Effects Of Csf3r Mutations On Myeloid Differentiation And Proliferation Of Hematopoietic Cells Of Congenital Neutropenia Patients

Over the last 25 years, with the clinical approval of recombinant human granulocyte colony-stimulating factor (rhG-CSF) remarkable progress has been achieved in the therapy of severe congenital neutropenia (CN). Nevertheless, CN patients have a high rate of transformation to myelodysplasia (MDS) or acute myeloid leukemia (AML). This risk is especially high in the group of patients who harbor acquired G-CSFR mutations, suggesting that these mutations are involved in the leukemogenesis. Deep sequencing of the intracellular part of G-CSFR allowed us to identify a subset of CN patients with a high mutant allele frequency (MAF) of G-CSFR mutations in the granulocytic compartment. We performed CSF3R mutation analysis of myeloid colonies from colony forming unit (CFU) assay of BM and CD34+ samples of 3 ELANE -CN patients, in which the percentage of granulocytes with G-CSFR mutation varied between 22.6% and 81%. Average number of sequenced colonies per CFU sample was 18. Intriguingly, we found that only cell clones with WT G-CSFR were grown in CFU assay. We hypothesized that lack of G-CSFR mutant clones in CFU assay is due to abnormal signaling downstream of truncated G-CSFR. To compare myeloid differentiation of G-CSFR mutant and WT G-CSFR hematopoietic cells, we generated ELANE -CN patient-derived induced pluripotent stem cells (CN-iPSCs) and performed CRISPR/Cas9 genome editing of CSF3R . Using 2 different sgRNAs specific for the intracellular region of G-CSFR, we introduced nucleotide indels at amino acid positions 740 and 735 (NP_000751.1) in CN-iPSCs. Subsequently, homozygous and heterozygous CN-iPSCs clones with truncated distal part of cytoplasmic domain of G-CSF receptor and subsequent loss of 3 out of 4 conserved tyrosine residues were generated.