The genotype-integrated gene expression profile at the single-cell level of bone marrow CD34-positive hematopoietic stem and progenitor cells (HSPCs) from a patient with myelodysplastic neoplasms (MDS) harboring germline and somatic DDX41 variants.

DDX41 is recognized as a major gene responsible for myeloid neoplasms associated with germline predisposition. Individuals typically inherit a germline pathogenic variant in one allele and later acquire a somatic variant—most commonly the p.R525H variant—in the other allele, leading to the development of myeloid neoplasms. In this study, we conducted single-cell RNA sequencing integrated with p.R525 site genotyping to investigate how acquiring the p.R525H variant influences gene expression changes in hematopoietic cells. Overall design: CD34-positive cells were isolated from the bone marrow of an MDS patient carrying germline and somatic DDX41 variants using a magnetic bead-based method. These cells were subjected to scRNA-seq using the terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) method (PMID: 35760847). To distinguish cells with and without the somatic p.R525H variant, amplicons for genotyping DDX41 p.R525 sites were generated from amplified whole transcripts. The genotyping data were then integrated with the transcriptomic profiles for comprehensive analysis.