CRISPR Excision and Long-Read Sequencing of BRCA1 and BRCA2 to Identify Previously Undetectable Classes of Mutations in Families with Ovarian Cancer
Abstract
The identification of BRCA1 and BRCA2 have had major impacts on prevention, diagnosis, and treatment of ovarian cancer, with sequencing of BRCA1 and BRCA2 now common practice in the care and treatment of ovarian cancer patients in the United States and many other parts of the world. Learning of inherited mutations in these genes before cancer is diagnosed can have a very substantial impact on ovarian cancer incidence. Our work has shown that, among ovarian cancer patients not selected for family history of cancer or age at diagnosis, approximately one in five patients carries an inherited damaging mutation in BRCA1, BRCA2, or related genes that normally repair damaged DNA. That is, 20% of ovarian cancer could disappear if women knew of inherited mutations in BRCA1, BRCA2, and their sister genes early enough to take preventive action. However, for a very large number of families severely affected with ovarian cancer, no causal mutation has been found. In our studies over 40 years, we have encountered many severely affected families with no detected mutation that could explain their ovarian cancer, even after evaluation using every state-of-the-art genomics approach. We hypothesize that, in many of these families, ovarian cancer is due to complex structural genomic mutations in “messy” regions of the genome that are hidden from current technologies, but that nonetheless lead to destruction of ovarian cancer genes. Such messy genomic regions comprise large swaths of tumor suppressor genes, especially BRCA1. To address this problem, we propose to combine two new technologies: CATCH (Cas9-Assisted Targeting of Chromosome segments) and SMRT (Single Molecule Real Time) sequencing. CATCH enables us to chemically clip BRCA1 and BRCA2 out of the DNA from cell lines of our patients, and SMRT enables us to sequence the clipped-out regions with new technology that yields very long continuous sequences. We validated our SMRT-CATCH approach, then tested DNA and cells from one family with four affected women, whom we have studied to no avail for 14 years. With our new approach, we identified a genomic insertion of ancient “junk DNA” into BRCA1, leading to abnormal BRCA1 RNA and protein. This mutation was not detectable by any other approach, even whole genome sequencing using conventional means. Success for one family may be beginners’ luck, but we are optimistic that our approach will resolve the genetic bases of inherited ovarian cancer for many of our participants. In this proposal, we will evaluate 225 families with ovarian cancer. Our approach could benefit all women who unknowingly carry inherited mutations predisposing to ovarian cancer. Our participants include military families at about their proportion in the general American population, or perhaps at slightly higher frequency, as the result of our frequent collaboration for referral of families with gynecologic oncologists working with the VA. Based on consent from our participants, results will be returned by our certified genetic counselor, Jessica Mandell, CGC. For women who carry actionable mutations, referral will be undertaken by our clinical colleague, Barbara Norquist, M.D. We hope that this project will lead to improvements in genetic testing for inherited predisposition to ovarian cancer.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010286
Entities
People
- Tomas Walsh
Organizations
- United States Army
- University of Washington