GATA2 Deficiency Syndrome Research Articles

AYA With GATA2 Deficiency Syndrome With Evolving Myeloid Neoplasia: Case Presentation

AYA With GATA2 Deficiency Syndrome With Evolving Myeloid Neoplasia: Case Presentation

Prognostic significance of GATA2 in patients with MDS/AML: a systematic review and meta-analysis.

GATA2 deficiency syndrome is a heterogeneous disorder characterized by a high risk of developing myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML). We conducted a meta-analysis of the literature to explore the prognostic significance of GATA2 mutations in patients diagnosed with MDS/AML, as previous studies have yielded conflicting findings regarding the impact of GATA2 mutations on patient outcomes. We conducted a comprehensive literature search of databases such as PubMed, Embase, the Cochrane Library, and the Web of Science to obtain studies on the prognostic significance of GATA2 mutations in patients with MDS/AML that were published through January 2024. We extracted the hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), disease-free survival (DFS), and event-free survival (EFS). The meta-analysis was conducted by choosing either a fixed-effect model or a random-effect model, depending on the variability observed among the studies. A total of 13 cohort studies were included in the final meta-analysis, including 2714 patients with MDS, of whom 644 had GATA2 mutations. The results revealed that GATA2 mutations had an adverse impact on OS (HR = 1.54, 95% CI = 1.08-2.18, P = 0.02) and EFS (HR = 1.32, 95% CI = 1.01-1.72, P = 0.04), but no significant effect on DFS (HR = 1.21, 95% CI = 0.89-1.64, P = 0.23). GATA2 mutations were associated with a significantly shorter OS in MDS patients (HR = 2.56, 95% CI = 1.42-4.06, P = 0.002) but not in AML patients (HR = 1.08, 95% CI = 0.92-1.26, P = 0.37). Our meta-analysis revealed that GATA2 mutations are associated with unfavourable outcomes in patients with MDS/AML. Furthermore, patients harbouring these mutations should be prioritized for aggressive therapeutic interventions.

GATA2 deficiency syndrome: A compensatory mechanism gone awry?

In their paper, using zebrafish models, Gioacchino etal. have demonstrated the GATA2 haploinsufficiency, the genetic hallmark of GATA2 deficiency syndrome, promotes erythroid and myeloid cytopenia, and have discovered a self-regulatory mechanism to compensate GATA2 levels and protein function. Commentary on: Gioacchino etal. GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia. Br J Haematol 2024;205:580-593.

Pathogenic GATA2 genetic variants utilize an obligate enhancer mechanism to distort a multilineage differentiation program

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

Noncoding GATA2 Genetic Variation As a Determinant of Hematopoietic Stem/Progenitor Cell Activity and Mobilization Efficiency

Germlinecoding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). We generated a mouse model of GATA2 deficiency syndrome by engineering mice with a single-nucleotide variant in an intronic enhancer (+9.5) from GATA2 deficiency patients combined with deletion of the enhancer from the second allele (compound heterozygous; CH). This system models patients harboring a germline pathogenic allele accompanied by epigenetic silencing of the normal allele. Unlike homozygous Gata2 coding or regulatory deletions that are embryonic lethal, CH represents a unique system to analyze postnatal GATA2 function. We demonstrated that adult germline Gata2 variation alters steady-state HSPCs, blocks HSPC expansion and differentiation upon chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS and attenuates HSPC mobilization in response to granulocyte colony stimulating factor (G-CSF) (Soukup et al., 2019, Soukup et al., 2021). As short-term G-CSF treatment induces HSPC metabolic gene signatures (Fast et al., 2021) and expansion and cell cycle entry (Schuettpelz et al., 2014), and CH have decreased MPPs (LSKCD150 -CD48 -) and common myeloid progenitors (CMPs) (Lin −Sca1 −Kit +FcγR −CD34 +) (Soukup et al., 2021), we asked if mobilization defects reflected decreased functional HSPCs in bone marrow. CH had 2.2-fold fewer colony forming units (CFU) from bone marrow (p = 0.007) than wild type (WT), reflecting a paucity of functional HSPCs. 16 hours following a single G-CSF dose, WT bone marrow immunophenotypic HSCs (LSKCD150 +CD48 -) and MPPs expanded 1.8-fold (p = 0.001 and 0.01, respectively); CH HSCs and MPPs were unchanged. CH led to decreased HSPCs and failed to induce rapid HSPC expansion post-G-CSF. We tested whether Gata2 variation impairs mobilization in response to different stimuli, or if select pathways are impacted via disrupting the CXCR4/CXCL12 chemokine axis with the CXCR4 antagonist plerixafor, stromal/neutrophil CXCR2 activation with rhIL-8, or VLA-4/VCAM1 binding with VLA-4 inhibitor BIO5192. IL-8 or BIO5192 resulted in 3.9- and 2.2-fold increased WT CFU, respectively (p < 0.0001 and 0.0014) without altering CH CFU. By contrast, mobilization with plerixafor resulted in a 4.3-fold increase in WT CFU (p < 0.0001) and 4.5-fold increase in CH CFU (p = 0.004). Targeting these pathways collectively with G-CSF was insufficient to restore normal mobilization in CH. Competitive transplantations with peripheral blood revealed G-CSF + plerixafor increased WT donor-derived hematopoiesis 24- and 20-fold at 8 and 12 weeks, respectively, versus vehicle control. Treatment of CH did not increase donor-derived contributions. Since GATA2 deficiency syndrome and somatic GATA2 variation involve heterozygous alleles, we tested whether monoallelic +9.5 Ets motif variation disrupts mobilization. G-CSF increased CFU 28.5-fold (p < 0.0001) relative to vehicle-treated WT. Mobilization of Gata2 Ets +/- HSPCs were reduced significantly (p = 0.003). CH variation severely disrupted mobilization, with 7.3-fold fewer CFU than Gata2 Ets +/- (p = 0.02). Synergistic G-CSF/plerixafor treatment increased CFU 89-fold in WT versus untreated (p < 0.0001), and Gata2 Ets +/- mobilization was reduced (p = 0.006) and not improved relative to Gata2 Ets -/- or CH, resembling G-CSF/BIO5192. Thus, Gata2 heterozygosity impaired G-CSF-induced HSPC mobilization with all regimens. Although GATA2 noncoding variation is not commonly evaluated, analysis of human data will be performed to assess relationships between variation and poor mobilization outcomes. Gata2 variation depletes functional HSPCs in the bone marrow, reducing the available HSPCs to mobilize and rendering most mobilization regimens inefficient. Ongoing studies are elucidating molecular mechanisms underlying the mobilization blockade. Our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms e.g., in the context of additional environmental and/or genetic insults. Future knowledge linking functionality of mobilization regimens in these contexts may unveil insights into mechanisms operational in poor mobilizers.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

The role of GATA2 in adult hematopoiesis and cell fate determination.

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

Gata2 noncoding genetic variation as a determinant of hematopoietic stem/progenitor cell mobilization efficiency

AbstractGermline genetic variants alter the coding and enhancer sequences of GATA2, which encodes a master regulator of hematopoiesis. The conserved murine Gata2 enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis during embryogenesis. Heterozygosity for a single-nucleotide Ets motif variant in the human enhancer creates a bone marrow failure and acute myeloid leukemia predisposition termed GATA2 deficiency syndrome. The homozygous murine variant attenuates chemotherapy- and transplantation-induced hematopoietic regeneration, hematopoietic stem and progenitor cell (HSPC) response to inflammation, and HSPC mobilization with the therapeutic mobilizer granulocyte colony–stimulating factor (G-CSF). Because a Gata2 +9.5 variant attenuated G-CSF–induced HSPC expansion and mobilization, and HSC transplantation therapies require efficacious mobilization, we tested whether variation affects mechanistically distinct mobilizers or only those operating through select pathways. In addition to affecting G-CSF activity, Gata2 variation compromised IL-8/CXCR2- and VLA-4/VCAM1-induced mobilization. Although the variation did not disrupt HSPC mobilization mediated by plerixafor, which functions through CXCR4/CXCL12, homozygous and heterozygous variation attenuated mobilization efficacy of the clinically used plerixafor/G-CSF combination. The influence of noncoding variation on HSPC mobilization efficacy and function is important clinically because comprehensive noncoding variation is not commonly analyzed in patients. Furthermore, our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms.

Prevalence and clinical expression of germ line predisposition to myeloid neoplasms in adults with marrow hypocellularity

AbstractSystematic studies of germ line genetic predisposition to myeloid neoplasms in adult patients are still limited. In this work, we performed germ line and somatic targeted sequencing in a cohort of adult patients with hypoplastic bone marrow (BM) to study germ line predisposition variants and their clinical correlates. The study population included 402 consecutive adult patients investigated for unexplained cytopenia and reduced age-adjusted BM cellularity. Germ line mutation analysis was performed using a panel of 60 genes, and variants were interpreted per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines; somatic mutation analysis was performed using a panel of 54 genes. Of the 402 patients, 27 (6.7%) carried germ line variants that caused a predisposition syndrome/disorder. The most frequent disorders were DDX41-associated predisposition, Fanconi anemia, GATA2-deficiency syndrome, severe congenital neutropenia, RASopathy, and Diamond-Blackfan anemia. Eighteen of 27 patients (67%) with causative germ line genotype were diagnosed with myeloid neoplasm, and the remaining with cytopenia of undetermined significance. Patients with a predisposition syndrome/disorder were younger than the remaining patients and had a higher risk of severe or multiple cytopenias and advanced myeloid malignancy. In patients with myeloid neoplasm, causative germ line mutations were associated with increased risk of progression into acute myeloid leukemia. Family or personal history of cancer did not show significant association with a predisposition syndrome/disorder. The findings of this study unveil the spectrum, clinical expressivity, and prevalence of germ line predisposition mutations in an unselected cohort of adult patients with cytopenia and hypoplastic BM.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Germline testing in haematology – who and why

With the inclusion for the first time of ‘Myeloid Neoplasms with Germline Predisposition’ in the most recent iteration of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, consideration of whether an underlying inherited factor may have contributed to malignancy development has become an essential part of the diagnostic work up. The disease groups which fall under this heading are broad; from the traditional inherited bone marrow failures such as Fanconi anaemia and dyskeratosis congenita, to the more recently described GATA2 deficiency syndrome and also some ‘non-syndromal’ conditions such as the predisposition to myeloid malignancy conferred by germline mutations in DDX41.

New Approach to Decipher GATA2 Deficiency Syndrome: Focus on the Recurrent GATA2 R396Q Mutation

Germline GATA2 mutations are identified in a complex disorder termed GATA2 deficiency syndrome. Clinical heterogeneous manifestations are associated with a wide diversity of molecular alterations (missense, frameshift, nonsense, intronic or splicing mutations). Truncating mutations decrease GATA2 expression suggesting a haploinsufficiency mechanism while molecular consequences of missense mutations are ill-known. We focus our research on the most recurrent GATA2 mutation, GATA2 R396Q.Our in vitro results indicate that the ectopic expression of GATA2 R396Q has a strong impact on the clonogenicity associated with an excessive granulocyte differentiation. This data motivates the elaboration of a more physiological approach through the development of a new knock-in mouse model with endogenous expression of Gata2 R396Q mutation. This model allowed us to address the question of the impact of this missense mutation on hematopoiesis at steady state and in challenging conditions.The Gata2R396Q/+ mice showed an abnormal distribution of the immature progenitors Lin - Sca-1 + Kit + (LSK) subpopulations, mainly defines by an increase of the number of aberrant Long-Term Hematopoietic Stem cells (LT-HSC) contrasting with the decrease of LT-HSC population in Gata2+/- mouse model. Functional assays on Gata2R396Q/+ LSK cells revealed also qualitative defects. Indeed, their reconstitution capacity and their response to inflammatory or chemotherapy stimuli seemed to be largely affected. To address at the molecular level the impact of the missense mutation in these progenitors, we combined gene expression analysis with chromatin gene accessibility. These analyses revealed a strong disruption of the cells' ability to respond to stimuli, such as IFN or TNFα signaling, confirming what we observed at the cellular level in functional assays. Furthermore, specific RNA sequencing of the LT-HSC, ST-HSC and MPP3-4 subpopulations showed that the majority of these molecular perturbations are detected in every subpopulation of the LSK cells. In addition, we are able to establish a precise genetic signature in LT-HSC that may be related to their major functional defects.Combination of in vitro and in vivo approaches leads to improve our understanding of GATA2 deficiency syndrome. Modelization of the most recurrent germline Gata2 mutation revealed a different phenotype than the haploinsufficient mouse model. Furthermore, expression of Gata2 R396Q mutation qualitatively impacts the LSK compartment mimicking functional and molecular defects observed in the GATA2 deficiency patients. DisclosuresDelabesse: Novartis: Consultancy; Astellas: Consultancy. Pasquet: Novartis: Consultancy.

Spacing Constraints of Neighboring Zinc Finger Modules within GATA2

Genomic analyses in clinical and experimental contexts have accelerated discoveries of human genetic variants. While elucidating the consequences of conspicuously loss-of-function variants is highly tractable, decoding the impact of missense or non-coding variants is considerably more challenging. Previously, we described a germline variant in GATA2 in a patient with GATA2-deficiency syndrome, which inserts nine amino acids between the two zinc fingers (9aa-Ins), one of which mediates sequence-specific DNA binding (Cavalcante de Andrade Silva M. et al., Leukemia, 2021). Unlike other GATA2 coding region and enhancer variants identified (Bresnick E.H. et al., Blood Adv., 2020), it was unclear whether the altered zinc finger spacing would be inhibitory, stimulatory or of no consequence. The 9aa-Ins variant was defective in activating several target genes (Hdc, Ear2 and Tpsb2) in a genetic complementation assay with Gata2 -77 enhancer-mutant (-77 -/-) primary hematopoietic progenitor cells. As only several target genes were tested, we used RNA-seq to conduct a genome-wide comparison of the capacity of GATA2 and 9aa-Ins to activate and repress transcription. To elucidate mechanisms, we considered the following models: 1) 9aa-Ins fails to regulate all genes normally controlled by GATA2; 2) 9aa-Ins fails to repress all genes normally controlled by GATA2; 3) 9aa-Ins fails to activate genes normally controlled by GATA2; 4) 9aa-Ins ectopically regulates genes not controlled by GATA2. Using a genetic complementation approach with -77 -/- cells that were immortalized by the Hoxb8 transcription factor (hi-77 -/-) (Wang G.G. et al., Nat. Methods, 2006; Johnson K.D. et al., JEM, 2020), we compared GATA2 and 9aa-Ins activities when expressed at a comparable level. This analysis revealed 2,138 GATA2-regulated, 525 GATA2 and 9aa-Ins-regulated, and 414 ectopically-regulated genes (at least two-fold change, adjusted P-value <0.05). A similar number of genes were GATA2-activated (1,061) and repressed (1,077). Only 144 out of the 1,061 (14%) were 9aa-Ins-activated and 381 out of 1,077 (35%) were 9aa-Ins-repressed, illustrating the severe consequences of this mutation and a greater impact on activation versus repression. Statistical analysis with a range of P-values constraints (0.01 to 0.1) verified that activation by 9aa-Ins was more significantly impaired than repression (86% were no longer activated, and 65% were no longer repressed, P = 5.4 x 10 -6). Gene ontology analysis revealed that the 9aa insertion impaired GATA2-mediated activation of genes related to GPCR signaling and GATA2-mediated repression of genes related to innate immune machinery. The ectopically-regulated genes did not conform to a particular mechanism or pathway.Since it was unclear whether the transcriptional defects of the 9aa-Ins mutant reflect a unique inhibitory activity imparted by the 9aa sequence, we systematically varied the inter-zinc finger spacer length to establish whether any alterations can be tolerated. Using the genetic complementation assay, 2, 4, 6, 8, and 9aa spacer variants were compared with GATA2 for their capacity to regulate GATA2-activated genes (Hdc, Il1rl1, Gata1 and S1pr1) and repressed genes (Irf8, Il6st, Il6ra and Tifab). GATA2-mediated activation was compromised by insertions of two amino acids or more, whereas repression tolerated two and four amino acid insertions; 6, 8 and 9 were more inhibitory. Quantitative analyses revealed that a 6aa insertion reduced activation of the GATA2-activated genes by >50% of the wild type value, whereas the GATA2-repressed genes were still repressed by at least 50% (18% retention of activation and 83% retention of repression, P = 0.001). Thus, zinc finger spacing alterations differentially impacted activation versus repression. These results provide a rigorous foundation for interpreting variants that alter zinc finger spacing without disrupting vital finger residues. In vitro and in vivo functional analyses and molecular modeling are ongoing to further dissect the underlying mechanisms and ascertain the importance of genetic networks and circuits that are sensitive or resistant to human disease variants. DisclosuresNo relevant conflicts of interest to declare.

Conditionally Pathogenic Genetic Variants of a Hematopoietic Disease-Suppressing Enhancer

Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis.Given the propensity of GATA2 deficiency syndrome patients to infection, we asked if CH mice could mount an appropriate response to the bacterial cell wall component LPS. In WT, LPS acts on HSCs to induce inflammatory signaling and proliferation (Caiado et al., 2021). Twenty-four hours after LPS (0.5 mg/kg) administration, CH LSK levels were 3.1-fold lower than WT (p = 0.009). Immunophenotypic LT-HSCs (LSKCD150 +CD34 -) were 1.9-fold lower in treated CH vs. WT mice (p = 0.019). LPS reduced all LSK subpopulations: CD34 -CD150 -, CD34 +CD150 -, and CD34 +CD150 + were 3.4-, 5.7-, and 5.3-fold lower, respectively (p = 0.007, 0.029, and 0.006). Thus, the CH mutation attenuated acute inflammation-induced HSPC expansion.G-CSF is among the cytokines/chemokines induced by infection (Quinton et al., 2002). As G-CSF is used to mobilize bone marrow HSCs for collection prior to transplantation, we tested the following models: 1) +9.5 mutation abrogates HSPC expansion and/or mobilization in response to diverse stimuli; 2) +9.5 mutation permits expansion/mobilization, yet compromises function; 3) HSPC expansion, mobilization and function are +9.5 mutation-insensitive. After 8 doses of G-CSF, circulating neutrophil numbers increased 2.6-fold in WT (p = 0.003), while neutrophils did not increase in the CH mice. G-CSF treatment increased bone marrow HSCs 2.6-fold in WT mice, whereas the CH mutation abrogated this response (p < 0.0001). As a metric of mobilization, splenic HSCs were quantified. After G-CSF treatment, WT splenic HSCs expanded 11-fold (p < 0.0001), while CH HSCs were unchanged. To functionally analyze mobilized HSPCs, colony forming unit (CFU) assays were conducted with peripheral blood. CFU increased 11-fold in WT blood post-treatment (p < 0.0001), and CH abrogated the increase.To test whether G-CSF signaling is impacted by the CH mutation, we asked whether G-CSF treatment of bone marrow ex vivo induced STAT3 phosphorylation using a phospho-flow cytometry assay. While G-CSF induced pSTAT3 8.5-fold in WT Lin -Kit + cells (p < 0.0001), the induction was attenuated to 4.9-fold in CH (p = 0.003). Signaling in Gr1 + granulocytes was indistinguishable between genotypes (p = 0.999). Thus, the CH mutation attenuated G-CSF-induced signaling and HSPC mobilization, abrogated HSPC expansion, and compromised HSPC function.The Gata2 CH bone marrow failure model is characterized by relatively normal developmental hematopoiesis and adult steady-state hematopoiesis, yet the mice are vulnerable to stressors. As the CH mice were hypersensitive to myeloablation and acute inflammation and exhibited an attenuated response to a clinical HSC-mobilizing agent, GATA2-driven regenerative hematopoiesis mitigates damage resulting from chemotherapy and inflammation and promotes G-CSF-induced HSC mobilization. As the enhancer variant controlled HSC responsiveness to a therapeutically important HSC-mobilizing drug, these results link non-coding genome variation with mobilization efficacy. Based on the prominent GATA2 activities described herein, we are investigating how the full ensemble of macromolecular components constituting the GATA2 regulatory network generate a vital protective system conferring resilience to the hematopoietic system in the context of diverse stressors and whether genetic variants in genes encoding components of these pathways resemble or differ from those of GATA2 variants. DisclosuresNo relevant conflicts of interest to declare.

Cover, Volume 42, Issue 11

The cover image is based on the Original Article GATA2 deficiency syndrome: A decade of discovery by Claire Homan et al., https://doi.org/10.1002/humu.24271.

GATA2 deficiency syndrome: A decade of discovery.

GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms, of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 480 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 240 of these confirmed to be familial and 24 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0–78 years) and myelodysplastic syndrome is the first diagnosis in 75% of these cases with acute myeloid leukemia in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss‐of‐function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing, and expression. In this review, we provide the first expert‐curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages

AbstractGermline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.

GATA2 Enhancer Modules Governing Hematopoietic Regeneration

The transcription factor GATA2 is essential for the generation and function of hematopoietic stem and progenitor cells (HSPCs), erythroid precursors, and endothelial cells. A conserved intronic GATA2 enhancer, 9.8 kb downstream of the transcriptional start site (+9.5 in the mouse), is mutated in patients with GATA2 deficiency syndrome. Patient mutations within this region include a c.1017+512del28 deletion, removing E-box and GATA motifs, c.1017+532T&amp;gt;A that disrupts the E-box, and, most frequently, C&amp;gt;T in a 3' Ets motif (c.1017+572C&amp;gt;T) (Soukup and Bresnick, 2020). Homozygous mutation of the Ets motif in mice allows normal developmental and steady-state hematopoiesis but impairs hematopoietic regeneration (Soukup et al., 2019). In addition to HSPCs, GATA2 is expressed in non-hematopoietic cells in the bone marrow niche, e.g. endothelial cells and neurons (Katsumura et al., 2017). As the +9.5(Ets) mutation is not hematopoietic cell-specific, we asked whether regenerative defects of +9.5(Ets)-/- mice reflect disruption of cell-intrinsic or -extrinsic activities. In a competitive transplant assay, +9.5(Ets)-/- HSPCs were 3-fold less effective at long-term reconstitution than WT, and mechanistic studies indicated that the motif functions in hematopoietic cells to promote regeneration (Soukup et al., 2019). We conducted a reciprocal transplant of WT HSPCs into irradiated WT or +9.5(Ets)-/- recipients and quantified reconstitution by peripheral blood counts 4, 8, 12, and 16 weeks post-transplant. This analysis revealed no significant differences between WT and mutant recipients. At week 16, donor-derived leukocytes were 92% (+9.5(Ets)-/- recipients) and 96% (WT recipients) of total; the contribution did not differ significantly at any time. After 16 weeks, animals were sacrificed and HSPCs analyzed, confirming no significant alterations in mutant recipients. These results rigorously establish the mutant microenvironment as competent to support WT HSPC functions, emphasizing the critical hematopoietic cell-intrinsic activity of the +9.5 Ets motif. As the +9.5 Ets motif promotes regenerative hematopoiesis, and the +9.5 E-box;GATA is essential for developmental hematopoiesis, we devised a strategy to leverage these activities to innovate new models for GATA2 function in adult HSPCs. We generated compound heterozygous (CH) mice containing a mutant E-box;GATA sequence on one allele and a mutant Ets motif on the other allele. CH mice survived past weaning, with adults exhibiting significant steady-state defects, including a 4.4-fold decrease in GATA2hi megakaryocytes (p &amp;lt; 0.0001) and 20% decrease (p = 0.02) in platelets. To test whether the CH mutations compromise regeneration, we quantified HSPC populations in bone marrow from mice treated with vehicle or 5-fluorouracil (FU) 9- and 10- days post treatment. Steady-state HSC (Lin−Sca1+Kit+CD48-CD150+) levels were unaltered in CH animals. Days 9 and 10 post-FU treatment, WT HSC levels increased 17- (p = 0.0006) and 18-fold (p = 0.0007) relative to vehicle-treated animals. CH HSCs did not expand and were &amp;lt;10% of the steady-state level. 7 days post-FU treatment, Gata2 expression increased 1.9-fold in WT HSCs (p = 0.029); this response was abrogated in CHs. We asked if CH HSCs were capable of reconstitution in a competitive transplant assay. Four weeks post-transplant, CH progeny were 40-fold lower than WT (p &amp;lt; 0.0001). At 8-, 12-, and 16-weeks post-transplant, CH contribution was reduced 90-, 266-, and 280-fold, respectively. Defects persisted upon secondary transplantation, demonstrating that the defects cannot be restored by passage through a WT microenvironment. Thus, CH and +9.5(Ets)-/- mice share phenotypes, but CH mutations more severely impair regeneration and long-term reconstituting activity. This supports a paradigm in which the Ets motif and additional +9.5 sequences are critical for regeneration. This study revealed molecular determinants for steady-state and regenerative enhancer functions to enable discovery of +9.5-like enhancers with common operating mechanisms. We predict that such enhancers reside at a GATA2-regulated gene cohort, including genes that will reveal new mechanisms in hematopoiesis. As CH mice are poised for hematopoietic collapse, but can be propagated as relatively normal adults, studies are underway with this unique model to identify triggers of bone marrow failure and leukemogenesis. Disclosures No relevant conflicts of interest to declare.

3115 – GATA2A DETERMINES THE SURVIVAL AND HOMEOSTATIC LINEAGE OUTPUT OF HAEMATOPOIETIC STEM AND PROGENITOR CELLS

The transcription factor Gata2 is key regulator of haematopoietic stem and progenitor cells (HSPCs) during embryonic development of vertebrates. We took advantage of a partial genome duplication in zebrafish that lead to the appearance of two Gata2 genes, Gata2a and Gata2b, to address how Gata2 programmes the haemogenic endothelium (HE) towards the HSPC cell fate. Through deletion of a conserved intronic enhancer (i4 enhancer) in the gata2a locus, we show that reducing gata2a levels in the endothelium leads to decreased expression of gata2b and runx1, thus demonstrating that Gata2a activity is required for HE programming in embryos. By contrast, loss of gata2b impaired cmyb expression in the HSPC population but did not affect HE programming, indicating that Gata2a programmes HE first and Gata2b modulates HSPC programming later. Single cell transcriptional profiling of the adult kidney marrow revealed that gata2a and gata2b are expressed in HSPCs, but in different subsets. Strikingly, homozygous Gata2a mutants are viable and fertile, but show hypocellularity in the marrow niche and neutropenia, resembling GATA2 deficiency syndrome in humans. Gata2a enhancer mutants show fewer HSPCs and a lineage skewing towards the erythroid and B cell fates at the expense of the neutrophilic and monocytic lineages. These differences are already detectable in HSPCs. In addition, myeloid lineages in gata2a enhancer mutants ectopically express B- and erythroid lineage markers and fail to express lineage-specific genes at wild type levels, suggesting that Gata2a activity in HSPCs initiates and maintains lineage commitment in haematopoietic cells.

Single-nucleotide human disease mutation inactivates a blood-regenerative GATA2 enhancer.

The development and function of stem and progenitor cells that produce blood cells are vital in physiology. GATA-binding protein 2 (GATA2) mutations cause GATA-2 deficiency syndrome involving immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA-2 physiological activities necessitate that it be strictly regulated, and cell type-specific enhancers fulfill this role. The +9.5 intronic enhancer harbors multiple conserved cis-elements, and germline mutations of these cis-elements are pathogenic in humans. Since mechanisms underlying how GATA2 enhancer disease mutations impact hematopoiesis and pathology are unclear, we generated mouse models of the enhancer mutations. While a multi-motif mutant was embryonically lethal, a single-nucleotide Ets motif mutant was viable, and steady-state hematopoiesis was normal. However, the Ets motif mutation abrogated stem/progenitor cell regeneration following stress. These results reveal a new mechanism in human genetics, in which a disease predisposition mutation inactivates enhancer regenerative activity, while sparing developmental activity. Mutational sensitization to stress that instigates hematopoietic failure constitutes a paradigm for GATA-2 deficiency syndrome and other contexts of GATA-2-dependent pathogenesis.