Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 14-14
Author(s):  
Rainer Nustede ◽  
Inna Kuznetsova ◽  
Karl Welte ◽  
Julia Skokowa
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia

Abstract Abstract 14 Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI. Disclosures: No relevant conflicts of interest to declare.

mRNA and Protein Expression Levels of Secretory Leukocyte Protease Inhibitor (SLPI) Are Severely Reduced in Patients with Severe Congenital Neutropenia (CN)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2165-2165
Author(s):  
Wienke Ellerbeck ◽  
Olga Klimenkova ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Protease Inhibitor ◽  
Cell Line ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Protein Levels ◽  
Myeloid Cell Line

Abstract Abstract 2165 Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-Neutrophil ELastase (NE), activities. Moreover, SLPI modulates intracellular signal transduction pathways such as NF-kB and Erk. The molecular interaction and the balance between NE and SLPI is tightly regulated. On the one side, NE upregulates the SLPI expression and at the other hand SLPI inhibits the NE-induced degradation of proteins. We identified severe diminished levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN) harbouring either ELANE or HAX1 mutations, as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. SLPI protein levels in plasma of CN patients were also significantly reduced. We further analysed whether diminished levels of SLPI are associated with the „maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI using lentivirus-based transduction of the myeloid cell line NB4 with SLPI-specific shRNA and analysed ATRA-triggered myeloid differentiation. Indeed, myeloid differentiation was severely affected in NB4 cells transduced with SLPI-specific shRNA, as compared to control shRNA transduced cells. Further, we analysed the mechanisms leading to SLPI downregulation. Previously, we identified severely reduced mRNA and protein levels of NE in myeloid cells and in plasma of CN patients with either ELANE or HAX1 mutations, as compared to healthy individuals. Knowing that NE induces SLPI expression, we assumed that diminished NE levels may be responsible for the low SLPI expression in CN patients. Indeed, inhibition of NE in the myeloid cell line NB4 using NE-specific shRNAs led to diminished expression of SLPI mRNA, as compared to ctrl shRNA transduced cells. At the same time, we also found that transduction of the myeloid cell line NB4 with wild type (WT) NE resulted in the increased expression of SLPI mRNA but mutated (MUT) forms of NE as found in CN patients were not able to induce SLPI mRNA, as compared to ctrl transduced cells. Taken together, both diminished NE levels and mutations in ELANE gene may cause downregulation of SLPI. In summary, SLPI is severely downregulated in CN patients due to defective NE protein levels and ELANE mutations. As a consequence, the anti-microbial and antiinflammatory activities of SLPI are diminished in CN patients. Disclosures: No relevant conflicts of interest to declare.

Aberrant G-CSFR Signaling in Congenital Neutropenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-45-SCI-45
Author(s):  
Julia Skokowa
Keyword(s):  
Transcription Factors ◽  
Myeloid Cells ◽  
Healthy Individuals ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Monosomy 7 ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Cd34 Cells

Abstract Clinical observations revealed that congenital neutropenia (CN) patients harboring either ELANE or HAX mutations have similar bone marrow morphology, responses to G-CSF therapy, requirements of G-CSF dosages, and the risk of developing leukemia. Therefore, we suggested a common pathomechanism of defective G-CSFR-triggered granulopoiesis downstream of both mutated genes in these patients. We identified severely diminished expression and functions of the transcription factors lymphoid enhancer binding factor-1 (LEF-1) and C/EBPa in myeloid cells of CN patients, in comparison to healthy individuals and patients with cyclic neutropenia (CyN). LEF-1 expression was abrogated in patients harboring either ELANE or HAX1 mutations, which suggested LEF-1 as a possible common candidate factor for defective G-CSFR signaling. We further identified a mechanism of the diminished LEF-1 expression downstream of HAX1 or ELANE mutations. HAX1 is HCLS1-Associated protein X1. HCLS1 is Hematopoietic Cell-Specific Lyn Substrate 1. We found that HCLS1 protein is expressed at high levels in human myeloid cells and is phosphorylated upon stimulation with G-CSF. HCLS1 interacted with LEF-1 protein, inducing nuclear translocation of LEF-1, LEF-1 autoregulation, C/EBPa activation, and granulocytic differentiation. In CN patients with HAX1 mutations, we found profound defects in the G-CSF-triggered phosphorylation of HCLS1, subsequently leading to abrogated nuclear transport and autoregulation of LEF-1. In CN patients with ELANE mutations we detected severely reduced levels of the natural inhibitor of neutrophil elastase (NE), and secretory leukocyte protease inhibitor (SLPI). We demonstrated the important role of SLPI in myeloid differentiation by activation of Erk1/2 phosphorylation and subsequent phErk1/2-triggered tyrosine phosphorylation and activation of the LEF-1 protein. Therefore, the direct link between ELANE mutations and diminished LEF-1 expression was established: in these patients LEF-1 protein expression is diminished due to the reduced levels of SLPI. We further evaluated how G-CSF treatment overcomes maturation arrest of granulopoiesis in CN patients despite the absence of LEF-1 and C/EBPa in myeloid cells. We identified nicotinamide phosphoribosyltransferase (NAMPT) as an essential enzyme mediating G-CSF-triggered granulopoiesis in healthy individuals and in CN patients. Treatment of healthy individuals with G-CSF resulted in upregulation of NAMPT levels in myeloid cells and in plasma. NAMPT and NAD+ amounts were even more dramatically elevated by G-CSF treatment of CN individuals. The molecular events triggered by NAMPT included elevation of NAD+, NAD+-dependent activation of protein deacetylase sirtuin-1 (SIRT1), binding of SIRT1 to the myeloid specific transcription factors C/EBPα and C/EBPβ, and activation of these transcription factors. In CN patients, C/EBPα expression is severely diminished; therefore “steady-state” granulopoiesis could not be activated. G-CSF treatment induces expression of C/EBPβ in these patients via NAMPT and SIRT1 and operated via the “emergency” pathway. We also investigated the patterns of acquisition of leukemia-associated-mutations in 31 CN patients developing leukemia using next-generation DNA deep sequencing. Intriguingly, 20 of the 31 patients (64.5%) demonstrated mutations within RUNX1. The majority of patients with RUNX1 mutations (85%) had acquired CSF3R mutations. Other leukemia-associated mutations in the patients with RUNX1 mutations were found infrequently. Cytogenetics of the leukemic cells revealed that 10 patients with RUNX1 mutations developed monosomy 7, and six patients had trisomy 21. Single cell analysis in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Functional studies demonstrated proliferative advantage of CD34+ cells transduced with mutated RUNX1 and CSF3R. By analysis of the leukemogenic role of the defective G-CSFR signaling in CN patients we identified a significant and sustained elevation in the levels of phospho-STAT5 in hematopoietic CD34+ cells of CN patients which were even higher in CN/ acute myeloid leukemia patients. The other possible reason for the leukemogenic transformation could be elevated NAMPT/SIRT-triggered deacetylation of tumor supressor protein p53, proto-oncogene FOXO3a and Akt proteins. Disclosures: No relevant conflicts of interest to declare.

Defective Expression of LEF-1 Transcription Factor mRNA and - Protein in Patients with Severe Congenital Neutropenia (Kostmann’S Syndrome).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 782-782
Author(s):  
Julia Skokowa ◽  
Gunnar Cario ◽  
Zheng Wang ◽  
Cornelia Zeidler ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Healthy Controls ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Abstract Severe congenital neutropenia (SCN) is characterized by “maturation arrest” of myeloid progenitor cells at the promyelocytic/myelocytic stage with the absence or only few mature neutrophils in the bone marrow and peripheral blood. Significant progress in the treatment of SCN patients has been achieved in the last 15 years by administration of granulocyte colony-stimulating factor (G-CSF), which significantly increases the number of neutrophils leading to an improvement of the quality of life. To date, the pathophysiology and underlying genetic defect in patients with congenital neutropenia is still under investigation. Wnt signalling pathway orchestrates a number of cellular programs such as proliferation, differentiation and cell fate determination in many tissues. In the present study we investigated the mRNA and protein expression patterns of Wnt signalling peptides, such as the High Mobility Group (HMG) box containing transcription factors such as lymphoid enhancer factor-1 (LEF-1) and T cell factors (TCFs), as well as β-catenin in CD33+ bone marrow myeloid progenitor cells from SCN patients (n = 6) in comparison to those of patients with cyclic neutropenia (n = 4) and G-CSF-treated healthy controls (n = 3). All SCN and cyclic neutropenia patients are under G-CSF therapy. mRNA expression of genes of interest was measured by quantitative real-time PCR. Protein expression was assessed by immunofluorescence staining, visualized and recorded by confocal microscopy. We found that CD33+ cells from patients with SCN exhibited 20 times lower or even absent expression of LEF-1 mRNA and protein, as compared to healthy G-CSF treated controls (mRNA expression ratio: SCN patients 0.83 ± 0.38 AU vs. healthy controls: 15.1 ± 0.4 AU; p < 0.0001). Intriguingly, LEF-1 mRNA expression levels on CD33+ cells from cyclic neutropenia patients were comparable to those of healthy controls. Immunostaining with anti-LEF-1 polyclonal antibody (kindly provided by Dr. R. Grosschedl) and confocal microscopy analysis revealed that LEF-1 protein was detectable at the expected level in CD33+ cells from healthy G-CSF treated controls. In patients with cyclic neutropenia LEF-1 protein expression in myeloid progenitor cells was comparable to healthy individuals. In contrast, in CD33+ cells from patients with SCN, LEF-1 protein was not detectable. mRNA expression of other TCFs: TCF-1, TCF-3, TCF-4 in SCN was not significantly different from healthy individuals. However, the expression level of LEF-1 binding partner in the Wnt pathway, β-catenin, was increased in SCN patients (SCN patients: 224.7 ± 42.4 AU vs. healthy controls: 107.2 ± 7.3 AU, p = 0.052). The defect in LEF-1 expression in SCN patients was further substantiated by the fact that mRNA expression of LEF-1 target genes such as c-myc, cyclin D1, survivin and neutrophil elastase were also significantly downregulated. In conclusion, our results suggest that defective LEF-1 expression might have an impact on the pathogenesis of SCN. In addition, it may help to distinguish SCN from cyclic neutropenia patients.

C/EBPα:C/EBPα and C/EBPα:Jun Leucine Zipper Complexes Are Detectable in Myeloid Cells Via Binding to Distinct DNA Elements.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1458-1458
Author(s):  
SunHwa Hong ◽  
Alan D. Friedman
Keyword(s):  
Cell Lines ◽  
Leucine Zipper ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Monocytic Differentiation ◽  
New Findings ◽  
Dna Elements ◽  
Gel Shift

Abstract Abstract 1458 Poster Board I-481 Mice lacking C/EBPα have reduced granulocyte-monocyte progenitors, and C/EBPα plays a role in the maturation of GMP along the granulocyte or monocyte lineages. C/EBPα is a member of the bZIP family of transcription factors, dimerizing with itself or other C/EBP proteins via its leucine zipper (LZ) domain to bind palindromic DNA elements (e.g. ATTGCGCAAT, designated αα sites) via the adjacent basic region (BR). AP-1 proteins such as c-Fos and c-Jun represent a distinct bZIP subfamily that heterodimerize amongst themselves to bind AP-1 sites (e.g. TGACTCA, designated JJ sites) to also mediate monocytic differentiation in hematopoietic cell lines or transduced marrow cells. In addition, we previously demonstrated that C/EBPα or C/EBPβ can zipper with c-Jun, JunB, or c-Fos (Cai et al 2008). To accomplish this we employed hybrid proteins in which, for example, the c-Jun LZ was swapped in place of the C/EBPα LZ and binding to a C/EBP site was then assessed after expression in 293T cells. We also provided data suggesting that C/EBPα:c-Jun heterodimers bind preferentially to hybrid αJ sites (e.g. TGACGCAAT). To accomplish this we forced heterodimerization by replacing the LZ in C/EBPα with an acidic zipper containing multiple glutamic acid residues (LZE) and replaced the LZ in c-Jun with a basic zipper containing multiple lysines (LZK). We have now extended these initial findings in two ways. First, we demonstrate that the intact proteins, not only those containing artificial acid or basic leucine zippers, maintain binding site specificity. In particular, when expressed in 293T cells or when generated by coupled in vitro transcription/translation, C/EBPα alone preferentially binds an αα site whereas C/EBPα co-expressed with c-Jun prefers the αJ site. Neither C/EBPα alone nor C/EBPα:c-Jun bound a JJ site. Consistent with the weak affinity of C/EBPα for c-Jun, formation of sufficient C/EBPα:c-Jun heterodimers to allow detection of preferential binding to the αJ site required a 4:1 c-Jun:C/EBPα ratio, with input proteins normalized via a shared N-terminal myc tag. Super-shift assays confirmed that a single gel shift band contains both proteins under these conditions. Co-expression of c-Jun with c-Fos yielded complexes that bound JJ sites, but interestingly c-Jun:c-Fos bound αJ sites with even greater affinity. Second, we have used biotinylated αα and αJ oligonucleotides to demonstrate the presence of both endogenous C/EBPα:C/EBPα and C/EBPα:c-Jun complexes in myeloid cell lines. When 32Dcl3 cells are placed in G-CSF for 24 hrs to stimulate granulopoiesis, Western blotting indicates that C/EBPα levels are induced while c-Jun levels remain constant, and binding of C/EBPα to the αα biotin-oligo increases, likely reflecting the presence of endogenous C:EBPα homodimers, as no interaction of c-Jun with the αα probe was detected. Both endogenous C/EBPα and c-Jun interact the αJ biotin-oligo, suggesting binding by an endogenous C/EBPα:c-Jun complex, and binding of each of these proteins to this probe also increases upon G-CSF induction of C/EBPα. Further support for the presence of an endogenous C/EBPα:c-Jun complex comes from the observation that interaction of C/EBPα with the αJ oligo is several-fold stronger than with the αα oligo, before or after G-CSF addition, whereas gel shift assay results described above indicate that C/EBPα homodimers prefer the αα probe. We previously found that C/EBPαLZK:C/EBPαLZE homodimers favor granulopoiesis whereas C/EBPαLZK:c-JunLZE heterodimers favor monopoiesis of marrow progenitors (Cai et al 2008). In summary, new data indicate that when c-Jun is present in excess, co-expressed C/EBPα and c-Jun preferentially binds hybrid αJ site, and C/EBPα:c-Jun complexes are detected endogenously in myeloid cells. These new findings provide biochemical support for the idea that C/EBPα:AP-1 heterodimers bind novel DNA elements to help mediate monopoiesis, with C/EBPα:C/EBPα homodimers potentially favoring granulopoiesis. Our future focus will be to further define the cellular levels of endogenous C/EBP, AP-1, and C/EBP:AP-1 complexes in immature and mature monocytic and granulocytic cells and to elucidate the spectrum of genes these complexes bind and regulate during myelopoiesis. Disclosures No relevant conflicts of interest to declare.

GATA2 Transcription Factor Mediates Myeloid Expansion Coupled with Lymphoid Block by Transcriptional Regulation of Key Target Genes.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2317-2317
Author(s):  
Satish Kumar Nandakumar ◽  
Geoffrey A. Neale ◽  
Derek A. Persons
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Cell Lines ◽  
Liquid Culture ◽  
Insertional Mutagenesis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Control Group ◽  
Mouse Bone Marrow

Abstract Abstract 2317 Functions of the transcription factor GATA2 in the hematopoietic system were difficult to explore due to the embryonic lethal phenotype on deletion (Tsai et al., Nature 1994) and HSC quiescence on overexpression (Persons et al., Blood 1999). To overcome this problem we developed a Tamoxifen inducible system by fusing the human GATA2 cDNA to the ligand binding domain of the estrogen receptor (GATA2-ERT). Previously we found that when competitive transplant experiments were performed with mouse bone marrow cells, GATA2-ERT transduced cells greatly outcompeted untransduced myeloid cells while at the same time not contributing to the lymphoid lineage. However, no leukemia was observed. Since these effects were observed in the absence of Tamoxifen, we speculated that GATA2-ERT protein leaked into the nucleus to provide low level tonic activity (known to occur in other ERT systems). Immunoblot analysis of BM cells transduced with the GATA2-ERT vector confirmed the presence of the protein in both nuclear and cytoplasmic fractions. In addition, GATA2-ERT transduced cells also out-competed mock transduced cells in liquid culture and could be serially replated in colony forming assays. GATA2-ERT and control GFP transduced cells were diluted with mock cells to achieve around 25% GFP+ cells. After 9 days in culture the percentage of GFP + cells in the GATA2-ERT group was 91%±6 compared to GFP group which remained at 38%±14. GATA2-ERT transduced cells could be cultured indefinitely while after 7 weeks the control GFP transduced cells differentiated to mast cells. These GATA2-ERT cell lines were dependent only on IL-3 and expressed myeloid cell surface markers (53%±1 Gr+, 87%±4 Mac1+ cells n=3). DNA binding mutants of GATA2-ERT completely abolished both serial colony replating ability and growth advantage in liquid culture. This suggested that GATA2-ERT mediates these effects through transcriptional regulation of target genes. To identify the targets of GATA2 responsible for myeloid expansion, gene expression profiling was done on three independently generated GATA2-ERT cell lines and compared to an immortalized myeloid cell line (due to insertional mutagenesis) which does not express GATA2. Genes involved in myeloid neoplasms like Nmyc, HoxA9 and Bcl11a were significantly elevated (HoxA9 –77-fold, Nmyc 60-fold, Bcl11a 36-fold) in the GATA2-ERT lines. To test if GATA2 mediates myeloid expansion through Nmyc, we knocked down Nmyc in GATA2-ERT cells and tested for growth defects. GATA2-ERT cells were transduced with lentiviral vectors harboring shRNA targeting GATA2-ERT (73%±10.2), Nmyc (77%±9.5) and scrambled (75%±7) and cultured for 18 days. As expected, transduced cell numbers dropped to 5%±1 for GATA2-ERT group, confirming GATA2 dependence, while transduced cells decreased to 40%±8 for the Nmyc group with the control group unchanged. This suggests that Nmyc could be one of the key targets of GATA2 responsible for myeloid expansion. Current studies are aimed at determining whether GATA2 directly regulates the Nmyc promoter. Disclosures: No relevant conflicts of interest to declare.

High Insta High Instability Of The CSF3R Gene In Severe Congenital Neutropenia Patients As Judged By Multiple CSF3R Mutations In The Majority Of Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2264-2264
Author(s):  
Maksim Klimiankou ◽  
Murat Uenalan ◽  
Siarhei Kandabarau ◽  
Lutz Wiehlmann ◽  
Anna-Lena Hagemann ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Conflicts Of Interest ◽  
Cell Clone ◽  
Stop Codon ◽  
Severe Congenital Neutropenia ◽  
Sequencing Data ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Extracellular Region ◽  
Inherited Mutations

Abstract It has been reported by us and others that approx. 30 % of all patients with congenital neutropenia (CN) acquire CSF3R mutations in their life time. More than 80 % of the CN patients who develop myeloid leukemia (CN/AML) harbour CSF3R mutations. This suggests that they are the first hit in leukemogenesis. However, detecting sequence changes e.g. by Sanger sequencing reveals only mutations presented in more than 20 % of the cells due to its technical detection limit. Therefore, we asked whether there is a systematic underestimation of cell clones harbouring CSF3R mutations, which might have been traditionally overlooked. We applied the deep-sequencing technology (SOLID 5500xl) to identify CSF3R mutations in myeloid cells from 158 patients with different types of neutropenia (86 severe congenital neutropenia (CN) patients with known inherited mutations (ELANE, HAX1, G6PC3), 21 cyclic neutropenia (CyN) patients, 28 patients with severe chronic neutropenia with so far unknown inherited mutations, 11 patients with SBDS-associated neutropenia) as well as a group of 12 healthy individuals. All neutropenia patients were treated with G-CSF and notably 21 CN patients developed leukemia or MDS. Deep sequencing data were processed according to our custom NGS pipeline (annotation of sequences and prediction of damaging effects on the coding sequence by Polyphen2, removal of known dbSNP variants, and accepting significant Phred-scores at the variant calling stage). Overall the read numbers ranged between 18 and 128069 (median 716), while only variants with at least two percent of the reads were accepted for further consideration (the statistically significant limit is between one and two percent of all reads). All together, we detected 92 CSF3R mutations in 42 CN patients leading to 49 distinct amino acid exchanges (38 missense and 11 stop-codon mutations). The frequency of the mutant alleles ranged from 2 to 96 %. In contrast, in CyN only five out of 21 patients harbour CSF3R mutations; interestingly, two of them in isoform IV of CSF3R (p.P752T). Most notably, whereas 18 patients displayed only one CSF3R mutation, 24 individuals had more than one CSF3R mutation (2-10 mutations, in total 74 mutations). During follow up of some patients, we could demonstrate that the number of mutations increased over time. The majority of mutations were located in the cytoplasmatic region (aa 651-831) of CSF3R, while 15 patients presented mutations within the extracellular region of CSF3R. Intriguingly, in 16 patients we detected 23 non-sense mutations, where 20 of these are stop-codon mutations affecting glutamine (Q) 768, 770, 776, and 781. This suggests that this part of CSF3R is highly instable. In two patients who did not respond to Filgrastim treatment, we detected a stop codon at aa 546 and 547, respectively, affecting the Fibronectin type-III like part of the CSF3R. Twelve patients who developed leukemia (CN/AML) had more than one CSF3R mutations (two to ten) , whereas eight with CN/AML harbored only one mutation. None of the healthy controls, only three neutropenia patients with unknown inheritance, and only one SBDS patient revealed mutations in CSF3R. Taken together, this data suggests that CSF3R is highly prone to genetic instability in severe congenital neutropenia, because more than one mutation in half of the patients was observed and various CSF3R mutations during the course of life accumulated. Once a cell clone harboring CSF3R mutation obtains a second hit (e.g. RUNX1 mutation), they are prone to undergo leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Contributions to Neutropenia from PFAAP5 (N4BP2L2), a Novel Protein Mediating Transcriptional Repressor Cooperation between Gfi1 and Neutrophil Elastase

2009 ◽  
Vol 29 (16) ◽  
pp. 4394-4405 ◽  
Author(s):  
Stephen J. Salipante ◽  
Meghan E. B. Rojas ◽  
Brice Korkmaz ◽  
Zhijun Duan ◽  
Jeremy Wechsler ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Target Genes ◽  
Transcriptional Repressor ◽  
Theoretical Models ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Reporter Assays ◽  
Neutrophil Differentiation ◽  
Novel Protein

ABSTRACT “Neutropenia” refers to deficient numbers of neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease neutrophil elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and neutrophil elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows neutrophil elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which neutrophil elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.

Inherited Biallelic Loss-Of-Function Mutations In CSF3R Define a Novel Type Of Severe Congenital Neutropenia With Full Myeloid Cell Maturation and Refractoriness To RhG-CSF

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1025-1025
Author(s):  
Alexa Triot ◽  
Päivi M Järvinen ◽  
Juan I. Arostegui ◽  
Tomas Racek ◽  
Jacek Puchalka ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Morphological Variability ◽  
Myeloid Cell ◽  
Cell Maturation ◽  
Morphological Evidence ◽  
Compound Heterozygous ◽  
Neutrophil Granulocytes ◽  
Severe Congenital Neutropenia ◽  
Loss Of Function ◽  
Congenital Neutropenia

Abstract Severe congenital neutropenia (SCN) is a heterogeneous group of disorders characterized by defective production and viability of neutrophil granulocytes and predisposition to life-threatening bacterial infections. Currently, OMIM lists five defined monogenic SCN: SCN1 ELANE, SCN2 GFI1, SCN3 HAX1, SCN4 G6PC3. Here, we describe a novel SCN subtype (SCN6) caused by recessively-inherited loss-of-function mutations in the gene encoding the granulocyte colony-stimulating receptor (CSF3R). We have identified four affected children in two distinct families. Family A had a homozygous missense mutation in close proximity of the highly conserved WSXWS motif (c.922T, p.Arg308Cys) and family B had two compound heterozygous small deletions provoking frameshift mutations (p.Gly316fs and p.Gly415fs). Mutated G-CSFR p.Arg308Cys protein was characterized by perturbed N-glycosylation and aberrant localization to cell surface. G-CSF induced phosphorylation of STAT3 and STAT5 was greatly diminished. In contrast to other SCN subtypes, all patients had morphological evidence of full myeloid cell maturation in bone marrow. However, none of the patients responded to granulocyte colony-stimulating growth factor (GCSF) treatment in vivo, confirming aberrant GCSF-receptor dependent signaling. Our studies highlight the genetic and morphological variability of SCN and provide evidence both for functional importance and redundancy of G-CSFR-mediated signaling in human granulopoiesis. Disclosures: No relevant conflicts of interest to declare.

Stop-Codon Mutation In The Extracellular Part Of G-CSFR Leading To G-CSF Unresponsiveness Due To Defects In Erk1/2 Phopsphorylation In a Patient With Severe Congenital Neutropenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3461-3461
Author(s):  
Olga Klimenkova ◽  
Cornelia Zeidler ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Bone Marrow ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Novel Mutation ◽  
Myeloid Cells ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Stop Codon Mutation ◽  
Codon Mutation

Abstract We identified a 10 year old female severe congenital neutropnia patient who did not respond to the treatment with G-CSF (Filgrastim) up to 100 µg/kg/day, s.c. By search of possible reasons of G-CSF unresponsiveness, we detected by deep sequencing of the CSF3R (SOLID 5500xl, 3125 reads) a novel mutation within the extracellular part of CSF3R. The mutation was also confirmed by convential Sanger sequencing. It is a heterozygous stop-codon mutation C1641T (NM_000760), p.W547* (NP_000751) which is localized proximal to the cell membrane in the fifth fibronectin type III domain of the CSF3R. This is a first report describing a stop-codon mutation in the extracellular part of CSF3R in severe congenital neutropenia. FACS analysis revealed severely diminished surface expression levels of G-CSFR on CD33+ bone marrow myeloid progenitor cells of this patient as compared to G-CSFR levels on CD33+ cells of healthy individuals. In line with reduced expression of G-CSFR, we found disturbed G-CSFR-triggered intracellular signaling pathways activated by G-CSFR. MAPK/Erk1/2 activation by phosphorylation is known to be crucial for G-CSF- induced myeloid differentiation and STAT5 signaling is important for proliferation and survival of myeloid cells upon stimulation with G-CSF. We demonstrated abrogated phosphorylation of ERK1/2 and of STAT5 in bone marrow CD33+ myeloid cells of CN patient treated with G-CSF in vitro for 2, 5, 10 and 15 minutes, comparing with healthy donor. Since this patient has no known inherited CN-associated mutations (e.g. ELANE, HAX1, G6PC3), it may represent a new subgroup of CN patients characterized by non-responsiveness to G-CSF due to lack of binding of G-CSF to its receptor. Since the majority of investigations on CSF3R mutations are targeting the cytoplasmic part of the receptor only, we suggest that CN patients who do not respond to G-CSF treatment should be tested also for mutations within the extracellular part of the CSF3R. Disclosures: No relevant conflicts of interest to declare.

LEF-1 Transcription Factor Regulates Proliferation and Differentiation of Myeloid Progenitors in Healthy Individuals and in Patients with Severe Congenital Neutropenia (CN).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 390-390 ◽  
Author(s):  
Julia Skokowa ◽  
Gunnar Cario ◽  
Vesna Bucan ◽  
Lan Dan ◽  
Zheng Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cyclin D1 ◽  
Target Genes ◽  
Healthy Controls ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Myeloid Progenitors

Abstract Wnt signaling pathway plays a key role in the signal transmission during cell growth, proliferation, differentiation and survival, especially in rapidly self-renewing tissues, such as bone marrow, skin, and intestinal mucosa. Regulation of target gene expression by Wnt signaling requires the presence of lymphoid enhancer factor 1 (LEF-1) and/or T-cell factors (TCFs) within the nucleus. Previously we reported 20 times lower or even absent expression of LEF-1 mRNA and protein in CD33+ myeloid progenitors from patients with severe congenital neutropenia (CN), as compared to healthy controls. Abolished LEF-1 expression in CD33+ progenitors from CN patients was accompanied by low levels of LEF-1 target genes, such as cyclin D1, c-myc, ELA2, and survivin. In the present study we aimed to characterize the role of LEF-1 transcription factor in myeloid cells from healthy individuals and from CN patients. Expression profile of LEF-1 mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR of different myeloid precursors (myeloblasts, promyelocytes, myelocytes/metamyelocytes, and mature granulocytes) from bone marrow smears of healthy controls and CN patients. LEF-1 mRNA was predominantly expressed in promyelocytes and myelocytes/metamyelocytes from healthy donors and was dramatically down-regulated in these cell types from CN patients. To investigate the functional consequence of LEF-1 downregulation, we specifically inhibitied LEF-1 expression in HL-60 promyelocytic cells and CD34+ cells from healthy individuals by lentiviral transduction of anti-LEF-1 shRNA. Interestingly, inhibition of LEF-1 resulted in down-regulation of LEF-1 target genes (cyclin D1, survivin, and c-myc), hematopoietic transcription factors C/EBPα and C/EBPε , and was accompanied by dramatically reduced proliferation and increased apoptosis in both HL-60 cells and CD34+ progenitors. Moreover, lentiviral transduction using vector containing LEF-1-GFP reporter into CD34+ cells from one CN patient led to normalization of expression of LEF-1 target genes (c-myc, survivin, cyclin D1), increased expression of C/EBPα , C/EBPε , and partial restoration of myelopoiesis. To investigate the cause of reduced LEF-1 expression in myeloid progenitors from CN patients, we sequenced the LEF-1 gene. We could not find any mutations in 15 CN patients studied. Therefore, regulatory mechanisms of LEF-1 expression, which are different from healthy controls, exist in CN. Interestingly, noggin, one of the activators of LEF-1 expression is significantly downregulated in CN patients. When exposed to noggin-containing medium, LEF-1 and LEF-1 target genes were induced in CD33+ cells from one CN patient, although the effect of noggin on myelopoiesis have to be analysed.Taken together, LEF-1 transcription factor regulate proliferation and differentiation of myeloid progenitors and is involved in the pathomechanism of CN.

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