Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3′ splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3′ splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples. [Display omitted] •GoT-Splice profiles single-cell genotype, expression, surface markers, and splicing•SF3B1mut cells show lineage skewing and stage-specific mis-splicing in MDS•SF3B1mut-specific isoform Bax-ω may provide MDS anti-apoptotic advantage•In CH, SF3B1mut cells display erythroid lineage bias and mirror MDS mis-splicing Cortés-López and colleagues develop GoT-Splice for the concurrent profiling of gene expression, surface proteins, somatic mutations, and RNA splicing in individual cells. By utilizing this method, they investigate the effects of SF3B1 mutations in patients with myelodysplastic syndrome and clonal hematopoiesis, unveiling splicing abnormalities that lead to lineage-specific clonal expansions.