Single Cell Transcriptome Analysis of CD34+ Cells from Patients with RUNX1-Fpdmm at Different Stages of Disease Progression

Germline pathogenic variants in RUNX1 lead to familial platelet disorder with associated myeloid malignancies (FPDMM), an autosomal dominant disease. Patients with this disorder have thrombocytopenia, defective megakaryocytic maturation, and an overall lifetime risk (35% to 45%) of developing hematologic malignancies, including MDS and AML. Patients with FPDMM suffer from early onset of clonal hematopoiesis of indeterminate potential (CHIP) and malignant transformation. However, it is unclear how germline RUNX1 variants and CHIP mutations lead to hematologic malignancies in the FPDMM patients.

Our group established the first natural history study of FPDMM patients in the NIH Clinical Center (https://clinicaltrials.gov/ct2/show/NCT03854318). From January 2019 to May 2024, we enrolled 91 families with confirmed pathogenic or likely pathogenic RUNX1 germline variants for a total of 218 RUNX1 variant carriers. So far, bone marrow exome sequencing results from at least one time point have been obtained for 96 RUNX1 variant carriers. Of these, 46% have only RUNX1 germline variants (44/96), 27% have CHIP (>2% variant allele frequency VAF, 26/96) and 27% have been diagnosed with a hematological malignancy (26/96).

To understand how RUNX1 germline variants may change the gene expression profile in the hematopoietic stem and progenitor cells (HSPCs) or push them toward malignancy in the presence of somatic mutations in CHIP or leukemia-related genes we performed single-cell RNA sequencing (scRNA-seq) of CD34+ HSPCs from 13 RUNX1-FPDMM patients and 4 healthy controls (130,000 cells). All the RUNX1-FPDMM patients had undergone exon sequencing and had CLIA-certified confirmation of germline RUNX1 pathogenic or likely pathogenic variants. Among the 13 RUNX1-FPDMM patients, 5 have either no somatic mutations or a somatic mutation with VAF <3% (group 1), 6 have multiple somatic mutations in CHIP or leukemia related genes with VAF ranging from 6-48% (group 2) and 2 have somatic mutations or genomic rearrangements and a diagnosis of chronic myelomonocytic leukemia (group 3).

Analysis of the scRNA-seq data showed that all the known CD34+ subpopulations in healthy controls are represented in the FPD patient samples. However differential expression analysis among the different patient groups allowed us to detect differentially expressed genes. Moreover, we observed that the proportion between myeloid vs erythroid progenitors is skewed toward the myeloid lineage in FPDMM patients, as compared to the healthy controls. More analysis is ongoing to better understand the common features but also the differences between the different RUNX1-FPDMM patient groups. Parallel single cell long-read genomic sequencing is ongoing to determine the cell populations that contain the somatic mutations, which will be correlated with transcriptomic profiles. Our unique cohort and access to primary samples at different stages of the disease offers a unique opportunity to identify previously unappreciated changes and signature genes that can help us understand mechanisms of malignant transformation and track disease progression.