Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: a , Design of the SGE experiment and the targeted regions. All possible SNVs were introduced and assessed in exon 15 (E15) to E26 encoding the BRCA2 DBD domain, along with 10 bp of adjacent intronic nucleotides for each exon. E18 and E25 were divided into 2 regions, which resulted in a total of 14 target regions. b , Schematic of the SGE workflow. In each target region, a SNV library that contained all possible SNVs was transfected with a corresponding Cas9–sgRNA construct into HAP1 haploid cells. gDNA was extracted at D5 and D14 after transfection, and the target region was amplified and barcoded for targeted gDNA sequencing. SNV abundance was evaluated and normalized to generate functional scores for all SNVs. An ACMG–AMP classification model was applied to formally classify SNVs based on the results of the MAVE functional assays and other evidence. The schematics in this figure were created using BioRender (credit: C.H., https://BioRender.com/u10b291 ; 2024).

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Transfection, Construct, Amplification, Sequencing, Functional Assay

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: Validation of the BRCA2 functional assay

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Biomarker Discovery, Functional Assay

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: A , Heatmap of functional categories (colour) for all possible amino acid substitutions encoded by SNVs. B , Cross-species sequence conservation from pufferfish to H omo sapiens (n = 10) relative to frequency of P_Strong missense variants (perfectly conserved: 100% identity across 10 species; highly conserved: 80% or 90% identity; poorly conserved: ≤70% identity). C – G , BRCA2 3-dimensional protein ribbon diagrams showing the frequency of P_Strong missense variants encoded by SNVs at each amino acid in the Helical ( C ), OB1 ( D ), OB2 ( E ), OB3 ( F ) domains, and the BRCA2-DSS1-ssDNA complex (PBD 1MJE) ( G ). Colour denotes the frequency of P_Strong missense alterations (green: 0%; yellow: <25%; orange: 25-49.9%; red: ≥50%). The subdomains were oriented to maximize views of the functionally pathogenic missense alterations. The BRCA2-DSS1-ssDNA complex is shown from N-terminus (left) to C-terminus (right).

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Functional Assay, Sequencing

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: a – d , Boxplots showing functional scores of SNVs encoding missense variants compared with a BRCA2 –/– V-C8 HDR assay ( a ), a DLD1 BRCA2 –/– olaparib sensitivity assay ( b ), a prime-editing-based haploid cell-survival assay ( c ) and a mouse Brca2 –/– embryonic stem cell complementation assay ( d ). The numbers of variants of each type resulting from the individual assays are shown. Functionally abnormal variants have significantly lower functional scores than functionally normal variants in a ( P = 1.6 × 10 −52 ), b ( P = 2.0 × 10 −11 ), c ( P = 3.4 × 10 −8 ) and d ( P = 1.1 × 10 −4 ), using two-sided Mann–Whitney–Wilcoxon tests. P values for all comparisons are shown. Boxes represent the interquartile range, the horizontal line is the median functional score, and whiskers show maximum and minimum values. Variants are shown as points and coloured by the functional strength of the evidence category. e , Comparison of the AUC values between MAVE and two in silico predictors (AlphaMissense and BayesDel) using ClinVar-classified missense standards ( n = 70). f , Comparison of the AUC values between MAVE and two in silico predictors (AlphaMissense and BayesDel) using missense variants characterized using a well-calibrated HDR assay ( n = 417).

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Functional Assay, Sensitive Assay, Clonogenic Cell Survival Assay, MANN-WHITNEY, Comparison, In Silico

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: A , B , Lifetime risk estimates for breast cancer ( A ) and ovarian cancer ( B ) associated with categories (P_Strong, P_Strong/Moderate/Supporting, B_Strong, B_Strong/Moderate/Supporting) of BRCA2 DNA binding domain SNVs from the BRCA2 MAVE study. Standards included known pathogenic (all protein truncating alterations), known benign (benign variants established by the ClinGen BRCA1/2 VCEP), general population breast/ovarian (age related risks of these cancers from the general SEER registry).

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Binding Assay

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: a , Sankey plot illustrating the clinical classification of SNVs after integration of BRCA2 MAVE functional data into the ClinGen BRCA1/2 VCEP ACMG–AMP classification framework. The numbers of SNVs for functional categories in each variant type are shown in the left-hand MAVE column. The numbers of variants in the classification category are shown in the right-hand ACMG column. b , Sankey plot illustrating the changes in variant classification status in ClinVar before (left) and after (right) incorporating BRCA2 MAVE functional results into the BRCA1/2 VCEP ACMG–AMP classification framework.

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Functional Assay, Variant Assay

Journal: Nature

Article Title: Functional evaluation and clinical classification of BRCA2 variants

doi: 10.1038/s41586-024-08388-8

Figure Lengend Snippet: Associations between variants in the BRCA2 DBD and risk of breast cancer and ovarian cancer

Article Snippet: Recently, guidelines from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) that incorporate multiple sources of evidence, including variant frequency in populations, in silico-sequence-based prediction and functional data, among others, have been utilized by clinical testing groups and the ClinGen BRCA1 and BRCA2 ( BRCA1/2 ) variant curation expert panel (VCEP) for the classification of variants .

Techniques: Control