70% of breast cancers exhibit aneuploidy, and this phenotype is used as an early prognostic factor because it usually indicates a high malignancy potential. Mutations in many different checkpoint genes result in aneuploidy and an increased risk of breast cancer including, BRCA1, PTEN, CHEK2, and ATM. However, the age of onset and type of cancer developed can vary among women with these mutations, which is partially attributed to the likelihood of the presence of unlinked genetic loci, which modify this risk. We have proposed to identify candidate modifier loci that impact genomic stability by screens in Saccharomyces cerevisiae. Budding yeast are commonly used organisms for modeling human processes, and many aspects of the DNA damage checkpoint pathway, in which BRCA1 is involved, are conserved between S. cerevisiae and humans. The S. cerevisiae Rad9 protein has similar functions and sequence motifs as BRCA1. We therefore developed a screen based on the hypotheses that rad9Δ/Δ mutations in S. cerevisiae will model BRCA1-/- mammary cells and haploinsufficiency at a second locus that alters the chromosome loss rate of our rad9Δ/Δ diploid strains serves as a potential modifier loci. To complete the screen we created a rad9Δ/Δ yeast strain carrying two marker systems for chromosome stability assays allowing a visible qualitative marker and a canavinine resistance quantitative marker of chromosome loss rate by fluctuation analysis. We utilize an insertional mutagenesis library to screen the yeast genome for heterozygous mutations, which alter chromosome loss rates. To date we have screened 7500 insertional mutants for genomic instability and qualitatively identified 10 candidates, 5 of which alter the chromosome loss rate of the rad9Δ/Δ yeast strain by ∼10 fold. The insertions include disruption of uncharacterized yeast ORFs as well as a gene implicated in chromatin silencing, which will need be further characterized via directed mutation of these genes and analysis for radiation sensitivity, DNA damage checkpoint function and genomic instability. Thus, our screening strategy is capable of identifying heterozygous mutations that alter genomic stability and the human homologs of genes identified in this screen can serve as candidate modifiers of genomic stability in mammary cells null for BRCA1 function. Funding thanks to: U.S. Army Breast Cancer Research Grant.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]