Multiplexed assay of variant effect reveals residues of functional importance in the BRCA1 coiled-coil and serine cluster domains

• Published in: PloS one Vol. 18; no. 11; p. e0293422
• Main Authors: Nagy, Gregory, Diabate, Mariame, Banerjee, Tapahsama, Adamovich, Aleksandra I, Smith, Nahum, Jeon, Hyeongseon, Dhar, Shruti, Liu, Wenfang, Burgess, Katherine, Chung, Dongjun, Starita, Lea M, Parvin, Jeffrey D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.11.2023; Public Library of Science (PLoS)
• Subjects: Amino acids; Analysis; Assaying; Biology and life sciences; BRCA1 protein; BRCA1 Protein - genetics; BRCA1 Protein - metabolism; Breast cancer; Cancer; Cancer therapies; Clusters; DNA; DNA repair; DNA Repair - genetics; Ethylenediaminetetraacetic acid; Genetic Predisposition to Disease; Health aspects; Homology; Humans; Medicine and Health Sciences; Multiplexing; Mutation, Missense; Ovarian cancer; Plasmids; Proteins; Recombinational DNA Repair; Research and Analysis Methods; Residues; Serine; Serine - genetics; Surveillance; Tumor suppressor genes; Tumor Suppressor Proteins - genetics; United States
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0293422

Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.