Saturation genome editing-based clinical classification of BRCA2 variants

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Bioinformatics

Background:

• Sequencing tests reveal numerous BRCA2 variants, but many are classified as variants of uncertain significance (VUS) due to insufficient data.
• Accurate classification of BRCA2 VUS is crucial for genetic counseling and patient management, particularly concerning cancer risk.

Purpose of the Study:

• To functionally characterize nearly all single nucleotide variants (SNVs) in the BRCA2 carboxylate-terminal DNA-binding domain using CRISPR-Cas9 saturation genome editing.
• To generate a comprehensive sequence-function map for BRCA2 variants to aid in clinical interpretation.

Main Methods:

• Utilized CRISPR-Cas9 saturation genome editing in a humanized mouse embryonic stem cell line to assess the functional impact of SNVs.
• Generated function scores for 6,551 SNVs across BRCA2 exons 15-26, covering 96.4% of possible variants in this region.
• Integrated functional data with existing clinical variant databases (ClinVar), orthogonal assays, and computational predictors.

Main Results:

• Assigned functional classifications to 6,551 SNVs, including 1,282 missense VUS from ClinVar, reclassifying 77.2% as benign and 20.4% as pathogenic.
• Identified 3,384 benign and 776 pathogenic SNVs within the studied region.
• Achieved high concordance with existing pathogenicity data and computational predictions.

Conclusions:

• The developed functional assay and resulting sequence-function map provide a robust resource for interpreting unidentified BRCA2 variants.
• This classification system aids physicians and genetic counselors in assessing BRCA2 VUS in patients, improving diagnostic accuracy and patient care.