De Novo Deep Intron ELANE Mutation Resulting in Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) comprises a diverse range of rare hematological disorders characterized by recurrent, often life-threatening infections that manifest within the first months of life. Mutations in the ELANE gene are the most prevalent cause of SCN. While over 230 mutations in ELANE have been documented, including substitutions, frameshifts, nonsense mutations, and splice site alterations, the occurrence of deep intronic mutations has not been previously reported. Herein, we present the case of a young girl who exhibited recurrent fever, respiratory infections, skin abscesses, and gingivitis shortly after birth. Laboratory analysis revealed markedly diminished neutrophil levels alongside elevated monocyte and eosinophil counts. Bone marrow examination disclosed a halt in myelopoiesis maturation. ELANE gene full-length sequencing identified a novel de novo deep intron mutation in ELANE (c.598 + 79G > T), subsequently confirmed by Sanger sequencing. cDNA sequencing of the patient demonstrated aberrant gene splicing. Utilizing a mini-gene splicing assay for ELANE intronic variants, we identified a mutant ELANE allele (c.597 + 1_597 + 83ins) leading to the creation of a premature termination codon (p.Gly200ValfsTer40). Confocal microscopy revealed heightened expression of myeloperoxidase and neutrophil elastase in the patient, suggesting a potential role for the unfolded protein response in the pathogenesis of the deep intron ELANE mutation. In summary, our findings illustrate the first reported instance of de novo deep intron ELANE mutations associated with SCN, underscoring the importance of exploring deep intronic regions in SCN patients lacking identifiable disease-causing gene mutations.

A neutrophil elastase-generated mature form of IL-33 is a potent regulator of endothelial cell activation and proliferative retinopathy.

Human interleukin-33 (IL-33) is a 270 amino acid protein that belongs to the IL-1 cytokine family and plays an important role in various inflammatory disorders. Neutrophil proteases (Cathepsin G and Elastase) and mast cell proteases (tryptase and chymase) regulate the activity of IL-33 by processing full-length IL-33 into its mature form. There is little evidence on the role of these mature forms of IL-33 in retinal endothelial cell signaling and pathological retinal angiogenesis. Here, we cloned, expressed, and purified the various mature forms of human IL-33 and then evaluated the effects of IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 on angiogenesis in human retinal microvascular endothelial cells (HRMVECs). We observed that IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 significantly induced HRMVEC migration, tube formation and sprouting angiogenesis. However, only IL-3399-270 could induce HRMVEC proliferation. We used a murine model of oxygen-induced retinopathy (OIR) to assess the role of these mature forms of IL-33 in pathological retinal neovascularization. Our 3'-mRNA sequencing and signaling studies indicated that IL-3399-270 and IL-33109-270 were more potent at inducing endothelial cell activation and angiogenesis than the other mature forms. We found that genetic deletion of IL-33 significantly reduced OIR-induced retinal neovascularization in the mouse retina and that intraperitoneal administration of mature forms of IL-33, mainly IL-3399-270 and IL-33109-270, significantly restored ischemia-induced angiogenic sprouting and tuft formation in the hypoxic retinas of IL-33-/- mice. Thus, our study results suggest that blockade or inhibition of IL-33 cleavage by neutrophil proteases could help mitigate pathological angiogenesis in proliferative retinopathies.

Absence of Neutropenia in Patients With Early Exon Nonsense Mutations in ELANE : Clinical Evidence to Support Gene Therapy Approaches for Severe Congenital Neutropenia.

Severe congenital neutropenia is an inherited bone marrow failure disorder characterized by profoundly low neutrophil counts and promyelocytic maturation arrest in bone marrow. Severe congenital neutropenia is most often caused by heterozygous ELANE mutations. In vitro and mouse xenograft studies using CRISPR/Cas9 have shown that introduction of frameshift/nonsense mutations in mutant ELANE may restore neutrophil counts, providing a model for gene therapy. Here, we present 2 children with inherited nonsense mutations in ELANE analogous to those proposed for gene therapy. Their normal peripheral blood neutrophil counts provide support for this approach through human "experiments of nature."

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

ELANE rs17223045C/T and rs3761007G/A variants: Protective factors against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for causing coronavirus disease 2019 (COVID-19). The development and severity of this infectious disease is influenced by a combination of environmental and genetic factors. Angiotensin-converting enzyme 2 (ACE2) facilitates SARS-CoV-2 entry into human cells, with transmembrane serine protease 2 (TMPRSS2) playing a crucial role in S protein priming. Other proteases, such as cathepsin L and elastase, neutrophil-expressed (ELANE), have the capability to prime the S protein and contribute to SARS-CoV-2 infection. ELANE variants have not been previously examined in COVID-19 patients. We aimed to assess the association of single nucleotide variants (SNVs) within ELANE with COVID-19 and biochemical markers. The study included 319 SARS-CoV-2-infected patients and 288 controls. Genotyping of ELANE rs17216663C/T (Pro257Leu), rs17223045C/T (As1n30Asn), and rs3761007G/A was conducted using a 5'-nuclease allelic discrimination assay (TaqMan assay). Our findings indicate that ELANE rs17223045C/T (C vs T: odds ratio [OR] 0.08, P = 0.005, and CC vs CT: OR 0.08, P = 0.005) and rs3761007G/A (G vs A: OR 0.38, P = 0.009, and GG vs GA: OR 0.40, P = 0.008) confer protection against COVID-19. However, these variants were not associated with biochemical markers. In conclusion, our data suggests that ELANE rs17223045C/T and rs3761007G/A SNVs may play a protective role against COVID-19.

Neutrophil Elastase Remodels Mammary Tumors to Facilitate Lung Metastasis.

Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE-/-) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE-/- mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE-/- mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE-/- tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.

Screening for ELANE, HAX1 and GFI1 gene mutations in children with neutropenia and clinical characterization of two novel mutations in ELANE gene.

BACKGROUND: Congenital neutropenia is a rare disease. Recurrent infections since young age are the presentation. The most common mutation causing severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) is the ELANE gene. The objectives of this study were to screen the three common genetic mutations of ELANE, HAX1 and GFI1 in children with chronic neutropenia and to describe the clinical characteristics of children who had the mutations. METHODS: Infants having ANC < 1,000/cu mm or children aged > 1 year having ANC < 1,500/cu mm at least 3 times in 3 months were enrolled in the study. Patients who had acquired neutropenia due to infection, immune deficiency, or drugs were excluded. The ELANE gene was first studied; and if mutations were not identified, the HAX1 and GFI1 genes were further examined. RESULTS: A total of 60 patients were enrolled in the study. The median (range) age, ratio of female to male, ANC, and last follow-up age were 9.2 (0.5-45.2) months, 1:1.2, 248 (0-1,101) /cu mm, and 19.9 (3.5-202.3) months, respectively. Infections were noted in 67.3% of all patients. ELANE gene mutation was found in only four patients (6.7%), and the rest (56 patients) showed no mutations in the HAX1 and GFI1 genes. In patients without mutations, 66.0% had normal ANC during the follow-up, with a median (range) age for normal ANC of 19.8 (4.0-60.0) months. Two novel mutations p. Ala79del (c.234_236del) and p. Val197GlufsTer18 (c.589_590insAGGCCGGC) were identified, and they respectively cause SCN and CyN. Patients with the two novel mutations presented with several episodes of infection, including pneumonia, sepsis, abscess, otitis media, and gum infection. CONCLUSION: The genetic screening for ELANE, HAX1, and GFI1 gene mutations in 60 patients with chronic neutropenia could identify four patients (6.7%) with ELANE gene mutation and two novel mutations, p. Ala79del in exon 3 and p. Val197GlufsTer18 in exon 4 causing SCN; and CyN, respectively.

Neutrophil elastase activates the release of extracellular traps from COPD blood monocyte-derived macrophages.

Neutrophil elastase (NE), a major inflammatory mediator in chronic obstructive pulmonary disease (COPD) airways, impairs macrophage function, contributing to persistence of airway inflammation. We hypothesized that NE activates a novel mechanism of macrophage-induced inflammation: release of macrophage extracellular traps (METs). The METs are composed of extracellular DNA decorated with granule proteinases and oxidants and may trigger persistent airway inflammation in COPD. To test the hypothesis, human blood monocytes were isolated from whole blood of subjects with COPD recruited following informed written consent. Patient demographics and clinical data were collected. Cells were cultured in media with GM-CSF to differentiate into blood monocyte derived macrophages (BMDMs). The BMDMs were treated with FITC-NE and unlabeled NE to determine intracellular localization by confocal microscopy and intracellular proteinase activity by DQ-Elastin assay. After NE exposure, released extracellular traps were quantified by abundance of extracellular DNA in conditioned media using the Pico Green assay. BMDM cell lysates were analyzed by Western analysis for proteolytic degradation of histone H3 or H4 or upregulation of peptidyl arginine deiminase (PAD) 2 and 4, two potential mechanisms to mediate extracellular trap DNA release. We observed that NE was taken up by COPD BMDM, localized to the cytosol and nucleus, and retained proteinase activity in the cell. NE induced MET release at doses as low as 50 nM. NE treatment caused histone H3 clipping but no effect on histone H4 nor PAD 2 or 4 abundance or activity. In summary, NE activated COPD MET release by clipping histone H3, a prerequisite for chromatin decondensation.

Case Report: Characterization of known (c.607G>C) and novel (c.416C>G) ELANE mutations in two Mexican families with congenital neutropenia.

The most common causes of congenital neutropenia are mutations in the ELANE (Elastase, Neutrophil Expressed) gene (19p13.3), mostly in exon 5 and the distal portion of exon 4, which result in different clinical phenotypes of neutropenia. Here, we report two pathogenic mutations in ELANE, namely, c.607G>C (p.Gly203Arg) and a novel variant c.416C>G (p.Pro139Arg), found in two Mexican families ascertained via patients with congenital neutropenia who responded positively to the granulocyte colony-stimulating factor (G-CSF) treatment. These findings highlight the usefulness of identifying variants in patients with inborn errors of immunity for early clinical management and the need to rule out mosaicism in noncarrier parents with more than one case in the family.

Causal Role for Neutrophil Elastase in Thoracic Aortic Dissection in Mice.

BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. METHODS: ß-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. RESULTS: NE aortic gene expression and plasma activity was significantly increased during ß-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents ß-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. CONCLUSIONS: We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.

Neutrophil elastase as a potential biomarker related to the prognosis of gastric cancer and immune cell infiltration in the tumor immune microenvironment.

Exploring biomarkers interrelated the tumor immune microenvironment (TIME) provides novel ideas for predicting the prognosis of gastric cancer (GC) and developing new treatment strategies. We analyzed the differential gene expression levels between the high and low StromalScore and ImmuneScore groups. Neutrophil elastase (ELANE) was evaluated as a potential biomarker by conducting intersection analysis of the protein-protein interaction network and univariate Cox regression analysis. The expression of ELANE was evaluated by immunohistochemistry. Its prognostic value was evaluated using Kaplan-Meier (K-M) survival curves and multivariate Cox regression analysis and its potential biological molecular mechanism was examined by gene set enrichment analysis (GSEA). We applied the CIBERSORT computing method to analyze the relationship between ELANE and tumor immune-infiltrating cells (TIICs). K-M survival curve showed that higher ELANE expression was closely related to shorter overall survival. The Cox regression analysis indicated that the high expression of ELANE was an independent prognostic risk factor in patients with GC. The GSEA revealed that genes in the ELANE high-expression group were involved in the signaling pathways regulating immune response; genes in the ELANE low-expression group were involved in the signaling pathways that regulate metabolism. ELANE might be participate in the change of TIME from immunodominant to metabolically dominant and its expression was closely related to tumor mutation burden and multiple TIICs. ELANE is a potential biomarker for predicting the GC patients' survival and prognosis. It influences the tumor immune cell infiltration in the TIME, and affects the TIME to maintain their immune status.

A high prevalence of neutrophil-specific antibodies in ELANE-mutated severe congenital neutropenia.

An assay for neutrophil-specific antibodies is frequently used in the workup of chronic severe neutropenia and is suggestive of autoimmune, or sporadically alloimmune neutropenia, rather than severe congenital neutropenia (SCN). We analyzed a neutropenia consortium database for the outcomes of antibody testing initiated before receiving genetic diagnosis in Polish SCN cohort. Test results, performed in a single reference laboratory, were available for 14 patients with ELANE-mutated SCN or cyclic neutropenia, and were frequently positive (36%). We note that the trigger for genetic studies in severe neutropenia should not be affected by antibody-positivity and should be clinically driven.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Severe congenital neutropenia caused by ELANE gene mutation: A case report and literature review.

RATIONALE: The Severe congenital neutropenia (SCN) is a rare genetic disease characterized by a deficiency of mature neutrophils in the bone marrow and peripheral blood disorders. After a BALF ACER examination, the patient was found to have a rareMycobacterium abscess infection. PATIENT CONCERNS: We report a case of SCN withMycobacterium abscess infection caused by ELANE gene mutation. Conventional antiinfection and granulocyte colony-stimulating factor (G-CSF) did not ameliorate patient's symptoms. The absolute neutrophil count (ANC) most of the time < 0.50 × 109/L. DIAGNOSES: According to Gene sequencing and other tests, the patient was diagnosed with SCN caused by ELANE gene mutation, severe pneumonia, Mycobacteriosis abscess, nutritional iron deficiency anemia, multiple abscesses of the skin, hypergammaglobuloemia, and thrush. INTERVENTIONS: Anti-infection agents, abscess incision and drainage, blood transfusion, G-CSF were treated. OUTCOMES: The fever subsided, the cough disappeared, the anemia improved, and the ANC improved (0.69 × 109/L). Currently, the patient has been followed up in the outpatient clinic for 20months, during which time fever, bone pain, gingivitis and thrush occasionally appeared. The ANC fluctuated between 0.20 and 1.27 × 109/L, suggesting the need for a timely hematopoietic stem cell transplant (HSCT). LESSONS: ELANE gene-related SCN is rare in children, and the possibility of this disease should be considered in children with recurrent severe bacterial infections and a significant reduction in neutrophils in the peripheral blood shortly after birth. In addition to strengthening nursing care and actively preventing and controlling infection, other rare bacterial infections should be considered in clinical practice.

Effects of leukocyte elastase in semen on sperm quality.

The study analyzed the effect of leukocyte elastase (LE) in 460 semen on sperm quality, and explore the reference interval of normal level of LE in semen. The differences of LE levels between normal semen and few, weak and abnormal semen were analyzed. Referring to domestic standards, the samples were divided into normal group (LE ≤ 250 ng/mL), occult infection (250 < LE ≤ 1000 ng/mL), and infection group (LE > 1000 ng/mL), and the differences in semen quality among the groups were compared. According to European standards, the samples were divided into normal group (≤600 ng/mL) and abnormal group (>600 ng/mL), and the differences in semen quality between the 2 groups were compared. The correlation between LE levels in semen and semen quality were analyzed. The positive rates of LE in the normal semen group and abnormal semen groups were 30.7% versus 34.7%, and there was no significant difference between the two groups (P > .05). When the semen divided into 3 groups, there was no significant difference between the physicochemical parameters, kinetic parameters, movement trajectory parameters, morphological parameters, and sperm DNA fragmentation index (DFI) (P > .05). There were significant differences in sperm morphology and sperm DFI between the two groups at 600 ng/mL (P < .05). Spearman correlation analysis showed that there was no significant difference between the level of LE in semen and physicochemical parameters, sperm kinetic parameters, sperm movement trajectory parameters, sperm morphological parameters, and sperm DFI (P > .05). It is appropriate to use 600 ng/mL as the threshold for the concentration of LE in semen; the correlation between the level of LE and sperm quality is not significant.

ELANE neutropenia and solid tumors: Four cases from the French severe chronic neutropenia registry.

Neutropenia related to ELANE gene mutations predisposes patients to infection and leukemia/myelodysplasia, but little is known about the predisposition to cancer. Among a cohort of 147 patients, we identified four with malignant solid tumors (papillary thyroid cancer, anal squamous cell cancer, papillary renal cell carcinoma, and adrenocortical carcinoma), all aged 25-50 years. Three occurred with cyclic neutropenia, and one occurred with severe chronic neutropenia. Previous radiotherapy was identified as a risk factor in one patient. No genetic predisposition was identified in the three other patients.