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 understanding the impact of dysregulated splicing in hematopoiesis remains challenging. To overcome technical limitations, we integrated Genotyping of Transcriptomes (GoT) with long-read single-cell transcriptome profiling and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to bone marrow progenitors from myelodysplastic syndrome (MDS) patients with mutations int 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 across progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid lineage bias and cell-type specific cryptic 3’ splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines cell-type specific impacts of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.EGA study EGAS00001007402