Single-cell profiling of ANKRD26 thrombocytopenia reveals progenitor expansion and polyploid apoptosis via JUNB-p21.

ANKRD26-related thrombocytopenia 2 (THC2) is a rare inherited platelet disorder caused by germ line variants in the 5' untranslated region (UTR) of ANKRD26. Although prior studies using in vitro models or isolated case reports have suggested impaired megakaryopoiesis as a central mechanism, detailed insights have remained elusive, primarily due to the rarity, fragility, and heterogeneity of megakaryocytes (MKs). Here, we present a comprehensive, crossvalidated analysis of bone marrow samples from 4 independent patients with THC2, integrating single-cell transcriptomics and ex vivo functional profiling. Across all patients, we analyzed CD34+ hematopoietic stem and progenitor cells (HSPCs; 47 281 THC2 HSPCs vs 51 907 control cells) and primary MKs (pMKs; 7309 THC2 pMKs vs 5077 control cells), uncovering a consistent pattern of MK progenitors (MkP) expansion and a marked reduction in polyploid MKs, indicating a conserved pathophysiologic phenotype. In our index patient, we identified the 5' UTR single-nucleotide variant in ANKRD26 that led to significantly elevated expression across 4 MK lineage subsets, spanning multipotent progenitors, common myeloid progenitors, MK-erythroid progenitors, and MkP as well as in terminally enriched pMKs. Confocal imaging localized ANKRD26 to the centrosome, implicating it in mitotic regulation during MK maturation. Mechanistically, we discovered that elevated ANKRD26 induces apoptosis in polyploid MKs via JUNB-mediated transcriptional activation of CDKN1A (p21), operating independently of the canonical p53-PIDDosome axis. This multipatient study provided the most comprehensive cellular and molecular portrait of ANKRD26-driven thrombocytopenia to date, offering novel insights into defective megakaryopoiesis and identifying candidate therapeutic targets to restore platelet production.