In this study we analyzed the biological effects

In this study, we analyzed the biological effects of two novel GATA2 mutations, which were identified in adult de novo AML patients.
Materials and methods The diagnosis of AML was based on the morphology, histopathology, the expression of leukocyte differentiation purchase monensin and/or the French–American–British (FAB) classification. GATA2 mutation was screened in 96 newly diagnosed de novo AML adult patients. Informed consent was obtained from all patients to use their samples for banking and molecular analysis, and approval was obtained from the ethics committee of Nagoya University School of Medicine. The full-length human wild-type (Wt)- and mutated (Mt)-GATA2 cDNAs were amplified from Wt- or Mt-GATA2-expressing leukemia cells, respectively. C-terminally Myc-tagged GATA2 cDNAs were cloned into pcDNA3.1 vector, and electrophoretic mobility shift assay (EMSA) and luciferase assay were performed as previously reported. C-terminally Myc-tagged Wt- and mutant-GATA2 cDNAs cloned in the pMX-IP vector were transduced into murine IL3-dependent myeloid progenitor cell line 32D cells as previously described. Stable Wt- and mutant-GATA2-expressing 32D cells were subjected to immunofluorescence, proliferation and differentiation analyses. Wt- and mutant-GATA2-expressed 32D cells were suspended in RPMI1640 medium containing 10% FCS with an increasing concentration of murine IL3 (R&D Systems), and 2×104 cells per well were seeded in 96-well culture plates. Cell viability was measured using the CellTiter96 Proliferation Assay (Promega). For the induction of myeloid differentiation, Wt- and mutant-GATA2-expressed 32D cells were cultured in RPMI1640 medium containing 10% FCS and 30ng/ml recombinant G-CSF (Kyowa-Kirin, Tokyo, Japan) without IL3. Luciferase assay and cell proliferation and differentiation analyses were performed three times independently. Differences in continuous variables were analyzed with unpaired t test for the distribution between two groups using Prism 5 (GraphPad Software Inc., La Jolla, CA). For all analyses, the P values were two-tailed, and a P value<0.05 was considered significant.
Results We identified two GATA2 gene mutations. One mutation (p.R308P-GATA2) was a p.Arg308Pro substitution within the N-terminal zinc finger (ZF)-1 domain in a FAB M6 patient (Fig. 1A and B). The other mutation (p.A350_N351ins8-GATA2) was a 24-nucleotide insertion resulting in an eight-amino-acid insertion between A350 and N351 residues within the C-terminal ZF-2 domain in a FAB M1 patient. This insertion mutation consisted of a one nucleotide insertion and a 23-nucleotide corresponding to N337-R344 residues duplication (Fig. 1A and C). In both patients, each GATA2 mutation was heterozygous. Unfortunately, we could not analyze germ-line sequence of both patients because of the lack of appropriate material. Therefore, we could not completely determine whether identified mutations were somatic or germ-line mutations. Both patients revealed normal karyotype and no mutation in FLT3, KIT, NRAS, TP53, NPM1, CEBPA, RUNX1, MLL-PTD and IDH1/2 genes. The expression ratios of Wt- and Mt-GATA2 transcripts of AML cells harboring the p.A350_N351ins8 mutation were semi-quantitatively examined using a gene scanning system with fluorescent-labeled PCR product. Although the p.A350_N351ins8 mutation was heterozygous, little expression of Wt-GATA transcript was observed in the primary AML cells (Fig. 1D). We transiently expressed Wt- and mutant-GATA2 in 293T cells, and nuclear lysates were extracted 48h later. In EMSA using an oligonucleotide containing GATA recognition sites, DNA-binding activity of p.A350_N351ins8-GATA2 was reduced to about 40% of that of Wt-GATA2, although that of p.R308P-GATA2 was almost the same as that of Wt-GATA2 (Fig. 2A). Consistent with the DNA-binding activity, the transcriptional activity of p.A350_N351ins8-GATA2 was significantly lower than that of Wt-GATA2 (P=0.006), while that of p.R308P-GATA2 was not affected (P=0.11) (Fig. 2B). Upon co-expression of Wt-GATA2 with p.A350_N351ins8-GATA2 at a 1:1 ratio, the transcriptional activity of Wt-GATA2 was not affected by p.A350_N351ins8-GATA, indicating that p.A350_N351ins8-GATA2 did not have a dominant-negative effect over Wt-GATA2 (Fig. 2C).