Insights into Hematopoiesis in GATA2 Deficiency Using Single-Cell Transcriptomics

GATA2 is an essential transcription factor in hematopoiesis. Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 deficiency results in varying clinical outcomes manifesting at either young or older age. Reason for the variation of the disease phenotype preceding hematological malignancy is not known. GATA2 mutations appear to cause loss of function of the mutated allele resulting in haploinsufficiency. Several different types of mutations in GATA2 have been identified; truncating, missense, large deletions, and regulatory region mutations. The most direct GATA2 control pathway is the effect it has on hematopoietic stem cells (HSCs). Through the progeny of the hematopoietic stem cells that function under GATA2 control, GATA2 indirectly has a great effect on immune system, both protection against external infection and internal tumor formation. We aimed at deciphering how do GATA2-mutated cells differ from wild type (wt) and what are the differences between GATA2 wt vs. GATA2-mutated cells' progeny and downstream signaling? One of the genes positively regulated by GATA2 is NFE2 . NFE2 encodes a component of the NF-E2 complex essential for regulating erythroid and megakaryocytic maturation and differentiation. As GATA2-positive cells are scarce in PB we focused in our analyses on NFE2-positive cells.

We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution (Zheng et al . Nature Communications, 2017) on peripheral blood (PB) samples from five GATA2 mutation carriers and two healthy controls. The studied GATA2 variants were p.Q328X (two cases), p.T354M, p.G237D, and a sample where a GATA2 mutation has not been identified, but only one allele GATA2 is expressed i.e. haploinsufficiency. Erythrocytes were lysed prior to washing, filtering and diluting the white blood cells in suitable concentration (1x10⁶ cells/mL) for the analysis. The gel beads in emulsion (GEM) generation was performed using the Chromium Single Cell 3' v2 Reagent Kit (10x Genomics) aiming for a 3000-cell capture per sample. The Illumina ready sequencing libraries were prepared according to the 10x Genomics instructions and the sample libraries were sequenced in Illumina HiSeq 2500 using Rapid mode. The Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3' RNA-sequencing barcode and read data to FASTQ files and to generate align reads and gene-cell matrices.

Significant gene expression differences in NFE2 expressing cells between patients carrying different variants in GATA2 were observed. Intriguingly, significant expression changes were noted e.g. in CD74, CFD, CTSS, CLEC1B, and CMTM5, all linked to cell signaling and immunological responses. Comparison of GATA2 -mutated vs. controls in NFE2 expressing cells revealed significant changes in expression patterns in hemoglobin genes HBA1, HBA2, and HBB, as well as in e.g. other erythrocyte-associated genes ALAS2 and AHSP. Further analyses also revealed significantly distinct expression patterns in PB eosinophils between study patients and controls in e.g. CEBPD, VCAN, and RGS2 . Also, when comparing the eosinophils from individual patients with controls and each other, we identified distinct expression patterns in e.g. CAMP, and NKG7, which play important roles in the immune system.

Following germline GATA2 mutation discovery in acute myeloid leukemia / myelodysplastic syndrome / immunodeficiency patients, we conducted whole transcriptome analysis to decipher the GATA germline mutation defects on single-cell level. Single-cell transcriptomics enables analyzing the role of e.g. GATA2 in a selected cell type and its' progeny. Phenotype of GATA2-deficient patients is multi-faced but generally manifested by challenges in immune functions and autoinflammation. Connection between our findings and patients' clinical manifestations is interesting and triggers us to investigate the possible link further. We will furthermore study the bone marrow sample single-cell transcriptomes to deepen the understanding of GATA2 deficiency on stem cell level and validate our transcriptome analysis findings. All this will hopefully increase our understanding on GATA2 deficiency disease mechanisms and improve patient (family) care.