Evidence that SF3B1 mutations are initiating mutations targeting rare HSCs in MDS-RS. (A) Computational prediction of fraction of cells with identified genomic lesions within total BM MNCs from SF3B1-mutated MDS-RS patients based on VAF. Error bars indicate 95% confidence intervals. Shown are cases where SF3B1 is the only identified recurrent mutation, SF3B1 is predicted to be the first of multiple recurrent mutations, or SF3B1 is predicted to be secondary to other mutations. Inconclusive results (overlapping 95% confidence intervals) are included in supplemental Figure 2. (B) Tracking of SF3B1 mutations in individually picked HSC-derived LTC-CFCs or MEP- and GMP-derived CFCs. 7 SF3B1 mutated and 1 SF3B1 wild-type (WT) patients were analyzed using pyrosequencing to screen for identified SF3B1 mutations in each case and scored as positive (gray), negative (white), or inconclusive (I) (see supplemental Methods for definitions and cutoffs). (C) Sequenom analysis from 4 MDS-RS samples of FACS-purified stem and progenitor cell populations to assess SF3B1 VAF. PAT, patient.