Single-Cell Whole Genome Sequencing Reveals Chromosomal Aberrations in Children with Myelodysplastic Syndrome
Authors: Van Leeuwen Anaïs J. C. N. , Van Der Werf Inge , Trabut Laurianne , Van Boxtel Ruben 
Published: December 2024

Introduction
The bone marrow (BM) carefully maintains hematopoiesis by controlling the proliferation, self-renewal, differentiation and migration of hematopoietic cells. This process ensures the continuous supply of healthy blood cells throughout life. However, perturbations in hematopoiesis can begin early in life, leading to a variety of malignancies. Pediatric myelodysplastic syndrome (MDS) is a group of early onset rare blood disorders where hematopoietic progenitors lose their ability to mature and function properly. Most MDS patients present with refractory cytopenia of childhood (RCC), which is characterized by persistent cytopenia, less than 5% blasts in the BM and less than 2% blasts in the peripheral blood. Unlike adult MDS, most MDS-RCC patients have BM hypocellularity, which complicates the classification of MDS and poses challenges for molecular diagnostics. Here, we aim to characterize the mutational landscape and the clonal composition of the hematopoietic system of MDS-RCC patients.With this in-depth characterization we aim to uncover potential therapeutic interventions of patients suffering from pediatric MDS.
Methods
We analyzed the genome-wide mutation burden of the bone marrow compartment of MDS-RCC patients (N=3, median age 17 years). We focused our cohort on MDS-RCC patients where no common driver mutations (monosomy 7, GATA2 or SAMD9/9L) were found. We performed whole-genome sequencing (WGS) of single hematopoietic stem and progenitor cells (HSPCs), including multipotent progenitors (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitors (CMP, CD34+CD38+CD45RA-CD90-) and granulocyte-monocyte progenitors (GMPs, CD34+CD38+CD45RA+CD90-). In addition to the mature lymphoid (T cells, CD45+CD20-CD14-CD3+ and B cells CD45+CD20+CD14-CD3-) and myeloid (Monocytes, CD45+CD20-CD14+CD3-) compartments. The DNA was amplified using primary template-directed amplification (PTA) followed by Illumina sequencing at 15x coverage. Mesenchymal stem cells (MSC) isolated from the same BM were used to filter germline variants. The mutation load was normalized using healthy donors to evaluate the impact of MDS on the hematopoietic system. Next, we performed mutational signature analysis to identify underlying mutational processes. In addition, we assessed the karyotype heterogeneity of the BM compartment using single-cell karyotype sequencing (scKaryoseq).
Results and conclusion
In our analysis, we found the mutation burden of HSPCs of MDS-RCC patients to be elevated (p= 0.001; p= 0.001; p= 0.012, t test) compared to age-matched healthy donors. Mutational signature analysis identified contributions from signatures 1 and 5, which are known to behave in a ‘mutational clock-like’ manner, accumulating linearly with age. These signatures have also been found to be elevated following chemotherapy exposure. Next, we used the detected somatic mutations to construct a phylogenetic lineage tree to assess the clonal relatedness of the stem cells and their progeny. We observed the emergence of clonal clades which are usually only observed in the hematopoietic system of people of old age. In accordance with our study cohort, the clonal expansions lacked known driver mutations associated with MDS.
Interestingly, we found cytogenetic aberrations in the BM compartment of all three MDS-RCC patients. These chromosomal abnormalities were non-recurrent and present in 10% of both the progenitor and myeloid cell compartment. The chromosome copy number variations were confirmed over a greater number of cells using scKaryoseq in one of the patients. In line with the detected chromosomal aberrations, we found an elevated proportion of MDS-RCC cells to be stalled in the G2M cell cycle phase compared to healthy BM, indicating upregulated DNA damage repair. Collectively, this leads to the hypothesis that MDS causes chromosomal instability that is not tolerated by the hematopoietic compartment leading to cell death and ultimately BM hypocellularity.