Evidence-based recommendations for gene-specific ACMG/AMP variant classification from the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel

Abstract

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants.

Keywords: ACMG/AMP variant curation guidelines; BRCA1; BRCA2; ClinGen; ClinVar; VCEP.

Graphical abstract

Figure 1

Timeline for migration from “external” expert panel to operation as a ClinGen Variant Curation Expert Panel following the ClinGen FDA-recognized protocol Discussions with key members of the SVI WG during 2019 relating to the strategy for alignment included the need to capture information sources used previously for BRCA1 and BRCA2 variant interpretation that are not explicitly designated under the baseline ACMG/AMP guidelines, especially information used in the context of multifactorial likelihood modeling. Revision of specifications at step 3 included updates in response to ClinGen SVI Splicing subgroup recommendations and re-calibration of bioinformatic prediction of missense impact in response to ClinGen SVI Computational subgroup recommendations. Details of ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel membership and biocurator workforce are available at https://clinicalgenome.org/affiliation/50087/. Pre-existing ENIGMA expert panel classification guidelines for BRCA1 and BRCA2 are shown in Data S1 as a point of reference (though no longer currently used). The final specifications first approved for internal ClinGen BRCA1/2 VCEP use are available via https://cspec.genome.network/cspec/ui/svi/affiliation/50087 and have been duplicated here as Data S2.

Figure 2

Overview of evidence supporting PM5 exon-specific weights application for premature termination codon variants in BRCA1 and BRCA2 Exon-specific points assigned were derived from per-exon evidence for BRCA1 (A) and BRCA2 (B), as detailed in Note S2. Information used to infer evidence and apply points were as follows: observed experimental impact on function for at least one premature termination codon (PTC) variant in an exon (Fxn PTC, assigned 4 points); observed experimental impact on function for at least one missense substitution variant (Fxn Missense, assigned 2 points); case-control odds ratio (OR) ≥4.0 estimated for PTC variants observed in a given exon, assigned at full strength for a statistically significant association (4 points), and at supporting strength for non-significant estimates (1 point); family history LR estimates from heterogeneity analysis (fam history LR, assigned points based on LR); standardized incidence ratio (SIR) ≥4.0 for PTC variants identified in non-Finnish European (NFE) probands with breast, ovarian, and/or pancreatic cancer compared to gnomAD NFE individuals (Ambry SIR/OR; SIR ≥4, p < 0.05 assigned 4 points); supporting strength for SIR ≥4 non-significant (1 point); and observation of ≥5 unique PTCs variant in ≥5 families from the CIMBA (https://cimba.ccge.medschl.cam.ac.uk/) highly ascertained cohort of individuals with a BRCA1 or BRCA2 pathogenic variant (CIMBA, assigned 1 point). The per-exon evidence was summed across the different evidence types (Fxn PTC, Fxn Missense, CaCo OR ≥4, fam history LR, Ambry SIR/OR, CIMBA), to derive an exon-specific evidence strength using a points-based approach (supporting = 1 point, moderate = 2 points, strong = 4 points). Based on the combined evidence, the PM5 (PTC) code can be applied as strong evidence in favor of pathogenicity for most exons. The PM5 (PTC) code can only be applied to germline variants that meet PVS1 codes, namely nonsense and frameshift changes, including large deletions and tandem duplications. Code weight is determined by the exon in which the predicted termination codon occurs. For example, a frameshift variant in BRCA2 exon 15 that is predicted to result in a PTC within exon 16 would use the code strength of BRCA2 exon 16 (PM5_Strong [PTC]). Variants in the AG-GT splice site positions, and variants with experimental splicing data demonstrating introduction of a PTC, do not qualify for PM5 (PTC) code. The points assigned are converted to a code weight using the points system proposed by Tavtigian et al., which is that 1 point assigned = PM5_Supporting (PTC), 2 points assigned = PM5 (PTC), and 4+ points assigned = PM5_Strong (PTC). Note that the maximum weight applied to PM5 (PTC) is strong, meaning all exons with ≥4 points are assigned PM5_Strong (PTC). The PM5 (PTC) code is considered not applicable for exons with <1 point.

Figure 3

Overview of variant classifications assigned during pilot of VCEP specifications Sankey diagram shows transition in classification categories for pilot variants from initial ClinVar classification to final classification assigned by the VCEP. The left column represents the initial ClinVar classification category/grouping of pilot variants, namely pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), conflicting (conflict = conflicting), likely benign (LB), and benign (B). The central column represents classification(s) by biocurators after the initial pilot phase. The right column represents the final classifications of pilot variants. For each category/grouping, the number of variants is shown in parentheses. Final VCEP curation resulted in increased certainty in classification for all variants with initial LP/P or B/LB category, movement of three of four variants with initial VUS classification outside of this category, and resolution in classification for the nine variants with initial conflicting classification—eight reaching classification other than VUS.