Mouse embryonic stem cell-based functional assay to evaluate mutations in BRCA2

Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, Maryland 21702, USA.
Nature medicine (Impact Factor: 27.36). 08/2007; 14(8):875-81. DOI: 10.1038/nm.1719
Source: PubMed


Individuals with mutations in breast cancer susceptibility genes BRCA1 and BRCA2 have up to an 80% risk of developing breast cancer by the age of 70. Sequencing-based genetic tests are now available to identify mutation carriers in an effort to reduce mortality through prevention and early diagnosis. However, lack of a suitable functional assay hinders the risk assessment of more than 1,900 BRCA1 and BRCA2 variants in the Breast Cancer Information Core database that do not clearly disrupt the gene product. We have established a simple, versatile and reliable assay to test for the functional significance of mutations in BRCA2 using mouse embryonic stem cells (ES cells) and bacterial artificial chromosomes and have used it to classify 17 sequence variants. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse ES cells. This technique may also serve as a paradigm for functional analysis of mutations found in other genes linked to human diseases.

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Available from: Sergey G Kuznetsov, Sep 09, 2014
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    • "Complementation tests to evaluate functions of VUS in BRCA1-and BRCA2-deficient cells have been conducted (Carvalho, et al., 2007). Mouse embryonic stem cell-based complementation functional assay to evaluate mutations in BRCA2 were established (Kuznetsov, et al., 2008), this method appearing robust in evaluating functions of BRCA2 variants. Human BRCA2 protein in mouse models, however, could in theory create "
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    ABSTRACT: The greatest interpretive challenge of modern medicine may be to functionally annotate the vast variation of human genomes. Demonstrating a proposed approach, we created a library of BRCA2 exon 27 shotgun-mutant plasmids including solitary and multiplex mutations to generate human knockin clones using homologous recombination. This 55-mutation, 13-clone syngeneic variance library (SyVaL) comprised severely affected clones having early-stop nonsense mutations, functionally hypomorphic clones having multiple missense mutations emphasizing the potential to identify and assess hypomorphic mutations in novel proteomic and epidemiologic studies, and neutral clones having multiple missense mutations. Efficient coverage of non-essential amino acids was provided by mutation multiplexing. Severe mutations were distinguished from hypomorphic or neutral changes by chemosensitivity assays (hypersensitivity to mitomycin C and acetaldehyde), by analysis of RAD51 focus-formation, and by mitotic multipolarity. A multiplex unbiased approach of generating all-human SyVaLs in medically important genes, with random mutations in native genes, would provide databases of variants that could be functionally annotated without concerns arising from exogenous cDNA constructs or interspecies interactions, as a basis for subsequent proteomic domain-mapping or clinical calibration if desired. Such gene-irrelevant approaches could be scaled up for multiple genes of clinical interest, providing distributable cellular libraries linked to public shared functional databases.This article is protected by copyright. All rights reserved
    Human Mutation 11/2014; 36(2). DOI:10.1002/humu.22736 · 5.14 Impact Factor
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    • "Aberrations in such common cellular processes, however, do not explain known p53-associated developmental defects in embryonic tissues of epidermal origin in female mice or a strong tissue-specific bias in the tumor spectrum. Trp53 knock-out mice mostly develop lymphomas and sarcomas234, while concurrent mutations in some DNA repair genes may, however, shift the tumor spectrum toward epithelia-derived carcinomas5. In addition, while cancer-associated point mutations in the TP53 gene are equally common in both lumenal and basal-like breast cancers, truncating mutations and large scale deletions in this gene are more prevalent in basal-like breast cancers compared with the lumenal subtypes suggesting that different cell types within mammary epithelia may have different requirements for TP536. "
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    ABSTRACT: Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.
    Scientific Reports 04/2014; 4:4663. DOI:10.1038/srep04663 · 5.58 Impact Factor
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    • "This highlights the fact that not a single assay can determine functional abrogation of a VUS, as different mechanisms may be affected by different variants irrespective of their location. The Sharan group developed elegant mouse models for examining VUS for both BRCA1 [42] and BRCA2 [45]. They engineered mouse embryonic stem cells with a knock-out allele and a conditional BRCA1 allele. "
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    ABSTRACT: The identification of variants of unknown clinical significance (VUS) in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.
    PLoS ONE 04/2014; 9(4):e93400. DOI:10.1371/journal.pone.0093400 · 3.23 Impact Factor
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