Functional characterization of germline ETV6 variants identified in pediatric ALL. (A) ETV6 transcription repressor activity was determined using a dual-luciferase reporter assay by cotransfecting an ETV6 expression construct, PF4 luciferase reporter construct, and Renilla luciferase reporter construct in HEK293T cells. Bars show the mean of triplicate experiments and repeated at least 3 times; error bars represent standard deviation (SD). (B) Electrophoretic mobility shift assays were performed using nuclear extracts from HEK293T cells ectopically expressing WT or variant ETV6. Bars show the mean of triplicate experiments; error bars represent SD. (C) Western blotting was performed after subcellular fractionation of HEK293T cells ectopically expressing WT or variant ETV6. Bars show the mean of nuclear to cytoplasmic ratio from 3 individual experiments; error bars represent SD. In panels A-C, a 1-way analysis of variance (ANOVA) with multiple comparison was performed to compare each variant with WT (*P < .01). (D) Germline ETV6 variants were classified as damaging or WT-like on the basis of functional characterization as shown in panels A-C.