Cause Of Severe Congenital Neutropenia Research Articles

Neutrophil Elastase Catalytic Activity Regulates Granulopoiesis

Heterozygous, autosomal dominantly inherited mutations in the gene, ELANE, encoding the neutrophil granule serine protease, neutrophil elastase, are the cause of cyclic neutropenia and the most common cause of severe congenital neutropenia. Many mutations throughout the protein occur in patients with congenital neutropenia, but they largely spare residues essential for enzymatic catalysis. Decades ago, neutrophil elastase was proposed to function as a chalone, in which its proteolytic activity feeds back to negatively regulate neutrophil production at steady state levels. To further determine if neutrophil elastase catalytic activity contributes to hematopoietic regulation and to evaluate how its mutations cause hereditary neutropenia, we have studied an induced pluripotent stem cell (iPSC) model of hematopoiesis, in which we have employed CRISPR/Cas9 to engineer a mutation not encountered in neutropenic patients. We inactivated neutrophil elastase's catalytic serine residue through substitution with alanine, generated CD34+ cells from iPSC, and directed their differentiation toward the granulocytic lineage. Compared to similarly edited isogenic controls in which the catalytic serine of neutrophil elastase remains intact, iPSC-derived CD34+ cells lacking the catalytic serine grew at a faster rate, failed to down-regulate ELANE expression while cyclically reactivating CD34 expression during granulocytic differentiation, produced greater quantities of neutrophils, and demonstrated residual progenitor cell populations in replating assays. Some, but not all, of these findings were reproducible upon treatment with small molecule inhibitors of neutrophil elastase. These observations suggest that neutrophil elastase's catalytic activity may help regulate granulopoiesis and could explain genotypic differences distinguishing cyclic neutropenia from severe congenital neutropenia.

Human 'knockouts' of CSF3 display severe congenital neutropenia.

Colony-stimulating factor 3 (CSF3) is a key factor in neutrophil production and function, and recombinant forms have been used clinically for decades to treat congenital and acquired neutropenia. Although biallelic inactivation of its receptor CSF3R is a well-established cause of severe congenital neutropenia (SCN), no corresponding Mendelian disease has been ascribed to date to CSF3. Here, we describe three patients from two families each segregating a different biallelic inactivating variant in CSF3 with SCN. Complete deficiency of CSF3 as a result of nonsense-mediated decay (NMD) could be demonstrated on RT-PCR using skin fibroblasts-derived RNA. The phenotype observed in this cohort mirrors that documented in mouse and zebrafish models of CSF3 deficiency. Our results suggest that CSF3 deficiency in humans causes a novel autosomal recessive form of SCN.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia

Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

A humanized Caenorhabditis elegans model for studying pathogenic mutations in VPS45, a protein essential for membrane trafficking, associated with severe congenital neutropenia.

VPS45, one of the essential membrane trafficking factors, has been identified as a cause of severe congenital neutropenia 5 (SCN5), but its pathophysiological role remains unknown. Here, we developed a humanized C. elegans model for three pathogenic VPS45 variants. We found that wild-type human VPS45 functionally complemented the loss of C. elegans VPS-45 , and the pathogenic human VPS45 variants functioned almost normally with respect to larval development and endocytosis in C. elegans . These results suggest that SCN5-associated mutations have little effect on the core function of VPS45, and/or that the degree of VPS45 requirement varies, depending on the cell/tissue.

Heterozygous variants of CLPB are a cause of severe congenital neutropenia

AbstractSevere congenital neutropenia is an inborn disorder of granulopoiesis. Approximately one third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 severe congenital neutropenia cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase that is implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the adenosine triphosphate-binding pocket. Both genetic loss of CLPB and expression of CLPB variants result in impaired granulocytic differentiation of human hematopoietic progenitor cells and increased apoptosis. These CLPB variants associate with wild-type CLPB and inhibit its adenosine triphosphatase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.

JAGN1 mutations in severe congenital neutropenia.

Severe congenital neutropenia (SCN) was first clinically described in 1956 by Swedish pediatrician Rolf Kostmann, who recognized that infants who suffered from severe bacterial infections with absolute neutrophil counts (ANC) persistently below 0.5 × 109/l represented an inherited genetic disorder.1 SCN is now known to be caused by mutations in at least 25 genes and has autosomal recessive (AR), autosomal dominant (AD) and X-linked forms.2 The most common cause is due to AD mutations in the neutrophil elastase gene, ELANE, while the original disease Kostmann described was found to be due to AR mutations in HAX1.3, 4 Many of the genes causing SCN appear to be involved in endoplasmic reticulum (ER) or mitochondrial function and their disruption is involved in increasing ER stress and enhancing neutrophil apoptosis. In this issue the paper by Khandagale et al.5 whole exome sequencing techniques were applied to 27 AR SCN patients and three were found to have homozygous mutations in jagunal homolog 1 (JAGN1) which was first described as a cause of SCN in 2014 by Boztug et al.6 Jagunal was originally described as being critical for oocyte development in Drosophila via its role in ER reorganization.7 This new study further enhances our knowledge of how JAGN1 mutations can lead to ER stress and intracellular calcium activation of calpain that leads to apoptosis of myeloid cells. The experiments reported here utilized patient neutrophils, human bone marrow and the HL-60 cell line (derived from a human acute promyelocytic leukemia) to demonstrate aberrant neutrophil granules and a predilection to higher cytosolic calcium upon stimulation and enhanced calpain-dependent apoptosis in the presence of the mutant JAGN1 which could be blocked by addition of a specific inhibitor (PD150606) or by silencing JAGN1. It is of note that knocking down JAGN1 with small interfering (si)-RNA did not affect susceptibility of HL-60 cells to cell death, suggesting a dominant pathophysiologic effect of patient mutant JAGN1 on a calpain-mediated process despite an autosomal recessive pattern of inheritance. Khandagale et al. have achieved a very important breakthrough in understanding how JAGN1 mutations promote a non-apoptotic, calpain-dependent form of cell death. The authors acknowledge that further work will be needed to fully understand this pathway. The mainstay of treatment for SCN has been filgrastim since its approval by the FDA in 1991.8 It is a recombinant human granulocyte-stimulating factor that must be injected. While improving quality of life and survival by enhancing numbers and function of neutrophils in most types of SCN, long-term use has been associated with patients being at increasing risk of clonal myeloid disorders and malignancies.9 Whether this is a direct toxicity or due to prolonged survival of the patients allowing evolution of malignant clones from the prolonged pathophysiologic stress from effects of the underlying mutation remains controversial. Other experimental treatments may be appropriate in carefully selected SCN patients such as small-molecule CXCR4 inhibitors such as plerixafor or mavorixafor for WHIM syndrome,10 empagliflozin for G6PT deficiency,11 and perhaps neutrophil elastase inhibitors such as alvelestat for ELANE mutations or JAGN1 inhibitors for mutant JAGN1. Bone marrow transplantation can cure SCN, but is limited by the availability of suitable donors, and is associated with the risk of graft-versus-host disease, infectious mortality and other complications which will be improved by utilizing more specific conditioning regimens.12 Donor availability has been made less of a concern since haploidentical transplant has become more common. Genetic correction of neutrophil gene defects has already been successfully undertaken13 and may be even more practical in the future. In AD SCN, recent evidence suggests deletion of the disease allele may be a simple and effective technique without need to specifically correct the gene defect. In the case of WHIM syndrome, evidence suggests that removal of one copy of CXCR4 enhances haematopoietic stem cell engraftment and survival without the need for conditioning.14 Similarly, editing and removal of ELANE may be curative in the future.15

Heterozygous germ line CSF3R variants as risk alleles for development of hematologic malignancies

AbstractColony-stimulating factor 3 receptor (CSF3R) encodes the receptor for granulocyte colony-stimulating factor (G-CSF), a cytokine vital for granulocyte proliferation and differentiation. Acquired activating heterozygous variants in CSF3R are the main cause of chronic neutrophilic leukemia, a hyperproliferative disorder. In contrast, biallelic germ line hypomorphic variants in CSF3R are a rare cause of severe congenital neutropenia, a hypoproliferative condition. The impact of heterozygous germ line CSF3R variants, however, is unknown. We identified CSF3R as a new germ line hematologic malignancy predisposition gene through analysis of 832 next-generation sequencing tests conducted in 632 patients with hematologic malignancies. Among germ line CSF3R variants, 3 were abnormal in functional testing, indicating their deleterious nature. p.Trp547* was identified in 2 unrelated men with myelodysplastic syndromes diagnosed at 76 and 33 years of age, respectively. p.Trp547* is a loss-of-function nonsense variant in the extracellular domain that results in decreased CSF3R messenger RNA expression and abrogation of CSF3R surface expression and proliferative responses to G-CSF. p.Ala119Thr is a missense variant found in 2 patients with multiple myeloma and acute lymphoblastic leukemia, respectively. This variant is located between the extracellular immunoglobulin-like and cytokine receptor homology domains and results in decreased G-CSF sensitivity. p.Pro784Thr was identified in a 67-year-old man with multiple myeloma. p.Pro784Thr is a missense variant in the cytoplasmic domain that inhibits CSF3R internalization, producing a gain-of-function phenotype and G-CSF hypersensitivity. Our findings identify germ line heterozygous CSF3R variants as risk factors for development of myeloid and lymphoid malignancies.

Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia

BackgroundThe molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease.ObjectiveOur aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN.MethodsWhole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow.ResultsWe identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient’s clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes.ConclusionSpecific mutations in SEC61A1 cause SCN through dysregulation of the UPR.

PML Plays a Key Role in Severe Congenital Neutropenia with Mutant ELANE Causing Neutrophil Elastase Protein Misfolding

Introduction: Severe congenital neutropenia (SCN) is a genetically heterogeneous disease characterized by recurrent infections and a predisposition for malignant transformation. A wide variety of autosomal dominant or sporadic mutations in ELANE encoding neutrophil elastase (NE) are the most frequent cause of SCN, whereas recessive mutations in HAX1 are responsible for the autosomal recessive form of SCN known as Kostmann syndrome. How ELANE and HAX1 mutations cause SCN is still unclear. A prevailing hypothesis is that cellular stresses either caused by protein misfolding or malfunction in the case of ELANE-SCN, or by mitochondrial dysfunction in the case of HAX1-SCN, are drivers of the neutropenia. We focused on the role of the promyelocytic leukemia protein (PML) because PML is implicated in controlling cellular stress responses caused by reactive oxygen species (ROS) and protein misfolding and may exert both oncogenic and tumor-suppressive functions. Aims: (1) To elucidate which cellular stress mechanisms are involved in different genetic subtypes of SCN. (2) To assess the role of PML in SCN with a predicted ELANE misfolding mutation. Methods: We generated induced pluripotent stem cells (iPSCs) from healthy control and SCN patients with non-overlapping mutations: ELANE-I60F, ELANE-R103L and HAX1-W44X. CD34+CD45+ Hematopoietic Stem and Progenitor cells (HSPCs) were derived from iPSCs using the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). PML-/- iPSCs were created by introducing a stop codon in exon 3, shared by all PML isoforms, using CRISPR/Cas9 mediated genome editing. Results: HSPCs derived from the SCN-iPSCs showed increased ROS levels as measured with CellROX Deep Red. Consequently, nuclear translocation of the antioxidant regulatory factor NRF2 was significantly elevated in both ELANE- and HAX1-mutant SCN HSPCs relative to controls. Mutation prediction analysis (Venselaar, BMC Bioinformatics 2010) showed that ELANE-I60F likely causes NE protein misfolding, whereas the ELANE-R103L mutation predictably causes NE malfunction by disrupting interactions with other proteins. The mutation in HAX1 was predicted to result in nonsense-mediated mRNA decay. Transcriptome analysis using Gene Set Enrichment Analysis (GSEA) confirmed upregulation of the nonsense mediated decay pathway in HAX1 mutant HSPCs and in line with previous studies (Klein et al, 2008), FACS analysis using TMRM and Mitotracker Red showed that loss of HAX1 protein reduced mitochondrial membrane integrity. Surprisingly, and in apparent conflict with the mutation prediction analysis, GSEA on ELANE-I60F HSPCs did not show increased expression of the classical unfolded protein response (UPR) pathway. Because PML has been implicated as an alternative player involved in degrading misfolded proteins (Guo, Mol Cell 2014), we investigated a possible link between ELANE-I60F and PML. Immunofluorescent staining showed increased numbers of PML nuclear bodies (PML-NBs) in ELANE-I60F derived HPSCs, but not in ELANE-R103L or HAX1-W44X HSPCs. Furthermore, GSEA showed upregulation of transcripts associated with PML chromatin binding in ELANE-I60F, but not in ELANE-R103L or HAX1-W44X cells. Deletion of PML by CRISPR-Cas9 revealed that PML enhanced MYC and mTORC1-induced transcription and cell cycle signatures in HSPCs from ELANE-I60F, suggestive of an oncogenic role of PML by inducing proliferation and metabolism in ELANE-I60F. In contrast, PML inhibited these pathways in HSPCs derived from healthy control iPSCs, indicative of its tumor-suppressive function in normal HSPCs. Finally, and perhaps most intriguingly, transcriptome analysis revealed that ELANE-I60F HSPCs expressed 5-fold higher levels of (mutant) ELANE transcripts than control HSPCs, which were reduced to basal levels after deletion of PML. Conclusion: HAX1 and ELANE mutations cause oxidative stress in SCN-HSPCs by distinct mechanisms. We provide evidence for a dual role of PML in the pathogenesis of SCN caused by an ELANE mutation (I60F) associated with NE misfolding: (1) NE misfolding and increased oxidative stress cause elevated formation of PML-NBs, leading to increased expression of proliferation, cell cycle and metabolism associated transcripts, (2) PML strongly enhances the levels of ELANE transcripts, thus driving the expression of the disease causative ELANE mutant through a feed-forward mechanism. Disclosures No relevant conflicts of interest to declare.

New Germline Syndrome Discovery: Heterozygous CSF3R Mutations May Predispose to Myeloid and Lymphoid Malignancies

Introduction Colony stimulating factor 3 receptor (CSF3R) plays an important role in granulocyte proliferation and differentiation. Acquired mutations in CSF3R are found in the majority of cases of chronic neutrophilic leukemia and in a minority of atypical chronic myeloid leukemia and acute myeloid leukemia patients, whereas inherited biallelic mutations have been identified as a cause of severe congenital neutropenia (SCN). CSF3R variants have also been detected in lymphoid cells in patients with SCN. However, whether germline monoallelic CSF3R variants predispose to myeloid and/or lymphoid malignancies is unknown. Methods We analyzed variants detected by a clinical next generation sequencing (NGS) panel of about 150 genes important in oncogenesis (OncoPlus) performed on peripheral blood and/or bone marrow of patients with hematopoietic malignancies at The University of Chicago Medical Center from June 2017 to February 2019. We prioritized genes for which variants were likely of germline origin based on variant allele fraction (VAF) > 0.4 and persistence over multiple tests. Variant germline status was determined by extracting DNA from cultured bone marrow-derived mesenchymal stromal cells and/or from cultured skin fibroblasts. Variants in CSF3R [transcript NM_000760.3] were ranked by predicted pathogenicity using standard American College of Medical Genetics criteria. Variants of uncertain significance (VUS) were evaluated using population frequency data, in silico functional prediction, and REVEL scores. Results A total of 620 OncoPlus tests were conducted on 496 patients (ages 1 month - 96 years; median 64 years), and 824 unique variants were identified within 33 genes. Among these patients, 89 (17.9%) had an invariant variant, one that was found at similar VAFs across multiple testing time points. These invariant variants involved 31 different genes with a total of 203 unique variants. Variants with a VAF less than 0.4 were excluded from further study, leaving 81 patients with 164 invariant variants among 30 genes (Figure 1). The genes with the most invariant variants included known germline predisposition genes, such as ATM, BRCA2, DDX41, ETV6, and TP53, as well as CSF3R, which was not previously recognized as an autosomal dominant germline cancer susceptibility gene. Among the 81 patients, 8 (9.9%) were found to have invariant variants in CSF3R, with 7 unique variants identified. Including patients for whom only a single OncoPlus test was conducted in addition to those with invariant variants, there were a total of 43 patients with 20 unique variants in CSF3R among the entire 496 patient cohort (Figure 2). Of these 20 CSF3R variants, 8 (40%) were found to be germline, and among those, one was classified as likely pathogenic, one was classified as a VUS, and six were classified as likely benign. We focused our attention on the likely pathogenic and VUS germline variants. These two variants were among those with the highest predicted pathogenicity scores. The likely pathogenic variant, W547*, is a nonsense mutation in the extracellular domain of CSF3R that was identified in a patient with a history of bladder cancer treated with surgery and chemotherapy who subsequently developed therapy-related myelodysplastic syndrome. W547* has previously been found to be pathogenic in a compound heterozygous state in an infant with SCN. The germline VUS, P784T, is a missense variant identified in a patient with multiple myeloma. This variant localizes to the cytoplasmic domain of CSF3R and is not listed in the COSMIC or gnomAD databases. Functional studies are underway to assess the oncogenic potential of the W547* and P784T variants. Conclusion Detection of invariant variants on clinical, tumor-based NGS panels can be used as a way to identify potential germline mutations to aid in new germline syndrome discovery. Using this approach, CSF3R was identified as a candidate gene for which monoallelic pathogenic variants may predispose to both myeloid and lymphoid malignancies. Future work is ongoing to assess the functional consequences of germline CSF3R variants and the frequency of such mutations. Disclosures Avalos: Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees. Godley:Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees; UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies .

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients

A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. “Maturation arrest,” the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and “maturation arrest” corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE. We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients’ primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.

PF343 CRISPR/CAS9 MEDIATED ELANE KNOCKOUT ENABLES NEUTROPHILIC MATURATION OF HSPCS AND IPSCS OF SEVERE CONGENITAL NEUTROPENIA PATIENTS

Background:Autosomal‐dominant ELANE mutations are the most common cause of severe congenital neutropenia (CN). Although the majority of CN patients respond to daily granulocyte colony stimulating factor (G‐CSF), approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15% of G‐CSF treated patients will develop myelodysplasia (MDS) or acute myeloid leukemia (AML).Aims:“Maturation arrest,” the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated CN. Because mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and “maturation arrest” corrected by CRISPR/Cas9 mediated gene editing.Methods:We applied CRISPR/Cas9‐sgRNA ribonucleoprotein (RNP) mediated ELANE knockout (ELANE KO) in induced pluripotent stem cells (iPSC) and primary hematopoietic stem and progenitor cells (HSPC).Results:We used iPSC from 2 CN patients and HSPC from 4 CN patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE. We observed that granulocytic differentiation of ELANE KO iPSC and HSPCs was comparable to healthy individuals and phagocytic function of the ELANE KO neutrophils was also normal. Knock down of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils.Summary/Conclusion:These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients’ HSPCs followed by autologous transplantation may be an alternative therapy for CN.

Small Molecule Inhibitor of Neutrophil Elastase Normalizes Myeloid Differentiation and Improves Impaired Cell Survival Triggered by Elastase Mutations In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

AbstractAbstract 386Heterozygous mutations in the neutrophil elastase gene ELANE have been identified as the primary cause of severe congenital neutropenia (SCN) associated with recurring severe infections and evolution to acute myeloid leukemia (AML). As of today, more than 50 substitution, truncation, insertion and deletion mutations have been identified. Animal studies based on knock-in or knockout of ELANE in mice failed to produce severe neutropenia phenotype. We and others previously reported that expression of various mutants but not wild type neutrophil elastase (NE) in human but not murine cells triggers accelerated apoptosis. We also reported that expression of mutant NE (del.145-152), identified in SCN patients one of whom evolved to develop MDS/AML, in human promyelocytic tet-off HL60 cells causes both accelerated apoptosis and characteristic block of myeloid differentiation similar to that seen in bone marrow of SCN patients. Examination of the tertiary structure of NE revealed that most of the mutations leave the active site of the mutant protease intact. We identified a small molecule inhibitor of neutrophil elastase, a derivative of L-malic acid (Merck, USA), that blocked the proteolytic activity of NE by approximately 80% and was capable of restoring impaired myeloid differentiation and normalizing production of myeloid cells expressing del145-152 NE mutant. It is important to note that block of proteolytic activity of NE with the NE-SMI had no adverse effect on control human myeloid progenitor cells expressing wild type NE, thus confirming the gain-of-function effect of NE mutants. More than 20% of SCN patients with NE mutations evolve to develop AML. Molecular modeling and analysis of the tertiary structures of NE available through the Protein Database revealed that 16 different mutations identified in AML patients affect predominantly the N95 or N144 glycosylation sites or the binding pocket of the protease suggesting that altered substrate specificity of the mutant enzyme is the cause of accelerated apoptosis and block of myeloid differentiation in SCN/AML. We sought to obtain bone marrow samples from 2 unrelated SCN/AML patients both on G-CSF treatment harboring either C122Y or insPQ94. Bone marrow purified CD34+ and/or CD34-/CD33+ myeloid progenitors from the patients showed basal level of apoptosis in a range of 20–25%, which gradually increased reaching 40–50% apoptosis by 3 days of culture. Importantly, treatment of primary bone marrow-derived cells with NE-SMI substantially reduced accelerated apoptosis to near initial rate with approximately up to 2-fold reduction of apoptosis by 3 days of culture as determined by flow cytometry. Thus, our findings demonstrate that 1) small molecule inhibitor of neutrophil elastase is effective in blocking accelerated apoptosis triggered by three different NE mutations identified in SCN patients evolved to develop MDS/AML and 2) the small molecule inhibitor of NE is a promising therapeutic agent that should be considered for testing in clinical trials in SCN/AML patients. Disclosures:Dale:Amgen: Consultancy, Research Funding; Merck: Patents & Royalties, Research Support.

Update On the Risk of Leukemia in Genetic Subgroups of Congenital Neutropenia (CN): Comparison of Patients with Known Gene Mutations (ELA2, HAX1, WASP, G6PC3, p14).

Abstract 3597Poster Board III-534An increased risk for malignant transformation (MDS or leukemia) is well documented in patients with congenital neutropenia (CN). In this study we assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with known gene mutations, e.g. ELA2, HAX1, G6PC3, p14, WAS or no identified mutation, respectively, by combining all available data from the European and US Branches of the Severe Chronic Neutropenia Registry (SCNIR) and the French Neutropenia Registry (FR).Data from mutational analysis were available for 407 patients. Mutations were identified in 259 CN patients, of whom 209 patients revealed ELA2 mutations, 20 HAX1 mutations, 18 WAS, 8 G6PC3 and 4 p14 mutations. In addition, in 57 patients neither ELA2 nor HAX 1 mutation were detectable and in another 91 patients ELA2 mutations could be excluded, but further genetic evaluation is not yet completed.Secondary malignancies occurred in 50 of the 407 CN patients. The distribution by genetic subtype is shown in the table below:CN subtype by gene mutationTotal patient number (n)MDS/Leukemia (n/%)ELA2-CN20931 (14. 8%)HAX1-CN204 (20%)ELA2 neg + HAX1 neg576 (10.5%)ELA2 neg/ HAX1 not tested915 (5.5%)WAS184 (22.2%)G6PC380p1440Total40750 (12.3%)All subgroups benefit from G-CSF treatment. Median G-CSF maintenance doses required during the years prior to leukemic transformation compared by genetic subtype is shown in the following table 2:CN subtype by gene mutationWithout leukemia (n)Median G-CSF dose (μg/kg/d)With leukemia (n)Median G-CSF dose (μg/kg/d)ELA2-CN755.01515HAX1-CN155.0413.4ELA2-/HAX1-367.2410.5WAS152.643.0G6PC383.90nap1445.10na ConclusionPatients with severe congenital neutropenia who have mutations in ELA2, HAX1, or WAS and also those with no recognized mutation are at risk of secondary leukemia. So far, progression to MDS leukemia has not yet been described in the small number G6PC3 or p14 CN cases in our database. ELA2-CN or HAX1-CN patients requiring higher doses of G-CSF are at greater risk. Mutational analysis is helpful to identify the genetic cause of severe congenital neutropenia but does not serve to identify patients at risk of leukemic transformation. Disclosures:No relevant conflicts of interest to declare.

Severe developmental delay and epilepsy in a Japanese patient with severe congenital neutropenia due to HAX1 deficiency

HAX1 deficiency has recently been identified as a cause of severe congenital neutropenia (SCN), but little is known about the phenotype. We described an SCN patient with a homozygous 256C-to-T transition causing an R86X mutation in the HAX1 gene. Notably, the patient has been complicated by epilepsy and severe delay of motor, cognitive, and intellectual development; each developmental quotient was 21-26 at 7 years old. Growth failure and dental development delay were also noted. Neurodevelopmental delay in this patient expands the clinical phenotype of HAX1 deficiency and suggests an important role of HAX1 on neural development as well as myelopoiesis.

X-Linked Neutropenia with a I294T Mutation of the Wiskott-Aldrich Syndrome Gene.

X-linked neutropenia (XLN, OMIM #300299) is a rare cause of severe congenital neutropenia and was first described in a three-generation Belgian family with 5 affected members. Reported features of XLN include severe congenital neutropenia and monocytopenia with recurrent bacterial infections, a decreased CD4/CD8 ratio and bone marrow maturation arrest at the promyelocyte/metamyelocyte stage. In the Belgian family, a L270P WAS mutation was identified, causing constitutive activity of the Wiskott-Aldrich-syndrome protein (WASP) toward actin polymerisation (Devriendt et al. Nat.Genet. 2001). Here, we report the clinical phenotype of a second large family with XLN and with a I294T WAS mutation. In this three-generation family, 10 affected males (7–45 y) and 8 female carriers were identified. Variable non-cyclic neutropenia is present in affected males (0.2–3.3*109/L in those not on G-CSF; 0.1–1.0*109/L on G-CSF). Five of 10 affected males in the I294T family have monocytopenia. A consistent feature in all cases is a reduced NK cell number. In fact, 3 of 3 tested L270P cases also had reduced NK cell counts. The severity of the clinical phenotype is variable without apparent correlation with the degree of neutropenia. Five of 10 affected males are receiving treatment with G-CSF because of recurrent infections. Four of 10 are reported healthy in the absence of G-CSF. One case with a borderline neutrophil count (2.4*109/L) is not on G-CSF, despite recurrent infections. In addition, two males with a history of recurrent infections, at least one of whom had neutropenia, died of infectious causes at age 5 and 18 years. Platelet counts are variably reduced in affected males, but with normal platelet volume. No consistent abnormalities in CD4/CD8 ratio are found. Available bone marrows have revealed no myelodysplastic features or cytogenetic abnormalities. Of note, female carriers show intermediate findings in neutrophil, platelet and NK cell counts. Only 1 female carrier is known with recurrent upper respiratory and ear infections and is treated with G-CSF. The T916C mutation we found in exon 9 of WAS, has recently been described (Ancliff et al. Blood online 2006) and results in a I294T mutation of the WASP GTPase-binding domain (GBD). The I294T GBD-VCA construct has a lower melting temperature (36°C) as measured by circular dichroism spectroscopy (78°C for wild-type). In the absence of Cdc42, I294T GBD-VCA is nearly completely active toward the Arp2/3 complex, contrary to wild-type GBD-VCA, but similar to the L270P construct. Thus, these data provide new and independent genetic evidence that mutations that disrupt the auto-inhibitory domain of WASP are the cause of XLN. In addition, based on this largest XLN kindred to date, reduced NK cell counts appear a consistent feature. None of the affected males presented with myelodysplasia. Finally, female carriers have moderately reduced neutrophil counts, mostly without clinical consequences. Thus, the presence of mild neutropenia in potential female carriers does not rule out the possibility of XLN in related males with neutropenia. Further investigation is needed to reveal how the L270P and the I294T WAS mutations lead to XLN.

Acquired Mutation and Reversion of the Neutrophil Elastase Gene, ELA2, in Kostmann Syndrome and Cyclic Neutropenia Suggest that Some Mutations Cause Neutropenia while Others Paradoxically Suppress It.

Heterozygous mutations in the gene, ELA2, encoding neutrophil elastase, cause cyclic neutropenia and are the commonest cause of severe congenital neutropenia, a genetically heterogeneous leukemia-predisposing condition also known as Kostmann syndrome. The cataloged mutations have divergent effects on the enzyme, defying singular explanations for how mutations cause disease. Until now, all mutations have appeared to be constitutional and heritable. We describe two exceptional patients with Kostmann syndrome and cyclic neutropenia who each respectively demonstrate somatic mosaicism in the bone marrow of multiple different ELA2 sequences representing acquired mutations of constitutionally wild type alleles or, conversely, acquired reversion of constitutionally mutant alleles. The first patient has Kostmann syndrome, lacks a family history of neutropenia, and comes from a consanguineous population where recessive inheritance is anticipated. Sequencing of PCR-amplified genomic DNA from peripheral blood revealed two novel, apparently heterozygous ELA2 mutations, V69L and V72L, absent in both parents. The mother did possess an adjacent intronic polymorphism inherited by the patient, and we employed this polymorphism to determine the parent of origin of the mutation by sequencing genomic PCR fragments subcloned in bacteria. Remarkably, either or both mutation, as well as a third (V72M), appeared on either maternal or paternal alleles. The mutations have thus arisen independently in multiple different cells of the bone marrow. We propose that Kostmann syndrome here results from inheritance of an unidentified second gene and that the resulting neutropenia paradoxically leads to the selection in the bone marrow of counteracting, “neutrophilic” ELA2 mutations. The second patient has cyclic neutropenia lessening in severity since birth. Sequencing PCR-amplified genomic DNA from peripheral blood revealed two different, apparently heterozygous ELA2 mutations, IVS4+1 G>A (ΔV161-F170), the commonest ELA2 mutation observed in cyclic neutropenia, and T99I, not previously reported. Both mutations were absent in the unaffected parents but present in the patient's buccal cells, demonstrating their de novo constitutional origins. Although the parents lacked distinguishing polymorphisms, the presence of two mutations allowed us to assess for somatic mosaicism. We subcloned genomic PCR fragments in bacteria and found that, in addition to wild type and both mutations in cis, some alleles contained just one or the other mutation, indicating that the mutations have undergone stepwise reversion in multiple, independent cells in the bone marrow. In colony formation assays of circulating stem cells, reversion to wild type occurred exclusively in CFU-GM, but not more primitive CFU-GEMM, suggesting selection in myeloid progenitors. Selection and counter-selection in these two complementary patients imply that there are two opposing categories of ELA2 mutations: some cause neutropenia and others suppress it. This may be the first description of an unexpected genetic phenomenon, where different mutations in a single gene can either cause or ameliorate the same disease, and may explain the divergent effect of various mutations.

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Clinical Relevance of Point Mutations in the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor Receptor Gene in Patients With Severe Congenital Neutropenia

AbstractRecently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF ) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.