NEW YORK – A pair of studies by independent research teams has demonstrated the potential for using saturation genome editing to systematically classify loss-of-function germline variants in the BRCA2 gene, which can increase the risk of developing breast or ovarian cancer.
"Owing to limited clinical, familial, and epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS)," senior and corresponding author Shyam Sharan, deputy director of the National Cancer Institute's mouse cancer genetics program, and his colleagues wrote in one of the studies published in Nature on Wednesday, adding that "many VUS are rare and might only be identified in a single person or family."
In an effort to systematically classify the functional impacts of BRCA2 variants, the team turned to a high-throughput "multiplexed assays for variant effects" (MAVE) strategy that involved CRISPR-Cas9 gene editing on humanized mouse embryonic stem cell lines, focusing on a carboxylate-terminal DNA-binding (CTDB) domain-coding region in BRCA2 exons 15 to 26 that contains many of the pathogenic missense variants already described in the ClinVar clinical variant database.
Using deep paired-end sequencing to assess variant dropout or enrichment patterns in edited cells grown with or without DNA-damaging drugs such as cisplatin or olaparib, the team was able to assess 6,551 single-nucleotide variants, including 1,282 missense variants from the VUS category in ClinVar.
In the process, the researchers classified 3,384 of the variants as benign and 776 variants as pathogenic. Those results coincided with pathogenic variant clues in ClinVar, they noted, adding that their saturation genome editing data was subsequently evaluated in the context of clinical classification guidelines from the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP).
"By integrating cellular fitness and response to DNA-damaging agents with structural predictions, we have established that our MAVE dataset can be integrated within the ACMG/AMP clinical framework," the authors explained, adding that the use of saturation genome editing in embryonic stem cell models made it possible to dial down the number of BRCA2 VUS.
For another paper appearing in Nature on Wednesday, researchers with the Mayo Clinic in Rochester, Minnesota, Ambry Genetics, and other centers in the US presented their own saturation genome editing-based MAVE analyses of VUSs in exons 15 to 26 of BRCA2, which they interrogated using CRISPR-Cas9 knock-in editing in human haploid HAP1 cells that were assessed with massively parallel sequencing at the five- and 14-day marks.
In that study, the team classified all but one of the 6,960 BRCA2 variants considered, using a VarCall Bayesian model to place the single-nucleotide changes in one of seven pathogenicity categories: "supporting," "moderate," or "strong" categories of pathogenic or benign variants as well as a VUS category.
The investigators went on to explore the potential roles in breast or ovarian cancer risk using case-control study data for hundreds of thousands of women with or without breast cancer or ovarian cancer who were tested for hereditary cancer variants at Ambry Genetics between 2012 and 2021, along with data for more than 164,320 participants in large population-based breast cancer studies.
Together with variant frequency data from version 4 of the GnomAD database, the case-control data supported the notion that BRCA2 variants classified as strongly pathogenic had ties to enhanced breast cancer or ovarian cancer risk, as did the combined set of variants in the supporting, moderate, and strong pathogenic groups.
"These data will prove useful in the future, through integration with other datasets, for the characterization and classification of all variants in this genetic location in individuals from all racial and ethnic backgrounds and for all BRCA2-associated forms of cancer," senior and co-corresponding author Fergus Couch, a laboratory medicine and pathology researcher at the Mayo Clinic, and his colleagues wrote.