High-Throughput and Highly Sensitive Single Cell Genotyping with Simultaneous Chromatin Accessibility Measurement in Myeloid Precursor Conditions
Authors: Motomura Masanori , Ochi Yotaro , Nakagawa Masahiro M , Nannya Yasuhito , Kakiuchi Nobuyuki , Kuroda Yutaka , Sezaki Nobuo , Nakagawa Tomoe , Zhao Lanying , Miyano Satoru , Matsuda Shuichi , Ogawa Seishi 
Published: December 2024

Age-related clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) frequently harbor mutations in genes encoding epigenetic regulators contributing to epigenetic dysregulation. However, it is not fully understood how CH/CCUS clones are positively selected and undergo phenotypic changes through these epigenetic alterations. In this regard, the analysis of the impact of these mutations on epigenesis at a single cell resolution would help understand the mechanism of clonal selection of CH and CCUS. Unfortunately, however, because of the frequent allele dropout in detecting mutations, few studies have addressed this with sufficient sensitivity to date. In the current study, we have developed a droplet-based simultaneous single cell gene mutations and DNA accessibility (ATAC-seq) profiling platform utilizing the Mission Bio Tapestri, which has achieved high sensitivity and accuracy in single cell genotyping and providing novel insight into the role of epigenetic mutations on chromatin accessibility.

First, we evaluated the performance of our platform through the analysis of a sample in which two cell lines, HL-60 and SKM-1, having a distinct variant in RUNX1 (heterozygous) and BCOR (hemizygous), respectively, were intentionally mixed. A total of 3,579 cells were clustered into two groups based on DNA accessibility data alone and annotated for identities based on gene accessibility profiles. We next assessed genotyping accuracy for the BCOR and RUNX1 variant. Our platform resulted in detection of only 3.7 % of doublets and genotype assignment of 74 % of cells with 99 % accuracy for the BCOR variant genotyping and 80 % of cells with 97 % accuracy for genotyping of the RUNX1 variant. These results suggest that our single cell platform can successfully integrate single cell ATAC-seq and genotyping of targeted loci, allowing for the simultaneous genotyping and ATAC with high accuracy and sensitivity for thousands of cells.

Then, to elucidate the epigenetic consequences by epigenetic mutations in primary CH/CCUS samples, we applied our platform to the analysis of whole bone marrow (WBM) cells from patients with CH (n = 3) and CCUS (n = 3) carrying single TET2 mutation. A total of 20,565 cells were clustered based on chromatin accessibility data and annotated by gene accessibility profiles. TET2 targeted sites were reliably genotyped in 81 (74-89) % of cells. TET2 mutations were highly enriched in myeloid cells, plasmacytoid dendritic cells (pDCs), and B cell progenitors. Within the hematopoietic stem and progenitor cell (HSPC) fraction, TET2-mutated HSPCs showed a higher accessibility to the binding motifs for transcription factors associated with myeloid differentiation (C/EBP family), lymphoid lineage priming (SPI1), and pDC differentiation (SPIB) compared with wild-type HSPCs. Compared with wild-type B cells, TET2-mutated B cells had a lower accessibility to the AP-1 family motif associated with B cell differentiation, including the BACH2 binding motif. To characterize the chromatin accessibility profiles of clones comprising multiple mutations, we next analyzed WBM cells from a CCUS patient with double TET2 mutations (TET2-D390fs and TET2-A1512V) and a ZRSR2 mutation. The ATAC-seq successfully profiled 3,725 cells, of which 2,468 (66.3 %) were analyzed for all three mutated loci, allowing for the reconstruction of clonal hierarchy. Linearly acquired, ZRSR2/TET2-D390fs double mutant and triple mutant clones were enriched in myeloid cells, pDCs and B cell progenitors, whereas they were depleted in T and plasma cells. In the ATAC-based pseudotime analysis, the fraction of ZRSR2/TET2-mutant and triple mutant clones decreased along B cell differentiation, suggesting the impaired B cell differentiation of these clones. Compared with wild-type B cells, ZRSR2/TET2-mutant B cells had lower ETS and POU family motif activity and the triple mutant B cells further lost the AP-1 family motif.

In summary, we have developed a novel single cell analysis platform enabling high-throughput simultaneous genotyping and ATAC analysis with high accuracy for primary human samples. Applying this to the analysis of CH/CCUS samples, we demonstrated how epigenetic mutations disrupt the transcriptional mechanism, contributing to the development of CH/CCUS.