RNA-seq analyses in HSPCs from patients with TBD and somatic U2AF1^S34 mutations. (A) UMAP plots of 5’ scRNA-seq analysis in CD34⁺ HSPCs isolated from NIH-39 harboring a germ line TERC r.116C>T mutation and a somatic U2AF1^S34 mutation (n = 3912 cells). Each dot represents 1 cell. Different colors indicate cluster identity (left) and the U2AF1^S34 mutational status (center). Pathway enrichment analysis of genes significantly downregulated in U2AF1^S34 mutant HSPCs compared with U2AF1^wild-type HSPCs (P adj ≤ .05). Hallmark gene sets are shown (right). Dotted lines indicate the annotated lineage clusters. (B) UMAP plots of the 3’ scRNA-seq analysis in CD34⁺ HSPCs isolated from an asymptomatic patient with TBD with a pathogenic TERT p.P704S germ line mutation (MDA-3) transduced with lentiviral vectors carrying the wild-type or mutant U2AF1 allele after in vitro culture for 3 days (n = 13 674 cells). Each dot represents 1 cell. Different colors indicate cluster identity (left) and the U2AF1 mutational status (middle). Pathway enrichment analysis of genes significantly downregulated in U2AF1^S34-transduced HSCs compared with those in U2AF1^{wild type}-transduced HSCs (P ≤.01). Reactome gene sets are shown (right). Dotted lines indicate the annotated lineage clusters. (C) Pathway enrichment analysis of genes significantly downregulated in U2AF1^S34-transduced CD34⁺ cells compared with those in U2AF1^{wild type} -transduced CD34⁺ cells from another asymptomatic patient with TBD with short telomeres harboring the TERT p.R774X pathogenic mutation (MDA-19). Cells were transduced with lentiviral vectors carrying the mutant or wild-type U2AF1 allele, and after in vitro culture for 3 days, bulk RNA-seq analysis was performed (P adj ≤ .001; fold change <1). Hallmark gene sets are shown. (D) Pathway enrichment analysis from bulk RNA-seq showing the genes that underwent aberrant exon skipping (top) and intron retention (bottom) in U2AF1^S34 HSPCs compared with U2AF1^{wild type} HSPCs from panel C (P adj ≤ .05). Reactome gene sets are shown. cDCs, classic dendritic cells; DCs, dendritic cells; Ery, erythroid; GMP, granulocytic-monocytic progenitors; LMPP, lymphoid-myeloid primed progenitors; Mk, megakaryocytic; Mono, monocytic; NK, natural killer cells; pDC, plasmacytoid dendritic cells; UMAP, uniform manifold approximation and projection.