Figure 2. Mutation A485P reduces expression and activity of the TOP2B protein and impairs early B cell development.

(A) Western blots showing expression of the TOP2B protein in primary dermal fibroblasts, induced pluripotent stem cells (iPSCs) dedifferentiated from dermal fibroblasts and T cell blasts derived from PBMC. Patients: A is III.1 in family A; B is IV.1 in family B. Fold change of band densitometry is shown. (B) Relaxation of negatively-supercoiled DNA by the purified recombinant TOP2B^WT and TOP2B^A485P proteins. (C) Decatenation of kinetoplast DNA by the purified recombinant TOP2B^WT and TOP2B^A485P proteins. (D) Decatenation of kinetoplast DNA by nuclear extracts from T cell blasts. (E, F) Decatenation of kinetoplast DNA by nuclear extracts from wild-type HEK-293 cells (E) or TOP2B-knockout HEK-293 cell (F), untransfected or transfected with plasmids encoding TOP2B^WT and TOP2B^A485P. P-values were calculated using two-tailed (E) and one-tailed (F) paired t-tests. Graphs show averages ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. (G) Bone marrow immunophenotyping of the BILU patient and a healthy unrelated control showing pro-B (i), pre-B (ii), immature B (iii) and mature B (iv) cells. (H) B cell development stages; red X shows defect in the BILU patients. HSC - hematopoietic stem cell, CLP - common lymphoid progenitor.