Figure 2.

Peripheral Blood Phenotype of Probands with VPS4A Mutations

(A) Flow cytometry analysis of probands’ peripheral RBCs (CD45⁻) showing presence of an unusual population positive for transferrin receptor (CD71⁺) and negative for RNA, as labeled by thiazole orange (TO).

(B) Quantification of immature RBC populations analyzed by flow cytometry and IFC (CD71⁺/TO⁺, CD71⁻/TO⁺, and CD71⁺/TO⁻) as a percentage of total CD71⁺ and/or TO⁺ cells (excluding all mature CD71⁻/TO⁻ RBCs since probands 1 and 2 are regularly transfused). Samples from individuals with sickle cell disease (SCD) were used as positive controls with increased reticulocyte counts. Data shown are mean ± SEM of n = 3 experiments for all but p2 (n = 2). ^∗p < 0.05 and ^∗∗p < 0.01 by a Student’s t test for samples with unequal variance.

(C) Relative expression of VPS4A and VPS4B in CD71⁺ reticulocytes isolated from whole blood and evaluated by qPCR. Data are mean ± SEM, n = 4 control; n = 3 p1&2 VPS4A, p2&3 VPS4B; n = 2 p1 VPS4B, p3 VPS4A, ^∗∗p < 0.01 by a Student’s t test for samples with unequal variance.