Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [WES]

Background: Familial platelet disorder (FPD) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. FPD patients show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPD is in a precancerous state. However, the DNA methylation status of FPD has not yet been elucidated due to no animal models for FPD and the difficulty in obtaining FPD patient-derived samples. Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPD-mimicking cells, which were established by genome editing for human iPS cells as a FPD-model, compared with those of wild-type cells. The FPD-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we demonstrated a putative causative transcription factor of the differential DNA hypermethylation, which are involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis. Overall design: To establish the human iPS cell lines with FPD-mimicking RUNX1 heterozygous mutations, we performed CRISPR-Cas9-mediated genome editing of RUNX1, followed by single-cell cloning of the resulting genome-edited cells. We performed whole exome sequencing (WES) analysis to confirm potential off-target editing on protein cording regions in the established cell lines.