping. Five candidate regulatory regions containing risk-associated SNPs were identified. Regulatory Region 1 in the first intron of WDR43 contains SNP rs4407214, which showed allele-specific interaction with the transcription factor USF1 in in vitro assays. CRISPR-mediated disruption of Regulatory Region 1 led to expression changes in the neighboring PLB1 gene, suggesting that the region acts as a distal enhancer. Regulatory Regions 2, 4, and 5 did not provide sufficient evidence for functionality in in silico and experimental analyses. Two SNPs (rs11680458 and rs1131880) in Regulatory Region 3, mapping to the seed region for miRNA-recognition sites in the 3' untranslated region of WDR43, showed allele-specific effects of ectopic expression of miR-376 on WDR43 expression levels. Taken together, our data suggest that risk of ER-negative breast cancer associated with the 2p23.2 locus is likely driven by a combinatorial effect on the regulation of WDR43 and PLB1.

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance.

PURPOSE: BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification. METHODS: We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification. RESULTS: Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach. CONCLUSION: High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

PALB2 Variants: Protein Domains and Cancer Susceptibility.

Since its discovery, partner and localizer of breast cancer 2 (BRCA2) (PALB2) has emerged as a major tumor suppressor gene linked to breast cancer (BC), pancreatic cancer (PC), and ovarian cancer (OC) susceptibility. Its protein product plays a pivotal role in the maintenance of genome integrity. Here we discuss the first functional evaluation of a large set of PALB2 missense variants of uncertain significance (VUSs). Assessment of 136 VUSs interrogating a range of PALB2 biological functions resulted in the identification of 15 variants with consistent loss of function across different assays. All loss-of-function variants are located at the PALB2 coiled coil (CC) or at the WD40 domain, highlighting the importance of modular domains mechanistically involved in the DNA damage response (DDR) and pinpointing their roles in tumor suppression.

Functional characterization of 84 PALB2 variants of uncertain significance.

PURPOSE: Inherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance. METHODS: The influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays. RESULTS: Four (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1-PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm. CONCLUSION: These findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.

A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor.

While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation.

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

The Role of PALB2 in the DNA Damage Response and Cancer Predisposition.

The deoxyribonucleic acid (DNA) damage response (DDR) is a major feature in the maintenance of genome integrity and in the suppression of tumorigenesis. PALB2 (Partner and Localizer of Breast Cancer 2 (BRCA2)) plays an important role in maintaining genome integrity through its role in the Fanconi anemia (FA) and homologous recombination (HR) DNA repair pathways. Since its identification as a BRCA2 interacting partner, PALB2 has emerged as a pivotal tumor suppressor protein associated to hereditary cancer susceptibility to breast and pancreatic cancers. In this review, we discuss how other DDR proteins (such as the kinases Ataxia Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR), mediators BRCA1 (Breast Cancer 1)/BRCA2 and effectors RAD51/DNA Polymerase η (Polη) interact with PALB2 to orchestrate DNA repair. We also examine the involvement of PALB2 mutations in the predisposition to cancer and the role of PALB2 in stimulating error-free DNA repair through the FA/HR pathway.

BRCA1 recruitment to damaged DNA sites is dependent on CDK9.

Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.

BRCA1 Circos: a visualisation resource for functional analysis of missense variants.

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.

Probing structure-function relationships in missense variants in the carboxy-terminal region of BRCA1.

Germline inactivating variants in BRCA1 lead to a significantly increased risk of breast and ovarian cancers in carriers. While the functional effect of many variants can be inferred from the DNA sequence, determining the effect of missense variants present a significant challenge. A series of biochemical and cell biological assays have been successfully used to explore the impact of these variants on the function of BRCA1, which contribute to assessing their likelihood of pathogenicity. It has been determined that variants that co-localize with structural or functional motifs are more likely to disrupt the stability and function of BRCA1. Here we assess the functional impact of 37 variants chosen to probe the functional impact of variants in phosphorylation sites and in the BRCT domains. In addition, we perform a meta-analysis of 170 unique variants tested by the transcription activation assays in the carboxy-terminal domain of BRCA1 using a recently developed computation model to provide assessment for functional impact and their likelihood of pathogenicity.

Characterization of LGALS3 (galectin-3) as a player in DNA damage response.

DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G 2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.

Charting the landscape of tandem BRCT domain-mediated protein interactions.

Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR by combining literature curation, yeast two-hybrid screens, and tandem affinity purification coupled to mass spectrometry. We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.

A guide for functional analysis of BRCA1 variants of uncertain significance.

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56-80% for breast cancer and 15-60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.

Identification of Filamin A as a BRCA1-interacting protein required for efficient DNA repair.

The product of the breast and ovarian cancer susceptibility gene BRCA1 has been implicated in several aspects of the DNA damage response but its biochemical function in these processes has remained elusive. In order to probe BRCA1 function we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. Here we report the identification of the actin-binding protein Filamin A (FLNA) as BRCA1 partner and demonstrate that FLNA is required for efficient regulation of early stages of DNA repair processes. Cells lacking FLNA display a diminished BRCA1 IR-induced focus formation and a delayed kinetics of Rad51 focus formation. In addition, our data also demonstrate that FLNA is required to stabilize the interaction between components of the DNA-PK holoenzyme, DNA-PKcs and Ku86 in a BRCA1-independent fashion. Our data is consistent with a model in which absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for the response to irradiation induced DNA damage.

Ectopic expression of histone H2AX mutants reveals a role for its post-translational modifications.

Recent evidence from a wide variety of biological systems has indicated important regulatory roles for post-translation histone modifications in cellular processes such as regulation of gene expression, DNA damage response and recombination. Phosphorylation of histone H2AX at serine 139 is a critical event in the response to DNA damage, but the functional implications of this modification are not yet clear. To investigate the role of H2AX phosphorylation we ectopically expressed epitope-tagged H2AX or mutants at the phosphorylation site. GFP-tagged wild type H2AX, H2AX Ser139Ala or H2AX Ser139Glu proteins were efficiently expressed, localizing exclusively to the interphase nucleus and to condensed chromosomes during mitosis. Biochemical fractionation indicated that epitope-tagged H2AX proteins are incorporated into nucleosomes. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site partially suppressed early G(2)/M arrest following ionizing radiation, and cells expressing this mutant were more sensitive to DNA damage. Conversely, expression of H2AX Ser139Glu, designed as phosphorylation mimic, induced a decrease in the number of cells in mitosis in the absence of DNA damage. Interestingly, this decrease induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and was partially suppressed in CHK2-deficient cells, suggesting a role for CHK2 in this process. Further analyses revealed that expression of either mutant lead to apoptosis and induced higher caspase-3/7 activity compared to expression of wild type H2AX. In addition, we also identified Lys119 as a site for ubiquitination that controls H2AX half-life. Phosphorylation of Ser139 and ubiquitination of K119 are not interdependent. Taken together these results demonstrate a role for H2AX Serine 139 phosphorylation in cell cycle regulation and apoptosis, and for Lysine 119 in the control of H2AX turnover.