Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation

Abstract

Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.

Integrated Single-Cell Genotyping and Chromatin Accessibility ChartsJAK2V617FHuman Hematopoietic Differentiation

“…To circumvent these limitations, single cell simultaneous capture of genotypes together with phenotypic information is required, enabling intra-sample, cell type-specific mutant to wildtype comparisons. Application of such single cell multi-omic approaches have shown that somatic mutations in human tissues exert a differing phenotypic effect as a function of cell state 5,6,24,25,28–33 .…”

“…Somatic driver mutations within HSPCs result in clonal expansions and the reshaping of differentiation landscapes. Altered HSPC differentiation landscapes range from subtle differentiation biases in clonal hematopoiesis 5,6 , through skewed accumulation of mature blood cells in myeloproliferative neoplasia (MPN) 7,8 , to impaired differentiation in leukemogenic transformation 9,10 . The recurrent V617F hotspot mutation in the Janus Kinase 2 (JAK2) gene serves as a prototypical example, associated both with clonal hematopoiesis [11][12][13][14] as well as overt differentiation skews in polycythemia vera (PV) and essential thrombocythemia (ET), and ultimately leading to bone marrow failure in myelofibrosis (MF) [15][16][17][18] .…”

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

“…Genotyping of single cells was carried out with the IronThrone (v.2.1) pipeline as previously described 15,110 . In brief, individual amplicon reads were assessed for the appropriate structure ( i .…”

“…The frequency of mutant cells, as determined by GoT, was assessed as previously performed in Nam et al 110 . Firstly, we used only cells with at least 1 UMI and only considered cell types with at least 300 genotyped cells.…”

“…Genotyping of single cells was carried out with the IronThrone (v.2.1) pipeline as previously described 15,110 . In brief, individual amplicon reads were assessed for the appropriate structure (i.e., presence of the primer sequence and the expected sequence between the primer and given mutation site) and all reads were assessed for a matching cell barcode to the list generated from the 10x paired GEX dataset.…”