Assessment of the Clinical Relevance of BRCA2 Missense Variants by Functional and Computational Approaches  Lucia Guidugli, Hermela Shimelis, David L. Masica, Vernon S. Pankratz, Gary B. Lipton, Namit Singh, Chunling Hu, Alvaro N.A. Monteiro, Noralane M. Lindor, David E. Goldgar, Rachel Karchin, Edwin S. Iversen, Fergus J. Couch  The American Journal of Human Genetics  Volume 102, Issue 2, Pages 233-248 (February 2018) DOI: 10.1016/j.ajhg.2017.12.013 Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 1 Influence of BRCA2-DBD Missense Variants on HDR Activity in Relation to Known Pathogenic and Neutral Variants Defined as Class 1, 4, or 5 in a Multifactorial Likelihood Model The model-based HDR fold change with SE is displayed on a logarithmic scale for the class 1, unclassified, class 4, and class 5 variants. Solid lines represent 99% probability of pathogenicity and 99% probability of neutrality (fold increase in GFP(+) cells < 1.66 for deleterious variants and > 2.41 for neutral variants). Dotted lines represent 95% probability of pathogenicity and 95% probability of neutrality (fold increase in GFP(+) cells < 1.78 for deleterious variants and > 2.25 for neutral variants). The American Journal of Human Genetics 2018 102, 233-248DOI: (10.1016/j.ajhg.2017.12.013) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 2 Comparison of HDR Activity and Align-GVGD Prediction for 139 BRCA2 Variants (A) HDR normalized fold changes for 139 BRCA2-DBD missense changes are displayed for each of the seven Align-GVGD classification grades (C0–C65). (B and C) 73 BRCA2-DBD missense changes with >95% probability in favor of neutrality (B) and 58 BRCA2-DBD missense changes with >95% probability in favor of pathogenicity (C) are displayed for each of the seven Align-GVGD classification grades (C0–C65). The American Journal of Human Genetics 2018 102, 233-248DOI: (10.1016/j.ajhg.2017.12.013) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 3 Plot of VarCall-Estimated Variant Effects Shows Individual Variants Plotted against HDR Activity Variants are shown as the natural log of the normalized fold change in HDR activity. Error bars represent SE within individual measurements for each variant. The probability of pathogenicity (Pr(eta\data)) based on the lower pathogenic component and upper neutral component is plotted in the right-hand margin. The wild-type (WT), p.Tyr3098His positive controls, p.Asp2723His negative control, known pathogenic variants, known neutral variants, and VUS are color coded as indicated. Variants in green (IARC < 3) are classes 1 and 2. Variants shown in pink (IARC > 3) are classes 4 and 5. The American Journal of Human Genetics 2018 102, 233-248DOI: (10.1016/j.ajhg.2017.12.013) Copyright © 2017 American Society of Human Genetics Terms and Conditions

Figure 4 ROC Curves for the Continuous-Valued ePOSE Score and Variants Defined as Pathogenic or Neutral For each of five variant groupings, P is the upper-bound HDR fold-change threshold for pathogenicity, and N is the lower-bound threshold for defining a variant as neutral. The color-coded inset shows the AUC at each of the five groupings. The American Journal of Human Genetics 2018 102, 233-248DOI: (10.1016/j.ajhg.2017.12.013) Copyright © 2017 American Society of Human Genetics Terms and Conditions