RNASeq of DNMT3A mutant K562

Donor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (> 100 life-time donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FD) compared to sporadic donors (< 10 life-time donations, 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and control donor (CD) cohorts. Functional analysis of FD enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive outgrowth potential upon stimulation with erythropoietin (EPO). In contrast, clones harboring leukemogenic DNMT3A R882 mutations exhibited a stronger response to inflammatory cues rather than EPO. Through concurrent mutational and immunophenotypic profiling of primary samples at single cell resolution as well as experiments in humanized mice, a myeloid bias of R882 mutant HSCs was detected. Remarkably, no significant lineage bias was observed in HSCs harboring EPO responsive DNMT3A variants. Our data demonstrate a nuanced ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain non-malignant DNMT3A mutations. Crucially, our findings robustly demonstrate that frequent blood donation over many years does not promote the selection of malignant clones, thus strongly reaffirming its safety. Overall design: Using CRISPR-mediated genetic engineering of K562 we generated K562 clones harbouring DNMT3A W305*, R882H or R882C mutations and compare their trnascriptome with wild type K562 cells.