Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs

Abstract

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

Keywords: BAC; BRCA2; CP: Cancer biology; CP: Genetics; DNA repair; VUS; bacterial artificial chromosome; breast cancer; cell viability; functional assay; mouse ES Cells; recombineering; variants of uncertain significance.

Graphical abstract

Figure 1

Schematic representation of the multiplexed mESC-based assay (A) Schematic representation of the mESC-based functional assay. The PL2F7 mESCs (containing a conditional allele containing two loxP sites and a knockout allele of Brca2) is complemented for the loss of Brca2 allele after Cre expression by the BAC DNA encoding the human BRCA2 gene containing one variant. The recombinants were selected in hypoxanthine, aminopterin, thymidine (HAT)-containing medium. Viable HAT^r cells show no impact of BRCA2 variants on function. Viable HAT^r cells were further tested to distinguish the variants that have moderate loss of function by their sensitivity to different DNA-damaging agents. A star in the BAC construct represents the variant. Solid arrows denote loxP sites, and the two halves of the Hypoxanthine-guanine phosphoribosyltransferase (HPRT) mini gene are marked in solid boxes as HP and RT on the conditional allele of Brca2. (B) Selection of two independent clones of mESCs that express BRCA2 variants. After introduction of BAC into PL2F7 cells, transfected cells were selected and analyzed for protein expression. Two clones were selected from each variant to eliminate positional bias of BAC integration. (C) Multiplexing of the downstream process by mixing mESC clones expressing different variants. After mixing, two independent experiments were performed from each mix, and at the indicated steps, samples were collected for DNA isolation and deep sequencing. (D) Schematic representation of loss of BRCA2 variants after selection with HAT, cisplatin, or olaparib. Genomic DNA was isolated, PCR amplified, and deep sequenced to quantify the relative abundance of variants. The ratio of abundance was normalized to the no-drug (M15) control. The relative viability data were further analyzed using the Bayesian statistical model to determine the probability of impact on function of each variant. Oval colored shapes represent cells with a variant, and solid-colored rectangles represent sequence reads.

Figure 2

Functional score (FS) distribution and the correlation of FS between three assays (A) The distribution of FSs calculated from HAT, cisplatin, and olaparib sensitivity of mESCs with different BRCA2 variants. The color codes demarcate controls and variants of uncertain significance (VUSs). Variants that were classified as benign or likely benign in ClinVar were used as neutral/functional controls, and those that were classified as pathogenic or likely pathogenic were used as pathogenic/non-functional controls. A total of 223 variants were analyzed. (B) Correlation of FSs from three different assays (sensitivity to HAT, cisplatin, or olaparib) are plotted to compare the results of different assays. The Spearman correlation coefficient is displayed in each plot.

Figure 3

Probability of impact on function (PIF) for the BRCA2 variants in the full dataset (n= 223), calculated using the main (semi-supervised learning) version of our PIF calculation algorithm The four subplots correspond to the four types of assays that provided FS data used in PIF calculation: HAT only (A), cisplatin only (B), olaparib only (C), and HAT, cisplatin, and olaparib combined (D). The dashed lines correspond to the variant classification thresholds of 0.05 and 0.99; a PIF value is considered indeterminate when 0.05 < PIF ≤ 0.99. In each subplot, the circles represent individual BRCA2 variants. Vertical lines show the 95% confidence intervals for each PIF. Values of confidence intervals are listed in Table S2.

Figure 4

Comparison of multiplexed mESC-based variant classification with other functional, multifactorial, and ClinVar classifications (A) Comparison of variants that were classified using the MANO-B functional assay. (B) Homologous recombination (HR)-based assay and classification using the mESC-based assay. (C) Plot showing the PIFs and classification using the mESC-based assay and classification of the variants in ClinVar. Overlapping dots are circled (dotted), and the number of dots in each circle is represented as n (numbers of variants on each circle). The dots that are not circled represent a single variant. (D) Side-by-side comparison of mESC assay-based classification of variants that have other functional assay data published. We used the data from the MANO-B (mixed-all nominated-in-one-BRCA [MANO-B]) method of classification, the mESC-based assay reported by Mesman et al., and the HR assay-based classification.^,

Figure 5

Position of classified variants in BRCA2 (A) BRCA2 domains are marked with colored rectangles, with the amino acid (aa) position marked in parentheses. The variants located in different regions are marked below. OB, oligonucleotide/oligosaccharides binding; CTRB, C-terminal RAD51 binding. (B) Position of the splicing variants analyzed in this study. Functional variants are marked in green, non-functionals in magenta, and indeterminants in saffron.