ELA2 Mutations Research Articles

Genetic and molecular diagnosis of severe congenital neutropenia

Severe congenital neutropenia has been a well known hematological condition for over 50 years. Over this long period of time, the variable genetic causes and associated sequelae of the disease have been ascertained, and successful treatment strategies developed. Over the past 2 years, however, new studies have added greatly to our understanding of the molecular basis of the disease, details of which are presented in this review. Recent studies have elucidated a role for the unfolded protein response in mediating the pathogenic effects of ELA2 mutations, the most common mutation in severe congenital neutropenia (SCN) as well as cyclic neutropenia. Genetic lesions in HAX1 have also been identified in the original Kostmann pedigree representing the autosomal recessive form of SCN. An emerging theme is the convergence of these and other genetic lesions underlying SCN in enhancing neutrophil apoptosis. Other studies have revealed the importance of multiple independent mutations in these and other genes in SCN. Finally, the key role for signal transducer and activator of transcription 5 in mediating the effects of granulocyte colony-stimulating factor receptor truncation mutations in the development of myelodysplastic syndrome/acute myeloid leukemia following SCN has been elucidated. As the full spectrum of molecular mutations causing neutropenia emerges, it is becoming possible to differentiate patients into subtypes with different prognoses, for whom tailored therapies are indicated.

Risk of Leukemia in Genetic Subgroups of Congenital Neutropenia (CN): Comparison of Patients with Mutations in HAX1 or ELA2

An increased risk for malignant transformation (myelodysplastic syndrome (MDS) or leukemia) is well documented in patients with congenital neutropenia (CN). However, the risk of defined genetic subgroups of CN remains unknown. We therefore assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with ELA2 and HAX1 mutations, respectively. The overall cumulative incidence of secondary leukemias in CN, as documented by long term follow up data from the European Severe Chronic Neutropenia Registry (SCNER) and the French Neutropenia Registry (FR) is 39 out of 343 patients (11.4 %). Up to now genetic testing has been performed in 177 of the 343 CN patients revealing ELA2 mutations in 99 (56%) and HAX1 mutations in 19 (11%) of tested CN patients. In 107patients no ELA2 mutation was detectable. Out of these 107 ELA2 negative patients 59 were also negative for HAX1 mutations (33% ELA2/HAX1 negative patients). In both, patients with ELA2-CN and HAX1-CN, the clinical phenotype is characterized by a maturation arrest of myelopoiesis and severe neutropenia and cannot be discriminated morphologically. There is also no difference in the gender distribution. Both subgroups benefit from G-CSF treatment and respond well to similar G-CSF doses (median G-CSF dose in ELA2-CN is 8,5 μg/kg/day versus 6 μg/kg/day in HAX1-CN. Secondary malignancies occurred in 17 (5 MDS, .11. AML, 1 bi-phenotypic leukemia) out of 99 (17%) ELA2-CN patients, 4 (2 MDS, 1 AML, 1 bi-phenotypic leukemia) out of 19 (21%) HAX1-CN patients, and 5 (1 MDS, 3 AML, 1 ALL) out of 59 (8 %) double negative patients. Mutations in the G-CSF receptor gene have been detectable in both groups of CN patients. The frequency of mutations increases over time with a significant higher frequency of G-CSFR mutations in the CN patients who developed leukemia indicative for an involvement of G-CSFR mutations in the process of leukemic transformation. Interestingly, G-CSFR mutations are detectable in 5 of 8 ELA2-CN leukemic patients tested and 1 of 2 HAX1-CN leukemic patients tested, but also in 1 out of 4 ELA2/HAX1 negative leukemic patients. Independent of the underlying genetic defect the dose response to G-CSF treatment seems to indicate the risk for leukemia. Patients being treated with less than 5 μg/kg/day had an leukemia incidence of 14% versus 26% in patients who received 5 μg/kg/day or more (p=0.027). Within the group of patients being treated with less than 5 μg/kg/day, 14% developed leukemia, whereas in the group of patients receiving = 5 μg/kg/day 26% developed leukemia (p=0.027), suggesting that those patients requiring higher doses of GCSF may be at higher risk for malignant transformation. Leukemia outcome is dependent on the success of hematopoietic stem cell transplantation. The outcome could be improved dramatically since SCT was initiated immediately after confirmation of the leukemia diagnosis. It would be interesting to further discuss why different genetic backgrounds (ELA2 and HAX1) reveal the same clinical phenotype in terms of morphology, response to G-CSF treatment, acquisition of G-CSFR mutations and the development of leukemia.

Incidence of SCN-Assoicated Gene Mutations in Severe Congenital Neutropenia Patients in North America

Severe congenital neutropenia is a genetically heterogeneous syndrome associated with mutations in several different genes including ELA2, HAX1, GFI1, WAS, and CSF3R. The goal of this study was to define the mutation frequency of these genes in the North American SCN patient population. We also sequenced SBDS, since mutations of SBDS have been associated with congenital and acquired neutropenia. A total of 159 patients were identified in the North American Severe Chronic Neutropenia International Registry (SCNIR) for whom informed consent and genomic DNA samples adequate for sequencing were available. To accommodate our semi-automated high-throughput sequencing pipeline, 94 samples were chosen for sequencing. Since ELA2 sequencing had already been performed in most cases, preference was given to those samples without known ELA2 mutation. Among the samples, 73 were from patients with SCN, 4 with cyclic neutropenia, 10 with idiopathic neutropenia, 2 with Shwachman-Diamond Syndrome (SDS), and 3 with Barth syndrome. Two samples were excluded because of poor sequence quality. Singleton cases with validated mutations of GFI1 (N382S) and WAS (L270P) were observed. The N382S GFI1 mutation was associated with striking monocytosis. A novel nonsense mutation of GFI1 (R412X) was detected in one additional case. As expected, compound heterozygous mutations of SBDS were present in the two cases of SDS. In addition, heterozygous mutations of SBDS (84Cfs3X and Q94X) were observed in two cases of SCN. Typical truncation mutations of CSF3R were detected in 4 cases, all developing MDS or AML. Surprisingly, no mutations of HAX1 were detected. Considering only patients with a diagnosis of SCN who were from North America (125 of the total 159 cases), the incidence of ELA2 mutations was 68%. Eleven novel ELA2 mutations were identified. In 28.8% of cases, no mutation of any gene were detected. Based on these data, we recommend that ELA2 genotyping be performed in all patients with suspected SCN. In the North American population mutations in HAX1, GFI1, SBDS, and WAS are rare and routine genotyping is not indicated. Finally, the data suggest that there are yet undiscovered genetic causes of SCN.

Induction of the Unfolded Protein Response but Normal Basal Granulopoiesis in Mice Expressing G192X ELA2

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.

The in Vivo Response of Patients Suffering from Severe Congenital Neutropenia (CN) to G-CSF Is Due to “Emergency” Granulopoiesis Induced by C/EBPβ

C/EBP transcription factors are crucial for the regulation of granulopoiesis in vitro and in vivo. C/EBPa is considered to be the master regulator of “steady state” granulopoiesis via upregulation of several myeloid genes (e.g. ELA2, CSFR3, etc.). The absence of C/ EBPa results in a complete loss of neutrophils. We were able to show that in patients with severe congenital neutropenia (CN) harbouring HAX-1 or ELA2 mutations C/EBPa is severely abrogated secondary to defective expression of LEF-1 (Skokowa J, et al. Nat Med. 12, 1191–7 ( 2006)). Therefore, we were interested, whether other transcription factors are capable of substituting C/EBPa, since these patients respond to G-CSF with slight increase in neutrophils from less than 200/ul to above 1500/ul depending on the dose of G-CSF. C/EBPβ has recently been shown to be required for cytokine induced “emergency” granulopoiesis (Hirai H, et al. Nat Immunol. 7, 732-9 ( 2006)). Therefore, we investigated the expression pattern of C/EBPβ during G-CSF treatment of CN patients. Indeed, C/EBPβ mRNA was upregulated 2.8-fold in CD33+ myeloid cells from CN patients by G-CSF treatment, as compared to healthy individuals. It was associated with upregulation of G-CSFR mRNA and –protein expression as well as ligand binding to G-CSFR in myeloid cells, and elevated levels of biologically active G-CSF in serum from CN patients. To confirm C/EBPβ-dependent activation of G-CSFR and G-CSF gene expression, we performed reporter gene assays in CD34+ bone marrow hematopoietic progenitor cells from two CN patients co-transfected with C/EBPβ and reporter constructs containing upstream regulatory regions of G-CSF or G-CSFR genes, −1470bp and −670bp, respectively. Indeed, C/EBPβ activated G-CSFR promoter 3.2-fold and G-CSF promoter 5.4-fold. These data demonstrate that in CN patients, G-CSF induces C/EBPa independent granulopoiesis and that C/EBPβ is required for the response to G-CSF treatment in these patients. C/EBPβ leads in response to G-CSF to induction of differentiation of neutrophil precursors to mature neutrophils in vivo. Our hypothesis is that in CN the steady state granulopoiesis is abrogated whereas the emergency granulopoiesis still leads to sufficient numbers of neutrophils.

Central nervous system involvement in severe congenital neutropenia: neurological and neuropsychological abnormalities associated with specific HAX1 mutations

Homozygous mutations in the HAX1 gene were recently identified in severe congenital neutropenia patients belonging to the original Kostmann family in northern Sweden. Our observations suggested that these patients also develop neurological and neuropsychological symptoms. Detailed clinical studies and mutation analyses were performed in the surviving patients belonging to the Kostmann kindred and in two patients not related to this family, along with studies of HAX1 splice variant expression in normal human tissues. Five of six Kostmann family patients and one other patient from northern Sweden harboured homozygous HAX1 mutations (568C-->T, Q190X) and one carried a heterozygous ELA2 gene mutation. One Swedish patient of Kurdish extraction carried alternative homozygous HAX1 mutations (131G-->A, W44X). All the three patients with Q190X mutations who were alive and available for evaluation developed neurological disease with decreased cognitive function, and three of four patients who reached 10 years developed epilepsy. In contrast, the patients with the ELA2 and W44X HAX1 mutations, respectively, showed no obvious neurological abnormalities. Moreover, two alternative HAX1 splice variants were identified in normal human tissues, including the brain. Both transcripts contained exon 5, harbouring the Q190X mutation, whereas the 5' end of exon 2 containing the W44X mutation was spliced out from the second transcript. We describe neurological and neuropsychological abnormalities for the first time in Kostmann disease patients. These central nervous system symptoms appear to be associated with specific HAX1 mutations.

Neurodevelopmental abnormalities associated with severe congenital neutropenia due to the R86X mutation in the HAX1 gene

Objective:Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the...

Granulopoïèse et leucémogenèse

Congenital neutropenia are extremely rare diseases, defined by a permanent or cyclic decrease of blood neutrophils. Molecular basis of several congenital neutropenia has been recently determined, involving gene coding for the neutrophil elastase gene (ELA2), GFI1, WAS protein and mitochondrial HAX1 protein. These mutations, dominant (ELA2, GFI1), X-linked (WAS) and autosomal recessive (HAX1), result in instability of the contents of the granules- particularly the neutrophil elastase- or in abnormalities of the cytoskeleton, and possibly, in an increased apoptosis. ELA2 mutations resulting both in profound and permanent neutropenia, and in cyclic--pseudo sinusoidal--neutropenia lead to consider that time pattern is very close in the two apparently distinct phenotypes. This observation suggests that temporal variations of neutrophils could be represented by non linear functions. Congenital neutropenia, specifically ELA2 mutated, are also characterized by a high rate of leukemia (about 15% at 20 years of age). Leukemia risk does not appear to be related to an oncogenic effect of ELA2 mutations, but much likely to the deepness of the neutropenia, and the intensity of G-CSF therapy.

Severe congenital neutropenia and the unfolded protein response

Severe congenital neutropenia is an inborn disorder of granulopoiesis. Mutations of ELA2, encoding neutrophil elastase, are present in approximately 50% of cases of severe congenital neutropenia and nearly all cases of cyclic neutropenia, a related disorder of granulopoiesis. Herein, we review recent studies of the molecular pathogenesis of severe congenital neutropenia, with an emphasis on those cases associated with mutations of ELA2. Genetic evidence suggests that ELA2 mutations act in a dominant, cell-intrinsic fashion to disrupt granulopoiesis. A unifying model that accounts for the different clinical phenotypes (severe congenital neutropenia versus cyclic neutropenia) and the diversity of ELA2 mutations (over 50 have been identified), however, is lacking. Recent studies suggest that mutations of ELA2 may cause disease through induction of the unfolded protein response. In this model, the ELA2 mutations result in the production of misfolded neutrophil elastase protein, activation of the unfolded protein response, and ultimately apoptosis of granulocytic precursors. The propensity of individual neutrophil elastase mutants to misfold may determine the magnitude of unfolded protein response-induced apoptosis and ultimately the clinical phenotype. Recent studies provide support for a unfolded protein response model of disease pathogenesis in cases of severe congenital neutropenia associated with ELA2 mutations and place severe congenital neutropenia in a growing list of human disease caused by misfolded proteins.

Chronic idiopathic neutropenias and severe congenital neutropenia

Chronic idiopathic and severe congenital neutropenias are rare disorders for which recent discoveries have highlighted mechanisms and consequences. An inflammatory bone marrow milieu has been shown to be a major contributor to the pathophysiology of chronic idiopathic neutropenia. Activated T-lymphocytes with myelosuppressive properties and pro-apoptotic mediators, such as IFNgamma, TNFalpha, Fas-ligand and TGFbeta1 result in accelerated apoptosis of granulocytic progenitor cells. Decreased levels of the anti-inflammatory cytokine IL-10 further disturb the balance between survival and pro-apoptotic mediators in chronic idiopathic neutropenia. Mutations in the HAX1 gene are associated with most cases of recessive autosomal severe congenital neutropenia, while ELA2 mutations are found in most cases of autosomal dominant and sporadic cases. The role of HAX-1 protein as a regulatory step in apoptosis provides further evidence for severe congenital neutropenia as a disorder of programmed cell death. The preleukemic character of severe congenital neutropenia, particularly for patients with need for high granulocyte colony stimulating factor dosage, was recently emphasized. Chronic idiopathic (or as recent data suggest, immunologic) and severe congenital neutropenias provide intriguing models for better understanding of regulation of myelopoiesis. Similarities and differences between the two disorders might help to dissect these regulatory events.

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

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.

Congenital dysgranulopoietic neutropenia

We investigated a 15-year-old female with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother who had recurrent stomatitis. In both patients, cells of the neutrophilic, eosinophilic, monocytic, megakaryocytic, and basophilic series were dysmorphic. Plasmacytoid lymphocytes and mild megaloblastic erythroid precursors were present. Bleeding times of both patients were prolonged. The mother had a secondary aggregation defect; the number of the plasmacytoid lymphocytes, dense granules of platelets, and dysmorphic neutrophils, neutrophil chemotaxis, and myeloperoxidase content fluctuated according to the presence or not of aphthae. The daughter's karyotype revealed 46,XX/46,XX, t(1;8). No ELA2 or G-CSFR mutation was detected. These findings support stem cell involvement in CDN.

Association of Neurodevelopmental Abnormalities Is a Common Clinical Character in Japanese Patients with Severe Congenital Neutropenia Due to HAX1 Deficiency.

Severe congenital neutropenia (SCN) is characterized by early childhood onset of profound neutropenia and recurrent life-threatening infections. The bone marrow usually shows a reduced number of mature myeloid cells with an arrest at the promyelocyte-myelocyte stage. SCN includes a variety of hematological disorders caused by the different genetic abnormality. Genetic analysis in individuals with SCN indicates that 60% of cases are attributable to heterozygous mutation in ELA2, encoding neutrophil elastase. Recently, homozygous mutation in HAX1 encoding HAX1, mitochondrial protein associates with HS1 which is a substrate for Src family tyrosine kinase, was identified in individuals with autosomal recessive forms of SCN and the original Kostmann family in Sweden. In this study, we examined involvement of HAX1 and ELA2 in 17 Japanese cases with SCN. Ten patients had heterozygous mutations, including two novel mutations in ELA2. Five patients had homozygous or compound heterozygous mutations in HAX1. A homozygous single-base-pair substitution at nucleotide 256 (256C>T) HAX1 mutation causing a nonsense change R86X was detected in three patients. Two sibling cases showed compound heterozygous mutation of 256C>T and 59-bp deletion at nucleotide 376–434. This 59-bp deletion was a novel mutation leading to a frameshift and a premature stop codon at nucleotide 441–443 (R126fsX128). All heterozygous carriers had no detectable phenotype. HAX1 protein was undetectable in both homozygous and compound heterozygous patients by immunoblot analysis of leukocytes. Notably, all patients with HAX1 deficiency showed failure to thrive and mild to severe developmental delay. Further, three patients carrying homozygous R86X mutation had epilepsy. Two of three patients had complex partial seizures, and another had atypical absence. An Iranian patient carrying homozygous R86X mutation of HAX1 was described to show failure to thrive in the original report of HAX1 deficiency, and three of six cases of the original Kostmann family showed neurological deficits. In contrast, no SCN patients with a heterozygous mutation in ELA2 displayed the association of neurodevelopmental abnormalities. Thus, R86X mutation in HAX1 is a common abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities as well as severe myelopoietic defects. Because HAX1 is a ubiquitously expressed gene, further accumulation of the patients should be needed to characterize the clinical features of HAX1 deficiency.

Granulocytic Precursors from Patients with ELA2-Mutant Severe Congenital Neutropenia Display a Transcriptional Profile Consistent with Activation of the Unfolded Protein Response.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of ELA2, encoding neutrophil elastase (NE), are present in approximately 50% of cases of SCN. To date, 52 distinct, mostly missense, mutations of ELA2 have been identified. The diversity of ELA2 mutations in SCN and lack of consistent effect of these mutations on NE enzyme activity led us to hypothesize that structural rather than functional perturbations in the NE protein might be responsible for the disruption in granulopoiesis. Specifically, we hypothesized that a shared feature of the different NE mutants is their propensity to misfold. In this model, accumulation of misfolded NE in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) and ultimately leads to apoptosis of granulocytic precursors. The UPR is an adaptive cellular program that ameliorates protein-folding defects in the ER by attenuating most new translation and increasing transcription of genes involved in productive refolding and ER-associated degradation of misfolded proteins. However, if the ER stress is severe and ER homeostasis cannot be restored, the UPR triggers cell apoptosis. In support of this model, we and others previously showed that expression of mutant but not wild-type NE in myeloid cells induces UPR activation. Moreover, expression of mutant NE induced apoptosis in a protease-independent fashion. Finally, we showed that BiP/GRP78 mRNA expression and XBP-1 splicing, two classic markers of UPR activation, were induced in primary SCN granulocytic precursors. These data, while supporting the UPR model of disease pathogenesis, are limited in their scope. Previous studies have shown that activation of the UPR induces the transcription of a characteristic set of genes involved in ER homeostasis. In the present study, we performed RNA profiling on primary SCN granulocytic precursors to determine whether these cells displayed a transcriptional profile consistent with the UPR. Fresh bone marrow samples were obtained from 8 patients with SCN (7 with ELA2 mutations), 5 healthy controls, 2 healthy controls treated with G-CSF for 5 days, or 4 patients with Shwachman-Diamond syndrome (as a control for stress response genes induced by neutropenia, per se). In addition, bone marrow from two healthy controls were cultured for 24 hours with or without tunicamycin (an established activator of the UPR). In each case, CD15+CD16lowCD14-CD9- cells were isolated by sorting to obtain a cell population highly enriched for promyelocytes/myelocytes (>85% in all cases). RNA profiling was performed using the Affymetrix HG U133 Plus 2.0 Array. Remarkably, the primary SCN granulocytic precursors, but not the other cohorts, exhibit a transcriptional program similar to that of normal granulocytic precursors treated with tunicamycin. Specifically, we detected similar upregulation of the following classes of genes that are transcriptionally activated by the UPR: ER resident chaperones (subclasses HSP40, HSP70, and HSP90), foldases (disulfide isomerase and peptidylprolyl isomerase subclasses), and mediators of apoptosis (CHOP/DDIT3 and pro-apoptotic members of the Bcl-2 family). These data provide strong new evidence in support of the UPR model of SCN disease pathogenesis.

Defective Expression of Neutrophil Serine Proteases in Myeloid Progenitors of Congenital Neutropenia Patients Carrying Either ELA2 or HAX1 Mutations.

Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis with two major types of inheritance:1.autosomal dominant CN defined by mutations in ELA2 gene encoding neutrophil elastase (NE) (Horwitz M., et al., Nat Genet. 1999; 23:433) and2.autosomal recessive CN (including Kostmann syndrome) carrying HAX-1 mutations (Klein C., et al., Nat Genet. 2007; 39:86), both characterized by a maturation arrest of granulopoiesis at the level of promyelocytes.In the present study we aimed to evaluate the expression profile of genes specifically expressed in the CD33+ bone marrow promyelocytes of both patient groups harbouring ELA2 or HAX1 mutations. In healthy individuals mRNA expression levels of neutrophil serine proteases (neutrophil elastase (ELA2), cathepsin G, cathepsin D, proteinase 3 and azurocidin) as well as of myeloperoxidase (MPO) and defensins reached highest levels in the azurophil granules at the promyelocytic stage of neutrophil differentiation (Borregaard N., et al., Curr Opin Hematol. 2001; 8:23). We found downregulation of mRNA expression levels of ELA2 (8.9 fold), cathepsin G (7.6 fold), cathepsin D (11.2 fold), proteinase 3 (9.2 fold) and defensin B1 (6.5 fold) in both groups of CN patients (with ELA2 or HAX1 mutations), in comparison to G-CSF-treated patients with idiopathic neutropenia (IN) and G-CSF-treated healthy controls. In contrast, there were no difference in mRNA expression levels of azurocidin and only slight decrease in the expression of MPO mRNA in CN patients. Additionally, we found significantly reduced protein levels of neutrophil elastase (NE) in plasma of CN patients irrespective of “ELA2 or HAX1” inheritance, in comparison to cyclic neutropenia (CyN) patients, IN patients and G-CSF-treated healthy controls. Taken together, both ELA2 and HAX1 mutations are associated with defective expression of neutrophil serine proteases such as NE, cathepsin G, cathepsin D, proteinase 3 as well as of defensin B1 in CD33+ myeloid progenitor cells of CN patients, suggesting a common pathway for both patient groups. Intriguingly, ELA2 expression is directly regulated by LEF-1, suggesting that abrogated LEF-1 expression in CN promyelocytes (Skokowa J., et al., Nat. Med. 2006; 12:1191) may be responsible for defective serine proteases expression in both groups, since all are regulated by a similar mechanism.

T-Cell Autoimmunity May Contribute to Neutropenia in a Patient with Cyclic Neutropenia (CN) and Double De-Novo Mutations in Gfi-1.

CN is a rare autosomal dominant disease characterized by predictable cycling of patients' neutrophils from normal levels to a nadir of ≤100 cells / μL. While heterozygous mutations in the neutrophil elastase gene (ELA2) occur in the majority of patients and altered subcellular trafficking of neutrophil elastase (NE) has also been demonstrated, it remains unknown how these defects lead to the CN phenotype. Similarly, mutations in the gene for the beta subunit of adapter protein 3 (AP3), which binds NE and shuttles it to endosomal vesicles, are present in severely neutropenic patients with Hermansky-Pudlak syndrome (type 2). Interestingly, separate single base-pair mutations in the zinc finger domains of Gfi-1, a gene that maintains the long-term potential of hematopoeitic stem cells and that negatively regulates ELA2 expression, were reported in two different patients with severe congenital neutropenia (SCN). Decreased immunity against recall antigens, and an increase in the proportion of T-cells bearing a mature memory phenotype have also been described in patients with Gfi-1 mutations. As yet, there is no unifying hypothesis linking these various abnormalities. We hypothesized that aberrantly expressed NE, resulting from mutations in ELA2 or associated regulatory genes, could predispose autologous T-cells to target myeloid precursors that aberrantly express NE. Such a reversal of immune tolerance could contribute to neutropenia in CN and SCN patients. We report in a 25 year-old man with CN from birth the simultaneous occurrence of heterozygous mutations in Gfi-1 (N382S and K403R), which were previously identified in separate patients with SCN. Neither mutation was found in this patient's parents however, and sequencing of mRNA encoding PRTN3, ELA2 and AP3 showed no mutations. Because Gfi-1 serves as a transcriptional repressor of ELA2, we hypothesized that NE may be overexpressed and / or mis-trafficked in neutrophils. To address this hypothesis, we analyzed granulocytes by FACS staining and confocal microscopy. NE was disproportionately expressed in the plasma membrane of 75% of the patient's granulocytes compared to only 3% of granulocytes from healthy donors (HD). We have previously demonstrated that cytotoxic T-lymphocytes (CTLs) that recognize proteinase 3 (P3) and NE-derived peptides, such as the HLA-A2 restricted peptide PR1 (VLQELNVTV), can kill leukemia but not normal myeloid cells due to aberrant expression of P3 and NE in the leukemia and that these PR1 specific CTLs (PR1-CTL) are not detected in healthy donors. Because this patient expressed the HLA-A2 allele, we used fluorescently labeled PR1/HLA-A2 tetramers to study whether PR1-CTL were present. Strikingly, 0.5% to 1% of total CD8+ cells from the patient were specific for PR1, as measured over 2 years, whereas no PR1-CTL were present in control HD or the patient's mother, who is also HLA-A2+. The PR1-CTLs co-expressed CCR7 and CD45, consistent with a naïve phenotype, and cytokine secretion after crosslinking the T-cell receptors was similar to controls. These data suggest that aberrant NE expression can lead to a reversal of immune tolerance and a corresponding increase in PR1-CTL. The mechanism of neutrophil cycling in CN patients is not known; however, an autoimmune response against peptides derived from aberrantly expressed granule proteins in myeloid precursors could lead to a CTL-mediated feedback loop that would also be consistent with the chalone hypothesis of feedback regulation already proposed for this disease.

The Human Severe Congenital Neutropenia-Associated Gfi1N382S Mutant Critically Requires Deregulated Expression of CSF1 To Block Granulopoiesis.

Severe Congenital Neutropenia (SCN) is characterized as a deficiency of mature neutrophils, which leads to recurrent bacterial and fungal infections. Mutations in the Ela2 gene predominate in SCN, but Ela2 mutation in mice does not recapitulate SCN. The Growth factor independent-1 (Gfi1) transcriptional repressor regulates Ela2. The N382S mutation in Gfi1 is associated with human SCN. Unlike Ela2, genetic deletion of Gfi1 results in murine neutropenia. Here we show that the human-SCN-associated Gfi1N382S mutant blocks murine granulopoiesis in a Csf1 dependent manner. Gfi1 functions as a rate-limiting granulopoietic molecular switch, and the DNA-binding-defective Gfi1N382S mutant acts as a dominant negative to block granulopoiesis by selectively derepressing a subset of Gfi1 target genes. One gene repressed by Gfi1 and derepressed by Gfi1N382S is the macrophage colony stimulating factor (Csf1). Chromatin immunoprecipitation and electrophoretic mobility shift analyses demonstrate direct binding of Gfi1 to the Csf1 promoter. Importantly, both antibody absorption of Csf1 and genetic deletion of Csf1 prevent the Gfi1N382S-mediated block to granulopoiesis. These data represent the first identified Gfi1 target gene to be critically required for SCN-like phenotypes engendered by Gfi1N382S, and suggest new potential treatment avenues for Gfi1-mutant SCN patients.

Cyclic Neutropenia Is Not Associated with Transformation to MDS and AML.

Cyclic neutropenia is a rare hematological disorder characterized by recurrent severe neutropenia, mouth ulcers, fever and infections beginning soon after birth. The diagnosis depends primarily on observing regular periods of severe neutropenia (neutrophils <500/μL, usually <200/μL) lasting for 2–4 days at approximately 21-day intervals, documented by serial blood counts (a minimum of 2–3 complete blood counts per week for at least six weeks). Severe congenital neutropenia is a similar, but usually more severe disease. Mutations of the ELA2 gene are regarded as the cause of most cases of both cyclic neutropenia and severe congenital neutropenia. However, only patients with severe congenital neutropenia, not cyclic neutropenia, are thought to be at risk of evolution to MDS/AML. Since its founding in 1994, the Severe Chronic Neutropenia International Registry (SCNIR) has utilized data from previous studies and enrolled patients with cyclic neutropenia based on the clinical information from their treating physicians. We reviewed 190 cases of cyclic neutropenia (including 30 families) enrolled for 0.5 to 19.9 years, (median 11 years) with a total of 2,035 patient years of observation. 173 of 190 patients were treated with G-CSF (median dose 2.82 mcg/kg/day). We identified three patients who were reported to have developed MDS/AML. Further analysis by three experts from the SCNIR revealed that all three patients lacked sufficient serial blood count data to show well-defined neutrophil cycles prior to G-CSF. Two of the three patients lacked a clinical history of mouth ulcers and fevers consistent with the diagnosis, and the bone marrow evaluation was not consistent with cyclic neutropenia in all three cases. One of two patients tested did not have an ELA2 mutation. Thus, using standard criteria for the diagnosis of cyclic neutropenia, none of the three patients fully satisfied the criteria for this diagnosis. In addition, previous family studies, reviews of the published literature, and personal experience of the authors have revealed only one case of leukemia. This case of CML emerged in a 35 year-old patient, who was never treated with G-CSF, 31 years after the diagnosis of cyclic neutropenia. Based upon the review of these cases, we believe it is extremely important to make the diagnosis of cyclic neutropenia by careful analysis of serial blood counts. When the diagnosis is made confidently, it is also important to assure patients that with or without G-CSF treatment, the possibility of evolution to leukemia is unlikely.

Identification of Two Novel Homozygous HAX1 Mutations in an Autosomal Recessive Jewish and Two Unrelated Sporadic British Families with Severe Congenital Neutropenia.

Severe chronic neutropenia (SCN) is a rare disorder characterized by profound neutropenia, recurrent bacterial infections and bone marrow arrest at the promyelocyte/myelocyte stage. Initially described as an autosomal recessive (AR) disease (Kostmann disease), autosomal dominant (AD), sporadic and X-linked forms have been reported. Pathogenic mutations in the neutrophil elastase ELA2 gene have been documented in both SCN and the related disorder cyclic neutropenia in a significant proportion of AD and sporadic but not AR patients, and mutations in the Wiskott-Aldrich syndrome (WAS) gene have been reported in X-linked SCN families. More recently, homozygous mutations resulting in AR disease were identified through linkage to chromosome 1p21 in Kurdish and the original Kostmann families, revealing mutations in the HAX1 gene that result in complete loss of this mitochondrial anti-apoptotic protein. In order to determine the frequency of these different mutations in SCN, we used denaturing HPLC WAVE analysis to investigate a cohort of 105 SCN kindreds seen in our institute or referred with a diagnosis of SCN of which 11 were presumed/known AD, 17 AR, 73 sporadic and 4 had X-linked disease. Overall, 25 different ELA2 mutations were detected in 31 kindreds (6 AD and 25 sporadic, 30% of the cohort). Mutations were distributed throughout the gene and were most frequent in exon 4 (42% of mutations), least frequent in exon 3 (10%). Two kindreds (2%) had WAS mutations as previously published, no new cases were identified. The remaining 72 cases were screened for all 7 HAX1 exons using mixtures of patient and control PCR products to detect homozygous substitutions. Four homozygous mutant kindreds were identified. One AR kindred of Kurdish descent had the previously reported p.Trp44X mutation. Two sisters of Jewish origin had a homozygous insertion of an extra G in a run of 5 Gs (nucleotides 121–125) in exon 2 resulting in a frameshift (p.Ser43LeufsX11); both parents were shown to be heterozygous for the mutation. Two unrelated sporadic SCN patients of Caucasian (British) origin had deletion of nucleotide G91 in exon 2 resulting in a frameshift (p.Glu31LysfsX54); to date, parents of one case have been confirmed as heterozygotes. Both frameshift mutations are predicted to result in a non-functional protein. The 4 HAX1-mutant kindreds have variable clinical severity (ranging from mild to severe) and G-CSF requirement (none to regular administration). All heterozygous parents are phenotypically normal. Our results indicate that HAX1 mutations are relatively uncommon (4% of our cohort) but not limited to patients with specific ethnic origin or apparent AR inheritance.

Constitutive Activation of STAT5a in Myeloid Progenitors of Severe Congenital Neutropenia Patients Leads to Downregulation of LEF-1.

Severe congenital neutropenia (CN) is a hematological disorder characterized by an early stage maturation arrest of granulopoiesis in bone marrow at promyelocytic stage. Previously we found that LEF-1 and its target gene C/EBPα are severely abrogated in promyelocytes of CN patients, which plays a decisive role in the pathomechanism of defective granulopoiesis (Skokowa J., et al., Nat. Med. 2006; 12:1191). Recently it has been shown that constitutively activated STAT5a inhibits myeloid differentiation of human CD34+ cells by downregulation of C/EBPα (Moore MA, et al., Exp. Hematol. 2007; 35:105). Therefore, we investigated whether the presence of constitutive activated STAT5a in CD34+ cells of healthy individuals leads to downregulation of LEF-1 and whether STAT5a is activated in myeloid progenitors of CN patients. A screen of the 10kb upstream region of LEF-1 gene revealed two putative STAT5a binding sites (−3913 bp to −3894 bp and −3728 bp to −3709 bp) and specificity of the STAT5a binding to the LEF-1 promoter in nuclear extracts of CD34+ cells was confirmed in a chromatin immunoprecipitation (ChIP) assay. Additionally, our data demonstrated that introduction of retroviral constructs expressing IRES-GFP and cDNA of constitutively activated STAT5a (mutant of STAT5a, (STAT5A[1*6]) into CD34+ cells of healthy individuals resulted in 20-fold downregulation of LEF-1 mRNA expression. To compare G-CSF- dependent phosphorylation of STAT5 in CD33+ bone marrow myeloid progenitors of CN patients and healthy individuals, we treated these cells with G-CSF in vitro and measured phosphorylation of STAT5 by intracellular staining of cells with phosphoSTAT5 (Y694) antibody. We found that G-CSF stimulation resulted in a significantly higher phosphorylation of STAT5 in myeloid progenitors of four CN patients, as compared to four healthy volunteers (mean fluorescence intensity (MFI) 436 ± 57 in CN patients vs 212 ± 30 in healthy controls). Interestingly, augmented phosphorylation of STAT5 has been observed in both groups of CN patients carrying either ELA2 or HAX1 mutations. These data confirm the hypothesis that downregulation of LEF-1 and LEF-1 target gene C/EBPα is at least in part a consequence of increased activation of STAT5a in both CD34+ cells of healthy individuals and in patients with CN.