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bioRxiv · 10.1101/2023.12.14.571713

AVENGERS: Analysis of Variant Effects using Next Generation sequencing to Enhance BRCA2 Stratification

Sahu, S.·Galloux, M.·Southon, E.·Caylor, D.·Sullivan, T.·Arnaudi, M.·Geh, J.·Chari, R.·Papaleo, E.·Sharan, S. K.
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Abstract

Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). To determine the functional impact of VUSs, here we develop AVENGERS: Analysis of Variant Effects using NGs to Enhance BRCA2 Stratification, utilizing CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain of BRCA2. We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessing BRCA2 VUSs in patients.

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Sahu, S., Galloux, M., Southon, E., Caylor, D., Sullivan, T., Arnaudi, M., Geh, J., Chari, R., Papaleo, E., Sharan, S. K.. 2023-12-15. AVENGERS: Analysis of Variant Effects using Next Generation sequencing to Enhance BRCA2 Stratification. https://doi.org/10.1101/2023.12.14.571713

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