Here, we present the first comprehensive assessment of the structural and functional consequences of 117 BRCA1 variants corresponding to the complete set of BRCT missense variants detected to date in the human population (100% coverage). Our protein structure assay, peptide binding, and transcriptional assays show a remarkable degree of agreement and give us confidence in the validity of these results. Correlation of this experimental data with the available clinical and family history data for a subset of these variants defines limits that can be used to tentatively predict functional effect that is likely to be reflected in the disease risk associated with these variants. In this way, we show that 42.7% of the variants fall into the “clear functional effect” or “moderate functional effect” categories () and are likely associated with an increased cancer risk, whereas 35.9% fall into the “no functional effect” or “low functional effect” categories. The remaining 21.4% falls into the uncertain category and could perhaps represent low-penetrance variants. The fact that a large proportion of variants tested displayed some functional effect lends support to the idea of applying higher prior probabilities for causality to variants found in the BRCT domains (35
The BRCA1 BRCT variant set provides a useful set of reagents for further detailed study of variants of particular interest. For example, variants that are moderately well folded based on proteolytic stability can often be purified and studied in more detail. Circular dicroism spectroscopy and differential scanning calorimetry have been used to quantitatively assess the fold stability of V1696L, R1699W, M1775K, M1775R, M1783T, V1808A, V1809F, and V1833M (49
). Although our proteolytic assay indicated that the stability of two of these variants, R1699W and V1808A, were indistinguishable from wild-type, the more sensitive thermodynamic studies of the purified forms of these variants reveal a small but significant reduction in fold stability. In addition, quantitative assays of phosphopeptide binding, such as isothermal titration calorimetry, can reveal the detailed thermodynamics of peptide-BRCT interactions (50
). The structural rearrangements induced by three of these variants, M1775R (47
), M1775K (37
), and V1809F (14
), have been visualized through crystallographic structural analysis. Each of the variants affect the +3 specificity pocket formed at the interface between the two BRCT repeats. For M1775R and M1775K, the substituted positively charged side chain adopts an orientation that fills the specificity pocket and blocks the binding of the Phe side chain at the +3 position of the target peptide. Intriguingly, the V1809F substitution, which occurs far from the +3 pocket, induces a series of conformational adjustments that results in the movement of Met1775 to fill the specificity pocket, explaining the loss of peptide binding specificity and transcriptional activity associated with this variant.
Nonsynonymous single nucleotide polymorphisms (SNP) that lead to amino acid substitutions are likely to have diverse phenotypic consequences and implications for human disease. Although computational methods have been proposed to assess the effect of individual SNPs (52
), relatively little has been done at the experimental level to provide insight into the effects of SNPs. The database of BRCA1/BRCA2 mutations provides an important first look at the kind of genetic diversity that exists in the human population. Another example of a large mutation database is that assembled for mutations found in p53 from human tumors. Detailed studies of the stability of the p53 DNA binding domain indicate that, like the BRCA1 BRCT domain, this relatively unstable domain is sensitive to mutations that destabilize the protein fold in addition to other classes of mutations that do not significantly affect protein fold stability but instead modify DNA-binding or protein-protein interaction surfaces (53
). Thus, rapid, general assays of protein structure and function such as the proteolysis assay used here could potentially prove useful for the rapid, high-throughput assessment of the effect of SNPs on protein function.
Ultimately, unambiguous classification of variants, as proposed by the IARC Unclassified Genetic variants Working Group (41
), will need a comprehensive integrative model to incorporate data from several sources. Robust integrative models have been proposed but they still lack a coherent framework to incorporate functional data. A major roadblock in the development of such a model was the lack of an established series of functional assays with standardized controls, validated against available genetic data. The present study fills this gap and allows for the development of better predictive models.
Although a systematic large-scale classification of all BRCA1 variants into the IARC classes has not yet been done, it is expected that a large number will fall into class 3 (uncertain), due to the lack of genetic-based data. Conceivably, even large-scale efforts may not be able to gather sufficient genetic data on individual variants because most are extremely rare. Thus, we anticipate that the use of structural and functional information will be instrumental to move variants from the class 3 into classes that provide a basis for better risk assessment and clinical decisions. In addition, the fact that variants can now also be functionally and structurally rationalized will provide an impulse for further functional dissection of the tumor-suppressive activities of BRCA1.