GATA2 gene encodes a member of the GATA family of zinc-finger transcription factors that play a pivotal role during the transition of primitive blood forming cells into white blood cells. Mutation in GATA2 results in the loss of function or even gain of function, including abnormal proliferation of white blood cells that may predispose to acute myeloid leukemia. Our results showed that the codon usage in GATA2 has been influenced by GC mutation bias where nature has highly favored fourteen most over represented codons but disfavored the ATA codon across five mammals. Purifying natural selection has affected GATA2 gene in human and other mammals to maintain its protein function during the period of evolution. Our findings report an insight into the codon usage patterns in gaining the clues for codon optimization to alter the translational efficiency as well as for the functional conservation of gene expression and the significance of nucleotide composition in GATA2 gene within mammals.

Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.

Catecholamine synthesized in the sympathoadrenal system, including sympathetic neurons and adrenal chromaffin cells, is vital for cardiovascular homeostasis. It has been reported that GATA2, a zinc finger transcription factor, is expressed in murine sympathoadrenal progenitor cells. However, a physiological role for GATA2 in adrenal chromaffin cells has not been established. In this study, we demonstrate that GATA2 is specifically expressed in adrenal chromaffin cells. We examined the consequences of Gata2 loss-of-function mutations, exploiting a Gata2 conditional knockout allele crossed to neural crest-specific Wnt1-Cre transgenic mice (Gata2 NC-CKO). The vast majority of Gata2 NC-CKO embryos died by embryonic day 14.5 (e14.5) and exhibited a decrease in catecholamine-producing adrenal chromaffin cells, implying that a potential catecholamine defect might lead to the observed embryonic lethality. When intercrossed pregnant dams were fed with synthetic adrenaline analogs, the lethality of the Gata2 NC-CKO embryos was partially rescued, indicating that placental transfer of the adrenaline analogs complements the lethal catecholamine deficiency in the Gata2 NC-CKO embryos. These results demonstrate that GATA2 participates in the development of neuroendocrine adrenaline biosynthesis, which is essential for fetal survival.

Keywords: Gata2; catecholamine; chromaffin cells; neural crest.

Germline mutations in GATA2 are associated with an inherited predisposition to bone marrow failure (BMF), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) remains the only curative therapy. However, patients may be at an increased risk for transplant-related toxicity (TRT) and mortality (TRM) due to their underlying disease biology. We performed a retrospective case-control study of pediatric BMF/MDS/AML patients with germline GATA2 mutations, comparing HSCT outcomes to randomly selected patients without germline GATA2 mutations and BMF/MDS (control A) and acute leukemia (control B). The 5-year overall and disease-free survival rates in the GATA2 cohort (65%, 51%) were similar to control A (58%, 49%) and B (45%, 43%) cohorts. In contrast, the 5-year event-free survival rate was significantly lower in the GATA2 cohort (7%±6%, 28%±10% and 33%±8% for GATA2, A, and B, respectively), due to an increased number of unique TRT. Specifically, neurologic toxicities occurred significantly more frequently in GATA2 patients than in the control groups and post-HSCT thrombotic events occurred only in the GATA2 cohort. There was no difference in TRM, infections or graft-versus-host disease (GVHD) across groups. The higher incidence of thrombotic and neurological events specific to GATA2 patients warrants further investigation and has potential treatment ramifications.

BACKGROUND

Leukemia cell lines are utilized as tools for molecular analysis. Their implementation in therapy will require standards for quality control, including appropriate selection criteria for functional analysis and efficacy determination.

METHODS

Characteristics of six human leukemia cell lines -Kasumi-1, NB-4, MOLM-13, MV-4-11, K562, and Jurkat cells-were investigated using multiple color analysis of surface antigen expression and comparative analysis of gene expression.

RESULTS

Differentiation states of Kasumi-1 and MOLM-13 cells are colony-forming units-granulocyte/macrophage equivalent cells to myeloblasts with comparatively high Growth factor independent-1(GFI1) and Transcription factor PU.1 (PU.1) expression, respectively. NB4 and MV-4-11 express high levels of CCAAT/enhancer-binding protein-alpha (CEBPα) and differentiate from myeloblasts to pro-monocytes and myeloblasts, respectively. K562 cells are colony-forming units-erythroid equivalent cells to erythroblasts, with the highest expression of GATA-binding factor 2 (GATA2), GATA1 and Friend of gata-1 (FOG1). Jurkat cells are pro-T to mature T-cells with the highest Neurogenic locus notch-1 homolog protein 1 (NOTCH1) expression.

CONCLUSIONS

Our study gives a useful guideline of standards for appropriate usage of leukemia cell lines for examining novel targets in vitro.

Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O₂) and compared to normoxia (21% O₂). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively. Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro.

Keywords: breast cancer; epigenetics; glycosylation; hypoxia; ovarian cancer.

Mast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage-determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes.

Molecular noise is a natural phenomenon that is inherent to all biological systems^1,2. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states-akin to a transcriptional priming phenomenon-the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness.

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.

Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates phosphoinositide-3-kinase (PI3K)/AKT signalling. This pathway is involved in a plethora of cellular functions including protein and lipid synthesis, cell migration, cell proliferation and apoptosis. In this study, we proposed to delineate the role of mTORC1 in haemopoietic lineage commitment using knock out (KO) mouse and cell line models. Mx1-cre and Vav-cre expression systems were used to specifically target Raptor^fl/fl (mTORC1), either in all tissues upon poly(I:C) inoculation, or specifically in haemopoietic stem cells, respectively. Assessment of the role of mTORC1 during the early stages of development in Vav-cre⁺Raptor^fl/fl mice, revealed that these mice do not survive post birth due to aberrations in erythropoiesis resulting from an arrest in development at the megakaryocyte-erythrocyte progenitor stage. Furthermore, Raptor-deficient mice exhibited a block in B cell lineage commitment. The essential role of Raptor (mTORC1) in erythrocyte and B lineage commitment was confirmed in adult Mx1-cre⁺Raptor^fl/fl mice upon cre-recombinase induction. These studies were supported by results showing that the expression of key lineage commitment regulators, GATA1, GATA2 and PAX5 were dysregulated in the absence of mTORC1-mediated signals. The regulatory role of mTOR during erythropoiesis was confirmed in vitro by demonstrating a reduction of K562 cell differentiation towards RBCs in the presence of established mTOR inhibitors. While mTORC1 plays a fundamental role in promoting RBC development, we showed that mTORC2 has an opposing role, as Rictor-deficient progenitor cells exhibited an elevation in RBC colony formation ex vivo. Collectively, our data demonstrate a critical role played by mTORC1 in regulating the haemopoietic cell lineage commitment.

Myelodysplastic syndromes (MDS) are hematopoietic disorders rare in childhood, often occurring in patients with inherited bone marrow failure syndromes or germinal predisposition syndromes. Among the latter, one of the most frequent involves the gene GATA binding protein 2 (GATA2), coding for a transcriptional regulator of hematopoiesis. The genetic lesion as well as the clinical phenotype are extremely variable; many patients present hematological malignancies, especially MDS with the possibility to evolve into acute myeloid leukemia. Variable immune dysfunction, especially resulting in B- and NK-cell lymphopenia, lead to severe infections, including generalized warts and mycobacterial infection. Defects of alveolar macrophages lead to pulmonary alveolar proteinosis through inadequate clearance of surfactant proteins. Currently, there are no clear guidelines for the monitoring and treatment of patients with GATA2 mutations. In patients with MDS, the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT) that restores normal hematopoiesis preventing the progression to acute myeloid leukemia and clears long-standing infections. However, to date, the donor type, conditioning regimen, and the optimal time to proceed to HSCT, as well as the level of chimerism needed to reverse the phenotype, remain unclear highlighting the need for consensus guidelines.

Keywords: GATA2 deficiency; cancer predisposition; childhood MDS; myelodysplastic syndromes; pediatric cancer.

The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disease characterized by moderate to severe macrocytic anemia and pathognomonic morphologic abnormalities of the erythroid precursors, including spongy heterochromatin. The disease is mainly caused by mutations in CDAN1 (encoding for Codanin-1). No patients with homozygous null type mutations have been described, and mouse null mutants die during early embryogenesis prior to the initiation of erythropoiesis. The cellular functions of Codanin-1 and the erythroid specificity of the phenotype remain elusive. To investigate the role of Codanin-1 in erythropoiesis, we crossed mice carrying the Cdan1 floxed allele (Cdan ^fl/fl ) with mice expressing Cre-recombinase under regulation of the erythropoietin receptor promoter (ErGFPcre). The resulting Cdan^ΔEry transgenic embryos died at mid-gestation (E12.5-E13.5) from severe anemia, with very low numbers of circulating erythroblast. Transmission electron microscopy studies of primitive erythroblasts (E9.5) revealed the pathognomonic spongy heterochromatin. The morphology of Cdan^ΔEry primitive erythroblasts demonstrated progressive development of dyserythropoiesis. Annexin V staining showed increases in both early and late-apoptotic erythroblasts compared to controls. Flow cytometry studies using the erythroid-specific cell-surface markers CD71 and Ter119 demonstrated that Cdan^ΔEry erythroid progenitors do not undergo the semi-synchronous maturation characteristic of primitive erythroblasts. Gene expression studies aimed to evaluate the effect of Cdan1 depletion on erythropoiesis revealed a delay of ζ to α globin switch compared to controls. We also found increased expression of Gata2, Pu.1, and Runx1, which are known to inhibit terminal erythroid differentiation. Consistent with this data, our zebrafish model showed increased gata2 expression upon cdan1 knockdown. In summary, we demonstrated for the first time that Cdan1 is required for primitive erythropoiesis, while providing two experimental models for studying the role of Codanin-1 in erythropoiesis and in the pathogenesis of CDA type I.

Keywords: Cdan1; Codanin-1; mice; primitive erythropoiesis; zebrafish.

BACKGROUND

Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.

RESULTS

In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.

CONCLUSIONS

These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

Although genetic predisposition to haematological malignancies has long been known, genetic testing is not yet the part of the routine diagnostics. In the last ten years, next generation sequencing based studies identified novel germline mutations in the background of familial aggregation of certain haematologic disorders including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). This is supported by the fact that the myeloid neoplasms with genetic predisposition represent a new category in the revised 2016 World Health Organization classification. According to the new classification, these disorders are subdivided based on the clinical and genetic features, including myeloid neoplasms with germline predisposition alone, or with pre-existing platelet disorder, cytopaenias or other organ failures. The predisposing genetic factors include mutations in the RUNX1, CEBPA, GATA2, ANKRD26, ETV6, DDX41, TERC or TERT and SRP72 genes. The genes affected in these syndromes are important regulators of haemopoiesis and are frequently implicated in leukaemogenesis, providing deeper insight into the understanding of normal and malignant haemopoiesis. Despite the growing knowledge of germline predisposing events in the background of familial myeloid malignancies, the germline genetic component is still unknown in a subset of these pedigrees. Here, we present the first study of inherited myeloid malignancies in Hungary. We identified three families with apparent clustering of myeloid malignancies with nine affected individuals across these pedigrees. All tested individuals were negative for CEBPA, GATA2, RUNX1, ANKRD26, ETV6, DDX41, TERC or TERT and SRP72 mutations, suggesting the presence of so far unidentified predisposing mutations.

When searching for genetic markers inherited together with musical aptitude, genes affecting inner ear development and brain function were identified. The alpha-synuclein gene (SNCA), located in the most significant linkage region of musical aptitude, was overexpressed when listening and performing music. The GATA-binding protein 2 gene (GATA2) was located in the best associated region of musical aptitude and regulates SNCA in dopaminergic neurons, thus linking DNA- and RNA-based studies of music-related traits together. In addition to SNCA, several other genes were linked to dopamine metabolism. Mutations in SNCA predispose to Lewy-body dementia and cause Parkinson disease in humans and affect song production in songbirds. Several other birdsong genes were found in transcriptome analysis, suggesting a common evolutionary background of sound perception and production in humans and songbirds. Regions of positive selection with musical aptitude contained genes affecting auditory perception, cognitive performance, memory, human language development, and song perception and production of songbirds. The data support the role of dopaminergic pathway and their link to the reward mechanism as a molecular determinant in positive selection of music. Integration of gene-level data from the literature across multiple species prioritized activity-dependent immediate early genes as candidate genes in musical aptitude and listening to and performing music.

Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/- aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor-related hematologic dysfunctions.

Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1 % of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.

Germline mutations in <i>GATA</i><i>2</i> are associated with a complex immunodeficiency and cancer predisposition syndrome. Somatic <i>GATA</i><i>2^mut</i> in myeloid malignancies may impart a similar phenotype. We reviewed adult patients with a diagnosis of <i>GATA</i><i>2^mut</i> hematological malignancy who were referred to our HHMC for genetic testing, and identified to have somatic <i>GATA</i><i>2^mut</i>. Nine patients with a median age of 63 years were included. Six patients (66.7%) were males. Atypical CML and acute myeloid leukemia were the most common initial presentation. The median overall VAF was 47.14%. Monocytopenia was pronounced when the <i>GATA</i><i>2^mut</i> involved the C-terminal ZFD. <i>GATA</i><i>2</i> N-terminal ZFD mutations tend to be co-mutated with <i>biCEBPA^mut</i>. Unlike germline <i>GATA</i><i>2</i> mutations, monocytopenia associated with somatic <i>GATA</i><i>2</i> mutations often resolved at remission. We concluded that similar to germline <i>GATA</i><i>2</i> mutations, a subset of somatic <i>GATA</i><i>2</i> mutations can impart a germline phenotype.

Monosomy 7 (-7) occurs in various types of paediatric myeloid disorders and has a poor prognosis. Recent studies have demonstrated that patients with germline gain-of-function SAMD9/9L variants and loss-of-function GATA2 variants are prone to developing myelodysplastic syndrome (MDS) associated with -7. However, the prevalence of the genetic variants among paediatric haematologic disorders with -7 is unknown. The present study screened germline variants of GATA2 and SAMD9/9L in 25 patients with various types of paediatric haematological disorders associated with -7. The diagnoses of the 25 patients included MDS (n = 10), acute myeloid leukaemia (AML) and myeloid sarcomas (n = 9), juvenile myelomonocytic leukaemia (n = 3) and other disorders (n = 3). Seven patients with a germline pathogenic GATA2 variant were found. For SAMD9/9L screening, next-generation sequencing was used to detect low-abundance variants and found four novel germline variants. Functional analysis revealed that three out of the four variants showed growth-restricting capacity in vitro and thus, were judged to be pathogenic. Cases with GATA2 mutation tended to be older, compared to those with SAMD9/9L mutations. In conclusion, GATA2 and SAMD9/9L were sequenced in 25 patients with paediatric haematologic disorders associated with -7, and 40% of them were found to have some pathogenic germline variants in the three genes.

Keywords: GATA2; SAMD9; SAMD9L; monosomy 7; pediatrics.

Human hematopoiesis is critically dependent on the transcription factor GATA2. Patients with GATA2 deficiency typically present with myelodysplastic syndrome, reduced numbers of monocytes, NK cells and B cells, and/or opportunistic infections. Here, we present two families that harbor distinct GATA2 mutations with highly variable onset and course of disease. We discuss the use of allogeneic hematopoietic cell transplantation in these patients, especially as treatment for pulmonary alveolar proteinosis.

Keywords: Allogeneic hematopoietic stem cell transplantation; GATA2 deficiency; Pulmonary alveolar proteinosis.

A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20 °C-35 °C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25 °C and 30 °C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.