Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3221-3228 ◽  
Author(s):  
David S. Grenda ◽  
Sonja E. Johnson ◽  
Jill R. Mayer ◽  
Morgan L. McLemore ◽  
Kathleen F. Benson ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Blood Analysis ◽  
Severe Congenital Neutropenia ◽  
Normal Numbers ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Related Disorder ◽  
Increased Risk

Abstract Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of theirEla2 gene (“V72M”) reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2317-2322 ◽  
Author(s):  
David C. Dale ◽  
Richard E. Person ◽  
Audrey Anna Bolyard ◽  
Andrew G. Aprikyan ◽  
Cindy Bos ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Rna Splicing ◽  
Bacterial Infections ◽  
Autosomal Dominant ◽  
Mononuclear Cells ◽  
Mutational Analysis ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Shwachman Diamond Syndrome

Abstract Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 316-316
Author(s):  
Kshama Gupta ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Malignant Transformation ◽  
Upstream Region ◽  
Dependent Manner ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Down Regulation ◽  
Increased Risk ◽  
Cd34 Cells

Abstract Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. For instance, Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90 % of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ~ 20 %). Since AML/MDS are not observed in cyclic or idiopathic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. STAT5 is activated by G-CSF signaling pathway and has also been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPa are a common pathologic mechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF-1 expression and functions. Indeed, we detected elevated phosphoSTAT5 expression in CD34+ cells from CN patients before G-CSF stimulation in vitro, as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34+ cells from healthy individuals resulted in significant downregulation of LEF1 levels in a dose dependent manner. A screen of 10 kb upstream region of LEF1 gene revealed two putative STAT5 binding sites (−3891bp to −3909bp and −3714bp to −3732bp) and the specificity of this binding was confirmed in the nuclear extracts of CD34+ cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34+ cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34+ cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1- dependent activation of LEF-1 promoter. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1. These downstream effects of activated STAT5a may contribute to the malignant transformation of myelopoiesis in CN.

Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease

Blood ◽  
2001 ◽  
Vol 98 (9) ◽  
pp. 2645-2650 ◽  
Author(s):  
Phil J. Ancliff ◽  
Rosemary E. Gale ◽  
Ri Liesner ◽  
Ian M. Hann ◽  
David C. Linch
Keyword(s):  
Neutrophil Elastase ◽  
Autosomal Recessive ◽  
Autosomal Dominant ◽  
Autosomal Recessive Disorder ◽  
Severe Neutropenia ◽  
Base Substitution ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Familial Form

Abstract Severe congenital neutropenia (SCN) was originally described as an autosomal recessive disorder. Subsequently, autosomal dominant and sporadic forms of the disease have been recognized. All forms are manifest by persistent severe neutropenia and recurrent bacterial infection. In contrast, cyclical hematopoiesis is characterized by periodic neutropenia inter-spaced with (near) normal neutrophil counts. Recently, linkage analysis on 13 affected pedigrees identified chromosome 19p13.3 as the likely position for mutations in cyclical hematopoiesis. Heterozygous mutations in the ELA2 gene encoding neutrophil elastase were detected in all families studied. Further work also demonstrated mutations in ELA2 in sporadic and autosomal dominant SCN. However, all mutations described to date are heterozygous and thus appear to act in a dominant fashion, which is inconsistent with an autosomal recessive disease. Therefore, the current study investigated whether mutations in ELA2could account for the disease phenotype in classical autosomal recessive SCN and in the sporadic and autosomal dominant types. All 5 exons of ELA2 and their flanking introns were studied in 18 patients (3 autosomal recessive, 5 autosomal dominant [from 3 kindreds], and 10 sporadic) using direct automated sequencing. No mutations were found in the autosomal recessive families. A point mutation was identified in 1 of 3 autosomal dominant families, and a base substitution was identified in 8 of 10 patients with the sporadic form, though 1 was subsequently shown to be a low-frequency polymorphism. These results suggest that mutations in ELA2are not responsible for classical autosomal recessive Kostmann syndrome but provide further evidence for the role of ELA2 in SCN.

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.

A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation

Blood ◽  
2014 ◽  
Vol 123 (8) ◽  
pp. 1239-1249 ◽  
Author(s):  
Olga Klimenkova ◽  
Wienke Ellerbeck ◽  
Maksim Klimiankou ◽  
Murat Ünalan ◽  
Siarhei Kandabarau ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Neutrophil Elastase ◽  
Secretory Leukocyte Protease Inhibitor ◽  
Myeloid Differentiation ◽  
Severe Congenital Neutropenia ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Key Points ◽  
Natural Inhibitor

Key Points The natural inhibitor of neutrophil elastase, SLPI, is severely reduced in severe congenital neutropenia patients. SLPI controls myeloid differentiation by regulation of NFκB, ERK1/2:LEF-1, and c-myc activation.

Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 493-500 ◽  
Author(s):  
Inga Köllner ◽  
Beate Sodeik ◽  
Sabine Schreek ◽  
Holger Heyn ◽  
Nils von Neuhoff ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Neutrophil Elastase ◽  
Protein Accumulation ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Mutant Proteins ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) are sporadic or inherited hematologic disorders of myelopoiesis. Heterozygous mutations in the gene encoding neutrophil elastase (ELA2) have been reported in both diseases. We used an inducible system to express a panel of ELA2 mutations and found for almost all mutants disruption of intracellular neutrophil elastase (HNE) protein processing at different levels. This disruption resulted in cytoplasmic accumulation of a nonfunctional protein, thereby preventing its physiologic transport to azurophil granules. Furthermore, the secretory capacity of the mutant proteins was greatly diminished, indicating alteration of the regulated and the constitutive pathways. Through analysis of primary granulocytes from SCN patients carrying ELA2 mutations, we found an identical pattern of intracellular accumulation of mutant HNE protein in the cytoplasm. Moreover, cells expressing mutant HNE protein exhibited a significant increase in apoptosis associated with up-regulation of the master ER chaperone BiP, indicating that disturbance of intracellular trafficking results in activation of the mammalian unfolded protein response.

Neutrophil elastase depends on serglycin proteoglycan for localization in granules

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4478-4486 ◽  
Author(s):  
Carsten U. Niemann ◽  
Magnus Åbrink ◽  
Gunnar Pejler ◽  
Rikke L. Fischer ◽  
Erik I. Christensen ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Neutrophil Elastase ◽  
Gram Negative Bacteria ◽  
Severe Congenital Neutropenia ◽  
Wild Type ◽  
Activity Assay ◽  
Congenital Neutropenia ◽  
Bacterial Killing ◽  
Cyclic Neutropenia ◽  
Granule Proteins

Abstract Granule proteins play a major role in bacterial killing by neutrophils. Serglycin proteoglycan, the major intracellular proteoglycan of hematopoietic cells, has been proposed to play a role in sorting and packing of granule proteins. We examined the content of major neutrophil granule proteins in serglycin knockout mice and found neutrophil elastase absent from mature neutrophils as shown by activity assay, Western blotting, and immunocytochemistry, whereas neutrophil elastase mRNA was present. The localization of other neutrophil granule proteins did not differ between wild-type and serglycin knockout mice. Differential counts and neutrophil ultrastructure were unaffected by the lack of serglycin, indicating that defective localization of neutrophil elastase does not induce neutropenia itself, albeit mutations in the neutrophil elastase gene can cause severe congenital neutropenia or cyclic neutropenia. The virulence of intraperitoneally injected Gram-negative bacteria (Klebsiella pneumoniae) was increased in serglycin knockout mice compared with wild-type mice, as previously reported for neutrophil elastase knockout mice. Thus, serglycin proteoglycan has an important role in localizing neutrophil elastase in azurophil granules of neutrophils, while localization of other granule proteins must be mediated by other mechanisms.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2317-2322 ◽  
Author(s):  
David C. Dale ◽  
Richard E. Person ◽  
Audrey Anna Bolyard ◽  
Andrew G. Aprikyan ◽  
Cindy Bos ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Rna Splicing ◽  
Bacterial Infections ◽  
Autosomal Dominant ◽  
Mononuclear Cells ◽  
Mutational Analysis ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Shwachman Diamond Syndrome

Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3044-3051 ◽  
Author(s):  
Julia Skokowa ◽  
John Paul Fobiwe ◽  
Lan Dan ◽  
Basant Kumar Thakur ◽  
Karl Welte
Keyword(s):  
Neutrophil Elastase ◽  
Gene Promoter ◽  
Severe Congenital Neutropenia ◽  
Granulocytic Differentiation ◽  
Congenital Neutropenia ◽  
Protein Levels ◽  
Cd34 Cells ◽  
Direct Binding ◽  
Myeloid Progenitors

Abstract Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis which follows an autosomal dominant or autosomal recessive pattern of inheritance. Genetic analyses indicate mutations in the ELA2 gene in most patients. We have identified LEF-1 as a decisive transcription factor in granulopoiesis controlling proliferation and granulocytic differentiation by direct activation of its target gene, C/EBPα. In patients with CN, the expression of LEF-1 and C/EBPα was abrogated in myeloid progenitors leading to maturation arrest of granulopoiesis. In the present study we demonstrated that ELA2 mRNA expression in myeloid progenitors and plasma protein levels of neutrophil elastase (NE) were markedly reduced in patients with CN harboring mutations in either ELA2 or HAX-1 genes. The ELA2 gene promoter is positively regulated by the direct binding of LEF-1 or C/EBPα, documenting the role of LEF1 in the diminished ELA2 expression. We found that transduction of hematopoietic cells with LEF-1 cDNA resulted in the up-regulation of ELA2/NE synthesis, whereas inhibition of LEF-1 by shRNA led to a marked reduction in the levels of ELA2/NE. LEF-1 rescue of CD34+ cells isolated from 2 patients with CN resulted in granulocytic differentiation of the cells which was in line with increased levels of functionally active ELA2/NE.

scholarly journals Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4179-4187 ◽  
Author(s):  
David S. Grenda ◽  
Mark Murakami ◽  
Jhuma Ghatak ◽  
Jun Xia ◽  
Laurence A. Boxer ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Strong Support ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Unfolded Protein ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins.

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