Single-cell analysis reveals the KIT D816V mutation in hematopoietic stem and progenitor cells in systemic mastocytosis: Supplementary data

Abstract

32Background: Systemic mastocytosis (SM) is a hematological disease characterized 33 by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation 34 in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution 35 and autoactivation of the receptor. Mast cells and CD34 + hematopoietic progenitors 36 can carry the mutation, however, in which progenitor cell subset the mutation arises is 37 unknown. We aimed to investigate the distribution of the D816V mutation in single 38 mast cells and single hematopoietic stem and progenitor cells. 39Methods: Fluorescence-activated single-cell index sorting and D816V mutation 40 assessment were applied to analyze mast cells and more than 10,000 CD34 + bone 41 marrow progenitors across 10 hematopoietic progenitor subsets. In vitro assays 42 verified cell-forming potential. 43Findings: We found that in SM 60-99% of the mast cells harbored the D816V 44 mutation. Despite increased frequencies of mast cells in SM patients compared with 45 control subjects, the hematopoietic progenitor subset frequencies were comparable. 46Nevertheless, the mutation could be detected throughout the hematopoietic landscape 47 of SM patients, from hematopoietic stem cells to more lineage-primed progenitors. In 48 addition, we demonstrate that FcεRI + bone marrow progenitors exhibit mast cell-49 forming potential, and we describe aberrant CD45RA expression on SM mast cells for 50 the first time. 51Interpretation: The KIT D816V mutation arises in early hematopoietic stem and 52 progenitor cells and the mutation frequency is approaching 100% in mature mast 53 cells, which express the aberrant marker CD45RA. 54 55