Validation of transactivation assays and characterization of RUNX1 variants of interest to support variant classification. Variant nomenclature refers to RUNX1 transcript variant 2 (NM_001001890.3) encoding for isoform RUNX1b. (A) Schematic overview of the investigated set of RUNX1b nonsense variants. Protein domains refer to Lam and Zhang.²¹  The bar graphs displays the firefly/renilla ratios relative to wild-type (WT) RUNX1b of applied reporter constructs rCSF1R, rETV1, and rMYL9 analyzing the set of RUNX1b nonsense variants in HEK293T cells (mean + standard deviation [SD]; 3 biological with 3 technical replicates; 1-way ANOVA in comparison with WT; Dunnett post hoc test; *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001). The threshold 115% and 20% of WT activity is highlighted regarding the MM-VCEP recommendations defining these thresholds for the application of BS3 and PS3, respectively.⁸  Differing results of variants for different reporters might be explained by effects of different coactivators/corepressors required. (B) Schematic overview of the analyzed set of RUNX1b variants of interest and used controls. Representative bar graphs displaying firefly/renilla ratios relative to WT RUNX1b of reporter constructs rETV1 in HEK293T cells and rMYL9 in HEL cells analyzing the set of RUNX1b variants of interest. Besides the potential impact of different coactivators/corepressors, differing results for individual reporters might be explained by effects of the cellular context (mean + SD; HEK293T: 3 biological with 6 technical replicates; HEL: 2 biological with 5 technical replicates; 1-way ANOVA in comparison with WT; Dunnett post hoc test; *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001). (C) At the top is a heat map summarizing analyses of RUNX1 variants scored relative to WT RUNX1b activity. Purple results were not included in the classification because of the detection limit of rCSF1R. Below the heat map, resulting PS3/BS3 criterion based on TA results is shown as functional class and concluded as follows: (1) PS3 moderate, ≥2 TA results with <20% of WT activity without conflicting results (ie, ≥80% of WT activity); (2) PS3 supporting, ≥2 TA results >115% of WT; and (3) BS3 supporting, ≥2 TA results with >80% to 115% of WT without conflicting results (ie, ≥1 with <20% or ≥2 with >115% of WT activity). Subsequently, we included already published results from secondary assays (eg, dimerization and DNA binding, for detailed references see supplemental Table 3) to adapt the strength of the criterion according to the MM-VCEP recommendations.⁸  Our data provide evidence for the applicability of PS3 supporting for His58Asn, but other studies demonstrate functionality of this variant.²²  No functional class was assigned (ie, variants of uncertain function) according to Brnich et al.¹¹  At the bottom, available ClinVar classifications (not following MM-VCEP guidelines, except for the likely benign classification of His58Asn), classification based on American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) and MM-VCEP guidelines without (w/o), and with (w/) applied functional criterion is shown. ANOVA, analysis of variance; aa, amino acid; TAD, transactivation domain.