New Yeast-Based Assay for Classifying BRCA1 Variants

Lifetime risk of developing ovarian or breast cancer is increased by germline mutations in the BRCA1 gene. While specific pathogenic variants have been well studied, new sequencing technologies continue to identify variants of uncertain significance (VUS). These variants are comparatively rare and cannot easily be studied in humans. Thus, a 2022 study in the International Journal of Molecular Sciences by Bellè et al. demonstrates a means to assess pathogenicity of a given variant in a cell-based assay in yeast. The new technique complements previous techniques (one in yeast, others computational) to improve the accuracy and sensitivity of assessing pathogenicity for the numerous variants of BRCA1.

From Bellè et al., 2022; red pathogenic, turquoise benign, black uncertain

Belle et al. approached their study by noting that BRCA1 mainly affects DNA repair and genome stability, and that yeast has a full toolbox for studying these processes. The team previously demonstrated that pathogenic BRCA1 variants increase the rates of intra- and interchromosomal homologous recombination (HR) and also gene reversion (GR) in yeast.

For the current study, they developed a diploid strain that allows simultaneous assessment of intra- and interchromosomal HR by use of two mutated markers, one that repairs by intrachromosomal exchange and the other by interchromosomal exchange. When they induced BRCA1 variants from a plasmid, they were able to compare rates of HR (compared to the WT BRCA1 gene) by the simple use of plate assays.

Similarly, a haploid strain with a different mutated marker that repairs by gene reversion was used to assess rates of GR upon induction of a BRCA1 variant versus wild type.

After multiple replications of the experiments, the authors plotted Waterfall distributions of the results to arrive at breakpoint values at which a variant could be called benign versus pathogenic for each assay. These results were then analyzed for rates of false positives and false negatives with respect to previous data.

From Bellè et al., 2022; red pathogenic, turquoise benign, gray undermined

The combination of the two yeast-based assays (HR/GR and SCP, together called yBRCA1) was evaluated for performance and shown to be highly accurate and reliable.

As full concordance of results was obtained for three out of ten VUS between the yeast-based combined method and other non-yeast methods, the authors conclude that no one technique is suitable for making a clinical assessment but that multiple techniques will reduce numbers of VUS and resolving conflicting interpretations. Measurement of DNA repair in yeast is a particularly potent example of how yeast can provide a whole-cell, functional assay that sheds light on cell-division disorders.