Spontaneous CH mutations in aged mice. (A) Frequency of aged (24 months) mice (n = 97) with CH mutations detected by ECTS in BM, or a mean of 10 months following transplantation into 2 lethally irradiated recipients (n = 48 donors). Mutations were only included if found to have an origin in the transplanted (CD45.2) rather than recipient (CD45.1) BM cells. ****P < .0001 Fisher’s exact test. (B-C) Distribution and characteristics of mutations detected by ECTS in BM of aged mice. Each circle in panel C represents 1 detected mutation, and the color indicates the type of mutation as indicated by the legend in panel B. (D-H) % VAF, as determined by ddPCR, of Tet2 and Asxl1 mutations detected by ECTS in aged steady-state BM (D), 2 Trp53 mutations, and 1 Asxl1 mutation in CD45.2 BM cells from 3 different donors in both recipients posttransplantation but not pretransplantation (E), Tet2 and Dnmt3a mutations with high VAF in 1 recipient and borderline VAF in both original aged donor and second recipient (F), mutations in CD45.2 BM from 1 recipient posttransplantation but not pretransplantation (G), and mutations detected only in 1 recipient posttransplant where a matched second recipient was missing (H). CD45.2 BM MNCs cells were sorted from the transplanted recipient mice. Error bars indicate 95% confidence interval, and each bar from posttransplanted mice indicates the individual recipients. #, not detected. ‡ indicates cases where ddPCR did not generate sufficient events to support confident detection (supplemental Methods).