Inherited predisposition syndromes (familial) are expected to account for up to 15% of breast cancers (Yamauchi & Takei, 2018),(Amaral et al., 2019). About 50% of the FBC predisposition genes remain unknown so far. Thus, my overarching goal of this project is to identify and unravel the function of novel putative FBC predisposition genes (Illustrated in Figure 1A). Currently, patients with suspected FBC are subjected to panel testing with known causal genes such as BRCA1/2. The gradual implementation of next generation sequencing in clinical settings is expected to provide a basis for identifying additional FBC predisposition genes. However, current technology is not enough to provide precise risk estimates for almost all developing FBC genes. This is because of the extensive locus heterogeneity within the genes and functional data at the gene and variant levels are missing. This creates challenges for the present cancer risk management and decision-making concerning patients and their families. It is incredibly difficult to gather reliable information regarding the pathogenic relevance of mutations in other novel FBC genes from patient cohorts because they are thought to be rare mutations. Therefore, in this line of thinking, we aimed to utilize clinical data, conduct gene and variant functional analyses and subsequently translate lab findings back to clinic. In this project, we proposed that the functional assays could serve as a novel tool for assessing variant's pathogenic potential. In order to advance current risk-assessment tools, my host lab developed a High-Throughput (HT) phenotype screening strategy to investigate novel FBC predisposition genes with the focus on genome maintenance pathways, as their dysfunction frequently underlie FBC. Notably, these newly identified genes are likely to be attractive targets for development of therapeutic drugs, since they play major role in DNA repair and support genome integrity. In collaboration with Dr. Finn Cilius Nielsen’s team (the major clinical unit at Rigshospitalet, in Copenhagen) we have carried out whole exome sequencing (WES) on genomic DNA purified from blood samples from 135 BRCA1/2 mutation-negative breast cancer patients of age less than 35 years. The age factor is crucial because early-onset breast cancer is highly indicative of FBC predispositions. By referring to exome sequencing data, 152 genes with so far no described DDR role were identified and they were enrolled for HT phenotype screen. The HT genome instability screening was performed to score phospho-histone H2A family member X (γ-H2AX, a marker for DNA damage), micronuclei formation (a marker for genomic stress) and Poly-(ADP-ribose) polymerase inhibitor (PARPi) sensitivity (Illustrated in Figure1B).
To utilize this preliminary clinical and phenotypic data, we focused on following specific objectives:
1. Ranking of novel FBC predisposition genes (hits) from HT-phenotypic screen
2. Elucidation of the molecular function of novel FBC predisposition gene(s) in genome maintenance (with the focus on BRCA1/2-like phenotypes)
3. Unravelling the role of the FBC candidate genes in somatic mouse models.
