GATA2 Germline Impair GATA2 Transcription, Causing : Functional Analysis of the p.Arg396Gln This information is current as of September 28, 2021. Xabier Cortés-Lavaud, Manuel F. Landecho, Miren Maicas, Leire Urquiza, Juana Merino, Isabel Moreno-Miralles and María D. Odero J Immunol 2015; 194:2190-2198; Prepublished online 26 January 2015; Downloaded from doi: 10.4049/jimmunol.1401868 http://www.jimmunol.org/content/194/5/2190 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2015/01/23/jimmunol.140186 Material 8.DCSupplemental References This article cites 29 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/194/5/2190.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Xabier Corte´s-Lavaud,*,†,1 Manuel F. Landecho,‡,1 Miren Maicas,* Leire Urquiza,* Juana Merino,x Isabel Moreno-Miralles,* and Marı´a D. Odero*,†

Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 mutations appear to cause loss of function of the mutated allele leading to haploinsufficiency; however, this postulate has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations that are translated into abnormal could affect the transcription of GATA2, triggering GATA2 deficiency. Chromatin

immunoprecipitation and luciferase assays showed that the human GATA2 activates its own transcription through Downloaded from a specific region located at 22.4 kb, whereas the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation. Accordingly, GATA2 expression was decreased to ∼58% in a patient with p.Arg396Gln, compared with controls. p.Arg396Gln is the second most common mutation in these syndromes, and no previous functional analyses have been performed. We therefore analyzed p.Arg396Gln. Our data show that p.Arg396Gln is a loss-of-function mutation affecting DNA- binding ability and, as a consequence, it fails to maintain the immature characteristics of hematopoietic stem and progenitor cells, which could result in defects in this cell compartment. In conclusion, we show that human GATA2 binds to its own promoter, http://www.jimmunol.org/ activating its transcription, and that the aforementioned mutations impair the transcription of GATA2. Our results indicate that they can affect other GATA2 target , which could partially explain the variability of symptoms in these diseases. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, which is unable to retain the progenitor phenotype in cells where it is expressed. The Journal of Immunology, 2015, 194: 2190–2198.

he GATA2 has an essential role in the So far, 50 different germline GATA2 mutations have been re- proliferation and differentiation of hematopoietic cells (1). ported; most families display mutations that are located within the Recently, germline mutations in GATA2 have been iden- highly conserved C-terminal zinc finger (ZF) domain, and they T by guest on September 28, 2021 tified as the cause of familial syndromes with autosomal-dominant correspond to missense mutations (Supplemental Tables I, II). It inheritance that share common symptoms. Severe monocytopenia, has been reported that mutations in a conserved intronic enhancer NK and B lymphopenia, near absence of dendritic cells, and a pre- element lead to decreased GATA2 transcript levels, indicating the disposition to develop , chronic myelo- necessity for both alleles to be functional to express the wild-type monocytic , and/or acute myeloid leukemia (AML) are (WT) phenotype, and suggesting GATA2 deficiency as the basis among the most prominent characteristics of these diseases (2–6). for these familial syndromes (7). In fact, Gata2+/2 mice express reduced levels of Gata2 in hematopoietic cells, and this has an impact on hematopoietic stem and progenitor cell (HSPC) ho- meostasis (8, 9). However, there is a considerable clinical het- *Hematology/Oncology Program, Center for Applied Medical Research, University erogeneity among patients (5), and GATA2 haploinsufficiency has of Navarra, Pamplona 31008, Spain; †Department of Biochemistry and Genetics, University of Navarra, Pamplona 31008, Spain; ‡Department of Internal Medicine, not been experimentally validated as the basis of these syndromes. University Clinic of Navarra, Navarra, Pamplona 31008, Spain; and xDepartment of We hypothesized that mutations affecting the C-terminal ZF, Immunology, University Clinic of Navarra, Pamplona 31008, Spain which are thought to allow production of a stable mRNA that is 1 X.C.-L. and M.F.L. contributed equally to this work. translated into an abnormal protein, could affect the transcription Received for publication July 22, 2014. Accepted for publication December 18, 2014. of GATA2, triggering GATA2 deficiency. In this study, we show This work was supported by Ministerio de Ciencia e Innovacio´n Grant PI11/02443, that the human GATA2 protein activates its own transcription and Departamento de Salud del Gobierno de Navarra Grant 78/2012, Instituto de Salud that three germline GATA2 mutations affecting the C-terminal ZF Carlos III–Red Tema´tica de Investigacio´n Contra el Ca´ncer Grant RD12/0036/0063, and by the Fundacio´n para Investigacio´nMe´dica Aplicada (Spain). impair GATA2 promoter activation. Moreover, we functionally ana- Address correspondence and reprint requests to Xabier Corte´s-Lavaud, Hematology/ lyze p.Arg396Gln, the second most common mutation in GATA2 Oncology Program, Center for Applied Medical Research, University of Navarra, deficiency syndromes, in the context of the clinical presentation of Avenida Pı´o XII 55, Pamplona 31008, Spain. E-mail address: [email protected] a patient with this mutation. The online version of this article contains supplemental material. Abbreviations used in this article: AML, acute myeloid leukemia; ATRA, all-trans Materials and Methods retinoic acid; ChIP, chromatin immunoprecipitation; G, granulocyte colony; GEMM, mixed colony; GM, granulocyte/macrophage colony; HSPC, hematopoietic stem and Cell lines and patient sample progenitor cell; IS, distal first exon; M, macrophage colony; MonoMAC, monocyto- penia and Mycobacterium avium complex infection; qPCR, quantitative PCR; qRT- HEL, TF1, MOLM13, and HeLa cell lines were cultured following the PCR, quantitative real-time RT-PCR; TPA, 12-O-tetradecanoylphorbol-13-acetate; German Collection of Microorganisms and Cell Cultures Cell Culture Bank TSS, transcription start site; WT, wild-type; ZF, zinc finger. recommendations (Braunschweig, Germany). Peripheral samples from patient and five healthy donors were obtained after written informed Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 consent by the University Clinic of Navarra. DNA was sequenced using www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401868 The Journal of Immunology 2191 specific primers for each GATA2 exon in a 3500DX genetic analyzer (Life particles following the protocol used for HL60. Infected lineage2 mouse Technologies, Carlsbad, CA). PBMCs were extracted for analysis using the bone marrow cells (1.5 3 105) were plated into p100 plates with Metho- Ficoll protocol. Cult GF M3434 (StemCell Technologies, Vancouver, BC, Canada) for puromycin selection at 2 mg/ml for 10 d. For the second and third platings, RNA analysis 5.5 3 104 cells were plated for 10 d. The numbers of CFU were analyzed Total RNAwas extracted from freshly isolated PBMCs of individuals or cell 10 d after each plating, whereas CFU types were analyzed 10 d after the lines using the TRIzol reagent (Life Technologies). Quantitative real-time second plating. RT-PCR (qRT-PCR) was performed in the ABI Prism 7500 (Life Tech- Ethics statement nologies) using SYBR Green master mix or TaqMan master mix (Life Technologies) with specific primers (sequences are available upon request). The study has been approved by the Comisio´ndeE´ tica de Investigacio´nde HPRT1 or GAPDH expression was used as an internal control for SYBR or la Facultad de Medicina de la Universidad de Navarra (no. 037/2008). All TaqMan methods, respectively. Data were analyzed using the comparative animal studies were performed in accordance with the guidelines of the cycle threshold (DDCt) method. Animal Care Committee of the University of Navarra (no. 063/12). Chromatin immunoprecipitation and quantitative PCR Statistical analysis Chromatin immunoprecipitation (ChIP) assays in HEL, TF1, and MOLM13 Statistical computations for quantitative PCR and luciferase experiments cells were performed as previously reported, using a GATA2 Ab (R&D were performed using GraphPad Prism version 5.1 (GraphPad Software, La Systems, Minneapolis, MN) or a nonspecific anti-goat IgG (Cell Signaling Jolla, CA). Comparisons were performed for data significance calculation Technology, Beverly, MA) (10). In the case of HeLa cells, 5 3 106 cells by using t tests. were transfected with 3 mg 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 Results Downloaded from (11). Quantitative PCR was used to analyze ChIP resulting material using Case report an unrelated genomic region of GAPDH as negative control (Diagenode, Denville, NJ). Fold enrichment value was calculated comparing GATA2 A 46-y-old female was referred to our institution for thrombopenia, immunoprecipitated and nonspecific IgG immunoprecipitated samples. leukopenia, and monocytopenia. At the age of 30 she gave birth to a premature infant (31 wk of gestation). In her second pregnancy, Plasmid construct cloning and mutagenesis 1 y later, she required urgent Cesarean delivery due to febrile

GATA2 regulatory regions were inserted in the pGL3-Basic vector preeclampsia. Since then, she has presented with a persistent http://www.jimmunol.org/ (Promega, Fitchburg, WI). GATA2 promoter and GATA2-expressing vector monocytopenia. Before these events, she had normal laboratory pCMV6-XL6-GATA2 (Origene, Rockville, MD) were mutated using a QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) tests. At 40 y old, she was diagnosed with a CMV infection that following the manufacturer’s protocol. resolved with antiviral treatment. Three years later, she was di- agnosed with pneumonia that resolved with levofloxacin mono- Transient transfection and luciferase reporter assays therapy. At that time, the peripheral leukocyte immunophenotype Luciferase reporter assays in HEL and HeLa cells were performed as pre- showed absence of NK cells as well as the previously men- viously reported (10). Each GATA2 promoter construct, the corresponding tioned monocytopenia. Bone marrow aspiration immunophenotype pCMV6-XL6 vector (only for HeLa cells), and pRL-SV40 (Promega) as showed an expansion of large granular lymphocytes. Additionally,

internal control were transfected. by guest on September 28, 2021 because anticardiolipin Abs were detected, the patient was diag- GATA protein modeling and viewing nosed with primary antiphospholipid syndrome. When she was 45 y The model of chicken GATA1 C-terminal ZF was downloaded from the old, she developed a skin in situ carcinoma (Bowen’s disease) that Protein Data Bank (2GAT). A GATA2 C-terminal ZF model was created required surgery. Four months later, she presented another pneu- using SWISS-MODEL software (http://swissmodel.expasy.org). Images monia that required prolonged treatment with quinolones. Since were created using the Swiss Protein Data Bank viewer software (http:// then, she has had multiple episodes of herpes labialis. Blood tests spdbv.vital-it.ch). at diagnosis (age 46) showed a mild leukopenia (4.0 3 109/l) Retroviral production (Table I). Autoimmunity markers were negative, with the excep- Retroviral particles were produced transfecting AmphoPack 293 cells with tion of mildly positive cryoglobulins (16 mg/dl, of which IgG was pMD2.G envelope vector and an empty, GATA2, or p.Arg396Gln cDNA 7.2 mg/dl, IgA was 0.22 mg/dl, and IgM was 0.03 mg/dl, but with containing pBabe-puro (12) or pLRT (13) vectors, using FuGene (Prom- a negative rheumatoid factor) and anti-B2 glycoprotein IgM ega). Particles were harvested 48-72 h after transfection, 0.45-mm filtered, (32.36 U/ml). Total complement activity and fractions and serum and used immediately for transduction. Igs were normal. NK cell perforin expression was measured HL60 cell line transduction and differentiation induction and within normal range. Karyotype was normal: 46,XX (30 meta- assessment phases). Bone marrow smear was hypocellular, with expansion of

7 large granular lymphocytes in the immunophenotype. Sequencing HL60 cells (10 ) were infected on 2 consecutive days with pLRT vector– → based particles by centrifugation at 3000 3 g for 3 h at 32˚C with 4 mg/ml of GATA2 revealed the presence of a heterozygous c.1187G A polybrene. Blasticidin at 1 mg/ml was added 24 h after the second infection mutation at exon 7a, resulting in a missense p.Arg396Gln muta- round for cell selection. Transduced cells (2.5 3 105 or 2.5 3 104) were tion. Therefore, the patient was diagnosed with monocytopenia induced to differentiate with 5 nM 12-O-tetradecanoylphorbol-13-acetate and Mycobacterium avium complex infection (MonoMAC) syn- (TPA) for 3 d or 2 mM all-trans retinoic acid (ATRA) for 6 d, respectively. Monocytic differentiation by TPA was analyzed by measuring CD14 with drome. Her mother did not carry the GATA2 mutation, and her a PE anti-CD14 Ab (BD Biosciences, Franklin Lakes, NJ) and granulo- father died of a stroke at the age of 60, with no signs of being cytic differentiation by ATRA with a PE-Cy7 anti-CD11b Ab (BD Bio- affected with the MonoMAC syndrome. Neither her sister, her sciences). brother, or her two children carry the mutation. Transduction of lineage-negative mouse bone marrow cells, Human GATA2 activates its own transcription through serial replating, and colony-forming assays a region located 2.4 kb upstream of the transcription start site Experiments were performed using C57BL/6 female mice of 8 wk of age, Previous studies in murine models found that Gata2 binds to its in accordance with the Ethics Committee of the University of Navarra. 2 2 Mice were treated with 5-fluorouracil at a 150 mg/kg dose for 4 d prior cis-regulatory elements located at 2.8 and 1.8 kb from the to lineage2 cell harvesting using autoMACS (Miltenyi Biotec, Bergisch distal first exon (IS) of GATA2 transcription start site (TSS), ac- Gladbach, Germany). Bone marrow cells were infected with pBabe-based tivating its own transcription in HSPCs (14, 15). Thus, we focused 2192 GATA2 GERMLINE–MUTATED PROTEINS IMPAIR GATA2 TRANSCRIPTION

Table I. Peripheral blood leukocyte analysis in a patient diagnosed with MonoMAC syndrome and p.Arg396Gln mutation in GATA2

Cell Type Percentagesa Status Neutrophils 59.5 Normal Lymphocytes 33.6 Normal B lymphocytes 0.6 Low T lymphocytes, of which: 32.9 Normal CD4+ 16.9 Low CD4+/CD8+ ratio CD8+ 22.1 Low CD4+/CD8+ ratio CD4+CD8+ 6.9 Low CD4+/CD8+ ratio DR+-activated T lymphocytes 13.8 Normal CD4+DR+-activated T lymphocytes 15.6 Normal CD8+DR+-activated T lymphocytes 13.8 Normal NK cells 0.007 Low Monocytes 0.2 Low Eosinophils 5.7 Normal Basophils 0 (0.00 3 109/l) Normal aPercentages are expressed relative to total leukocytes. Downloaded from on these regions to study the GATA2 autoregulatory loop in hu- gion alone (P3). The HeLa cell line was chosen to perform these man cells in the context of GATA2 deficiency syndromes. To studies owing to its low difficulty to transfect successfully com- search for homologous GATA2 binding sites, we first aligned the pared with hematopoietic cells in suspension. We first tested the murine and human GATA2 promoters using the ECR Browser dose at which we could transfect .90% of cells and detect activity software. We found five putative GATA2 binding sites at 23410, of the GATA2 promoter in response to WT-GATA2. Interestingly,

23390, 22292, 22418, and 22409 bp from the IS TSS in the we found a dose-dependent effect under these conditions (Fig. http://www.jimmunol.org/ human GATA2 promoter (Supplemental Fig. 1). Comparison of 2A). The luciferase activity in HeLa cells was notably lower than core sequences showed total homology in four of these sites. Next, in HEL cells, which could be explained by their different cellular we examined whether these conserved GATA2 binding sites were background. Next, we transfected 40 ng of each pCMV6-XL6 bound by GATA2 in the human promoter. ChIP–quantitative PCR vector to analyze the effect induced by the mutations compared (qPCR) assay was performed in two GATA2-expressing AML cell with WT-GATA2 (Fig. 2B). WT-GATA2 activated the P3 con- lines (HEL and TF1), as well as in the nonexpressing MOLM13 struct, whereas p.Thr354Met, p.Thr355del, and p.Arg396Gln did cell line as negative control (Fig. 1A). Our results showed that not activate it. We also cotransfected 20 ng WT-GATA2 and 20 ng GATA2 binds at the 23.4 and 22.4 kb regions in HEL and TF1 empty vector or the corresponding mutation, mimicking hetero- cells. As expected, no difference in fold enrichment was found in zygosity, and found no activation of the GATA2 promoter (data by guest on September 28, 2021 MOLM13 (Fig. 1B). These results confirm the specific binding of not shown). Thus, our results suggest that these mutations could GATA2 to its own promoter in human cells. contribute to the GATA2 haploinsufficiency in these diseases. To assess the activity of the GATA2 promoter in response to Next, we analyzed the expression of GATA2 and other 26 genes GATA2, we performed luciferase reporter assays with different involved in hematopoiesis and related to GATA2 in a sample from GATA2 promoter constructs in the AML cell line HEL. P1, P2, a patient with MonoMAC syndrome and the p.Arg396Gln muta- and P3 constructs were similarly activated in HEL cells (Fig. 1C). tion. We found that GATA2 expression was decreased to ∼58% in Because the deletion of the 23410, 23390, and the 22992 the PBMCs of the patient (Fig. 2C). Moreover, FLT3, LMO2, binding sites did not alter the activation of the GATA2 promoter, MEF2C, and SPI1 were downregulated, and CDK4, CTNNB1, we mutated the 22418 and 22409 sites either individually or DNMT3A, FOG1, FOXM1, GATA1, GATA3, KLF1, MEIS1, MPL, simultaneously in the P3 construct. Mutation of the 22418 RUNX1, STAT3,andTAL1 were upregulated (Fig. 2D). There were binding site decreased GATA2 promoter activation by 44%, no significant changes in the expression of CD44, CEBPA, CXCR4, whereas the mutation of the 22409 site showed an 80% decrease. EGFL7, ETS1, FLI1, KIT, MYC,andNOTCH1 (not shown). Mutation of both sites showed 87% of activity lost. Taken to- p.Arg396Gln acts as a loss-of-function mutation in human cells gether, our results show that the activation of GATA2 in HEL cells is driven by the 22.4 kb region, with a main activation role As indicated above, p.Arg396Gln is the second most common triggered by the 22409 binding site and secondarily by the 22418 mutation in GATA2 deficiency syndromes, and it affects the GATA2 binding site. C-terminal ZF of GATA2. It has been reported in seven families, including a total of 15 individuals, and there is considerable p.Thr354Met, p.Thr355del, and p.Arg396Gln germline variability in the overall phenotype (Supplemental Table III) mutations of GATA2 impair the transcription of GATA2 (4, 5, 7, 17–21). However, there have been no functional studies Next, we assessed whether mutated GATA2 proteins had an im- in this mutation. To understand the functional consequences paired effect in the transcription of GATA2, altering the GATA2 of p.Arg396Gln mutation in GATA2, we first used molecular autoregulatory loop. For this study, we chose p.Thr354Met and simulations to predict the structural changes introduced by p.Arg396Gln, which to date are the most common mutations the mutation. Polyphen2 and MutationTaster analyses showed associated with GATA2 deficiency syndromes (Supplemental p.Arg396Gln as a probably damaging or disease-causing muta- Table I), and p.Thr355del, which has a severe loss of function tion, respectively. Hence, to check whether the arginine to gluta- compared with WT-GATA2 (16). We mutated the pCMV6-XL6- mine mutation altered the interaction with DNA, we used the GATA2 vector to generate the p.Thr354Met-, p.Thr355del-, and crystallized structure of the C-terminal domain of chicken Gata1 p.Arg396Gln-expressing vectors and performed luciferase reporter bound to DNA (22). GATA1 and GATA2 are evolutionarily con- assays with the promoter construct that contains the 22.4 kb re- served between species, and they share high homology in the ZF The Journal of Immunology 2193 Downloaded from http://www.jimmunol.org/

FIGURE 1. GATA2 protein expressing human AML cell lines show GATA2 binding to its own promoter and activation of its own transcription through the 2.4 region. (A) Western blot of GATA2 protein in human AML cell lines. (B) GATA2 ChIP-qPCR on GATA2 promoter in the GATA2-expressing HEL

and TF1 cell lines. Sequence enrichment for the 23.4 kb and for the 22.4 kb region is shown. Data are represented as the GATA2/normal IgG sequence by guest on September 28, 2021 enrichment in each cell line. The GATA2 nonexpressing MOLM13 AML cell line was used as a negative control. (C) Three serially deleted GATA2 promoter regions and three promoter mutants, including the IS, which is transcribed in the hematopoietic tissue, were cloned into the pGL3-Basic luciferase reporter vector [firefly luciferase coding sequence (Luc)]. GATA2 binding sites are noted as black boxes, with numbers in the upper part showing their relative location from the TSS; mutated sites are in gray. The 22418 binding site sequence was mutated from 59-cagataag-39 to 59-cCCGCGag-39, and the 22409 site was mutated from 59-cttatcag-39 to 59-cCCGCGag-39. The construct name is written on the left. HEL cells were cotransfected with the cor- responding GATA2 promoter construct or pGL3-Basic vector and the internal standard pRL-SV40 vector for luciferase reporter assays. The relative lu- ciferase value calculated is the quotient between the activity of the promoter and the activity of a promoterless vector. Means and SD are shown and t test comparisons were made. *p , 0.05. Experiments were performed in triplicate. regions (23), which allowed us to locate the homologous arginine HeLa cells with the pCMV6-XL6 vector and performed the ChIP- residue in the chicken Gata1. This approach showed that the qPCR. As expected, WT-GATA2 bound to its own promoter positively charged guanidinium group of the equivalent arginine (Fig. 4A). Conversely, p.Arg396Gln not only did not bind to the residue would be close to the negatively charged phosphate group promoter, but also abrogated the binding of endogenous WT- in the backbone of DNA, suggesting electrostatic interaction (Fig. GATA2, binding significantly less than when an empty vector 3A). However, the substitution of the arginine for a glutamine, was transfected. which has a shorter polar uncharged amide group, would abrogate Finally, we compared the effects of the WT-GATA2 and the interaction with the phosphate group. Moreover, the carbonyl p.Arg396Gln proteins by transducing the human leukemia HL60 group of the side chain of the glutamine would be able to form a cell line with a retroviral vector to express WT-GATA2 or 3.13 A˚ hydrogen bond with the nitrogen of the peptidic bond of p.Arg396Gln. HL60 cells are sensitive to ATRA and TPA, which neighboring asparagine. Analysis of the predicted structure of the induce granulocytic and monocytic differentiation, respectively C-terminal ZF domain of human GATA2 showed that the mutation (24), and previous studies used the granulocytic differentiation from arginine to glutamine favored a 3.01 A˚ hydrogen bond be- model to demonstrate the role of other GATA2 germline mutations tween the nitrogen from the amide group of Arg396 and the oxygen (16). In the presence of ATRA, the granulocytic CD11b marker from the carbonyl group in the peptidic bond of Gln394 (Fig. 3B), increased more with the empty vector and with p.Arg396Gln than predictably hampering the binding to phosphate groups of DNA. in the presence of WT-GATA2 (Fig. 4B). Likewise, in TPA-treated Taken together, our in silico analysis suggests that p.Arg396Gln cells, the monocytic marker CD14 increased more with the empty ablates the interaction with phosphate groups of DNA, favoring the vector and with p.Arg396Gln than in the presence of WT-GATA2 formation of hydrogen bonds ∼3A˚ long with neighboring residues. (Fig. 4C). The expression level achieved with these retroviral Next, we analyzed whether this DNA binding ability loss sug- vectors was comparable to the endogenous WT-GATA2 dose of gested in silico was actually occurring in vitro. We transfected untransduced cells, duplicating the resulting expression in trans- 2194 GATA2 GERMLINE–MUTATED PROTEINS IMPAIR GATA2 TRANSCRIPTION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. p.Thr354Met, p.Thr355del, and p.Arg396Gln do not activate GATA2 transcription, and the p.Arg396Gln-harboring patient shows altered expression. (A) Luciferase activity of the GATA2 promoter region P3 measured in HeLa cells, transfecting increasing quantities of pCMV6-XL6- GATA2 into HeLa cells to determine an appropriate range of activation of the GATA2 promoter. Subsequent luciferase reporter assays were performed using 40 ng pCMV6-XL6 vector. (B) Luciferase activity of the GATA2 promoter region P3 measured in HeLa cells to detect functional changes of GATA2 mutations compared with wild-type GATA2. Upper panel shows the relative luciferase activity. Middle panel shows GATA2 or mutant protein expression upon pCMV6-XL6 expression vector transfection in HeLa, using b-actin as an internal control. The lower panel shows the corresponding pCMV6-XL6 expression vector in each case, as well as the GATA2 P3 promoter for all cases. The relative luciferase value calculated is the combined quotient between the activity of the promoter and the activity of the promoterless vector, and the activity in the presence or absence of ectopic GATA2 or mutant expression. Means and SD are shown and t test comparisons were made. *p , 0.05. Experiments were performed in triplicate. (C) GATA2 mRNA expression in PBMCs of a patient with the p.Arg396Gln GATA2 mutation, and in five normal controls, measured by qRT-PCR. (D) mRNA expression of differentially expressed genes of the patient and normal controls in PBMCs as measured by qRT-PCR. duced cells (Fig. 4D). This allowed us to compare the effect of extracted and HSPCs were selected and retrovirally transduced p.Arg396Gln in an environment resembling heterozygosity, as with WT-GATA2, p.Arg396Gln, or an empty vector. Colony for- supposedly happens in patients with missense mutations. Collec- mation ability was analyzed after puromycin selection. There tively, these results indicate that p.Arg396Gln loses DNA binding was a great decrease in colony numbers after WT-GATA2 over- ability and this is reflected as a loss of function in vitro, where expression. In contrast, p.Arg396Gln-transduced cells formed it was unable to maintain the immature phenotype as did WT- a number of colonies similar to empty vector-transduced control GATA2 in HL60 cells. cells (Fig. 5A). Examination of colony types revealed that only WT-GATA2 p.Arg396Gln lacks the capacity of maintaining the immature significantly increased the CFU proportion corresponding to the phenotype of hematopoietic stem and progenitor cells more immature cell compartment compared with empty vector, Afterward, we examined whether this could happen in primary namely CFU–mixed colony (GEMM) and CFU–granulocyte/ HSPCs. To address this question, murine bone marrow cells were macrophage colony (GM) (Fig. 5B). Conversely, p.Arg396Gln The Journal of Immunology 2195

FIGURE 3. Arg396 interaction with phos- phate and neighboring residues. (A) Crystal- lized structure of chicken GATA1 C-terminal ZF domain bound to DNA shows the short distance between the negatively charged phosphate and positively charged arginine side chain, suggesting electrostatic inter- action. Shorter, polar uncharged glutamine would favor the interaction with neighbor- ing asparagine. (B) Predicted structure of human GATA2 C-terminal ZF domain shows interaction of mutated p.Arg396Gln with neigh- boring Gln394. Downloaded from

did not increase CFU-GEMM, and CFU-GM showed a variable Emberger syndrome. These syndromes are now recognized as trend. The more mature cell compartments were affected both in different manifestations of a single with protean the presence of WT-GATA2 and p.Arg396Gln: WT-GATA2 in- disease manifestations (5). However, there is considerable clinical duced a moderate increase in the proportion of CFU–granulocyte heterogeneity among patients with GATA2 deficiency, and the http://www.jimmunol.org/ colony (G) and a decrease in CFU–macrophage (M) colony. molecular basis of these diseases remains undetermined. A recent Surprisingly, p.Arg396Gln greatly increased the proportion of study suggested GATA2 haploinsufficiency as the basis of these CFU-G, whereas the effect on CFU-M was similar to WT-GATA2. syndromes because of the finding that mutations in intronic reg- However, CFU-GEMM and CFU-GM were less crowded, and ulatory regions of GATA2 decreased GATA2 transcript levels (7). CFU-M was smaller in the case of p.Arg396Gln expression In the present study, we show that the human GATA2 protein (Fig. 5C). Taken together, these results indicate that p.Arg396Gln activates its own transcription through a specific region located does not retain the progenitor phenotype, and it favors the de- at 22.4 kb from the IS TSS, and that the p.Thr354Met, p.Thr355del, velopment of granulocytic colonies. and p.Arg396Gln germline mutations impair GATA2 promoter ac- tivation, confirming that GATA2 haploinsufficiency would be the by guest on September 28, 2021 Discussion basis for these familial syndromes. Heterozygous mutations in GATA2 have been identified as the Initial efforts to characterize the GATA2 autoregulatory loop cause of four previously described clinical syndromes: MonoMAC were performed in murine models, and they found that the regions syndrome; dendritic cell, monocyte, and B and NK lymphoid located 2.8 and 1.8 kb upstream of the IS TSS individually con- deficiency; familial myelodysplastic syndrome and AML; and tributed to the transcription of Gata2 (14, 15). Our results indicate

FIGURE 4. p.Arg396Gln loses DNA binding ability, and its function in the HL60 cell line under differentiation stimuli is impaired. (A) GATA2 ChIP- qPCR on GATA2 22.4 region in HeLa cells transfected with pCMV6-XL6 vector (empty, or expressing GATA2 or p.Arg396Gln). Data are represented as the GATA2/normal IgG sequence enrichment, and a sequence of the E2F3 gene with no GATA binding sites was used as control. (B) HL60 cells transduced with retroviral particles to express GATA2, p.Arg396Gln, or an empty vector, treated with 2 mM ATRA for 6 d and granulocytic differentiation mea- surement by CD11b. (C) The same HL60 cells as in (B), treated with 5 nM TPA for 3 d and monocytic differentiation measurement by CD14. Experiments were performed three times; means, SD, and Student t test comparisons are shown. *p , 0.05. (D) Ectopic expression of WT GATA2 or p.Arg396Gln in HL60 cells. HL60 cells were transduced to achieve expression of GATA2 or p.Arg396Gln and to perform subsequent treatment with ATRA or TPA for granulocytic or monocytic differentiation, respectively. Densitometry values in the middle indicate that vector expression matched that of endogenous GATA2 expression, doubling normal GATA2 expression in the case of WT GATA2, and resembling a heterozygous environment in the case of p.Arg396Gln. Blots of two of three experiments are shown (first experiment in odd lanes, second experiment in even lanes, beginning from the left). 2196 GATA2 GERMLINE–MUTATED PROTEINS IMPAIR GATA2 TRANSCRIPTION Downloaded from http://www.jimmunol.org/

FIGURE 5. Colony formation ability of retrovirally transduced murine bone marrow cells and colony type assessment. (A) Colony numbers upon transgene expression in murine HSPC bone marrow cells in colony-forming medium supplemented with myeloid and erythroid growth factors. (B) Colony types were assessed on day 10 after the second plating. (C) Representative colony type depiction at 340 original magnification. Empty retroviral vector was ,

used as control. Experiments were performed three times; means, SD, and t test comparisons are shown. *p 0.05. by guest on September 28, 2021 that human GATA2 activates its own promoter through the 22.4 ported with the p.Arg396Gln mutation developed a myeloid kb region, homologous to the murine 21.8 region, with a main malignancy (Supplemental Table I), and mice carrying hypo- activation role triggered by the 22409 binding site and second- morphic Spi1 alleles, which reduce Spi1 expression to 20% of arily by the 22418 GATA2 binding site. Moreover, we show that normal levels, developed AML (25). Taken together, in addition some of the most common GATA2-mutated proteins impair this to the insufficient GATA2 expression, the putative dysregulation activation, which would cause GATA2 deficiency. As indicated of specific GATA2 targets caused by mutated proteins could also above, GATA2 haploinsufficiency perturbs normal hematopoietic contribute to the disease, and symptoms could differ from a pure homeostasis in murine models (8). Of note, a compre- haploinsufficiency phenotype. Along these lines, it has been hensive examination of the clinical features of 57 patients with reported that frameshift mutations favor an earlier onset age, GATA2 deficiency found high prevalence of cytopenias and bone compared with missense mutations (6). Therefore, further stud- marrow failure, supporting the notion that a similar defect exists in ies are needed to elucidate the pathogenesis of GATA2-related human GATA2 haploinsufficiency (5), although it remains unclear syndromes. why monocytes, dendritic cells, B cells, and NK cells are pref- In this study we have focused on the p.Arg396Gln mutation, for erentially depleted. The finding that our patient with a p.Arg396Gln which no previous functional analyses had been reported. This mutation showed a reduction of ∼42% in GATA2 expression missense mutation was reported previously in a total of 15 indi- compared to controls also supports the hypothesis of the viduals, and there was a considerable variability in the overall haploinsufficiency. These results would match the previously re- phenotype (Supplemental Table III). The mechanisms that could ported phenotype in Gata2 haploinsufficient mice, which pre- alter hematopoietic homeostasis and trigger the disease are un- sented 80% of the normal Gata2 levels in total marrow, and 50% known. Dickinson et al. (6) suggested that disease evolution may in the hematopoietic stem cell population, where Gata2 is pre- be consistent with both cell-intrinsic (progressive deterioration of dominantly expressed (8). Interestingly, our results also point out the HSPC compartment irrespective of environmental factors) and that GATA2 germline mutations could affect other GATA2 target cell-extrinsic (immunocompromising events such as infections) genes and, together with previous analyses that found differentially mechanisms. Our patient showed the first symptoms of the disease affected genes depending on the mutation (16), explain in part the the same year she became pregnant and had febrile preeclampsia. clinical heterogeneity among patients with GATA2 deficiency (5). Whether pregnancy is an immunocompromising condition is a In fact, we show that the expression of several genes related to matter of discussion (26). The case presented resembles a typical GATA2 is impaired in the patient sample (Fig. 2D). The finding that manifestation of the disease, with recurrent infections and altered the expression of SPI1 in our patient was ∼17% of normal leukocyte numbers beginning in early or middle adulthood. To levels could be of special interest. Thirteen of the 15 cases re- date, she has not displayed chronic neutropenia or lymphedema. The Journal of Immunology 2197

Our bioinformatic approach indicates that the substitution of the would increase the risk of severe infections in patients with these arginine for glutamine in the p.Arg396Gln mutation could impair diseases. binding to DNA. Several studies propose that cationic regions of proteins that contain arginine or lysine mediate the nonspecific Disclosures interaction between the protein and the negatively charged phos- The authors have no financial conflicts of interest. phate group of DNA backbone (27). Accordingly, Arg396 would stabilize the non–sequence-specific binding to DNA, mediating electrostatic interactions with the phosphate group. Although References p.Arg396Gln would not necessarily alter the specificity for 1. Tsai, F. Y., G. Keller, F. C. Kuo, M. Weiss, J. Chen, M. Rosenblatt, F. W. Alt, and S. H. Orkin. 1994. An early haematopoietic defect in mice lacking the tran- WGATAR sequences, it would probably affect the stability of scription factor GATA-2. Nature 371: 221–226. GATA2 binding to DNA, which could lead to a loss of function. 2. Bigley, V., M. Haniffa, S. Doulatov, X.-N. Wang, R. Dickinson, N. McGovern, Moreover, it is possible that the interaction of Gln396 with L. Jardine, S. Pagan, I. Dimmick, I. Chua, et al. 2011. The human syndrome of 394 397 dendritic cell, monocyte, B and NK lymphoid deficiency. J. Exp. Med. 208: 227–234. neighboring residues (presumably Gln or Asn by our models) 3. Dickinson, R. E., H. Griffin, V. Bigley, L. N. Reynard, R. Hussain, M. Haniffa, could also alter protein–protein interactions, as has been shown for J. H. Lakey, T. Rahman, X.-N. Wang, N. McGovern, et al. 2011. Exome se- quencing identifies GATA-2 mutation as the cause of dendritic cell, monocyte, B p.Thr354Met and p.Thr355del (16). Nevertheless, these deductions and NK lymphoid deficiency. Blood 118: 2656–2658. are confirmed with the ChIP assay in the HeLa cell line, which 4. Hsu, A. P., E. P. Sampaio, J. Khan, K. R. Calvo, J. E. Lemieux, S. Y. Patel, clearly shows that the p.Arg396Gln is unable to bind the 22.4 re- D. M. Frucht, D. C. Vinh, R. D. Auth, A. F. Freeman, et al. 2011. Mutations in GATA2 are associated with the autosomal dominant and sporadic mono- gion of the GATA2 promoter. The finding that this mutation abro- cytopenia and mycobacterial infection (MonoMAC) syndrome. Blood 118: gated the binding of endogenous WT-GATA2 in HeLa cells 2653–2655. Downloaded from suggests that a dominant-negative effect could be involved. 5.Spinner,M.A.,L.A.Sanchez,A.P.Hsu,P.A.Shaw,C.S.Zerbe,K.R.Calvo, D. C. Arthur, W. Gu, C. M. Gould, C. C. Brewer, et al. 2014. GATA2 deficiency: In vitro results in HL60 cells show that p.Arg396Gln is not able a protean disorder of hematopoiesis, lymphatics and immunity. Blood 123: 809–821. to function as WT-GATA2 does, which, in the context of these 6. Dickinson, R. E., P. Milne, L. Jardine, S. Zandi, S. I. Swierczek, N. McGovern, S. Cookson, Z. Ferozepurwalla, A. Langridge, S. Pagan, et al. 2014. The evo- syndromes, could affect the maintenance of the progenitor com- lution of cellular deficiency in GATA2 mutation. Blood 123: 863–874. partment. It has been reported that the population of granulocyte/ 7. Hsu, A. P., K. D. Johnson, E. L. Falcone, R. Sanalkumar, L. Sanchez, +/2 D. D. Hickstein, J. Cuellar-Rodriguez, J. E. Lemieux, C. S. Zerbe, macrophage progenitor cells in Gata2 mice was diminished (9), http://www.jimmunol.org/ E. H. Bresnick, and S. M. Holland. 2013. GATA2 haploinsufficiency caused by suggesting that appropriate WT-GATA2 expression levels main- mutations in a conserved intronic element leads to MonoMAC syndrome. Blood tain the characteristics of the progenitor cell stage. A similar ap- 121: 3830–3837. proach found that p.Thr354Met was able to inhibit differentiation 8. Rodrigues, N. P., V. Janzen, R. Forkert, D. M. Dombkowski, A. S. Boyd, S. H. Orkin, T. Enver, P. Vyas, and D. T. Scadden. 2005. Haploinsufficiency of in the same way that WT-GATA2 does, whereas p.Thr355del was GATA-2erturbs adult hematopoietic stem-cell homeostasis. Blood 106: 477–484. unable to do so (16), indicating that each mutation exerts different 9. Rodrigues, N. P., A. S. Boyd, C. Fugazza, G. E. May, Y. Guo, A. J. Tipping, D. T. Scadden, P. Vyas, and T. Enver. 2008. GATA-2 regulates granulocyte- activities. In this context, the activity of p.Arg396Gln would be macrophage progenitor cell function. Blood 112: 4862–4873. similar to p.Thr355del. 10. Maicas, M., I. Va´zquez, C. Vicente, M. A. Garcı´a-Sa´nchez, N. Marcotegui, Alternatively, previous reports have shown that ectopically L. Urquiza, M. J. Calasanz, and M. D. Odero. 2012. Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: by guest on September 28, 2021 overexpressed GATA2 is able to induce hematopoietic cell qui- RUNX1 and ELK1 directly regulate its transcription. Oncogene 32: 2069–2078. escence, reducing colony output compared with cells with normal 11. Pippa, R., L. Espinosa, G. Gundem, R. Garcı´a-Escudero, A. Dominguez, GATA2 expression levels (28). Thus, physiological GATA2 ex- S. Orlando, E. Gallastegui, C. Saiz, A. Besson, M. J. Pujol, et al. 2012. p27Kip1 represses transcription by direct interaction with p130/E2F4 at the promoters of pression levels allow a healthy balance between quiescent and target genes. Oncogene 31: 4207–4220. cycling hematopoietic cell populations at stem and progenitor 12. Morgenstern, J. P., and H. Land. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complemen- stages (28). Our analysis in murine HSPCs suggests that, although tary helper-free packaging cell line. Nucleic Acids Res. 18: 3587–3596. the results with WT-GATA2 are in agreement with the afore- 13. Watsuji, T., Y. Okamoto, N. Emi, Y. Katsuoka, and M. Hagiwara. 1997. Con- mentioned study (which suggested that WT-GATA2 induces trolled gene expression with a reverse tetracycline-regulated retroviral vector (RTRV) system. Biochem. Biophys. Res. Commun. 234: 769–773. quiescence in stem and progenitor cells), p.Arg396Gln would 14. Grass, J. A., M. E. Boyer, S. Pal, J. Wu, M. J. Weiss, and E. H. Bresnick. 2003. be unable to stop immature cell division. Moreover, the increase GATA-1-dependent transcriptional repression of GATA-2 via disruption of in immature colony proportion with WT-GATA2 and not with positive autoregulation and domain-wide chromatin remodeling. Proc. Natl. Acad. Sci. USA 100: 8811–8816. p.Arg396Gln further suggests that although WT-GATA2 retains 15. Snow, J. W., J. J. Trowbridge, K. D. Johnson, T. Fujiwara, N. E. Emambokus, the immature phenotype, p.Arg396Gln fails to do so. It has been J. A. Grass, S. H. Orkin, and E. H. Bresnick. 2011. Context-dependent function of “GATA switch” sites in vivo. Blood 117: 4769–4772. suggested that hyperstimulation of the stem cell pool that pro- 16. Hahn, C. N., C.-E. Chong, C. L. Carmichael, E. J. Wilkins, P. J. Brautigan, duces hematopoietic progenitor cells with reduced expansion X.-C. Li, M. Babic, M. Lin, A. Carmagnac, Y. K. Lee, et al. 2011. Heritable potential may lead to stem cell exhaustion in MonoMAC and GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia. Nat. Genet. 43: 1012–1017. dendritic cell, monocyte, and B and NK lymphoid deficiency 17. Holme, H., U. Hossain, M. Kirwan, A. Walne, T. Vulliamy, and I. Dokal. 2012. syndromes (29). Our results would indicate that, in the context of Marked genetic heterogeneity in familial myelodysplasia/acute myeloid leu- these syndromes, p.Arg396Gln would force HSPCs to divide and kaemia. Br. J. Haematol. 158: 242–248. 18. Pasquet, M., C. Bellanne-Chantelot, S. Tavitian, N. Prade, B. Beaupain, differentiate prematurely, which would gradually drain the stem O. LaRochelle, A. Petit, P. Rohrlich, C. Ferrand, E. van den Neste, et al. 2012. cell pool. High frequency of GATA2 mutations in patients with mild chronic neutropenia evolving to MonoMac syndrome, myelodysplasia and acute myeloid leukemia. In summary, we show that human wild-type GATA2 contributes Blood 121: 822–829. to its own transcription through a specific region located at 22.4 kb 19. Ishida, H., K. Imai, K. Honma, S.-I. Tamura, T. Imamura, M. Ito, and from IS TSS, and that p.Thr354Met, p.Thr355del, and p.Arg396Gln S. Nonoyama. 2012. GATA-2 anomaly and clinical phenotype of a sporadic case of lymphedema, dendritic cell, monocyte, B- and NK-cell (DCML) deficiency, fail to do so. In agreement with this finding, a patient with a and myelodysplasia. Eur. J. Pediatr. 171: 1273–1276. p.Arg396Gln mutation and MonoMAC syndrome displayed lower 20. Vinh, D. C., S. Y. Patel, G. Uzel, V. L. Anderson, A. F. Freeman, K. 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Supplementary Figure 1. Murine and human GATA2 promoter alignment shows highly conserved regions. Brackets delimit the highly conserved regions in both genomes, as graphics obtained from ECR Browser show: large areas marked in red indicate highly similar intergenic sequences, repetitive sequences in green, exonic sequences in yellow, and intronic sequences in salmon color. Horizontal axis represents base positions in the genome and the vertical axis represents the percentage identity between the human and mouse genomes. GATA2 binding sites are indicated in red within the brackets, and they were located using the TFSearch, Match and MatInspector transcription factor binding site finder algorithms. Comparison of GATA2 binding sites between both genomes and several surrounding nucleotides is shown in the middle, where GATA2 binding site core sequences are shown in italic and underlined. Two GATA2 binding sites at -2.8 belonged to a highly conserved region spanning 389 bp, with 87.7% homology with the human GATA2 promoter. Similarly, other two GATA2 binding sites at -1.8 were located within a 282 bp region with 75.5% homology. Additionally, a third 142 bp region was taken into account containing the -2992 GATA2 binding site, which showed 68.3% homology.

Supplementary Table I: List of reported germline GATA2 mutations and associated syndromes.

Mutation (DNA)* Affected exon Mutation (protein) Mutation type Affected ZF Reference Cases/families Associated syndrome c.1-200_871+527del2033 4, 5 p.Met1_Ser290del In-frame deletion None 4,7,20,S1 2/1 MonoMAC c.121C>G 4 p.Pro41Ala Missense (NC) None 17 3/1 Familial MDS/AML c.230-1_230insC 5 p.Arg78ProfsX107 Frameshift Not translated S3 3/1 Emberger syndrome c.243_244delAinsGC 5 p.Gly81GlyfsX103 Frameshift Not translated 4,7,20,21 1/1 MonoMAC c.257_258delGC 5 p.Arg86ProfsX98 Frameshift Not translated 6 1/1 GATA2 deficiency c.302delG 5 p.Gly101AlafsX16 Frameshift Not translated 7 1/1 MonoMAC 17 2/1 Familial MDS/AML c.310_311insCC 5 p.Leu105ProfsX15 Frameshift Not translated S2 5/1 Emberger syndrome c.318_319insT 5 p.Ala106CysfsX78 Frameshift Not translated 6 2/1 GATA2 deficiency c.579_580insA 5 p.Ala194SerfsX8 Frameshift Not translated S2 1/1 Emberger syndrome c.586_593dup 5 p.Gly199LeufsX20 Frameshift Not translated 7 1/1 MonoMAC c.594delG 5 p.Gly200ValfsX19 Frameshift Not translated 6 3/1 GATA2 deficiency c.599_600insG 5 p.Ser201X Nonsense Not translated 3,7 2/1 DCML c.610C>T 5 p.Arg204X Nonsense Not translated 18 1/1 MonoMAC c.670G>T 5 p.Glu224X Nonsense Not translated 18 1/1 MonoMAC c.735_736insC 5 p.Ile246HisfsX36 Frameshift Not translated 6 1/1 GATA2 deficiency c.751C>T 5 p.Pro254Leu Missense (NC) None 4,7 1/1 MonoMAC c.769_778dup 5 p.Tyr260CysfsX24 Frameshift Not translated 4,7,21 1/1 MonoMAC c.941_951dup 6 p.Asn317fsX11 Frameshift 1 4,7 1/1 MonoMAC 18 4/1 MonoMAC c.988C>T 6 p.Arg330X Nonsense 1 5 2/1 GATA2 deficiency c.992_993insGACC 6 p.Leu332ThrfsX53 Frameshift 1 S1 1/1 MonoMAC/DCML S2 1/1 Emberger syndrome c.1009C>T 6 p.Arg337X Nonsense 1 5,7 1/1 MonoMAC c.1017+512del28 None None Intron 5 deletion None 7,20,S3 3/1 MonoMAC c.1017+572C>T None None Intron 5 SNV None 6,7 10/4 MonoMAC 4 1/1 MonoMAC c.1018-1G>T 7a p.Thr340_Thr381del In-frame deletion 1, 2 3 1/1 DCML S2 1/1 Emberger syndrome c.1018-3_1031del17 7a p.Ala341ArgfsX38 Frameshift 1 6 1/1 GATA2 deficiency c.1019_1022delCGGC 7a p.Ala341ProfsX45 Frameshift 1 S2 1/1 Emberger syndrome c.1024G>A 7a p.Ala342Thr Missense (NC) 1 S4 3/1 Pediatric de novo AML-M0

Supplementary Table I (cont.)

Mutation (DNA)* Affected exon Mutation (protein) Mutation type Affected ZF Reference Cases/families Associated syndrome 4,7,20,21,S5 5/3 MonoMAC S1 3/1 MonoMAC/DCML 16 18/3 Familial MDS/AML c.1061C>T 7a p.Thr354Met Missense (C) 2 17 4/1 Familial MDS/AML 3 1/1 DCML S6 6/1 GATA2 deficiency 6 5/1 GATA2 deficiency c.1063_1065delACA 7a p.Thr355del In-frame deletion 2 16 2/1 Familial MDS/AML 5,7 1/1 MonoMAC c.1081C>T 7a p.Arg361Cys Missense (NC) 2 6 1/1 GATA2 deficiency c.1082G>C 7a p.Arg361Leu Missense (NC) 2 S2 1/1 Emberger syndrome c.1083_1094del12 7a p.Arg362_Asn365del In-frame deletion 2 4,20 1/1 MonoMAC c.1099_1100insG 7a p.Asp367GlyfsX15 Frameshift 2 5 2/1 GATA2 deficiency c.1103_1104insG 7a p.Val369CysfsX13 Frameshift 2 7 1/1 MonoMAC c.1113C>G 7a p.Asn371Lys Missense(NC) 2 4,20 1/1 MonoMAC c.1114G>A 7a p.Ala372Thr Missense (NC) 2 6 1/1 GATA2 deficiency c.1116_1130del15 7a p.Cys373del5 In-frame deletion 2 5 3/1 GATA2 deficiency c.1117T>C 7a p.Cys373Arg Missense (NC) 2 S2 1/1 Emberger syndrome c.1162A>G 8 p.Met388Val Missense (NC) 2 7,S1 2/1 MonoMAC/DCML c.1163T>C 8 p.Met388Thr Missense (C) 2 5,7 3/1 MonoMAC c.1168_1170delAAG 8 p.Lys390del In-frame deletion 2 6 1/1 GATA2 deficiency 4,5,7,20 2/1 MonoMAC c.1186C>T 8 p.Arg396Trp Missense (NC) 2 S7 1/1 MonoMAC 4,21 1/1 MonoMAC 5,7 6/2 MonoMAC c.1187G>A 8 p.Arg396Gln Missense (NC) 2 19 1/1 MonoMAC/Emberger syndrome 17 3/1 Familial MDS/AML 18 4/1 MonoMAC 4,7,20,21 6/5 MonoMAC c.1192C>T 8 p.Arg398Trp Missense (NC) 2 3,6 4/2 DCML c.1193G>A 8 p.Arg398Gln Missense (NC) 2 6 5/1 GATA2 deficiency 18 1/1 MonoMAC 1/1 MonoMAC/DCML Complete deletion† All Complete deletion Complete deletion Not translated S1 1/1 MonoMAC/DCML 1/1 MonoMAC/DCML C indicates conserved amino acid type in missense mutation; NC non-conserved amino acid type; MonoMAC, monocytopenia and mycobacterial infection syndrome; DCML, dendritic cell, monocyte, B and NK lymphoid deficiency; SNV, single nucleotide variant. *The RefSeq sequence used as reference was NM_032638.4. †Although deletions are different in each case, all include a heterozygous deletion of the entire GATA2 locus.

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S6. Bödör C., A. Renneville, M. Smith, A. Charazac, S. Iqbal, P. Etancelin, J. Cavenagh, M.J. Barnett, K. Kramarzová, B. Krishnan, A. Matolcsy, C. Preudhomme, J. Fitzgibbon, and C. Owen. 2012. Germline GATA2 p.THR354MET mutation in familial myelodysplastic syndrome with acquired monosomy 7 and ASXL1 mutation demonstrating rapid onset and poor survival. Haematologica 97: 890-894.

S7. Camargo J.F., S.A. Lobo, A.P. Hsu, C.S. Zerbe, G.P. Wormser, and S.M. Holland. 2012. MonoMAC Syndrome in a Patient With a GATA2 Mutation: Case Report and Review of the Literature. Clin. Infect. Dis. 57: 697-699.

Supplementary Table II. Summary of germline GATA2 mutations.

Complete Missense Frameshift Non-sense In-frame deletion Intronic mutation deletion of Total GATA2 locus 15 17 5 6 2 4 50 2 conservative 1 deletion of 28 bp 13 non-conservative 1 single nucleotide variant 2 before ZF1 11 before ZF1 3 before ZF1 1 before ZF1 1 located in ZF1 4 located in ZF1 2 located in ZF1 1 overlaps ZF1 and ZF2 12 located in ZF2 2 located in ZF2 4 located in ZnF2

38 families reported 17 families reported 7 families reported 7 families reported 5 families reported 4 families reported

Supplementary Table III. Revised literature showing the data of patients with p.Arg396Gln mutation.

Case Reference Diagnosis Clinical history 1 Female, she died of AML-M2, no apparent accessory phenotype. Son of case 1, he developed AML-M2 at 14 years old (30% blasts on bone marrow), no apparent accessory 2 (17) Familial MDS/AML phenotype. 3 Son of case 1, he was diagnosed with myelodysplastic syndrome at 26 years old. Female, she had a history of recurrent gingivitis and skin infection, with chronic neutropenia and monocytopenia. Twelve years later, she developed AML-M2 with normal karyotype, and myelodysplasia-related 4 changes. She had three sons and two miscarriages. She died from cytomegalovirus pneumonia without achieving a complete remission. Son of case 4, history of chronic neutropenia with a chronic EBV replication in gut, and severe respiratory distress syndrome at 14 years old. He was diagnosed with a refractory cytopenia with multilineage dysplasia 5 with normal karyotype. Low number of CD14+ monocytes, CD19+ B-cell lymphocytes and CD3-/CD56+ NK cells, but normal CD3+ T-cell lymphocyte count. He underwent hematopoietic stem cell transplantation with a matched (18) MonoMAC unrelated donor. Son of case 4. He had a history of recurrent infections and mild neutropenia. He developed an AML-M2 at 14 years old, with myelodysplasia-related changes (similar cytological presentation than his mother). Karyotype 6 showed trisomy 11 and partial 7q deletion. He received in first complete remission matched unrelated hematopoietic stem cell transplantation. Son of case 4. He presented recurrent fever and chronic EBV replication in the blood. Blood count and bone marrow examination were normal. Diagnosed of unclassifiable myelodysplastic syndrome. Karyotype showed 7 monosomy 7. Matched unrelated bone marrow transplantation was performed, with a complication by a grade IV cutaneous graft-versus-host disease, requiring immunosuppressive therapy. Female, first presentation at 16 years-old. Suffered from Mycobacterium abscessus infection, nocardiosis, Developed MDS with trisomy of 8, hypocellularity with atypical megakaryocytes and focal blasts. 8 (4, 5, 21) MonoMAC Received bone marrow transplantation from a matched unrelated donor, recovering trilineage hematopoiesis and normal cytogenetics, but suffering from graft-versus-host disease in the skin. Last visit to National Institutes of Health (NIH) at 25 years-old. Female with a severe Varicella zoster infection at the age of 2. At 4 years old, mild neutropenia, deficiency of MonoMAC dendritic, B and NK cells, and profound monocytopenia in peripheral blood was detected, with a low CD4+/CD8+ 9 (19) Emberger Syndrome ratio. She suffered a prolonged Salmonella infection at 8 years of age. She developed lymphedema at the age of 13. Finally, she presented trilineage dysplasia at the age of 19, with normal karyotype.

Supplementary Table III (cont.).

Case Reference Diagnosis Clinical history Case with mild phenotype. B lymphopenia at the age of 54, and no reports of monocytopenia or recurrent 10 (5, 7) infections, but with a skewed allelic expression. Last visit to NIH at 55 years-old. Son of case 10, with a first presentation at 18 years-old, had suffered from human papillomavirus infection. He 11 has developed myelodysplastic syndrome with trisomy 8. Last visit to NIH at 23 years-old. Son of case 10, with a first presentation at 16 years-old, had suffered from human papillomavirus infection, 12 desmoid tumor, deep vein thrombosis and pulmonary embolism. He developed myelodysplastic syndrome with trisomy 8. Last visit to NIH at 21 years-old. MonoMAC, GATA2 Son of case 10, with a first presentation at 14 years-old, had suffered from human papillomavirus and varicella (5) deficiency 13 zoster virus infection and hypothyroidism. He has developed myelodysplastic syndrome with trisomy 8. Last visit to NIH at 17 years-old. Male with a first presentation at 27 years-old, had suffered from human papillomavirus infection, granulomatous 14 lymphadenitis, invasive aspergillosis and a sarcoidosis-like pulmonary process. He developed myelodysplastic syndrome. Last visit to NIH at 32 years-old. Brother of case 14, first presentation at 29 years-old, has developed myelodysplastic syndrome. Last visit to 15 NIH at 29 years-old.