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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.

SUBMITTER: Wu Z

PROVIDER: S-EPMC7322959 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Wu Zhijie Z Gao Shouguo S Diamond Carrie C Kajigaya Sachiko S Chen Jinguo J Shi Rongye R Palmer Cindy C Hsu Amy P AP Calvo Katherine R KR Hickstein Dennis D DD Holland Steven M SM Young Neal S NS

Blood advances 20200601 12

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 hema ...[more]

PMID: 32556286

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Project description:Background and Purpose: Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutation has a broad spectrum of clinical phenotypes including systemic infections, lymphedema, cytopenias, MDS and AML. A comprehensive profiling of transcriptome of hematopoiesis in GATA2 deficiency is currently lacking. Methods: We performed single-cell RNA sequencing of sorted bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) from eight GATA2 deficiency patients, who had various well characterized GATA2 mutations and clinically manifest myelodysplasia. We characterized transcriptomes in lineages, computationally defined cells with chromosomal abnormalities, and described gene expression of these cells. Results: Mapping patients’ cells onto normal hematopoiesis, we observed preferred deficiency in lymphoid and myeloid progenitors, which also was evidenced in loss of heterogeneity in gene correlations. HSPCs in patients exhibited distinct gene expression pattern and gene coexpression pattern compared with its counterparts in healthy donors. Distinct lineages show different transcriptional profiles resulting from GATA2 mutations. HSCs in patients exhibited dysregulated genes in apoptosis, cell cycle and quiescence, and had increased expression of erythroid/megakaryocytic priming programs and decreased lymphoid priming programs. Thus, the prominent deficiency in myeloid/lymphoid lineages in GATA2 deficiency was partly due to expression of aberrant gene programs in HSCs prior to lineage commitment. We computationally defined cells with chromosomal abnormalities and described gene expression of these cells. DNA repair genes were downregulated in trisomy 8 cells, possibly rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetics had defects in multi-lineage differentiation and cell cycle. Conclusion: Germline GATA2 mutations modulate gene expression and change gene coexpression patterns. Distinct lineages show different transcriptional profiles resulting from GATA2 mutations. The prominent deficiency in myeloid/lymphoid lineages in GATA2 deficiency was partly due to expression of aberrant gene programs in HSCs prior to lineage commitment.

Project description:Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48?hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600), the master regulator of sexual development, from an epigenetically silenced state during asexual replication. AP2-G expression during this 'commitment cycle' prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1-30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling. Here we use highly parallel, single-cell RNA sequencing of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.

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

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

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