Abstract P4-05-19: Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy
Authors: Grageda Andre , Sarkar Debasree , Kuznetsov Vladimir A 
Published: March 2020

Abstract
An estimated 35,000 patients with breast cancer (BC) have pathogenic BRCA1/2 variants. Unfortunately, in the current state of medical practice, 50-80% of pathogenic mutation carriers remain undetected and undiagnosed. These are largely women with moderate penetrance mutations, including women with BRCA1 mutations, which may not have been identified. There is an unmet challenge to improve the identification and risks of patients who have an inherited increased lifetime risk of BC. BRCA1 mutation carriers (BRCA1-mc) consist ~8% of woman suffering from BCs. A transcriptional R-loop is a guanine-rich 3-stranded nucleic acid structure that plays key roles in (dis)regulation of transcription events, DNA damage, and genome (in)stability. R-loops can be involved in cancer cell precursor development in BRCA1-mc, but it is not clear how they affect luminal precursor differentiation to mature basal-like BCs associated with BRCA1-mc. The role of R-loops in basal-like cells in normal-like tissue of BRCA1-mc have not been studied, limiting the biological interpretation of previous observations. At a genome wide scale, accuracy and resolution of experimentally defined R-loop boundaries is still a challenge. Integrative statistical and genomic approaches have demonstrated that it is possible to computationally predict R-loop forming sequences (RLFSs) in the human genome at an accuracy of 84-92%. In these regards, integrative analysis of available datasets could be useful. We hypothesize that such analyses of RLFS-positive RNA:DNA hybrids/R-loops and gene expression profiles of BRCA1 wild-type and BRCA1 mutation non-cancer epithelial cells could distinguish normal individuals and pre-malignant risks in BRCA1-mc. To test this hypothesis, we used publicly available datasets and carried out a comprehensive genome-wide analysis for BRCA1-mc and non-carriers without BC malignancy. We identified differentially expressed genes (DEGs) for non-cancer breast epithelium cells from publicly available 10 non-cancer individuals with and 10 without BRCA1 mutations. Our QmRLFS finder, DRIP-seq dataset (GEO:GSE96672) and our R-loop database (http://rloop.bii.a-star.edu.sg/) were used to identify RLFS-positive R-loops genome wide. A majority of DEGs, 1813, in non-cancer epithelial cells of BRCA1-mc are downregulated; Gene Ontology terms include metabolism, immune system, cell signaling, cell adhesion, transcription, and stress response. But, 597 DEGs were upregulated; splicing events. 662 downregulated and 185 upregulated DEGs contained RLFS-positive R-loops. In BRCA1 mutation carrier and non-carrier genomes, RLFS and R-loops were commonly located upstream and downstream promoter regions of many genes regardless of breast cell types. Interestingly, RLFS-positive R-loops were observed most often in luminal cells, however they were also found in mature basal-like cells. In particular, RLFS-positive R-loops were found only in BRCA1 locus (in 1st intron) of mature basal-like cells of non-BRCA1 mutation breast tissue samples, but not in luminal cells. However, for BRCA1-mc, we observed R-loops in BRCA1 locus of luminal precursors and mature luminal cells, but not in basal-like cells. Also, in BRCA1 pathway BACH1 promoter region, the RLFS-positive R-loops were observed in all breast cell types for both BRCA1-mc and non-carriers. This study suggests strong association of RLFS-positive R-loop formation and its enrichment variations within mature basal-like breast cells. We highlight the potential clinical significance of early detection of RLFS-positive R-loops in non-cancer breast
Citation Format: Andre Grageda, Debasree Sarkar, Vladimir A Kuznetsov. Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-19.