Phenotypic characteristics of monoallelic THPO variants and expression in HEK293T cells. (A) Sex-stratified graphs of PLT and mean platelet volume obtained using a Sysmex XN-Series hematology analyzer from 48 345 blood donors from the INTERVAL study.¹⁷  The arrows indicate the platelet parameters of the cases harboring THPO variants. (B) Representative May-Grünwald-Giemsa–stained peripheral blood films from THPO variant cases confirming thrombocytopenia and indicating that some platelets (arrows) were enlarged. Scale bars, 5 μM. (C) Serum TPO concentration plotted against platelet counts in healthy controls or hemato-oncology patients with thrombocytopenia caused by bone marrow suppression after chemotherapy. Corresponding data from 4 cases with THPO variants (A I.1 and II.2, B I.2, C II.1) are also shown. The solid and dotted lines represent the line of best-fit and 95% confidence intervals, respectively, from a nonlinear regression of control data. (D) Effects of THPO variants in HEK293T cells: Flag-tagged WT, E204Gfs*123, L269P*58, or R99W substituted TPO was expressed in HEK293T cells by transient transfection. Secretion of FLAG-tagged TPO in cell supernatant (D) and lysate (E) was measured using an ELISA and normalized against total protein in the cell lysate. Results are mean ± standard error from ≥5 independent experiments. The P values were calculated using 1-way analysis of variance. (F) Representative images from HEK293T cells transfected with WT and variant THPO cDNAs and stained with an antibody against the FLAG epitope (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Images were captured using a Leica SP5 II confocal microscope with a 63×/1.4 NA lens and analyzed with ImageJ/Fiji. All images are shown with the same intensity range. (G) Quantification of FLAG-tagged protein by mean fluorescence intensity in representative cells (n = 5).