Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants

Author:

Sahu SounakORCID,Sullivan Teresa L.,Mitrophanov Alexander Y.ORCID,Galloux MélissaORCID,Nousome Darryl,Southon Eileen,Caylor Dylan,Mishra Arun PrakashORCID,Evans Christine N.,Clapp Michelle E.,Burkett SandraORCID,Malys Tyler,Chari Raj,Biswas Kajal,Sharan Shyam K.ORCID

Abstract

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

Funder

NIH Intramural Research Program

Center for Cancer Research

National Cancer Institute

U.S. National Institutes of Health

National Cancer Institute, National Institutes of Health

Publisher

Public Library of Science (PLoS)

Subject

Cancer Research,Genetics (clinical),Genetics,Molecular Biology,Ecology, Evolution, Behavior and Systematics

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