Figure 6: Functional validation of BRCA1 missense and truncation variants.
From: Patterns and functional implications of rare germline variants across 12 cancer types
(a) 68 rare missense and 4 truncation variant sites were tested by HDR assay. All samples were depleted of endogenous BRCA1 by transfection of a siRNA targeting the 3′-untranslated region. Indicated in the legend are the plasmids transfected to test for rescue of BRCA1 activity. ‘pcDNA3’ is empty vector and ‘WT’ represents wild-type BRCA1 plasmid. The y-axis denotes the HDR activity relative to the wild-type BRCA1 protein. Error bars depict s.d. from the mean. Dots on the x-axis represent LOH status, each dot corresponding to one case. Blue, red, dark grey and light grey denote statistical significance, non-significance, unknown LOH (due to lack of sufficient coverage) and untested, respectively. Variants in different functional domains are indicated with colours as follows: orange, RING domain; green, nuclear localization signal (NLS); blue, DNA-binding region; purple, a SQ/TQ cluster domain (SCD); and red, BRCA1 C-terminal domain (BRCT). All the HDR assays were tested in triplicate. (b) Crystal structure of the BRCA1 RING (left) domain in complex with the BARD1 RING domain (labelled in grey) and BRCT domain (right panel) are displayed, with HDR-defective variants labelled in red and partial HDR-defective variants tagged in orange. Variants in yellow are functional in the HDR assay.
