Abstract
Although many causative genes have been identified in inherited bone marrow failure syndromes (IBMFS), the genetic etiology remains elusive in some cases, limiting accurate diagnosis and informed clinical decision-making.
Recently, our collaborator, together with other research groups, reported that pathogenic variants in SLF2 and SMC5 cause an autosomal recessive developmental disorder termed Atelis Syndrome, which is characterized by hematological abnormalities, most notably anemia and lymphopenia, as well as microcephaly and short stature. However, its disease concept remains undefined in hematology.
Strikingly, our re-evaluation of Atelis Syndrome cases revealed that some patients developed myelodysplastic syndromes (MDS) or MDS-like features at a young age, underscoring the critical need to define its disease concept within hematology.
SLF2 and SMC5 cooperate in a shared genome maintenance pathway to facilitate DNA double-strand break repair and regulate chromatin organization. Interestingly, our analysis of public datasets (GSE104406 and GSE188889) revealed that SLF2 expression increases with aging in normal hematopoietic stem cells (HSCs), suggesting the significance of these pathways in HSC biology and their potential relevance to age-associated hematopoietic regulation. However, their roles in hematopoiesis and the pathogenic mechanisms underlying hematological defects in Atelis Syndrome remain poorly understood.
Given this context, we generated induced pluripotent stem cell (iPSC) lines from a patient with Atelis Syndrome carrying compound heterozygous variants of SLF2. To generate isogenic controls, we successfully corrected one of the mutated alleles using CRISPR-Cas9. Both the patient-derived and gene-corrected iPSCs were subsequently differentiated into hematopoietic lineages. In colony-forming unit (CFU) assays, hematopoietic stem and progenitor cells (HSPCs) derived from patient iPSCs (mutant HSPCs) exhibited significantly reduced colony-forming capacity in both myeloid and erythroid lineages. Upon erythroid induction, the mutant cells showed significant impairment of erythroid differentiation.
Knockdown of SMC5 in human cord blood-derived CD34+ cells also led to reduced colony-forming capacity in CFU assays.
Next, we investigated the transcriptional profiles of mutant HSPCs using RNA sequencing. RNA sequencing revealed activation of the p53/p21 pathway in response to DNA damage in the mutant cells. Consistently, the protein levels of p53 and p21 were also elevated, and increased apoptosis was observed in these mutant cells. A significant increase in γ-H2AX foci was observed in the mutant cells following mitomycin C treatment, directly indicating accumulated DNA damage and genomic instability.
Furthermore, RNA sequencing revealed reduced multilineage differentiation potential, along with the upregulation of genes associated with myeloid differentiation and senescence in mutant HSPCs. In cell cycle analysis, a significant reduction in the quiescent state (G0 phase) was observed, indicating loss of stem cell dormancy associated with premature aging and functional exhaustion of the HSC pool.
ATAC sequencing revealed hallmark epigenetic features of HSC aging in mutant HSPCs, including reduced global promoter accessibility, increased accessibility at p53-binding regions, and PU.1 motif enrichment in open chromatin. These alterations suggest altered fate commitment and a shift toward myeloid-biased differentiation.
In conclusion, SLF2 and SMC5 are newly defined causative genes for IBMFS. These dysfunctions in HSC drive activation of the p53/p21 pathway, epigenetic remodeling, and premature HSC aging, accompanied by HSC dysfunction, a loss of multilineage differentiation potential, and altered fate commitment. Clinically, these aging-associated alterations lead to cytopenia and myeloid-biased hematopoiesis, and the accompanying sustained genomic instability suggests a predisposition to MDS.
These results define Atelis Syndrome as a distinct entity within the IBMFS spectrum and underscore the need for its incorporation into diagnostic frameworks, potentially contributing to improved genetic diagnosis, informed patient management, and appropriate therapeutic strategies. Furthermore, our findings highlight the significance of SLF2 and SMC5 in maintaining functional HSC integrity, thereby advancing our understanding of HSC aging and IBMFS pathogenesis.
