Effect of R174Q and R139X RUNX1 mutations on HSC and progenitor capacities. (A-C) CD34⁺CD38⁻ peripheral blood cells from FPD/AML patients with R174Q (AII-1, AII-2) or R139X (BII-2, BII-3, BIII-1) RUNX1 mutation, and from healthy individuals (C) were analyzed. (A) Assessment of colony forming cells (CFC). CD34⁺CD38⁻ cells were plated in methylcellulose. The number of colonies (BFU-E, CFU-GM, and CFU-GEMM) was expressed per 1 × 10³ of plated CD34⁺ cells, pedigree A (top), and pedigree B (bottom). (B) Assessment of B/NK/myeloid potentialities. Analysis of the progeny of single CD34⁺CD38⁻ cells. CD34⁺CD38⁻ cells were plated at one cell per well in 96-well plates on MS-5 cells in presence of IL-3, SCF, FLT3-L, TPO, IL-7, IL-15, and IL-2. The experiment was performed once for each patient or control sample. Histograms indicate the number of positive wells in 200 plated wells containing 1 (B, NK, M), 2 (B/M, B/NK, M/NK), or 3 (B/NK/M) lineages. Myeloid cells correspond to CD15⁺ cells, B cells to CD19⁺ cells, and NK cells to CD56⁺ cells. *P < .02 (calculated as described in “Statistical analyses”). (C) Assessment of LTC-IC frequency. Analysis of the progeny of single CD34⁺CD38⁻ cells. (C, Mean ± SD). Histograms: number of positive wells in 200 plated wells. *P < .02 (calculated as described in “Statistical analyses”). (D) FPD/AML CD34⁺ cell ability to grow in liquid medium. CD34⁺CD38⁻ peripheral blood cells from AII-1, AII-2 (R174Q mutation) patients were grown in liquid medium containing cytokine cocktail. May-Grünwald-Giemsa (top panels) and myeloperoxidase (bottom panels) staining were performed at 10 weeks of culture (i). Flow cytometric analysis was performed at 4 months of culture (ii).