BRCA1 frameshift variants leading to extended incorrect protein C termini

Abstract

Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus constitute loss-of-function variants and underscore the need for comprehensive assessment of individual variants.

Figure 1

Functional assessment of BRCA1 EITs (A) Protein amino acid sequence alignment of human BRCA1 C-terminal wild-type sequence (aa 1787–1863; NP_009225.1) and frameshift variants assessed in this study. Vertical red highlight line indicates position of codon 1853. Residues in red represent incorrect protein sequences. Highlighted positions indicate hydrophobic residues (yellow) and tryptophan 1837 (blue) conserved in BRCT domains. Premature protein termination (PPT) variants are shown in red font. Inset, PDB image 1JNX of BRCA1 tandem BRCT highlighting features in the amino acid sequence. (B) Transcription activity of EITs and PPT controls (c.5363dup, c.5464dup, c.5530del, c.5534del, and c.5542del) relative to wild-type BRCA1 in HEK293FT cells. (C) BRCA1 protein levels in whole-cell extracts from transfected HEK293FT cells by immunoblotting using anti-GAL4 DNA-binding domain (DBD) and anti-β-actin. (D) Small colony phenotype assay in AH109 yeast cells overexpressing a Myc-tagged GAL4 DBD fusion of wild-type BRCA1, c.5578dup, c.5578del, c.5579_∗2del16, or c.5534del (pathogenic control). Overexpression of wild-type BRCA1, but not pathogenic variants, leads to a small colony phenotype. (E) Number of colony-forming units (CFUs) per mL observed in plates shown in (D). BRCA1 protein levels of constructs in whole-cell extracts from transformed AH109 cells by immunoblotting using anti-Myc.

Figure 2

VarCall estimated variant-specific effects High-resolution graph depicting boxplots summarizing the marginal posterior distributions of the variant-specific effect parameters generated by VarCall for all BRCA1 EITs and PPTs (blue boxes) tested in the current study. To facilitate visualization, this is a zoomed-in version of the complete graph (shown in Figure S2).

Figure 3

Homologous recombination (HR) assay and family history (FHx) analysis (A) Relative HR activities of the BRCA1 variants studied. Variants were introduced into full-length BRCA1 with 3XMyc tag at the N terminus and their activities assessed in U2OS/DR-GFP cells depleted of the endogenous BRCA1. Activity values were normalized against that of the WT BRCA1. Error bars represent standard deviations from 3 independent experiments. (B) Expression levels of the variants studied and their binding capacities to known BRCA1 interacting partners in 293T cells. Cells were transfected with an empty vector (EV) or 3XMyc-tagged BRCA1 constructs. Whole-cell extracts (WCEs) were subjected to direct western blotting or immunoprecipitation (IP) with anti-Myc antibody followed by western blotting analysis as indicated. (C) FHx likelihood model assessment of aggregate FHx for 23 carriers of c.5578dup (blue vertical line) plotted relative to the distribution of simulated control groups for benign (green curve) and pathogenic (red curve) variants. Dashed and solid vertical lines represent the 95^th and 99^th percentiles for the control curves. One count refers to one simulated dataset with the same number of probands as the test dataset. Each curve in the graph, benign and pathogenic, is comprised of 100,000 simulated datasets with the same number of probands as the test dataset, and count refers to the number of those simulated datasets. LLR, log likelihood ratio.