Comprehensive Analysis of Missense Variations in the BRCT Domain of BRCA1 by Structural and Functional Assays

Department of Biochemistry, School of Systems Molecular Medicine, University of Alberta, Edmonton, Alberta, Canada.
Cancer Research (Impact Factor: 9.33). 06/2010; 70(12):4880-90. DOI: 10.1158/0008-5472.CAN-09-4563
Source: PubMed


Genetic screening of the breast and ovarian cancer susceptibility gene BRCA1 has uncovered a large number of variants of uncertain clinical significance. Here, we use biochemical and cell-based transcriptional assays to assess the structural and functional defects associated with a large set of 117 distinct BRCA1 missense variants within the essential BRCT domain of the BRCA1 protein that have been documented in individuals with a family history of breast or ovarian cancer. In the first method, we used limited proteolysis to assess the protein folding stability of each of the mutants compared with the wild-type. In the second method, we used a phosphopeptide pull-down assay to assess the ability of each of the variants to specifically interact with a peptide containing a pSer-X-X-Phe motif, a known functional target of the BRCA1 BRCT domain. Finally, we used transcriptional assays to assess the ability of each BRCT variant to act as a transcriptional activation domain in human cells. Through a correlation of the assay results with available family history and clinical data, we define limits to predict the disease risk associated with each variant. Forty-two of the variants show little effect on function and are likely to represent variants with little or no clinical significance; 50 display a clear functional effect and are likely to represent pathogenic variants; and the remaining 25 variants display intermediate activities. The excellent agreement between the structure/function effects of these mutations and available clinical data supports the notion that functional and structure information can be useful in the development of models to assess cancer risk.

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    • "Case control studies are made difficult, however, by the rarity of specific mutations in the population, while segregation studies suffer from uncertainties generated by the high likelihood of phenocopies among the affected, and the potential for late onset cancer in the unaffected. In cases where clinical data are not available to classify VUS, several functional assays have been developed to assess the effects of individual mutations on specific BRCA1 functions, including a transcriptional activation assay [33], phosphopeptide binding assay [34], ubiquitin ligase activity assay [35] and an embryonic stem-cell based functional assay [36]. However these functional assays can be technically complex and are limited to mutations in specific domains impacting particular functions. "
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    ABSTRACT: The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.
    PLoS ONE 06/2014; 9(6):e100068. DOI:10.1371/journal.pone.0100068 · 3.23 Impact Factor
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    • "The method is based on the ectopic expression of BRCA1 C-terminal fragments fused to GAL4-DBD and the ability of the resulting chimeric protein to activate transcription of a reporter gene [27], [28]. Interestingly, there is a strong correlation of transcriptional activation results and other biochemical activities assigned to BRCA1 such as the specific recognition of phosphorylated peptides [17] indicating that the transcriptional assay functions as a monitor of the structural integrity of the BRCA1 C-terminal region. Importantly, the TA displays a strong correlation with pathogenicity indicating that the assay is specific and sensitive for BRCA1 missense variants in the C-terminus [17]. "
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    ABSTRACT: 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.
    PLoS ONE 05/2014; 9(5):e97766. DOI:10.1371/journal.pone.0097766 · 3.23 Impact Factor
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    • "For example two cancer associated mutations in the BRCT domain, the M1775R and Y1853X, have been shown to also affect the translocation of the protein into the nucleus [35], thus also affecting the nuclear shuttling of the protein controlled by the two nuclear localization signals (NLS) [36]. Likewise the phosphopeptide binding assay is also domain specific, as it only examines the ability of the BRCT domains to interact with small phosphorylated peptides [37], [38]. "
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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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