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.

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.

DNA damage response and repair in perspective: Aedes aegypti, Drosophila melanogaster and Homo sapiens.

BACKGROUND: The maintenance of genomic integrity is the responsibility of a complex network, denominated the DNA damage response (DDR), which controls the lesion detection and DNA repair. The main repair pathways are base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HR) and non-homologous end joining repair (NHEJ). They correct double-strand breaks (DSB), single-strand breaks, mismatches and others, or when the damage is quite extensive and repair insufficient, apoptosis is activated. METHODS: In this study we used the BLAST reciprocal best-hit methodology to search for DDR orthologs proteins in Aedes aegypti. We also provided a comparison between Ae. aegypti, D. melanogaster and human DDR network. RESULTS: Our analysis revealed the presence of ATR and ATM signaling, including the H2AX ortholog, in Ae. aegypti. Key DDR proteins (orthologs to RAD51, Ku and MRN complexes, XP-components, MutS and MutL) were also identified in this insect. Other proteins were not identified in both Ae. aegypti and D. melanogaster, including BRCA1 and its partners from BRCA1-A complex, TP53BP1, PALB2, POLk, CSA, CSB and POLß. In humans, their absence affects DSB signaling, HR and sub-pathways of NER and BER. Seven orthologs not known in D. melanogaster were found in Ae. aegypti (RNF168, RIF1, WRN, RAD54B, RMI1, DNAPKcs, ARTEMIS). CONCLUSIONS: The presence of key DDR proteins in Ae. aegypti suggests that the main DDR pathways are functional in this insect, and the identification of proteins not known in D. melanogaster can help fill gaps in the DDR network. The mapping of the DDR network in Ae. aegypti can support mosquito biology studies and inform genetic manipulation approaches applied to this vector.

Germline variants in cancer genes in high-risk non-BRCA patients from Puerto Rico.

Inherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1/2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2, MUTYH, and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA-negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.

No Evidence for the Pathogenicity of the BRCA2 c.6937 + 594T>G Deep Intronic Variant: A Case-Control Analysis.

BACKGROUND: The role of deep intronic variants in hereditary cancer susceptibility has been largely understudied. Previously, the BRCA2 c.6937 + 594T>G variant has been shown to preferentially promote the inclusion of a 95 nucleotide cryptic exon and to introduce a premature termination codon. Our objective was to further assess the pathogenicity of the BRCA2 c.6937 + 594T>G deep intronic variant. PATIENTS AND METHODS: We examined the association between BRCA2 c.6937 + 594T>G and breast cancer (BC) risk in 464 BC cases and 497 noncancer controls from Puerto Rico. RESULTS: The overall frequency of the G allele was 2.1% in this population. There was no association between the TG/GG genotypes and BC risk in the uncorrected model and after correcting for confounders. There was only one carrier of the GG genotype. This individual did not have personal or family history of cancer and did not meet the National Comprehensive Cancer Network criteria for hereditary cancer genetic testing. CONCLUSIONS: Although previous work has demonstrated that the BRCA2 c.6937 + 594T>G variant affects splicing, this association study does not support a pathogenic role for the BRCA2 c.6937 + 594T>G intronic variant in breast and ovarian cancer syndrome susceptibility. Furthermore, it emphasizes the need to take into account multiple diverse populations in association studies for the assessment of variant pathogenicity.

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.

DNA repair genes PAXIP1 and TP53BP1 expression is associated with breast cancer prognosis.

Despite remarkable advances in diagnosis, prognosis and treatment, advanced or recurrent breast tumors have limited therapeutic approaches. Many treatment strategies try to explore the limitations of DNA damage response (DDR) in tumor cells to selectively eliminate them. BRCT (BRCA1 C-terminal) domains are present in a superfamily of proteins involved in cell cycle checkpoints and the DDR. Tandem BRCT domains (tBRCT) represent a distinct class of these domains. We investigated the expression profile of 7 tBRCT genes (BARD1, BRCA1, LIG4, ECT2, MDC1, PAXIP1/PTIP and TP53BP1) in breast cancer specimens and observed a high correlation between PAXIP1 and TP53BP1 gene expression in tumor samples. Tumors with worse prognosis (tumor grade 3 and triple negative) showed reduced expression of tBRCT genes, notably, PAXIP1 and TP53BP1. Survival analyses data indicated that tumor status of both genes may impact prognosis. PAXIP1 and 53BP1 protein levels followed gene expression results, i.e., are intrinsically correlated, and also reduced in more advanced tumors. Evaluation of both genes in triple negative breast tumor samples which were characterized for their BRCA1 status showed that PAXIP1 is overexpressed in BRCA1 mutant tumors. Taken together our findings indicate that PAXIP1 status correlates with breast cancer staging, in a manner similar to what has been characterized for TP53BP1.

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.

Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.

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.

Tandem BRCT Domains: DNA's Praetorian Guard.

The cell's ability to sense and respond to specific stimuli is a complex system derived from precisely regulated protein-protein interactions. Some of these protein-protein interactions are mediated by the recognition of linear peptide motifs by protein modular domains. BRCT (BRCA1 C-terminal) domains and their linear motif counterparts, which contain phosphoserines, are one such pair-wise interaction system that seems to have evolved to serve as a surveillance system to monitor threats to the cell's genetic integrity. Evidence indicates that BRCT domains found in tandem can cooperate to provide sequence-specific binding of phosphorylated peptides as is the case for the breast and ovarian cancer susceptibility gene BRCA1 and the PAX transcription factor-interacting protein PAXIP1. Particular interest has been paid to tandem BRCT domains as "readers" of signaling events in the form of phosphorylated serine moieties induced by the activation of DNA damage response kinases ATM, ATR, and DNA-PK. However, given the diversity of tandem BRCT-containing proteins, questions remain as to the origin and evolution of this domain. Here, we discuss emerging views of the origin and evolving roles of tandem BRCT domain repeats in the DNA damage response.

The accidental cancer geneticist: hilário de gouvêa and hereditary retinoblastoma.

In 1872 a Brazilian ophthalmologist performed an enucleation in a young boy with retinoblastoma. This boy survived and married a woman without any family history of cancer. The couple had two daughters with bilateral retinoblastoma also seen by the same ophthalmologist, Hilário de Gouvêa. This case became the first documented report of a family with retinoblastoma in more than one generation. Here we examine the life of de Gouvêa and his contribution which raised the possibility that cancer had a genetic basis. We discuss how de Gouvêa's mind had been prepared to realize the importance of this observation. We attempt to define the conditions that allowed not only his discovery, but also the report of the findings and a dogged pursuit for credit over many years in a country which had virtually no research tradition and was still grappling with its recent colonial history.

Prevalence of BRCA1 and BRCA2 mutations in breast cancer patients from Brazil.

The contribution of BRCA1 and BRCA2 to breast cancer incidence in Brazil has not yet been explored. In order to estimate the proportion of breast cancers due to BRCA1 and BRCA2 mutations in Brazil, we conducted a study of unselected breast cancer patients from Rio de Janeiro, Brazil. We enrolled 402 women with breast cancer from a large public hospital and two private medical clinics in the city. A detailed family history was obtained from each patient and a blood sample was obtained for DNA analysis. Mutations in BRCA1 and BRCA2 were sought using a combination of techniques, but all mutations were confirmed by direct sequencing. Overall, nine mutations were identified (six in BRCA1 and three in BRCA2) representing 2.3% of the total. The most common mutation, 5382insC in BRCA1, was seen five times and accounted for 56% of all identified mutations. A second mutation, in BRCA2 (6633del5) was seen in two unrelated women. In summary, BRCA1 and BRCA2 mutations are not uncommon in Brazilian women with breast cancer. It appears that a small number of founder mutations may be predominant. Moreover, a small number of founder mutations may be prevalent in Brazil, raising the possibility that a rapid and inexpensive genetic test may be developed to screen for inherited susceptibility to breast cancer in Brazil.

Long-term culture of cholangiocytes from liver fibro-granulomatous lesions.

BACKGROUND: Extensive bile duct proliferation is a key feature of the tissue reaction to clinical and experimental forms of liver injury. Experimental infection of mice by Schistosoma mansoni is a well-studied model of liver fibrosis with bile duct hyperplasia. However, the regulatory mechanisms of bile duct changes are not well understood. In this study we report the reproducible isolation of long-term cultures of cholangiocytes from mice livers with schistosomal fibrosis. METHODS: We have isolated a cholangiocyte cell line from Schistosoma-induced liver granulomas using a combination of methods including selective adhesion and isopyknic centrifugation in Percoll. RESULTS: The cell line was characterized by morphological criteria in optical and transmission electron microscopy, ability to form well differentiated ductular structures in collagen gels and by a positive staining for cytokeratin 18 and cytokeratin 19. To our knowledge, this is the first murine cholangiocyte cell line isolated from schistosomal fibrosis reported in the literature. CONCLUSION: After 9 months and 16 passages this diploid cell line maintained differentiated characteristics and a high proliferative capacity. We believe the method described here may be a valuable tool to study bile duct changes during hepatic injury.