Myelodysplastic syndromes are multiclonal diseases derived from hematopoietic stem and progenitor cells

<jats:title>Abstract</jats:title><jats:p>Myelodysplastic syndromes (MDS) are generally considered as a group of clonal diseases derived from hematopoietic stem cells, but a number of studies have suggested that they are derived from myeloid progenitor cells. We aimed to identify the cell of origin in MDS by single-cell analyses. Targeted single-cell RNA sequencing, covering six frequently mutated genes (<jats:italic>U2AF1</jats:italic>, <jats:italic>SF3B1</jats:italic>, <jats:italic>TET2</jats:italic>, <jats:italic>ASXL1</jats:italic>, <jats:italic>TP53</jats:italic>, and <jats:italic>DNMT3A</jats:italic>) in MDS, was developed and performed on individual cells isolated from the CD34<jats:sup>+</jats:sup> and six lineage populations in the bone marrow of healthy donors (HDs) and patients with MDS. The detected mutations were used as clonal markers to define clones. By dissecting the distribution of clones in six lineages, the clonal origin was determined. We identified three mutations both in HDs and patients with MDS, termed clonal hematopoiesis (CH) mutations. We also identified fifteen mutations only detected in patients with MDS, termed MDS mutations. Clonal analysis showed that CH clones marked by CH mutations and MDS clones marked by MDS mutations were derived from hematopoietic stem cells as well as various hematopoietic progenitor cells. Most patients with MDS showed the chimeric state with CH clones and MDS clones. Clone size analysis suggested that CH mutations may not contribute to clonal expansion of MDS. In conclusion, MDS comprise multiple clones derived from hematopoietic stem and progenitor cells.</jats:p>

section_content: To the editor, Myelodysplastic syndromes (MDS) were proposed to arise from a stepwise acquisition of mutations [1, 2] and are reported as hematopoietic stem cells (HSCs)-origin diseases [3]. However, an increasing number of studies have suggested myeloid progenitor cells are the cell of origin [4, 5]. Additional mutations can be acquired and confer the self-renewal ability to hematopoietic progenitor cells (HPCs) in the progression of MDS [3, 4]. Thus, the cell of origin in MDS remains controversial. In principle, mutant clones that originate from HSCs should be detected in all their descendent blood lineages, enabling the reconstruction of clonal structures in cancer [6]. Single-cell sequencing but not bulk sequencing detects the co-occurrence of mutations in the same cells, permitting to distinguish individual clones from one another. In this study, we took advantage of lineage tracing techniques by using targeted single-cell RNA sequencing to address this issue [7]. 

BM cells were obtained from five healthy donors (HDs) and two AML patients in complete remission (HD group), as well as fourteen MDS patients (MDS group) S1 ). Single cells were sorted from CD34 + HPCs and six lineage populations: neutrophils, monocytes, erythroblasts, megakaryocytes, B cells, and T cells (Additional file 2: Fig. S1 ). These populations were then subjected to a targeted single-cell RNAsequencing mutation analysis, covering U2AF1, SF3B1, TET2, ASXL1, TP53, and DNMT3A, the most frequently mutated genes in MDS [1, 2] (Additional file 2: Fig. S2A-C ). After filtration, we obtained 3,549 single cells (Additional file 2: Fig. S3A-C ). A total of eighteen mutations were detected from all donors. All mutations were reported in COSMIC database. Three mutations detected in both the HD and MDS groups were termed CH mutations while the remaining fifteen mutations only detected in MDS group were termed MDS mutations (Fig. 1A, Additional file 1: Table S2 ), although the border between CH related mutations and MDS/AML related mutations is still blurry [8]. The fraction of cells carrying CH mutations was similar to that carrying MDS mutations (Fig. 1B ). Greater mutation number in the MDS group than that in the HD group was observed (Fig. 1C ).

section_name: Open Access

section_content: Clones were defined as cells with identical combinations of mutations [9] (Fig. 1D ). A total of eighty-nine clones were detected in all donors. CH clones were marked with CH mutations while MDS clones were marked with MDS mutations alone or both CH mutations and MDS mutations (Additional file 2: Fig. S4A ). Greater clone number and clonal diversity in the MDS group than that in the HD group were observed (Fig. 1E, F ), suggesting the accumulation of genetic lesions with more complexity during the progression to malignant diseases [10]. 

Most patients with MDS presented the mixture of multiple CH clones and MDS clones (Fig. 2A ). The clone size of CH clones was significantly greater than that of MDS clones (Additional file 2: Fig. S4B ), even within the MDS group (Fig. 2B ), suggesting the acquisition of CH mutations occurs earlier than that of MDS mutations [11]. No statistical difference in the clone size of CH clones between HD and MDS groups was observed (Fig. 2C ). Both CH mutant clones and MDS mutant clones were larger in clone size than double mutant clones from MDS group, suggesting that CH mutations do not provide a significant growth advantage to MDS mutations (Fig. 2D ). 

CH clones and MDS clones were detected in a variety of lineage combinations (Fig. 2E ). The number of cell populations involved in CH clones was significantly greater than that in MDS clones (Fig. 2F ). When the same clones were detected in all six lineage populations regardless of H population, they were considered to originate from HSCs, otherwise from HPCs. 27% CH clones were derived from HSCs while 10% MDS clones were derived from HSCs (Fig. 2G ). Of note, exact HPC types could not be defined by our strategy. 10 out of 37 HPC-derived MDS clones were found in MDS-EB1 patients and 19 out of 37 HPC-derived MDS clones was found in MDS-EB2 and sAML patients. MDS with higher blasts seems to have more chance to have HPC origin (Additional file 1: Table S3 ). But the percentage of blasts was not related to clone distribution (Additional file 2: Fig. S4C ). A small number of single cells such as 18-26 cells per population were examined in previous single-cell analysis of MDS/ AML [12]. In two MDS patients (PT2. 13 and PT2. 14), more single cells were analyzed, confirming the HSC and HPC origins for most MDS clones. 

The number of HSC-derived clones might be underestimated, because a limited number of cells per population was analyzed. Nevertheless, if not all, MDS appeared to be the mixture of CH clones and MDS clones originating from HSCs and HPCs.
section_name: Experimental Hematology & Oncology

section_name: Acknowledgments

section_content: The authors thank Toshio Suda for critical reading of the paper, and Peng Wu, Wanjun Lei, Haizhao Fan, and Jingyuan Tong for advice on bioinformatics analysis.
section_name: Acknowledgments

section_name: Funding

section_content: This work was supported by grants from the National Key R&D Program of China ( 2019YFA0110203 and 2021YFA1100900 ), the National Natural Science Foundation of China ( 81970119, 82070112, 81890990, and 82170118 ), the Haihe Laboratory of Cell Ecosystem Innovation Fund ( HH22KYZX0002 ), the PUMC Project for Building Subjects (201920101401), and the CAMS Initiative Fund for Innovative Medicine ( 2021-I2M-1-073 and 2021-I2M-1-040 ).
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section_name: Supplementary Information

section_content: Abbreviations MDS: Myelodysplastic syndromes; BM: Bone marrow; CH: Clonal hematopoiesis; sAML: Secondary acute myeloid leukemia; HD: Healthy donor; HSC: Hematopoietic stem cell; HPC: Hematopoietic progenitor cell; RT-PCR: Reverse transcription-polymerase chain reaction.

section_content: The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40164-022-00280-3. 

Additional file 1: Table S1. BM donors. Table S2. Comparison of mutations detected by targeted bulk DNA sequencing and targeted single cell RNA sequencing. Table S3. The number of HSC-and HPC-derived clones in individual donors. Table S4. Primers. Table S5. Barcode, index and adaptor sequences. Table S6. Targeted genes in DNA sequencing. 

Additional file 2: Figure S1. Gating strategy for seven cell populations. FACS strategy used to isolate single cells from the seven populations: H, CD34 + CD45 low cells; E, CD45 -CD235a + CD71 + cells; M, CD45 -CD235a -CD41 + cells; m, CD45 + CD33 + CD14 + CD16 -cells; n, CD45 + CD33 + CD14 -CD16 + cells; B, CD45 + CD19 + CD20 + cells; and T, CD45 + CD3 + cells. The upper panels represent HD data, and the lower panels represent data from a patient with MDS. Figure S2. Targeted single-cell RNA-seq method. A Schematic diagram of the targeted singlecell RNA-seq method. Single H, n, m, E, M, B, and T were sorted by flow cytometry into a 96-well plate. One-step RT-PCR was performed with the first-round primer mix. The second round of PCR was performed with PCR primer mix 1 or mix 2. Finally, a sequencing library was constructed with p5 and p7 adaptors. B Frequency at which the DNMT3A p. R301W mutation was detected in 32 single JURKAT cells. The positive rate was 78. 1%. C Electrophoretogram of the representative libraries from single cells and 20 cells from a patient with MDS. One pair of each primer (U2AF1, SF3B1, ASXL1, TP53, TET2, and DNMT3A) from the PCR primer mix (PCR primer mix 1 or PCR primer mix 2) was used for the second round of PCR, followed by library construction. PCR products were separated on a gel. Library construction performed without cells served as a negative control (neg). Fourteen patients with MDS were classified into low blasts (1%-9%) group (n = 8) mainly consisting of EB1 and high blasts (>9%) group (n = 6) mainly consisting of EB2. ns, p > 0. 05 (Mann-Whitney U test).
section_name: Supplementary Information

section_content: BL and HE designed the study. BL, FD, QZ, HB, and JW performed experiments and analyzed data. YJ analyzed targeted bulk DNA sequencing data. TQ and ZX provided patient materials and clinical data. SM and EJ helped to obtain heathy donor materials. TC, ZX, and HE supervised the study. BL and HE wrote the paper. All the authors have read and approved the final manuscript.
section_name: Author contributions

section_content: Supplementary information including Additional file 2: Figs. S1-S4 and Additional file 1: Tables S1-S6 are provided with the online version of this paper. The datasets analyzed during the current study are available from the corresponding author on reasonable request.
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section_content: Ethics approval and consent to participate Human samples were approved by the Ethics Committee, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College and met all requirements of the Declaration of Helsinki.
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