Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay

Abstract

BRCA1 (Breast Cancer 1, early onset) is linked to breast and ovarian cancer predisposition. Still, the risks conferred by a significant portion of BRCA1 variants identified in the population remains unknown. Most of these variants of uncertain significance are missense alterations. However, the functional implications of small in-frame deletions and/or insertions (indels) are also difficult to predict. Our group has previously evaluated the functional impact of 347 missense variants using an extensively validated transcriptional activity assay. Here we show a systematic assessment of 30 naturally occurring in-frame indels located at the C-terminal region of BRCA1. We identified positions sensitive and tolerant to alterations, expanding the knowledge of structural determinants of BRCA1 function. We further designed and assessed the impact of four single codon deletions in the tBRCT linker region and six nonsense variants at the C-terminus end of BRCA1. Amino acid substitutions, deletions or insertions in the disordered region do not significantly impact activity and are not likely to constitute pathogenic alleles. On the other hand, a sizeable fraction of in-frame indels at the BRCT domain significantly impact function. We then use a Bayesian integrative statistical model to derive the probability of pathogenicity for each variant. Our data highlights the importance of assessing the impact of small in-frame indels in BRCA1 to improve risk assessment and clinical decisions for carriers.

Figure 1

Transcription activation assay for indels that result in missense substitutions. (A) Schematic representation of BRCA1, with its domains. CC: Coiled-coil motif. The studied region (aa 1396–1863) is enlarged with schematic representation of the secondary structures and connecting loops in the BRCT domains. (B) Transcription activity of indels that result in missense substitutions (light gray bars) shown as a percentage of wild type activity (± Standard error of three independent experiments). The wild type control is represented with a black bar. The known benign p.(S1613G) variant is represented with a dark gray bar. Light red and blue backgrounds represent values below 30% and above 80%, respectively. (C) GAL4 DBD-BRCA1 protein levels in HEK293FT cells. Immunoblot using anti-GAL4DBD and anti-β-actin.

Figure 2

Transcription activation assay for small in-frame deletions that result in amino acid residue deletions. (A) schematic representation of the studied region (aa 1396–1863) depicting the secondary structures and connecting loops in the BRCT domains. (B) Transcription activity of small in-frame deletions (light gray bars) shown as a percentage of wild type activity (± Standard error of three independent experiments). The wild type control is represented with a black bar. The known benign p.(S1613G) variant is represented with a dark gray bar. Light red and blue backgrounds represent values below 30% and above 80%, respectively. (C) GAL4 DBD-BRCA1 protein levels in HEK293FT cells. Immunoblot using anti-GAL4DBD and anti-β-actin.

Figure 3

Transcription activation assay for indels that result in amino acid residue insertions. (A) schematic representation of the studied region (aa 1396–1863) depicting the secondary structures and connecting loops in the BRCT domains. (B) Transcriptional activity of small in-frame deletions (light gray bars) shown as a percentage of wild type activity (± Standard error of three independent experiments). The wild type control is represented with a black bar. The known loss-of-function M1775R variant is represented in red. Light red and blue backgrounds represent values below 50% and above 80%, respectively. Regions enclosing the variants are depicted on the top of the bar graph. CC: Coiled-coil. DR: Disordered region. (C) GAL4 DBD-BRCA1 protein levels in HEK293FT cells. Immunoblot using anti-GAL4DBD and anti-β-actin.

Figure 4

Transcription activation assays for single codon deletions at the linker region and nonsense variants at the C-terminus border of BRCA1. (A) structure of BRCA1 tandem BRCT domains (PDB 1JNX). The linker region and the C-terminal border are depicted with dashed boxes. The linker is rotated (90°) and enlarged to show amino acid residue positions tested. (B) Transcriptional activity of single codon deletions located at the linker region between BRCT domains (light gray bars) shown as a percentage of wild type activity (± Standard error of three independent experiments). The wild type control is presented with a black bar. (C) BRCA1 C-terminal end variants alignment (D) Transcriptional activity of nonsense variants located at the C-terminus of BRCA1 (light gray bars). The wild type control is represented with a black bar. Previously evaluated variants are represented with red bars. Light red and blue backgrounds represent values below 30% and above 80%, respectively. (E) GAL4 DBD-BRCA1 protein levels in HEK293FT cells. Immunoblot using anti-GAL4DBD and anti-β-actin.