Early clonal dominance, linear accumulation of mutations, and clonal selection during myeloid differentiation. (A) Clones from sorted BM HSC (CD34⁺/CD38⁻/CD90⁺, noted with an asterisk) or peripheral blood immature cells (CD34⁺/CD38⁻) of 28 patients (detailed in Table 1) were cultured at 1 per well in liquid medium for 12 days in the presence of cytokines, then collected, and assessed by allele discrimination analysis of each individual mutation initially identified in peripheral blood CD14⁺ cells. Proportion of clones with black: wild type, yellow: 1 mutation, orange: 2 mutations, red: 3 mutations, purple: 4 mutations. Numbers on the top of the bars indicate the number of clones analyzed. (B) Examples of Sanger sequences in CD14⁺ and CD3⁺ sorted cells for 2 mutations in a representative sample (UPN #524). (C) Putative evolutionary trees of sorted BM CD34⁺ populations generated by genetic classification of individual cells. Simple linear trees from 2 samples (top: UPN #531: TET2 Q810×; SRSF2 P95H; JAK2 V617F; bottom: UPN #632: TET2 N1156I; SRSF2 P95L; NRAS A59G) with all mutations heterozygous in all clones (white: no mutation, yellow: 1 mutation, orange: 2 mutations, red: 3 mutations). (D) Repartition of 193 CD34⁺/CD38⁻ clones showing a unique somatic mosaicism in patient UPN #752 harboring 2 independent subclones with NRAS G12D and KRAS G12A genotypes. (E) Proportion of mutated clones in HSC or MPP, CMP, and GMP fractions from 14 patients (10 with 2 or more mutations, 4 with 1 mutation); ns, nonsignificant, *P < .05 (HSC or MPP vs GMP, Wilcoxon matched-pair signed-rank test for 11 and 8 pairs, respectively). Bar: median percentage.