Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation

Abstract

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

Keywords: BRCA1; BRCT domains; VarCall; breast cancer; breast cancer risk; cancer prevention; clinical annotation; clinical risk assessment; human genetics; protein conformation.

Figure 1.

BRCA1 variant functional annotation. A, overview of BRCA1 variants. B, diagram of BRCA1 C-terminal region (aa 1396–1863) and location of variants. Variants tested in previous studies and in the current study are shown as blue and red sticks, respectively. Missense VUS tested are grouped according to their structural feature.

Figure 2.

Transcription activation assay for BRCA1 missense VUS at the N- and C-terminal borders of the BRCT domains. A, transcription activity of missense variants located at the transition between the disordered region and the BRCT domain. B, transcription activity of missense variants located at the C-terminal border of the BRCT domain. Western blots indicate steady state levels of expression of the fusion proteins containing C-terminal deletions. Regions of interest are indicated in the crystal structure diagram (Protein Data Bank 1Y98_A) by a dashed circle. Variants with significantly reduced activity (<80% WT) are denoted in red font.

Figure 3.

Transcription activation assays for BRCA1 missense VUS at the BRCT linker region. A, structure of BRCA1 tandem BRCT domains (PDB 1JNX). The linker region is shown as a magenta structure perpendicular to the plane of the page (red box). The linker is rotated (90°) and enlarged to show amino acid residue positions tested. B and C, transcription activity of missense variants located at the linker region between BRCT domains. D, transcription activity of missense variants located at the disordered region, and intervening segments and α-helices of BRCT domains. Variants with significantly reduced activity (< 80% WT) are denoted in red font.

Figure 4.

Transcription activation assays for BRCA1 missense VUS normalized by internal vector and protein levels. A–E, transcription activity of missense variants located at the transition between the disordered region and the BRCT domain normalized against vector control and protein levels (Fig. S1). For comparison, squares denote whether the variant displayed activity similar to WT (open squares) or reduced (<80% WT; filled squares) activity when normalizing against vector control only.

Figure 5.

VarCall analysis of missense variants in the C-terminal region of the BRCA1 protein. A, in this study, transcriptional assays were performed for 99 missense variants (blue bars). Results of these transcriptional assays were combined with previous data (25) (combined dataset of 347 variants) and analyzed using VarCall to predict the likelihood of pathogenicity. See Fig. S3, a high resolution version, for additional details and variant labels. B, circos plot illustrates how the new series of variants improves classification for all variants. Variants assigned to fClasses (1–5) in Woods et al. (25) (denoted as Previous fClass 1–5) were re-assessed by VarCall (denoted as New fClass 1–5) after including the set of variants in the present study. C, fraction of variants tested that displayed impact on function grouped by structural features.

Figure 6.

High concordance between VarCall classification and other functional assays. A, diagram showing classification of all missense variants in aa 1315–1863 as defective (red squares) or not (blue squares according to VarCall (Track 8) or to functional assays based on homologous recombination (Tracks 18–23 in Table S6). First row depicts IARC Class using the multifactorial model (Table S4) with red squares (Classes 4–5) and blue squares (Classes 1–2). Discordant results between VarCall and any HR assay are indicated by a red star. Green stars indicate discordant from any assay but in agreement with another HR assay. Variants are in bold font. B, diagram showing classification of variants tested in this study as defective (red squares) or not (blue squares) according to VarCall (Track 8) or to a saturation mutagenesis study (Track 30; Findlay et al. (28)) based on cell viability. Discordant results are indicated by a red star. C, fraction of missense variants with disagreements between assays.