Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay

Abstract

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

Keywords: BRCA2; Classification; Functional assay; Inherited cancer; Next-generation sequencing; Variants of unknown clinical significance (VUS).

Fig. 1

Functional analysis of BRCA2 variants in Brca2^cko/ko mES cells.a Schematic representation of the mES cell-based functional assay. b Expression of BRCA2 variants in mES cells by Western blotting. Two independent clones were generated for each variant. Vinculin was used as loading control. c Expression of BRCA2 P3039P by RT-PCR using primers from exons 20 (5′-AGGAAGAAAAGGAAGCAGCAAAATATGTGG-3′) and 25 (5′-TCTCCAGCAAATAAAGTAAGAAGG-3′) revealed lack of full-length transcript. Two alternatively spliced transcripts were observed. d Sequence analysis of the two transcripts revealed major alternatively spliced transcript skipped exon 23 (lower band) and a minor form that skipped exon 23 but retained 63pb of intron 22 (upper band). e Quantification of viability of Brca2^ko/ko mES cell by various BRCA2 variants. Two independent BAC clones expressing the variants were analyzed. Average numbers of viable cells from two independent clones were plotted. mES cell clone expressing WT BRCA2 was used as control. f Clonogenic survival assay confirming sensitivity of mES cells expressing A262T to olaparib (P value < 0.001 at 100 nM and < 0.0001 at 500 nM concentrations using a multiple t-test). g Representative images showing γH2AX and RAD51 foci at 3, 6, and 10 h post 10 Gy IR in mES cells expressing WT and A262T BRCA2. h Quantification of γH2AX and RAD51 foci at 3, 6, and 10 h post 10 Gy IR in mES cells expressing WT and A262T BRCA2. i Quantification of homologous recombination using a GFP-based HR reporter in mES cells expressing WT and A262T BRCA2 (P values are 0.008 for Clone 1 and 0.01 for Clone 2 using paired t-test)