Abstract
Germline mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome. Germline and somatic BRCA1/2 mutations may define therapeutic targets and refine cancer treatment options. However, routine BRCA diagnostic approaches cannot reveal the exact time and origin of BRCA1/2 mutation formation, and thus, the fine details of their contribution to tumor progression remain less clear. Here, we establish a diagnostic pipeline using high-resolution microscopy and laser microcapture microscopy to test for BRCA1/2 mutations in the tumor at the single-cell level, followed by deep next-generation sequencing of various tissues from the patient. To demonstrate the power of our approach, here, we describe a detailed single-cell-level analysis of an ovarian cancer patient we found to exhibit constitutional somatic mosaicism of a pathogenic BRCA2 mutation. Employing next-generation sequencing, BRCA2 c.7795G>T, p.(Glu2599Ter) was detected in 78% of reads in DNA extracted from ovarian cancer tissue and 25% of reads in DNA derived from peripheral blood, which differs significantly from the expected 50% of a hereditary mutation. The BRCA2 mutation was subsequently observed at 17-20% levels in the normal ovarian and buccal tissue of the patient. Together, our findings suggest that this mutation occurred early in embryonic development. Characterization of the mosaic mutation at the single-cell level contributes to a better understanding of BRCA mutation formation and supports the concept that the combination of single-cell and next-generation sequencing methods is advantageous over traditional mutational analysis methods. This study is the first to characterize constitutional mosaicism down to the single-cell level, and it demonstrates that BRCA2 mosaicism occurring early during embryogenesis can drive tumorigenesis in ovarian cancer.