Variance in age of onset, type of cancer developed and outcome occur in populations of women who carry the same BRCA1 mutations. One explanation for these different cancer risks relates to the potential for other genetic loci, termed modifier genes, to influence cancer risk. Commonly used methodologies to discover modifier genes rely either on prior knowledge of gene function or are discovered through genome-wide association studies, which are capable of determining loci of interest but not specific genes. These methodologies have therefore resulted in limited discovery of secondary genetic loci impacting cancer incidence. Identification of modifier loci has the potential to save lives and the healthcare system money, aiding in development of diagnostic tests for preemptive treatment, as with BRCA1 testing, and identifying mechanistic cause as a starting point for therapeutic treatments. Therefore we have sought to overcome the limitations of current approaches for identifying novel cancer susceptibility genes, through development of an innovative approach utilizing the budding yeast, S. cerevisiae, to screen the genome for heterozygous mutations that modify genomic instability in strains that are deficient for genes that encode proteins functionally related to BRCA1. The utilization of a budding yeast model is appropriate in this situation as eukaryotes possess well-conserved pathways to monitor genomic stability and respond to DNA damage, and furthermore are genetic malleable and tractable, have high speeds of growth, are easy of handle and are highly accessible to undergraduate students. In this application we propose studies that will help to determine the mechanism of action of these candidate modifiers thereby creating a ranked pipeline of genes whose homologs may be investigated for modifier status in human studies. Two particular groups of genes were identified based on their haploinsufficient effects on genomic instability, those impacting nutrient availability and those whose impact is checkpoint-deficient dependent. In studying nutrient availability we seek to determine the scope of genes involved in these responses and their dosage sensitivities and then test our hypothesis that TOR pathway and autophagy regulation are altered leading to chromosome loss events. Modifiers shown to have checkpoint-deficient dependency where identified based on a rad9-deficiency in the DNA damage checkpoint pathway; to investigate the mechanism of this phenotype we will test double mutant combination with other DNA damage checkpoint genes and other point specific checkpoint deletions. Ultimately, we will identify orthologous mammalian genes that can then serve as candidate genetic modifiers of cancer risk in ongoing epidemiologic studies. This information will provide new insights into cancer predisposition in susceptible individuals as well as identify factors that may mediate a cancer cell's response to treatment modalities that damage DNA or interfere with chromosome segregation. By engaging undergraduate research students in genetic and molecular based projects, the planned proposal meets the goals of the NIH AREA program.
The objective of the proposed research is relevant to public health because it seeks to identify candidate cancer susceptibility modifier loci, which will aid in diagnosis and uncover new targets for therapeutic development. Compared to currently used methods, we are able to detect heterozygous mutations with haploinsufficiency effects on genome instability using the Saccharomyces cerevisiae model system. In this application we propose studies that will help to determine the mechanism of action of these candidate modifiers thereby creating a ranked pipeline of genes whose homologs may be investigated for modifier status in human studies. In addition to advancing the field of modifier loci identification, this multi-disciplinary project mets the stated objectives of the AREA program by offering undergraduate students hands-on training in classical genetics and modern molecular techniques and strengthens the research environment at Northern Kentucky University.
