Sequencing of RNA in single cells reveals a distinct transcriptome signature of hematopoiesis in GATA2 deficiency

Abstract

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes, including systemic infections, lymphedema, cytopenias, and myeloid neoplasms. Genotype–phenotype correlation is not well understood mechanistically in GATA2 deficiency. We performed whole transcriptome sequencing of single hematopoietic stem and progenitor cells from 8 patients, who had pathogenic GATA2 mutations and myelodysplasia. Mapping patients’ cells onto normal hematopoiesis, we observed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. HSPCs of patients exhibited distinct patterns of gene expression and coexpression compared with counterparts from healthy donors. Distinct lineages showed differently altered transcriptional profiles. Stem cells in patients had dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. The prominent deficiency in lympho-myeloid lineages in GATA2 deficiency appeared at least partly due to the expression of aberrant gene programs in stem cells prior to lineage commitment. We computationally imputed cells with chromosomal abnormalities and determined their gene expression; DNA repair genes were downregulated in trisomy 8 cells, potentially rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetic abnormalities showed defects in genes related to multilineage differentiation and cell cycle. Single-cell RNA sequencing is powerful in resolving transcriptomes of cell subpopulations despite a paucity of cells in marrow failure. Our study discloses previously uncharacterized transcriptome signatures of stem cells and progenitors in GATA2 deficiency, providing a broad perspective of potential mechanisms by which germline mutations modulate early hematopoiesis in a human disease. This trial was registered at www.clinicaltrials.gov as NCT01905826, NCT01861106, and NCT00001620.

Comparative Transcriptomic Analysis of the Hematopoietic System between Human and Mouse by Single Cell RNA Sequencing

“…Thus, a joint measurement of gene expression and chromatin accessibility of the same cells in human and mouse will enable a deep comparison of the regulatory and transcriptomic landscape of hematopoiesis [ 46 ]. The current study only analyzed protein coding genes; inclusion of miRNA and lncRNA will add more layers of complexity to gene regulation to assess species conservation of haematopoiesis [ 15 , 47 , 48 ].…”

“…Lineage − CD117 + cells were sorted from bone marrow of C57BL/6 mice ( Figure 1 a). The Chromium Single Cell 3′ platform (10× Genomics) was used to prepare scRNA-seq cDNA libraries [ 14 , 15 ]. RNA-seq libraries were sequenced with paired-end reads of 75-bp on Illumina HiSeq 3000 System.…”

“…Cells with a high percentage of mitochondrion gene reads (>10%) were also excluded. Raw and processed data from all experiments were deposited in the NCBI Gene Expression Omnibus with GSE135194 and GSE142235 [ 14 , 15 ]. Downstream analyses were performed using the R software package Seurat ( , v2.3.4, accessed on 18 June 2018).…”

Why Single-Cell Sequencing Has Promise in MDS

“…Wu et al looked at the single-cell transcriptome of bone marrow cells from 8 patients with pathogenic GATA2 mutations and myelodysplasia. Deficiency in lymphoid/myeloid progenitors was observed and stem cells in patients had dysregulated gene expression implying GATA2 mutation impacts hematopoiesis prior to lineage commitment (83). Izzo et al analyzed the loss of the DNA methylation genes DNMT3A and TET2 (which are among the most frequently mutated founding genes in MDS) through scRNA-seq, chromatin accessibility, and methylome; showing TET2 KO hypermethylation led to myelomonocytic skews in HSC priming.…”

“…looked at the single-cell transcriptome of bone marrow cells from 8 patients with pathogenic GATA2 mutations and myelodysplasia. Deficiency in lymphoid/myeloid progenitors was observed and stem cells in patients had dysregulated gene expression implying GATA2 mutation impacts hematopoiesis prior to lineage commitment ( 83 ). Izzo et al.…”