GATA2 Germline Mutations Impair GATA2 Transcription, Causing Haploinsufficiency: Functional Analysis of the p.Arg396Gln Mutation

The GATA2 transcription factor has an essential role in the proliferation and differentiation of hematopoietic cells (1). Recently, germline mutations in GATA2 have been identified as the cause of familial syndromes with autosomal-dominant inheritance that share common symptoms. Severe monocytopenia, NK and B lymphopenia, near absence of dendritic cells, and a predisposition to develop myelodysplastic syndrome, chronic myelomonocytic leukemia, and/or acute myeloid leukemia (AML) are among the most prominent characteristics of these diseases (2–6).

So far, 50 different germline GATA2 mutations have been reported; most families display mutations that are located within the highly conserved C-terminal zinc finger (ZF) domain, and they correspond to missense mutations (Supplemental Tables I, II). It has been reported that mutations in a conserved intronic enhancer element lead to decreased GATA2 transcript levels, indicating the necessity for both alleles to be functional to express the wild-type (WT) phenotype, and suggesting GATA2 deficiency as the basis for these familial syndromes (7). In fact, Gata2^+/− mice express reduced levels of Gata2 in hematopoietic cells, and this has an impact on hematopoietic stem and progenitor cell (HSPC) homeostasis (8, 9). However, there is a considerable clinical heterogeneity among patients (5), and GATA2 haploinsufficiency has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations affecting the C-terminal ZF, which are thought to allow production of a stable mRNA that is translated into an abnormal protein, could affect the transcription of GATA2, triggering GATA2 deficiency. In this study, we show that the human GATA2 protein activates its own transcription and that three germline GATA2 mutations affecting the C-terminal ZF impair GATA2 promoter activation. Moreover, we functionally analyze p.Arg396Gln, the second most common mutation in GATA2 deficiency syndromes, in the context of the clinical presentation of a patient with this mutation.

HEL, TF1, MOLM13, and HeLa cell lines were cultured following the German Collection of Microorganisms and Cell Cultures Cell Culture Bank recommendations (Braunschweig, Germany). Peripheral blood samples from patient and five healthy donors were obtained after written informed consent by the University Clinic of Navarra. DNA was sequenced using specific primers for each GATA2 exon in a 3500DX genetic analyzer (Life Technologies, Carlsbad, CA). PBMCs were extracted for analysis using the Ficoll protocol.

Total RNA was extracted from freshly isolated PBMCs of individuals or cell lines using the TRIzol reagent (Life Technologies). Quantitative real-time RT-PCR (qRT-PCR) was performed in the ABI Prism 7500 (Life Technologies) using SYBR Green master mix or TaqMan master mix (Life Technologies) with specific primers (sequences are available upon request). HPRT1 or GAPDH expression was used as an internal control for SYBR or TaqMan methods, respectively. Data were analyzed using the comparative cycle threshold (ΔΔCt) method.

Chromatin immunoprecipitation (ChIP) assays in HEL, TF1, and MOLM13 cells were performed as previously reported, using a GATA2 Ab (R&D Systems, Minneapolis, MN) or a nonspecific anti-goat IgG (Cell Signaling Technology, Beverly, MA) (10). In the case of HeLa cells, 5 × 10⁶ cells were transfected with 3 μg pCMV6-XL6 vector (empty, WT-GATA2, or p.Arg396Gln) using Lipofectamine 2000 (Life Technologies), and ChIP was performed as previously reported, using the above mentioned Abs (11). Quantitative PCR was used to analyze ChIP resulting material using an unrelated genomic region of GAPDH as negative control (Diagenode, Denville, NJ). Fold enrichment value was calculated comparing GATA2 immunoprecipitated and nonspecific IgG immunoprecipitated samples.

GATA2 regulatory regions were inserted in the pGL3-Basic vector (Promega, Fitchburg, WI). GATA2 promoter and GATA2-expressing vector pCMV6-XL6-GATA2 (Origene, Rockville, MD) were mutated using a QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer’s protocol.

Luciferase reporter assays in HEL and HeLa cells were performed as previously reported (10). Each GATA2 promoter construct, the corresponding pCMV6-XL6 vector (only for HeLa cells), and pRL-SV40 (Promega) as internal control were transfected.

The model of chicken GATA1 C-terminal ZF was downloaded from the Protein Data Bank (2GAT). A GATA2 C-terminal ZF model was created using SWISS-MODEL software (http://swissmodel.expasy.org). Images were created using the Swiss Protein Data Bank viewer software (http://spdbv.vital-it.ch).

Retroviral particles were produced transfecting AmphoPack 293 cells with pMD2.G envelope vector and an empty, GATA2, or p.Arg396Gln cDNA containing pBabe-puro (12) or pLRT (13) vectors, using FuGene (Promega). Particles were harvested 48-72 h after transfection, 0.45-μm filtered, and used immediately for transduction.

HL60 cells (10⁷) were infected on 2 consecutive days with pLRT vector–based particles by centrifugation at 3000 × g for 3 h at 32°C with 4 μg/ml polybrene. Blasticidin at 1 μg/ml was added 24 h after the second infection round for cell selection. Transduced cells (2.5 × 10⁵ or 2.5 × 10⁴) were induced to differentiate with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 d or 2 μM all-trans retinoic acid (ATRA) for 6 d, respectively. Monocytic differentiation by TPA was analyzed by measuring CD14 with a PE anti-CD14 Ab (BD Biosciences, Franklin Lakes, NJ) and granulocytic differentiation by ATRA with a PE-Cy7 anti-CD11b Ab (BD Biosciences).

Experiments were performed using C57BL/6 female mice of 8 wk of age, in accordance with the Ethics Committee of the University of Navarra. Mice were treated with 5-fluorouracil at a 150 mg/kg dose for 4 d prior to lineage⁻ cell harvesting using autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Bone marrow cells were infected with pBabe-based particles following the protocol used for HL60. Infected lineage⁻ mouse bone marrow cells (1.5 × 10⁵) were plated into p100 plates with MethoCult GF M3434 (StemCell Technologies, Vancouver, BC, Canada) for puromycin selection at 2 μg/ml for 10 d. For the second and third platings, 5.5 × 10⁴ cells were plated for 10 d. The numbers of CFU were analyzed 10 d after each plating, whereas CFU types were analyzed 10 d after the second plating.

The study has been approved by the Comisión de Ética de Investigación de la Facultad de Medicina de la Universidad de Navarra (no. 037/2008). All animal studies were performed in accordance with the guidelines of the Animal Care Committee of the University of Navarra (no. 063/12).

Statistical computations for quantitative PCR and luciferase experiments were performed using GraphPad Prism version 5.1 (GraphPad Software, La Jolla, CA). Comparisons were performed for data significance calculation by using t tests.

A 46-y-old female was referred to our institution for thrombopenia, leukopenia, and monocytopenia. At the age of 30 she gave birth to a premature infant (31 wk of gestation). In her second pregnancy, 1 y later, she required urgent Cesarean delivery due to febrile preeclampsia. Since then, she has presented with a persistent monocytopenia. Before these events, she had normal laboratory tests. At 40 y old, she was diagnosed with a CMV infection that resolved with antiviral treatment. Three years later, she was diagnosed with pneumonia that resolved with levofloxacin monotherapy. At that time, the peripheral leukocyte immunophenotype showed absence of NK cells as well as the previously mentioned monocytopenia. Bone marrow aspiration immunophenotype showed an expansion of large granular lymphocytes. Additionally, because anticardiolipin Abs were detected, the patient was diagnosed with primary antiphospholipid syndrome. When she was 45 y old, she developed a skin in situ carcinoma (Bowen’s disease) that required surgery. Four months later, she presented another pneumonia that required prolonged treatment with quinolones. Since then, she has had multiple episodes of herpes labialis. Blood tests at diagnosis (age 46) showed a mild leukopenia (4.0 × 10⁹/l) (Table I). Autoimmunity markers were negative, with the exception of mildly positive cryoglobulins (16 mg/dl, of which IgG was 7.2 mg/dl, IgA was 0.22 mg/dl, and IgM was 0.03 mg/dl, but with a negative rheumatoid factor) and anti-B2 glycoprotein IgM (32.36 U/ml). Total complement activity and fractions and serum Igs were normal. NK cell perforin expression was measured within normal range. Karyotype was normal: 46,XX (30 metaphases). Bone marrow smear was hypocellular, with expansion of large granular lymphocytes in the immunophenotype. Sequencing of GATA2 revealed the presence of a heterozygous c.1187G→A mutation at exon 7a, resulting in a missense p.Arg396Gln mutation. Therefore, the patient was diagnosed with monocytopenia and Mycobacterium avium complex infection (MonoMAC) syndrome. Her mother did not carry the GATA2 mutation, and her father died of a stroke at the age of 60, with no signs of being affected with the MonoMAC syndrome. Neither her sister, her brother, or her two children carry the mutation.

Previous studies in murine models found that Gata2 binds to its cis-regulatory elements located at −2.8 and −1.8 kb from the distal first exon (IS) of GATA2 transcription start site (TSS), activating its own transcription in HSPCs (14, 15). Thus, we focused on these regions to study the GATA2 autoregulatory loop in human cells in the context of GATA2 deficiency syndromes. To search for homologous GATA2 binding sites, we first aligned the murine and human GATA2 promoters using the ECR Browser software. We found five putative GATA2 binding sites at −3410, −3390, −2292, −2418, and −2409 bp from the IS TSS in the human GATA2 promoter (Supplemental Fig. 1). Comparison of core sequences showed total homology in four of these sites. Next, we examined whether these conserved GATA2 binding sites were bound by GATA2 in the human promoter. ChIP–quantitative PCR (qPCR) assay was performed in two GATA2-expressing AML cell lines (HEL and TF1), as well as in the nonexpressing MOLM13 cell line as negative control (Fig. 1A). Our results showed that GATA2 binds at the −3.4 and −2.4 kb regions in HEL and TF1 cells. As expected, no difference in fold enrichment was found in MOLM13 (Fig. 1B). These results confirm the specific binding of GATA2 to its own promoter in human cells.

To assess the activity of the GATA2 promoter in response to GATA2, we performed luciferase reporter assays with different GATA2 promoter constructs in the AML cell line HEL. P1, P2, and P3 constructs were similarly activated in HEL cells (Fig. 1C). Because the deletion of the −3410, −3390, and the −2992 binding sites did not alter the activation of the GATA2 promoter, we mutated the −2418 and −2409 sites either individually or simultaneously in the P3 construct. Mutation of the −2418 binding site decreased GATA2 promoter activation by 44%, whereas the mutation of the −2409 site showed an 80% decrease. Mutation of both sites showed 87% of activity lost. Taken together, our results show that the activation of GATA2 in HEL cells is driven by the −2.4 kb region, with a main activation role triggered by the −2409 binding site and secondarily by the −2418 GATA2 binding site.

Next, we assessed whether mutated GATA2 proteins had an impaired effect in the transcription of GATA2, altering the GATA2 autoregulatory loop. For this study, we chose p.Thr354Met and p.Arg396Gln, which to date are the most common mutations associated with GATA2 deficiency syndromes (Supplemental Table I), and p.Thr355del, which has a severe loss of function compared with WT-GATA2 (16). We mutated the pCMV6-XL6-GATA2 vector to generate the p.Thr354Met-, p.Thr355del-, and p.Arg396Gln-expressing vectors and performed luciferase reporter assays with the promoter construct that contains the −2.4 kb region alone (P3). The HeLa cell line was chosen to perform these studies owing to its low difficulty to transfect successfully compared with hematopoietic cells in suspension. We first tested the dose at which we could transfect >90% of cells and detect activity of the GATA2 promoter in response to WT-GATA2. Interestingly, we found a dose-dependent effect under these conditions (Fig. 2A). The luciferase activity in HeLa cells was notably lower than in HEL cells, which could be explained by their different cellular background. Next, we transfected 40 ng of each pCMV6-XL6 vector to analyze the effect induced by the mutations compared with WT-GATA2 (Fig. 2B). WT-GATA2 activated the P3 construct, whereas p.Thr354Met, p.Thr355del, and p.Arg396Gln did not activate it. We also cotransfected 20 ng WT-GATA2 and 20 ng empty vector or the corresponding mutation, mimicking heterozygosity, and found no activation of the GATA2 promoter (data not shown). Thus, our results suggest that these mutations could contribute to the GATA2 haploinsufficiency in these diseases.

Next, we analyzed the expression of GATA2 and other 26 genes involved in hematopoiesis and related to GATA2 in a sample from a patient with MonoMAC syndrome and the p.Arg396Gln mutation. We found that GATA2 expression was decreased to ∼58% in the PBMCs of the patient (Fig. 2C). Moreover, FLT3, LMO2, MEF2C, and SPI1 were downregulated, and CDK4, CTNNB1, DNMT3A, FOG1, FOXM1, GATA1, GATA3, KLF1, MEIS1, MPL, RUNX1, STAT3, and TAL1 were upregulated (Fig. 2D). There were no significant changes in the expression of CD44, CEBPA, CXCR4, EGFL7, ETS1, FLI1, KIT, MYC, and NOTCH1 (not shown).

As indicated above, p.Arg396Gln is the second most common mutation in GATA2 deficiency syndromes, and it affects the C-terminal ZF of GATA2. It has been reported in seven families, including a total of 15 individuals, and there is considerable variability in the overall phenotype (Supplemental Table III) (4, 5, 7, 17–21). However, there have been no functional studies in this mutation. To understand the functional consequences of p.Arg396Gln mutation in GATA2, we first used molecular simulations to predict the structural changes introduced by the mutation. Polyphen2 and MutationTaster analyses showed p.Arg396Gln as a probably damaging or disease-causing mutation, respectively. Hence, to check whether the arginine to glutamine mutation altered the interaction with DNA, we used the crystallized structure of the C-terminal domain of chicken Gata1 bound to DNA (22). GATA1 and GATA2 are evolutionarily conserved between species, and they share high homology in the ZF regions (23), which allowed us to locate the homologous arginine residue in the chicken Gata1. This approach showed that the positively charged guanidinium group of the equivalent arginine residue would be close to the negatively charged phosphate group in the backbone of DNA, suggesting electrostatic interaction (Fig. 3A). However, the substitution of the arginine for a glutamine, which has a shorter polar uncharged amide group, would abrogate the interaction with the phosphate group. Moreover, the carbonyl group of the side chain of the glutamine would be able to form a 3.13 Å hydrogen bond with the nitrogen of the peptidic bond of neighboring asparagine. Analysis of the predicted structure of the C-terminal ZF domain of human GATA2 showed that the mutation from arginine to glutamine favored a 3.01 Å hydrogen bond between the nitrogen from the amide group of Arg³⁹⁶ and the oxygen from the carbonyl group in the peptidic bond of Gln³⁹⁴ (Fig. 3B), predictably hampering the binding to phosphate groups of DNA. Taken together, our in silico analysis suggests that p.Arg396Gln ablates the interaction with phosphate groups of DNA, favoring the formation of hydrogen bonds ∼3 Å long with neighboring residues.

Next, we analyzed whether this DNA binding ability loss suggested in silico was actually occurring in vitro. We transfected HeLa cells with the pCMV6-XL6 vector and performed the ChIP-qPCR. As expected, WT-GATA2 bound to its own promoter (Fig. 4A). Conversely, p.Arg396Gln not only did not bind to the promoter, but also abrogated the binding of endogenous WT-GATA2, binding significantly less than when an empty vector was transfected.

Finally, we compared the effects of the WT-GATA2 and p.Arg396Gln proteins by transducing the human leukemia HL60 cell line with a retroviral vector to express WT-GATA2 or p.Arg396Gln. HL60 cells are sensitive to ATRA and TPA, which induce granulocytic and monocytic differentiation, respectively (24), and previous studies used the granulocytic differentiation model to demonstrate the role of other GATA2 germline mutations (16). In the presence of ATRA, the granulocytic CD11b marker increased more with the empty vector and with p.Arg396Gln than in the presence of WT-GATA2 (Fig. 4B). Likewise, in TPA-treated cells, the monocytic marker CD14 increased more with the empty vector and with p.Arg396Gln than in the presence of WT-GATA2 (Fig. 4C). The expression level achieved with these retroviral vectors was comparable to the endogenous WT-GATA2 dose of untransduced cells, duplicating the resulting expression in transduced cells (Fig. 4D). This allowed us to compare the effect of p.Arg396Gln in an environment resembling heterozygosity, as supposedly happens in patients with missense mutations. Collectively, these results indicate that p.Arg396Gln loses DNA binding ability and this is reflected as a loss of function in vitro, where it was unable to maintain the immature phenotype as did WT-GATA2 in HL60 cells.

Afterward, we examined whether this could happen in primary HSPCs. To address this question, murine bone marrow cells were extracted and HSPCs were selected and retrovirally transduced with WT-GATA2, p.Arg396Gln, or an empty vector. Colony formation ability was analyzed after puromycin selection. There was a great decrease in colony numbers after WT-GATA2 overexpression. In contrast, p.Arg396Gln-transduced cells formed a number of colonies similar to empty vector-transduced control cells (Fig. 5A).

Examination of colony types revealed that only WT-GATA2 significantly increased the CFU proportion corresponding to the more immature cell compartment compared with empty vector, namely CFU–mixed colony (GEMM) and CFU–granulocyte/macrophage colony (GM) (Fig. 5B). Conversely, p.Arg396Gln did not increase CFU-GEMM, and CFU-GM showed a variable trend. The more mature cell compartments were affected both in the presence of WT-GATA2 and p.Arg396Gln: WT-GATA2 induced a moderate increase in the proportion of CFU–granulocyte colony (G) and a decrease in CFU–macrophage (M) colony. Surprisingly, p.Arg396Gln greatly increased the proportion of CFU-G, whereas the effect on CFU-M was similar to WT-GATA2. However, CFU-GEMM and CFU-GM were less crowded, and CFU-M was smaller in the case of p.Arg396Gln expression (Fig. 5C). Taken together, these results indicate that p.Arg396Gln does not retain the progenitor phenotype, and it favors the development of granulocytic colonies.

Heterozygous mutations in GATA2 have been identified as the cause of four previously described clinical syndromes: MonoMAC syndrome; dendritic cell, monocyte, and B and NK lymphoid deficiency; familial myelodysplastic syndrome and AML; and Emberger syndrome. These syndromes are now recognized as different manifestations of a single genetic disorder with protean disease manifestations (5). However, there is considerable clinical heterogeneity among patients with GATA2 deficiency, and the molecular basis of these diseases remains undetermined. A recent study suggested GATA2 haploinsufficiency as the basis of these syndromes because of the finding that mutations in intronic regulatory regions of GATA2 decreased GATA2 transcript levels (7). In the present study, we show that the human GATA2 protein activates its own transcription through a specific region located at −2.4 kb from the IS TSS, and that the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation, confirming that GATA2 haploinsufficiency would be the basis for these familial syndromes.

Initial efforts to characterize the GATA2 autoregulatory loop were performed in murine models, and they found that the regions located 2.8 and 1.8 kb upstream of the IS TSS individually contributed to the transcription of Gata2 (14, 15). Our results indicate that human GATA2 activates its own promoter through the −2.4 kb region, homologous to the murine −1.8 region, with a main activation role triggered by the −2409 binding site and secondarily by the −2418 GATA2 binding site. Moreover, we show that some of the most common GATA2-mutated proteins impair this activation, which would cause GATA2 deficiency. As indicated above, GATA2 haploinsufficiency perturbs normal hematopoietic stem cell homeostasis in murine models (8). Of note, a comprehensive examination of the clinical features of 57 patients with GATA2 deficiency found high prevalence of cytopenias and bone marrow failure, supporting the notion that a similar defect exists in human GATA2 haploinsufficiency (5), although it remains unclear why monocytes, dendritic cells, B cells, and NK cells are preferentially depleted. The finding that our patient with a p.Arg396Gln mutation showed a reduction of ∼42% in GATA2 expression compared to controls also supports the hypothesis of the haploinsufficiency. These results would match the previously reported phenotype in Gata2 haploinsufficient mice, which presented 80% of the normal Gata2 levels in total marrow, and 50% in the hematopoietic stem cell population, where Gata2 is predominantly expressed (8). Interestingly, our results also point out that GATA2 germline mutations could affect other GATA2 target genes and, together with previous analyses that found differentially affected genes depending on the mutation (16), explain in part the clinical heterogeneity among patients with GATA2 deficiency (5). In fact, we show that the expression of several genes related to GATA2 is impaired in the patient sample (Fig. 2D). The finding that the expression of SPI1 in our patient was ∼17% of normal levels could be of special interest. Thirteen of the 15 cases reported with the p.Arg396Gln mutation developed a myeloid malignancy (Supplemental Table I), and mice carrying hypomorphic Spi1 alleles, which reduce Spi1 expression to 20% of normal levels, developed AML (25). Taken together, in addition to the insufficient GATA2 expression, the putative dysregulation of specific GATA2 targets caused by mutated proteins could also contribute to the disease, and symptoms could differ from a pure haploinsufficiency phenotype. Along these lines, it has been reported that frameshift mutations favor an earlier onset age, compared with missense mutations (6). Therefore, further studies are needed to elucidate the pathogenesis of GATA2-related syndromes.

In this study we have focused on the p.Arg396Gln mutation, for which no previous functional analyses had been reported. This missense mutation was reported previously in a total of 15 individuals, and there was a considerable variability in the overall phenotype (Supplemental Table III). The mechanisms that could alter hematopoietic homeostasis and trigger the disease are unknown. Dickinson et al. (6) suggested that disease evolution may be consistent with both cell-intrinsic (progressive deterioration of the HSPC compartment irrespective of environmental factors) and cell-extrinsic (immunocompromising events such as infections) mechanisms. Our patient showed the first symptoms of the disease the same year she became pregnant and had febrile preeclampsia. Whether pregnancy is an immunocompromising condition is a matter of discussion (26). The case presented resembles a typical manifestation of the disease, with recurrent infections and altered leukocyte numbers beginning in early or middle adulthood. To date, she has not displayed chronic neutropenia or lymphedema.

Our bioinformatic approach indicates that the substitution of the arginine for glutamine in the p.Arg396Gln mutation could impair binding to DNA. Several studies propose that cationic regions of proteins that contain arginine or lysine mediate the nonspecific interaction between the protein and the negatively charged phosphate group of DNA backbone (27). Accordingly, Arg³⁹⁶ would stabilize the non–sequence-specific binding to DNA, mediating electrostatic interactions with the phosphate group. Although p.Arg396Gln would not necessarily alter the specificity for WGATAR sequences, it would probably affect the stability of GATA2 binding to DNA, which could lead to a loss of function. Moreover, it is possible that the interaction of Gln³⁹⁶ with neighboring residues (presumably Gln³⁹⁴ or Asn³⁹⁷ by our models) could also alter protein–protein interactions, as has been shown for p.Thr354Met and p.Thr355del (16). Nevertheless, these deductions are confirmed with the ChIP assay in the HeLa cell line, which clearly shows that the p.Arg396Gln is unable to bind the −2.4 region of the GATA2 promoter. The finding that this mutation abrogated the binding of endogenous WT-GATA2 in HeLa cells suggests that a dominant-negative effect could be involved.

In vitro results in HL60 cells show that p.Arg396Gln is not able to function as WT-GATA2 does, which, in the context of these syndromes, could affect the maintenance of the progenitor compartment. It has been reported that the population of granulocyte/macrophage progenitor cells in Gata2^+/− mice was diminished (9), suggesting that appropriate WT-GATA2 expression levels maintain the characteristics of the progenitor cell stage. A similar approach found that p.Thr354Met was able to inhibit differentiation in the same way that WT-GATA2 does, whereas p.Thr355del was unable to do so (16), indicating that each mutation exerts different activities. In this context, the activity of p.Arg396Gln would be similar to p.Thr355del.

Alternatively, previous reports have shown that ectopically overexpressed GATA2 is able to induce hematopoietic cell quiescence, reducing colony output compared with cells with normal GATA2 expression levels (28). Thus, physiological GATA2 expression levels allow a healthy balance between quiescent and cycling hematopoietic cell populations at stem and progenitor stages (28). Our analysis in murine HSPCs suggests that, although the results with WT-GATA2 are in agreement with the aforementioned study (which suggested that WT-GATA2 induces quiescence in stem and progenitor cells), p.Arg396Gln would be unable to stop immature cell division. Moreover, the increase in immature colony proportion with WT-GATA2 and not with p.Arg396Gln further suggests that although WT-GATA2 retains the immature phenotype, p.Arg396Gln fails to do so. It has been suggested that hyperstimulation of the stem cell pool that produces hematopoietic progenitor cells with reduced expansion potential may lead to stem cell exhaustion in MonoMAC and dendritic cell, monocyte, and B and NK lymphoid deficiency syndromes (29). Our results would indicate that, in the context of these syndromes, p.Arg396Gln would force HSPCs to divide and differentiate prematurely, which would gradually drain the stem cell pool.

In summary, we show that human wild-type GATA2 contributes to its own transcription through a specific region located at −2.4 kb from IS TSS, and that p.Thr354Met, p.Thr355del, and p.Arg396Gln fail to do so. In agreement with this finding, a patient with a p.Arg396Gln mutation and MonoMAC syndrome displayed lower GATA2 expression than did normal controls. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, possibly triggered by a defective DNA binding, which as a consequence is unable to retain the progenitor phenotype in cells where it is expressed. This effect would cause HSPC pool damage, triggering cytopenias that would increase the risk of severe infections in patients with these diseases.

The authors have no financial conflicts of interest.