GATA2-deficient Patients Research Articles

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.

GATA2-TAL1 Axis Is Essential for Human NK Cell Development and Function

Introduction: Natural killer (NK) cells, as the major subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity (1-3). Since NK cells are not limited by clonotypic receptors, they can be utilized in cell therapies against a broad spectrum of malignancies. Irrespective of the clinical potentials, the transcriptional regulation of the development and functions of human NK cells is far from fully understood. A dysregulated NK cell development is a hallmark of GATA2 haploinsufficiency (4)(eg. GATA2 T354M mutation). The specific loss of the CD56 bright NK cell population is a significant striking feature in GATA2-deficient patients (4), with or without reduced total CD56 dim NK cells (4, 12, 13). Consequently, the effector functions of these NK cells are significantly reduced, including inflammatory cytokine production and anti-tumor cytotoxicity (4, 12). However, a mechanistic understanding of GATA2 functions is lacking. In humans, GATA2 forms a core heptad complex with six other transcription factors (TF), including TAL1, regulating more than 1000 target genes. However, the unique functions of the GATA2-TAL1 axis in immature and mature NK cell has not been established. Based on these, we hypothesize that GATA2-dependent and independent functions of TAL1 play an essential role in the development and functions of human NK cells. Results: To explore the mechanism of co-transcriptional regulation of GATA2 and TAL1, we transduced NK-92 cells with lentiviral vectors containing GATA2 WT or GATA2 T354M plasmid and quantified TAL1 mRNA levels by qPCR and protein levels by WB. TAL1 is upregulated in GATA2 T354M-expressing NK-92 cells both at mRNA ( Fig. 1A) and protein levels ( Fig. 1B). TAL1 is a TF activating GTPase of immunity-associated protein (GIMAP) family super-enhancer regions in HSC and T cells (11). As TAL1 target genes, we further quantify GIMAP mRNA levels by qPCR. The result indicates that genes from the GIMAP family were notably upregulated in GATA2 T354M-expressing NK-92 cells ( Fig. 1C). Knocking down TAL1 results in the significant downregulation of TAL1 and GIMAPs in GATA2 T354M-expressingcells ( Fig. 1D). To further validate the GATA2-TAL1 axis, sorted NK cells from PBMCs of three GATA2 T354M patients and five age-matched healthy controls (HC) were analyzed with single-cell RNA-seq. We identified the genes from GIMAP family were significantly upregulated in all three GATA2 T354M patients. The representative data from one patient is shown in Fig. 1E. To explore how GATA2 and TAL1 mediate transcriptional regulation, we performed GATA2 CUT&Tag experiments with human NK cells, in which TAL1 motif was predominately enriched in 63.2% of GATA2 targets among NK cells through HOMER motif enrichment analyses ( Fig. 1F). On the other hand, the TAL1 ChIP-seq data from the K562 cells (download from ENCFF101DBG) reveals that the GATA2 motif is enriched in approximately 44% of TAL1 targets ( Fig. 1G). Moreover, both GATA2 and TAL1 expression in NK cells are modified by H3K27Me3 instead of H3K4Me3, suggesting a repressive methylenation signature in their expression ( Fig. 1H). Conclusion: Based on this study, we uncover a novel role for the GATA2-TAL1 axis in human NK cells. we define the transcriptional regulation role of GATA2 and TAL1 complex and provide direct evidence for the mechanism of NK cell deficiency among GATA2 T354M patients. Importantly, through our current study, we will determine the unique role of TAL1 in NK development and function and its transcriptional network.

Applicability of T cell receptor repertoire sequencing analysis to unbalanced clinical samples - comparing the T cell receptor repertoire of GATA2 deficient patients and healthy controls.

T cell receptor repertoire sequencing (TCRseq) has become one of the major omic tools to study the immune system in health and disease. Multiple commercial solutions are currently available, greatly facilitating the implementation of this complex method into translational studies. However, the flexibility of these methods to react to suboptimal sample material is still limited. In a clinical research context, limited sample availability and/or unbalanced sample material can negatively impact the feasibility and quality of such analyses. We sequenced the T cell receptor repertoires of three healthy controls and four patients with GATA2 deficiency using a commercially available TCRseq kit and thereby (1) assessed the impact of suboptimal sample quality and (2) implemented a subsampling strategy to react to biased sample input quantity. Applying these strategies, we did not find significant differences in the global T cell receptor repertoire characteristics such as V and J gene usage, CDR3 junction length and repertoire diversity of GATA2-deficient patients compared with healthy control samples. Our results prove the adaptability of this TCRseq protocol to the analysis of unbalanced sample material and provide encouraging evidence for use of this method in future studies despite suboptimal patient samples.

Somatic genetic alterations predict hematological progression in GATA2 deficiency.

Germline GATA2 mutations predispose to myeloid malignancies resulting from the progressive acquisition of additional somatic mutations. Here we describe clinical and biological features of 78 GATA2-deficient patients. Hematopoietic stem and progenitor cell phenotypic characterization revealed an exhaustion of myeloid progenitors. Somatic mutations in STAG2, ASXL1 and SETBP1 genes along with cytogenetic abnormalities (monosomy 7, trisomy 8, der(1;7)) occurred frequently in patients with GATA2 germline mutations. Patients were classified into three hematopoietic spectra based on bone marrow cytomorphology. No somatic additional mutations were detected in patients with normal bone marrow (spectrum 0), whereas clonal hematopoiesis mediated by STAG2 mutations was frequent in those with a hypocellular and/or myelodysplastic bone marrow without excess blasts (spectrum 1). Finally, SETBP1, RAS pathway and RUNX1 mutations were predominantly associated with leukemic transformation stage (spectrum 2), highlighting their implications in the transformation process. Specific somatic alterations, potentially providing distinct selective advantages to affected cells, are therefore associated with the clinical/hematological evolution of GATA2 syndrome. Our study not only suggests that somatic genetic profiling will help clinicians in their management of patients, but will also clarify the mechanism of leukemogenesis in the context of germline GATA2 mutations.

Prospective and Retrospective Observational Study in Adult and Pediatric Patients with GATA2 Variants (GATA2 Deficiency)

Heterozygous germline GATA2 variants are associated with a broad spectrum of manifestations, encompassing immuno-haematological alterations, severe/recurrent infections and extra-haematological phenomena. We here describe 25 patients (11 males) with molecular diagnosis of GATA2 deficiency identified in 9 AIEOP (Associazione Italiana di Ematologia Oncologia Pediatrica) centers in Italy (Table 1). Patients' median age at onset of symptoms was 12.5 years. The main signs/symptoms at onset were severe/recurrent infections (40%), cytopenias (20%) and myelodysplasia (MDS)/acute myeloid leukemia (AML) (20%). The prevalence of these manifestations increased during the follow-up and further clinical manifestations, i.e.hepatosplenomegaly, haemorrhagic diathesis, thrombosis, bone marrow failure, lymphedema, pulmonary alveolar proteinosis and sensorineural deafness, occurred. Once the diagnosis was established, congenital deafness was considered part of the clinical picture of GATA2 deficiency in two individuals. We identified a rare (myelofibrosis) and two possible new manifestations (pilonidal cyst/sacro-coccygeal fistula and cholangiocarcinoma). Complete blood count with differential and lymphocyte subsests revealed that monocytopenia (83%) and CD19+ lymphocytopenia (70%) were the most frequent and distinctive changes in patients without hematological malignancies or in whom we obtained data before the onset of MDS/AML (Table 2). Different myeloid neoplasms were observed: refractory cytopenia in childhood (RCC) (24%), AML (12%), MDS with excess blasts (MDS-EB) (12%), MDS-EB in transformation (MDS-EBt) (4%) and MDS with multilineage dysplasia(MDS-MLD) (8%). Monosomy 7 (8%) and trisomy 8 (16%) were the two most frequent karyotype abnormalities. Targeted next-generation sequencing (NGS) analysis for somatic mutations associated with myeloid neoplasms was performed in 48% of the patients, resulting positive in 8% of cases (i.e. EZH2 and MECOM in one individual each). The majority of GATA2 variants were located at zinc-finger 2 (ZF2) domain of the GATA2 protein. Missense and null mutations were the most frequent (46% each). Intronic variants were found in 8% of the cohort. We report 3 missense mutations out of the 4 cases of lymphedema described.Median age at molecular diagnosis was 18 years. The diagnostic delay (time between age at onset of symptoms and molecular diagnosis) had a median of 4 years, but, excluding who had onset of the disease before the first description of GATA2 deficiency (2011) the median was 1 year. Hematopoietic stem cell transplantation (HSCT) was the most widely used therapeutic option (52%). HSCT was performed in case of myeloid neoplasia (8/13) and bone marrow failure (3/13), but also in patients with immunodeficiency (i.e. hypogammaglobulinemia with immunoglobulin replacement therapy, immunodeficiency with previous severe infections). Time between molecular diagnosis and HSCT was1 year. The event free survival (EFS) was 20% at the age of 20 and 15% at the age of 40. The overall survival (OS) was 90% at the age of 20 and 70% at the age of 40. Family screening was essential to identify asymptomatic patients (8% in our series). In conclusion, our series represents the third largest national cohort after the ones described by Donadieu and Spinner (79 and 57 patients, respectively)1,2. GATA2 deficiency should be included in targeted gene panels for congenital deafness and we recommend to perform the sequencing of the regulatory region. Our data disproves the hypothesis that lymphedema is exclusively associated with null and regulatory mutations3, as we here describe three patients with lymphedema and missense mutations. HSCT should be considered in case of patients with immunodeficiency without myeloid neoplasms. Family screening should be offered to first degree relatives, as identification of asymptomatic GATA2 deficient patients could allow to exclude potential HSC donors and to investigate risk factors that may explain the phenotypic difference.

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.

ASXL1 and STAG2 are common mutations in GATA2 deficiency patients with bone marrow disease and myelodysplastic syndrome

AbstractPatients with GATA2 deficiencyharbor de novo or inherited germline mutations in the GATA2 transcription factor gene, predisposing them to myeloid malignancies. There is considerable variation in disease progression, even among family members with the same mutation in GATA2. We investigated somatic mutations in 106 patients with GATA2 deficiency to identify acquired mutations that are associated with myeloid malignancies. Myelodysplastic syndrome (MDS) was the most common diagnosis (∼44%), followed by GATA2 bone marrow immunodeficiency disorder (G2BMID; ∼37%). Thirteen percent of the cohort had GATA2 mutations but displayed no disease manifestations. There were no correlations between age or sex with disease progression or survival. Cytogenetic analyses showed a high incidence of abnormalities (∼43%), notably trisomy 8 (∼23%) and monosomy 7 (∼12%), but the changes did not correlate with lower survival. Somatic mutations in ASXL1 and STAG2 were detected in ∼25% of patients, although the mutations were rarely concomitant. Mutations in DNMT3A were found in ∼10% of patients. These somatic mutations were found similarly in G2BMID and MDS, suggesting clonal hematopoiesis in early stages of disease, before the onset of MDS. ASXL1 mutations conferred a lower survival probability and were more prevalent in female patients. STAG2 mutations also conferred a lower survival probability, but did not show a statistically significant sex bias. There was a conspicuous absence of many commonly mutated genes associated with myeloid malignancies, including TET2, IDH1/2, and the splicing factor genes. Notably, somatic mutations in chromatin-related genes and cohesin genes characterized disease progression in GATA2 deficiency.

Germline GATA2 variant disrupting endothelial eNOS function and angiogenesis can be restored by c-Jun/AP-1 upregulation.

GATA2 is a transcription factor with key roles in hematopoiesis. Germline GATA2 gene variants have been associated with several inherited and acquired hematologic disorders, including myelodysplastic syndromes. Among the spectrum of GATA2 deficiency- associated manifestations thrombosis has been reported in 25% of patients, but the mechanisms are unknown. GATA2 was shown to be involved in endothelial nitric oxide synthase (eNOS) regulation and vascular development. We assessed eNOS expression and angiogenesis in patients with GATA2 deficiency. Platelets and blood outgrowth endothelial cells (BOEC) from GATA2 variant carriers showed impaired NO production and reduction of eNOS mRNA and protein expression and of eNOS activity. GATA2 binding to the eNOS gene was impaired in BOEC from GATA2-deficient patients, differently from control BOEC. GATA2 deficiency BOEC showed also defective angiogenesis, which was completely restored by treatment with the NO-donor Snitroso- N-acetylpenicillamine (SNAP). Atorvastatin, but not resveratrol, largely restored eNOS expression, NO biosynthesis and neoangiogenesis in GATA2-deficient BOEC by a mechanism involving increased expression of the eNOS transcription factor AP-1/c-JUN, replacing GATA2 when the latter is inactive. Our results unravel a possible thrombogenic mechanism of GATA2 mutations, definitely establish the regulation of eNOS by GATA2 in endothelial cells and show that endothelial angiogenesis is strictly dependent on the eNOS/NO axis. Given the ability of atorvastatin to restore NO production and angiogenesis by GATA2-deficient endothelial cells, the preventive effect of atorvastatin on thrombotic events and possibly on other clinical manifestations of the syndrome related to deranged angiogenesis should be explored in patients with GATA2 deficiency in an ad hoc designed clinical trial.

Allogeneic Hematopoietic Stem-Cell Transplantation for GATA2 Deficiency Using a Busulfan-Based Regimen

Background: GATA2 deficiency is an increasingly recognized bone marrow failure syndrome responsible for: MonoMAC, monocytopenia with atypical mycobacterial infection (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). GATA2 deficiency can be reversed by hematopoietic stem cell transplantation (HSCT). However, the intensity of conditioning required to ensure engraftment and reverse the myelodysplastic syndrome (MDS) characteristic of the disease remains controversial.Methods: We carried out a prospective study of allogeneic HSCT in 22 patients (mean age 26 years; range 17 to 45 years) with GATA2 deficiency, or the MonoMac syndrome, using a myeloablative, busulfan-based conditioning regimen. All 22 patients had MDS and two had AML. Eleven patients had clonal cytogenetic changes in the bone marrow. There were two matched related donor (MRD) recipients, 13 matched unrelated donor (URD) recipients, and 7 haploidentical related donor recipients. MRD and URD recipients received conditioning with 4 days of busulfan (days -6 to -3) to target an AUC between 3600 and 4800 min*microm/L and 4 days of fludarabine 40 mg/m2/day (days -6 to -3). Haploidentical related donor recipients received low-dose cyclophosphamide for two days (days -6 and -5), fludarabine 30 mg/m2/day for 5 days (days -6 to -2), 2-3 days of busulfan depending upon cytogenetics, along with 200 cGY total body irradiation (TBI) on day -1. MRD and URD recipients received tacrolimus and short course methotrexate for post-transplant graft-versus-host disease (GVHD) prophylaxis, whereas haploidentical recipients received high-dose post-transplant cyclophosphamide (PT/CY) followed by tacrolimus and mycophenolate mofetil for GVHD prophylaxis.Results: Nineteen of the 22 patients are alive with resolution of the clinical phenotype and correction of the bone marrow MDS with eradication of the cytogenetic changes at a median follow-up of 23 months (range, 6 to 48 months) giving an overall disease-free survival of 86 percent. The three deaths all occurred in the URD group. Of the three deaths, one resulted from refractory acute myelogenous leukemia (AML), and two from complications related to treatment for GVHD. There was a 26 percent incidence of grade III-IV acute GVHD (aGVHD) in the MRD and URD groups, and no grade III-IV aGVHD in the haploidentical related donor cohort. Similarly, there was a 46 percent incidence of chronic GVHD (cGVHD) in the MRD and URD cohort, whereas two of the 7 (28 percent) of the haploidentical related donor recipients had cGVHD.Conclusions: Despite overall disease-free survival of 86 percent with eradication of the clonal cytogenetic abnormalities in the bone marrow, GVHD remains a limitation in the MRD and URD cohorts treated with standard post-transplant tacrolimus/methotrexate prophylaxis for GVHD. We are currently de-escalating the busulfan dosing based on the AUC in all cohorts and incorporating PT/CY in the MRD and URD cohorts to decrease toxicity and reduce the incidence of aGVHD and cGVHD. Together with our previous studies using a nonmyeloablative regimen, it appears that when GATA2 deficiency patients are transplanted when they have a hypocellular bone marrow with MDS and without cytogenetic changes, a nonmyeloablative regimen results in reliable engraftment in GATA2 deficiency since the GATA2 deficient marrow has a proliferative disadvantage. However, with clonal progression and unfavorable cytogenetic changes and/or a hypercellular marrow, where the malignant clone has a proliferative advantage, a higher dose regimen results in more reliable engraftment and eradication of the clone. Studies to identify the precise level of conditioning for the stages of the disease are ongoing. DisclosuresNo relevant conflicts of interest to declare.

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. DisclosuresNo relevant conflicts of interest to declare.

Inherited GATA2 Deficiency Is Dominant by Haploinsufficiency and Displays Incomplete Clinical Penetrance.

Germline heterozygous mutations of GATA2 underlie a variety of hematological and clinical phenotypes. The genetic, immunological, and clinical features of GATA2-deficient patients with mycobacterial diseases in the familial context remain largely unknown. We enrolled 15 GATA2 index cases referred for mycobacterial disease. We describe their genetic and clinical features including their relatives. We identified 12 heterozygous GATA2 mutations, two of which had not been reported. Eight of these mutations were loss-of-function, and four were hypomorphic. None was dominant-negative in vitro, and the GATA2 locus was found to be subject to purifying selection, strongly suggesting a mechanism of haploinsufficiency. Three relatives of index cases had mycobacterial disease and were also heterozygous, resulting in 18 patients in total. Mycobacterial infection was the first clinical manifestation in 11 patients, at a mean age of 22.5years (range: 12 to 42years). Most patients also suffered from other infections, monocytopenia, or myelodysplasia. Strikingly, the clinical penetrance was incomplete (32.9% by age 40years), as 16 heterozygous relatives aged between 6 and 78years, including 4 older than 60years, were completely asymptomatic. Clinical penetrance for mycobacterial disease was found to be similar to other GATA2 deficiency-related manifestations. These observations suggest that other mechanisms contribute to the phenotypic expression of GATA2 deficiency. A diagnosis of autosomal dominant GATA2 deficiency should be considered in patients with mycobacterial infections and/or other GATA2 deficiency-related phenotypes at any age in life. Moreover, all direct relatives should be genotyped at the GATA2 locus.

Single-Cell RNA Sequencing Reveals a Distinct Transcriptome Signature of Hematopoiesis in GATA2 Deficiency

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes including systemic infections, lymphedema, cytopenias, myelodysplasia, and a high risk of developing myeloid leukemias. GATA2 deficiency is recognized as a major MDS predisposition syndrome, germline GATA2 mutations are found in 7% of primary MDS cases in children and adolescents. Monosomy 7 and trisomy 8 are frequent chromosomal abnormalities in GATA2 deficiency and features of disease prognosis and malignant transformation. GATA2 mutations mostly affect two zinc finger domains and broadly classify into three major categories: missense, null, and regulatory region mutations. Profound monocytopenia, B-cell and NK-cell lymphopenias, and dendritic cell deficiency are specific for GATA2 deficiency. Absence of multi-lymphoid progenitor and decreased granulocyte -macrophage progenitors occur early in disease. There are limited studies of gene expression profiles in GATA2 deficiency and these usually in bulk populations, partly due to the paucity of cells in patients. How GATA2 deficiency negatively affects hematopoiesis, especially the preferential loss of specific lineages, is poorly understood. 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 pathogenic GATA2 mutations and clinically manifest myelodysplasia (Fig A). We characterized transcriptomes in lineages, computationally defined cells with chromosomal abnormalities, and described gene expression of these cells. We first used gene expression signatures of single HSPCs from healthy donors to impute identity of stem cells or progenitors of erythroid/megakaryocytic (MEP), myeloid and lymphoid lineages, and then proceeded to reconstruct hematopoietic differentiation by pseudotemporal ordering. Mapping patients' cells onto normal hematopoiesis, we observed marked deficiency in granulocyte/monocyte/lymphocyte differentiation and relatively preserved MEP (Fig B, top). Based on signature genes of each differentiated lineage, we computed a score of gene expression to corresponding lineage in individual patient and found a significantly lower score for granulocyte/monocyte and B cell progenitors in patients, MEP scores tended to be higher in patients without significance (Fig B, bottom). Upregulated genes in patients' HSPCs related to erythroid differentiation, and cell cycle and proliferation (Fig C), while downregulated gene sets were highly enriched in immune responses (Fig D). Differential co-expressed genes were defined by weighted correlation network analysis; they were enriched in immune response and DNA repair (genes exhibited higher correlation in MTOR and T cell receptor signaling pathway in patients [Fig E]). We observed differential effects of GATA2 mutations in distinct lineages, which could only be resolved with the single cell method in rare cell populations. In HSCs, upregulated genes were related to apoptosis and downregulated genes were involved in maintenance of quiescence and cell cycle; there was increased expression of erythroid/megakaryocytic priming programs and decreased expression of lymphoid priming programs. We also observed lower GATA2 expression levels at initial stages of hematopoiesis, likely contributing to disturbed stem cell homeostasis in GATA2 deficiency. 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. In 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 is partly due to expression of aberrant gene programs in HSCs prior to lineage commitment. Disclosures No relevant conflicts of interest to declare.

Prognostic Value of IPSS-R Score, Cytogenetic Aberrations, and Cytopenias on Outcome of Allogeneic Hematopoietic Stem Cell Transplant in Myelodysplastic Syndrome in GATA2 Deficiency

Sporadic or familial germline mutations in GATA2 frequently result in a bone marrow failure syndrome characterized by recurrent life-threatening infections, cytopenias, and progression to myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), or chronic myelomonocytic leukemia (CMML). Hematopoietic stem cell transplantation (HSCT) represents the only definitive therapy for GATA2 deficiency. However, the highly variable disease penetrance in different families, and in mutation positive individuals within a single family, make the timing of HSCT challenging. We assessed the utility of scoring patient characteristics according to the Revised International Prognostic Scoring System (IPSS-R) to predict survival after HSCT in a cohort of 51 patients with GATA2 deficiency who had progressed to MDS. Higher IPSS-R scores significantly correlated with reduced overall survival after transplant. In evaluating the components of the IPSS-R score for patients with GATA2 deficiency, cytopenias, including anemia, neutropenia, and thrombocytopenia, were important variables in placing these patients in higher risk IPSS-R categories. Since GATA2 deficiency patients have very low peripheral blood monocyte, B cell, and NK cell counts, we evaluated these cytopenias in conjunction with IPSS-R scoring. These cytopenias also had prognostic value for outcome after HSCT in patients with GATA2 deficiency. Cytogenetics, another component of the IPSS-R score, also placed patients with GATA2 deficiency into higher risk categories. In particular, patients harboring monosomy 7 had poorer survival outcomes than patients with normal cytogenetics or trisomy 8. Of note, all five patients with GATA2 deficiency and trisomy 1q successfully underwent HSCT. Clonal cytogenetic progression with the development of additional cytogenetic aberrations was a harbinger of myeloid progression and led to HSCT. In a cohort of four patients who underwent HSCT following progression to AML/CMML, there was only one survivor. Thus, HSCT is recommended in patients with GATA2 deficiency and intermediate or high-risk IPSS-R scores before the development of AML or CMML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2535: Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients

Abstract GATA2 is an essential transcription factor in the proliferation and maintenance of hematopoietic stem and progenitor cells. Germline GATA2 mutations cause hematological malignancies, especially myelodysplastic syndrome and acute myeloid leukemia. Pre-leukemic phenotype of GATA2 deficient patients varies markedly. However, peripheral blood (PB) lymphopenia is characteristic for almost all the patients and encounters most prominently B and natural killer (NK) cells. We aimed to gain understanding on the effects of GATA2 deficiency in hematopoiesis by comparing expression patterns in lymphocytes in GATA2 wild type and GATA2 deficient individuals. We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution on PB samples from three healthy controls and three GATA2 deficient patients (p.Q328X, p.T354M, and one haploinsufficiency due to unidentified reason). Erythrocytes were lysed prior to washing, filtering and resuspending white blood cells in 0.04% BSA/PBS (1x106 cells/mL). The gel beads in emulsion were generated using Chromium Single Cell 3' v2 Reagent Kit. The sample libraries, prepared according to 10x Genomics instructions, were sequenced in Illumina HiSeq 2500. Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3' RNA-sequencing barcode; read data to FASTQ files; and generate align reads and gene-cell matrices. The data were analyzed using Seurat, an R package designed for single cell analysis. Cell type clusters were identified through known markers from Seurat's graph-based clustering. Significant gene expression differences in lymphocytes (GATA2 deficient patients vs. controls) were observed (Wilcoxon rank sum test, Bonferroni-corrected p <0.05). In NK cells the distinction was found e.g. in KLRB1, CD74, CCL5, STAT1, and LAG3, all linked to immune responses, and BTG1 and DDIT4, regulating cell proliferation and differentiation. Expression changes in immune system-associated genes were also noted in T cells e.g. in STAT1, IL7R, CD48, and CXCR4, as well as in NPM1, TPT1, and TXNIP, genes identified to participate in diverse cellular processes. Also, several genes encoding ribosomal proteins were differentially expressed between patients' and controls' T cells. Comparison of B cells revealed changes in e.g. GZMH, PPBP, and LCN2, which, too, play roles in the immune system. We observed significant dissimilarities in gene expression pattern in GATA2 deficient patients' vs. controls' lymphocytes. These were reflected in several genes involved in immune system and cellular processes. Connection between our findings and patients' clinical manifestations is intriguing and may have an impact in the development of future patient care. We will extend our material and include also bone marrow sample single-cell transcriptomes and healthy mutation carriers to the analysis. Citation Format: Elina A. Hirvonen, Inari Natri, Pirkko Mattila, Jenni Mattila, Esa Pitkänen, Kimmo Porkka, Outi Kilpivaara, Ulla Wartiovaara-Kautto. Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2535.

Bacille Calmette-Guerin Complications in Newly Described Primary Immunodeficiency Diseases: 2010-2017.

Bacille Calmette–Guerin (BCG) vaccine is widely used as a prevention strategy against tuberculosis. BCG is a live vaccine, usually given early in life in most countries. While safe to most recipients, it poses a risk to immunocompromised patients. Several primary immunodeficiency diseases (PIDD) have been classically associated with complications related to BCG vaccine. However, a number of new inborn errors of immunity have been described lately in which little is known about adverse reactions following BCG vaccination. The aim of this review is to summarize the existing data on BCG-related complications in patients diagnosed with PIDD described since 2010. When BCG vaccination status or complications were not specifically addressed in those manuscripts, we directly contacted the corresponding authors for further clarification. We also analyzed data on other mycobacterial infections in these patients. Based on our analysis, around 8% of patients with gain-of-function mutations in STAT1 had mycobacterial infections, including localized complications in 3 and disseminated disease in 4 out of 19 BCG-vaccinated patients. Localized BCG reactions were also frequent in activated PI3Kδ syndrome type 1 (3/10) and type 2 (2/18) vaccinated children. Also, of note, no BCG-related complications have been described in either CTLA4 or LRBA protein-deficient patients; and not enough information on BCG-vaccinated NFKB1 or NFKB2-deficient patients was available to drive any conclusions about these diseases. Despite the high prevalence of environmental mycobacterial infections in GATA2-deficient patients, only one case of BCG reaction has been reported in a patient who developed disseminated disease. In conclusion, BCG complications could be expected in some particular, recently described PIDD and it remains a preventable risk factor for pediatric PIDD patients.

Melanoma in patients with GATA2 deficiency.

GATA2 deficiency is a recently described genetic disorder affecting hematopoietic stem cells and is associated with immunodeficiency, hematologic malignancy, and various cutaneous pathologies including cutaneous tumors. To explore the incidence and clinical course of melanoma in patients with germline GATA2 deficiencies, we conducted a retrospective chart review of 71 such patients and identified two with invasive melanoma. One melanoma was diagnosed early because it was associated with pruritus due to a graft-versus-tumor effect following bone marrow transplantation. The other one, a lentigo maligna melanoma, was locally excised but progressed to widespread metastasis and death several years later. Our observations and published studies of melanoma biology suggest an association between decreased GATA2 expression and melanoma progression. These findings suggest that GATA2 deficient patients may have an increased risk of melanoma and should be observed closely for new or changing skin lesions.

Acquired Senescent T-Cell Phenotype Correlates with Clinical Severity in GATA Binding Protein 2-Deficient Patients.

GATA binding protein 2 (GATA2) deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity caused by spontaneous or autosomal dominant mutations in the GATA2 gene. Clinical manifestations range from neutropenia, lymphedema, deafness, to severe viral and mycobacterial infections, bone marrow failure, and acute myeloid leukemia. Patients also present with monocytopenia, dendritic cell, B- and natural killer (NK)-cell deficiency. We studied the T-cell and NK-cell compartments of four GATA2-deficient patients to assess if changes in these lymphocyte populations could be correlated with clinical phenotype. Patients with more severe clinical complications demonstrated a senescent T-cell phenotype whereas patients with lower clinical score had undetectable changes relative to controls. In contrast, patients’ NK-cells demonstrated an immature/activated phenotype that did not correlate with clinical score, suggesting an intrinsic NK-cell defect. These studies will help us to determine the contribution of T- and NK-cell dysregulation to the clinical phenotype of GATA2 patients, and may help to establish the most accurate therapeutic options for these patients. Asymptomatic patients may be taken into consideration for hematopoietic stem cell transplantation when dysregulation of T-cell and NK-cell compartment is present.

Heterogeneity of GATA2-related myeloid neoplasms.

The GATA2 gene codes for a master hematopoietic transcription factor that is essential for the proliferation and maintenance of hematopoietic stem and progenitor cells. Heterozygous germline mutations in GATA2 have been initially associated with several clinical entities that are now collectively defined as GATA2 deficiency. Despite pleiotropic clinical manifestations, the high propensity for the development of myelodysplastic syndromes (MDS) constitutes the most common clinical denominator of this major MDS predisposition syndrome. The immunological phenotypes can be variable and mostly include deficiency of monocytes and/or B cells. Thus far, nearly 380 GATA2-deficient patients had been reported, with a roughly estimated prevalence of myeloid neoplasia of at least 75%. The most common abnormal karyotypes associated with GATA2-related MDS are monosomy 7, der(1;7) and trisomy 8. The overall clinical penetrance seems to be nearly complete for this transcriptopathy disorder. The high-risk MDS subtypes and karyotypes, and the underlying immunodeficiency guide decision-making toward timely stem cell transplantation.

GATA2 deficiency and related myeloid neoplasms

The GATA2 gene codes for a hematopoietic transcription factor that through its two zinc fingers (ZF) can occupy GATA-DNA motifs in a countless number of genes. It is crucial for the proliferation and maintenance of hematopoietic stem cells. During the past 5 years, germline heterozygous mutations in GATA2 were reported in several hundred patients with various phenotypes ranging from mild cytopenia to severe immunodeficiency involving B cells, natural killer cells, CD4+ cells, monocytes and dendritic cells (MonoMAC/DCML), and myeloid neoplasia. Some patients additionally show syndromic features such as congenital deafness and lymphedema (originally defining the Emberger syndrome) or pulmonary disease and vascular problems. The common clinical denominator in all reported cohorts is the propensity for myeloid neoplasia (myelodysplastic syndrome [MDS], myeloproliferative neoplasms [MPN], chronic myelomonocytic leukemia [CMML], acute myeloid leukemia [AML]) with an overall prevalence of approximately 75% and a median age of onset of roughly 20 years. Three major mutational types are encountered in GATA2-deficient patients: truncating mutations prior to ZF2, missense mutations within ZF2, and noncoding variants in the +9.5kb regulatory region of GATA2. Recurrent somatic lesions comprise monosomy 7 and trisomy 8 karyotypes and mutations in SETBP1 and ASXL1 genes. The high risk for progression to advanced myeloid neoplasia and life-threatening infectious complications guide decision-making towards timely stem cell transplantation.

GATA2 Deficiency Causes Severe, Persistent HPV Infections [19E

INTRODUCTION: Immunocompromised females are at increased risk for recurrent, multifocal Human Papillomavirus (HPV)-associated dysplasia and malignancy. Deficiency of GATA2, a hematopoietic transcription factor, is associated with myeloid malignancy, chronic leukocytopenia and intracellular infections. We sought to determine the risk of persistent, severe HPV disease in GATA2 deficient female patients. METHODS: NIH medical records of females with GATA2 deficiency, including laboratories, histopathology and cytopathology were retrospectively reviewed. RESULTS: Of 35 females with GATA2 deficiency, 27 (77%) were diagnosed with HPV: 23 (66%) with genital warts and 19 (54%) with extragenital warts. Median age of onset was 12 y (r: 6–39 y) for genital warts and 10 y (r: 6–39 y) for extragenital warts. Overall 18 (67%) of the HPV+ patients developed dysplasia: vulvar in 12 (67%) and cervical in 15 (83%). Median age of diagnosis for dysplasia was 27 y (r: 15–59 y). Median age of genital cancer diagnosis was 34 y (r: 22–40 y). 83% of patients with dysplasia (n=15) had laser ablation or surgical excision, undergoing multiple procedures (r: 1–40) by the age of 40. CONCLUSION: GATA2 deficiency causes persistent, severe, multifocal HPV disease in women. GATA2 deficiency requires earlier, more frequent HPV disease surveillance to prevent disease progression and thereby limit the need for repeated surgery. GATA2 deficiency should be considered in young women with unusually severe HPV disease.