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With the expanding catalogue of novel disease-genes, there is an increasing need to establish the significance of potential disease-causing variants. Based on the idea that pathogenic variants in structured protein domains disturb folding and association with macromolecular assemblies, we employed Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS) to assess in vivo protein complex formation. Since the molecular underpinning of BRCA-associated breast and ovarian cancers is well defined and data from a recent genome editing screening allowed us to compare variant binding data with a reliable functional HRD test in addition to ClinVar and AlphaMissense data, we examined the binding of mutated full-length BRCA1 or isolated RING and BRCT domains to BARD1 and RBBP8, respectively. The results demonstrate that FCCS, whether applied to full-length BRCA1 in live cells and/or to isolated domains in cellular lysates, identified pathogenic BRCA1 RING or BRCT domain variants. We moreover demonstrate the feasibility of employing FCCS for analysis of HNPCC-related factor MSH2 and MEN1 factor Menin variants in combination with DNA mismatch repair factor MSH6 and transcription factor JUND, respectively. We propose that FCCS may be an appealing complement to current clinical procedures for classifying variants for many monogenic diseases, given its generic nature and ease of use.

BACKGROUNDDecoding the clinical impact of genetic variants is particularly important for precision medicine in cancer. Genetic screening of mainly patients with breast and ovarian cancer has identified numerous BRCA1/BRCA2 variants of uncertain significance (VUS) that remain unclassified owing to a lack of pedigrees and functional data.METHODSHere, we used CRISPR-Select - a technology that exploits unique inbuilt controls at the endogenous locus - to assess 54 rare ClinVar VUS located in the PALB2-binding domain of BRCA2. Variant deleteriousness was examined in the absence and presence of PARPi, cisplatin, or mitomycin C.RESULTSMarked functional deficiency was observed for variants in the exon 2 donor splice region (A22 = c.66A>C, A22 = c.66A>G, A22 = c.66A>T, and D23H) and Trp31 aa (W31G, W31L, and W31C), both critical for BRCA2 function. Moreover, T10K and G25R resulted in an intermediate phenotype, suggesting these variants are hypomorphic in nature. Combining our functional results with the latest ClinGen BRCA1/2 Variant Curation Expert Panel recommendations, we classified 49 of the 54 VUS as either likely benign (n = 45) or likely pathogenic (n = 4).CONCLUSIONTherefore, CRISPR-Select is an important tool for efficient variant clinical classification. Application of this technology in the future will ultimately improve patient care.FUNDINGDanish Cancer Society, Novo Nordisk Foundation, Sygeforsikring Danmark, Børnecancerfonden, Neye-Fonden, Roche, Novartis, Pfizer, AstraZeneca, MSD, and Daiichi Sankyo Europe GmbH.

Purpose Decades of research have identified multiple genetic variants associated with breast cancer etiology. However, there is no database that archives breast cancer genes and variants responsible for predisposition. We set out to build a dynamic repository of curated breast cancer genes. Methods A comprehensive literature search was performed in PubMed and Google Scholar, followed by data extraction and harmonization for downstream analysis. Results Using a subset of 345 studies, we cataloged 652 breast cancer-associated loci across the genome. A majority of these were present in the non-coding region (i.e., intergenic (101) and intronic (345)), whereas only 158 were located within an exon. Using the odds ratio, we identified 429 loci to increase the disease risk and 198 to confer protection against breast cancer, whereas 25 were identified to both increase disease risk and confer protection against breast cancer. Chromosomal ideogram analysis indicated that chromosomes 17 and 19 have the highest density of breast cancer loci. We manually annotated and collated breast cancer genes in which a previous association between rare-monogenic variant and breast cancer has been documented. Finally, network and functional enrichment analysis revealed that steroid metabolism and DNA repair pathways were predominant among breast cancer genes and variants. Conclusions We have built an online interactive catalog of curated breast cancer genes ( https://cbcg.dk ). This will expedite clinical diagnostics and support the ongoing efforts in managing breast cancer etiology. Moreover, the database will serve as an essential repository when designing new breast cancer multigene panels.

Loss-of-function mutations in genome maintenance genes fuel tumorigenesis through increased genomic instability. A subset of these tumor suppressors are challenging to identify due to context dependency, including functional interactions with other genes and pathways. Here, we searched for potential causal genes that impact tumor development and/or progression in breast cancer through functional-genetic screening of candidate genes. MYH4 , encoding a class II myosin, emerged as a top hit impacting genomic stability. We show that MYH4 suppresses DNA replication stress by promoting replication licensing and replication fork progression. Moreover, we observed a strong synergistic relationship among class II myosins in suppressing replication-associated DNA damage. Genomic analysis of Pan-Cancer Analysis of Whole Genomes project breast cancer samples revealed frequent concomitant loss of TP53 with MYH4 and class II myosins on chromosome 17p. Notably, Myh4 disruption accelerated mouse mammary tumorigenesis in a Trp53 -deficient background. In conclusion, our results suggest an unanticipated function of MYH4 in p53-mediated tumor suppression that can explain their combined loss in breast cancer.

Besides mutations in BRCA1 / BRCA2 , heterozygous defects in PALB2 are important in breast cancer predisposition. PALB2 heterozygosity increases the risk of malignancy about sixfold. PALB2 interacts with BRCA1 and BRCA2 to regulate homologous recombination and mediate DNA damage response. Here we show, by analysing lymphoblastoid cell lines from heterozygous female PALB2 mutation carriers, that PALB2 haploinsufficiency causes aberrant DNA replication/damage response. Mutation carrier cells show increased origin firing and shorter distance between consecutive replication forks. Carrier cell lines also show elevated ATR protein, but not phosphorylation levels, and a majority of them display aberrant Chk1-/Chk2-mediated DNA damage response. Elevated chromosome instability is observed in primary blood lymphocytes of PALB2 mutation carriers, indicating that the described mechanisms of genome destabilization operate also at the organism level. These findings provide a new mechanism for early stages of breast cancer development that may also apply to other heterozygous homologous recombination signalling pathway gene mutations in hereditary cancer predisposition. PALB2 is a BRCA1-/BRCA2-interacting protein and heterozygous mutations in PALB2 are associated with hereditary breast cancer predisposition. Here the authors show that human lymphoblastoid cells from heterozygous PALB2 mutation carriers display abnormal DNA replication dynamics and DNA damage response.

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37 °C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ~80% of DOK cell killing was achieved as compared to ~60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.

Mutations in genome maintenance factors drive sporadic and hereditary breast cancers. Here, we searched for potential drivers based on germline DNA analysis from a cohort consisting of patients with early-onset breast cancer negative for BRCA1/BRCA2 mutations. This revealed candidate genes that subsequently were subjected to RNA interference–based (RNAi-based) phenotype screens to reveal genome integrity effects. We identified several genes with functional roles in genome maintenance, including Glucose-6-Phosphatase Catalytic Subunit 3 ( G6PC3 ), SMC4 , and CCDC108 . Notably, G6PC3-deficient cells exhibited increased levels of γH2AX and micronuclei formation, along with defects in homologous recombination (HR) repair. Consistent with these observations, G6PC3 was required for the efficient recruitment of BRCA1 to sites of DNA double-strand breaks (DSBs). RNA-Seq analysis revealed that G6PC3 promotes the expression of multiple homologous recombination repair genes, including BRCA1 . Through CRISPR-Select functional-genetic phenotype analysis of G6PC3 germline mutations, we identified 2 germline G6PC3 variants displaying partial loss of function. Furthermore, our study demonstrated that G6pc3 deficiency accelerates mammary tumor formation induced by Trp53 loss in mice. In conclusion, our cohort-based functional analysis has unveiled genome maintenance factors and identified G6PC3 as a potential candidate tumor suppressor in breast cancer.

Genetic variants perturbing BRCA1 and BRCA2 function are linked to hereditary breast and ovarian cancers. As BRCA1 and BRCA2 are critical for homologous recombination-mediated DNA repair, interpretation of variants of uncertain significance is essential. Here, we review established and emerging assays used to functionally characterize BRCA1 and BRCA2 variants and highlight the strengths and limitations of each approach, as well as their general relevance to variant classification and clinical applications.

Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. One significant pathway that is disabled as a result is homologous recombination repair (HRR). With the aim of identifying new candidate genes, we examined early-onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene), which mediates HRR through the end resection of DNA double-strand breaks (DSBs). Notably, these patients exhibited a number of rare germline RBBP8 variants. Functional analysis revealed that these variants did not affect DNA DSB end resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to that of cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which, when compromised, may predispose to the development of early-onset breast cancer.

Loss-of-function mutations in genome maintenance genes fuel tumorigenesis through increased genomic instability. A subset of these tumor suppressors are challenging to identify due to context dependency, including functional interactions with other genes and pathways. Here, we searched for potential causal genes that impact tumor development and/or progression in breast cancer through functional-genetic screening of candidate genes. MYH4, encoding a class II myosin, emerged as a top hit impacting genomic stability. We show that MYH4 suppresses DNA replication stress by promoting replication licensing and replication fork progression. Moreover, we observed a strong synergistic relationship among class | myosins in suppressing replication-associated DNA damage. Genomic analysis of Pan-Cancer Analysis of Whole Genomes project breast cancer samples revealed frequent concomitant loss of TP53 with MYH4 and class || myosins on chromosome 17p. Notably, Myh4 disruption accelerated mouse mammary tumorigenesis in a Trp53-deficient background. In conclusion, our results suggest an unanticipated function of MYH4 in p53-mediated tumor suppression that can explain their combined loss in breast cancer.