Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays

Abstract

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.

Figure 1

A, ribbon diagram representation of the BRCA1 BRCT tandem repeats (PDB ID 1T15; ref. 13). Blue, α-helices; orange, β-strands. Red, the linker between the N-BRCTs and C-BRCTs. Green, the bound BACH1 decapeptide; sticks, the pSer and Phe(+3) residues. Black spheres along the polypetidic chain, the positions of the studied mutations. B, the complete BRCA1 BRCT missense variant set. Shown is the sequence of the human BRCA1 BRCT domain with the protein secondary structure indicated. Below the sequence are colored boxes indicating the level of sequence identity of each residue within a 13 species alignment (http://agvgd.iarc.fr/alignments.php). Neutral and disease-associated variants (Table 1) are highlighted as are residues that contact the pSer and Phe at the +3 position of the phosphopeptide target.

Figure 2

Functional analysis of 19 variants plotted against the combined log₁₀ likelihood ratio (LLR) score as calculated. Points, mean binding activity of each of the variants as percent of wild-type activity; bars, SD. Outliers are labeled. A, structural stability. B, phosphopeptide binding activity. C, phosphopeptide binding specificity. D, transcription activation activity.

Figure 3

Cross-validation of structural and functional assays. The results for protease sensitivity (PS), peptide binding activity (BA), peptide binding specificity (PS), and transcriptional activity (TA) are tabulated using blue circles to indicate no defect, yellow circles to indicate an intermediate activity, and red circles to indicate a large defect. Also shown are the predicted align-GVGD scores (AG) with circles colored from blue to red to indicate no predicted effect, to a large effect, respectively.

Figure 4

Variants classified with no functional effect (A) or strong functional effect (B) by structure/function assays (see Table 2) were analyzed with the GV-GD algorithm and sorted in one of the seven grades (C0–C65).