A Novel Clinical Syndrome Associating Severe Congenital Neutropenia and Complex Developmental Aberrations Caused by Deficiency of G6PC3

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5-5
Author(s):  
Kaan Boztug ◽  
Giridharan Appaswamy ◽  
Angel Ashikov ◽  
Alejandro A Schäffer ◽  
Ulrich Salzer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bacterial Infections ◽  
Myeloid Cells ◽  
Missense Mutations ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Increased Susceptibility

Abstract We here describe a previously unrecognized nosological entity in 12 patients from 8 unrelated pedigrees. All patients presented with severe congenital neutropenia and severe invasive bacterial infections. In addition, patients had a variety of additional syndromic features such as congenital heart disease (8/12), urogenital malformations (5/12), inner ear hearing loss (2/12), and myopathy (1/12). Furthermore, most patients (10/12) showed increased visibility/angiectasia of subcutaneous veins. The bone marrow smear was characterized by a typical “maturation arrest” due to premature apoptosis of mature neutrophils. Similar to Kostmann’s disease secondary to mutations in HAX1, myeloid cells from patients with this novel syndrome showed increased susceptibility to apoptosis. Myeloid progenitor cells revealed an abnormally enlarged rough endoplasmic reticulum and increased endoplasmic reticulum stress evidenced by increased expression of BiP. A genome-wide linkage study, performed in two consanguineous pedigrees, gave statistical evidene of a linkage interval on chromosome 17q21 (LOD score 5.74). We identified homozygous missense mutations in G6PC3, a ubiquitously expressed paralog of glucose-6-phosphatase. Biochemical studies confirmed deficient enzymatic activity. Using retroviral G6PC3-gene transfer into primary hematopoietic stem cells and in vitro differentiation into myeloid cells, the phenotype of increased susceptibility to apoptosis could be reverted. Eight distinct biallelic mutations were found, including missense and nonsense mutations. G6PC3-deficient myeloid cells showed a predominance of the unphosphorylated form of GSK3beta, a key molecule controlling cellular differentiation and apoptosis. As a consequence of increased GSK3beta activity, increased phosphorylation of the antiapoptotic molecule Mcl1 was detected, explaining increased susceptibility to apoptosis in neutrophils. In summary, our study describes a novel disease, determines its molecular etiology, and sheds light on the role of glucose-dependent pathways in controlling the homeostasis of the endoplasmic reticulum and control of apoptosis.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 315-315
Author(s):  
Lan Dan ◽  
Basant Kumar Thakur ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Transcription Factors ◽  
Steady State ◽  
Myeloid Cells ◽  
Biologically Active ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Mrna And Protein Expression ◽  
Induction Of Differentiation

Abstract 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.

scholarly journals Efficient Correction of ELANE mutations in Primary HSPCs of Severe Congenital Neutropenia Patients Using CRISPR/Cas9 and rAVV6 HDR Repair Templates

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1036-1036
Author(s):  
Malte U Ritter ◽  
Benjamin Secker ◽  
Maksim Klimiankou ◽  
Masoud Nasri ◽  
Narges Aghaallaei ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Healthy Donor ◽  
Hot Spot ◽  
Bone Marrow Failure ◽  
Patient Specific ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
Exon 2

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood (<500/µl) leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. The majority of patients harbor heterogenous mutations in ELANE, coding for Neutrophil Elastase. Up to now, the only curative therapy for CN patients that do not respond to G-CSF or with overt AML remains hematopoietic stem cells transplantation with its associated risks. A clinical need for gene therapy for these patients is imminent. We recently described the CRISPR/Cas9 mediated ELANE knockout as a possible gene therapy approach for CN patients with ELANE mutations (ELANE-CN) (Nasri et al. 2019). As an alternative, we wanted to test if specific target therapy for individual ELANE-CN patients could be an option. Here we describe the correction of ELANE mutations using CRISPR/Cas9 to edit the ELANE gene and recombinant adeno-associated virus 6 (rAAV6) to deliver a template for homology directed repair (HDR). We selected ELANE mutations p.A57V or p.A57T in exon 2, and p.G214R or p.G214RV in exon 5, both known hot spot mutations observed in G-CSF non-responders or in CN/AML patients (Makaryan et al. 2015). We used SpCas9 V3 and chemically modified sgRNA. For exon 2, we choose the highly efficient sgRNA (Nasri et al. 2019) yielding the benefit, that double-strand breaks (DB) that do not result in HDR correction are producing ELANE knockout. For exon 5, we established a sgRNA that produced average 87% (± 6%) editing in healthy donor cells. Two HDR donor template backbones (DTB) were generated. DTB1 is spanning exons 1-3 and DTB2 exons 4-5 of ELANE. Silent mutations were introduced in the repair templates for both ELANE mutations between the cut site and mutation to enhance HDR. To test the knock-in efficacy, we electroporated healthy donor CD34+cells with CRISPR/Cas9 RNP and transduced them with rAAV6 containing the templates at MOI 105. We achieved 34,5% (± 4,5%) knock-in (KI) and 35,6% (± 2,5%) indels for exon 2, or 39,2% KI (± 12,8%) and 18,85% indels (± 4,25%) for exon 5. Edited cells showed high viability, expanded and differentiated well into neutrophils in vitro. We further applied this approach to primary HSPCs from 4 CN patients harboring selected ELANE mutations. For p.A57, we achieved 14% (±2,3%) KI and 44,7% (±1,9%) indels. For p.G214, the KI was 59,9% (± 0,1%) and indels 28,8% (± 0,6%). To assess the effect of ELANE correction on the neutropenic phenotype in vitro, we performed CFU and liquid culture neutrophilic differentiation assays. We compared the corrected cells to cells from the same patient that were edited in the AAVS1 safe harbor, as isogenic controls. We observed a significant (p < 0,05) increase in number of CFU-GMs for CRISPR/Cas9 edited HSPCs from two CN patients with p.A57V/T mutations and of CFU-G or CFU-GM for two CN patients with p.G214R/V ELANE mutation. Morphological assessment of Wright-Giemsa stained cytospins of cells derived on day 14 of differentiation revealed significant increases of mature neutrophils for all four edited patient samples ascompared to the respective controls. Further we performed live cell imaging of neutrophil extracellular trap (NET) formation after PMA stimulation and chemotaxis. NET formation was either improved or comparable between control- and ELANE- edited cells. Chemotaxis showed no difference between control- and ELANE-edited cells. For a patient with p.G214V ELANE mutation, we were able to evaluate chemotaxis and phagocytosis in vivo in zebrafish embryos at 48hpf, as described in Nasri et al 2019. This showed a qualitative improvement of ELANE- corrected cells ascompared to control AAVS1 edited cells. This indicates that our manipulation does not alter the functionality of produced neutrophils while increasing the number of mature cells being produced. Taken together, we established a protocol for efficient correction of ELANE mutations in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. We reached high enough editing to correct the dominant negative effects of mutations, as assessed by markedly improved neutrophilic differentiation in vitro. Generated repair constructs allow fast adaptation to patient-specific mutations in all exons of ELANE. This approach is enticing to be investigated further for clinical translation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel HAX1 Gene Mutation in a Vietnamese Boy with Severe Congenital Neutropenia

2018 ◽  
Vol 2018 ◽  
pp. 1-4
Author(s):  
Tham Thi Tran ◽  
Quang Van Vu ◽  
Taizo Wada ◽  
Akihiro Yachie ◽  
Huong Le Thi Minh ◽  
...  
Keyword(s):  
Early Onset ◽  
Bacterial Infections ◽  
Complete Blood Count ◽  
Hereditary Diseases ◽  
Severe Neutropenia ◽  
Sequencing Analysis ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Direct Dna Sequencing ◽  
Life Threatening

Severe congenital neutropenia (SCN) is a rare disease that involves a heterogeneous group of hereditary diseases. Mutations in the HAX1 gene can cause an autosomal recessive form of SCN-characterized low blood neutrophil count from birth, increased susceptibility to recurrent and life-threatening infections, and preleukemia predisposition. A 7-year-old boy was admitted due to life-threatening infections, mental retardation, and severe neutropenia. He had early-onset bacterial infections, and his serial complete blood count showed persistent severe neutropenia. One older sister and one older brother of the patient died at the age of 6 months and 5 months, respectively, because of severe infection. Bone marrow analysis revealed a maturation arrest at the promyelocyte/myelocyte stage with few mature neutrophils. In direct DNA sequencing analysis, we found a novel homozygous frameshift mutation (c.423_424insG, p.Gly143fs) in the HAX1 gene, confirming the diagnosis of SCN. The patient was successfully treated with granulocyte colony-stimulating factor (G-CSF) and antibiotics. A child with early-onset recurrent infections and neutropenia should be considered to be affected with SCN. Genetic analysis is useful to confirm diagnosis. Timely diagnosis and suitable treatment with G-CSF and antibiotics are important to prevent further complication.

In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia

2018 ◽  
Vol 29 (4) ◽  
pp. 146-152 ◽  
Author(s):  
Hamid Farajifard ◽  
Mahdi Zavvar ◽  
Taraneh Rajaei ◽  
Farshid Noorbakhsh ◽  
Mahin Nikougoftar-zarif ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Therapeutic Target ◽  
In Vitro Study ◽  
Vitro Study ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Viral Transduction

scholarly journals Complement sensitivity of paroxysmal nocturnal hemoglobinuria bone marrow cells

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1040-1046 ◽  
Author(s):  
J Tumen ◽  
LB Kline ◽  
JW Fay ◽  
DC Scullin ◽  
EG Reisner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Erythroid Cell ◽  
Hematopoietic Stem ◽  
Increased Sensitivity ◽  
Complement Lysis ◽  
Increased Susceptibility

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which erythrocytes, granulocytes, and platelets are defective, as shown by increased susceptibility of RBCs, WBCs, and platelets to complement- mediated lysis in vitro. The purpose of this study is to determine the sensitivity to complement lysis of PNH and non-PNH erythroid and myeloid precursors using the release of 59Fe and myeloperoxidase as specific markers to monitor the lytic action of complement on erythroid and myeloid cell precursors, respectively. Erythroid cell precursors in four of four PNH patients demonstrated increased sensitivity to complement-mediated lysis. Myeloid cell precursors in four of five PNH patients also exhibited increased sensitivity to complement and antibody. In addition, CFU-c growth was below normal in the marrow of seven PNH patients. These findings support the hypothesis that the defect in PNH occurs at the level of the hematopoietic stem cell.

scholarly journals Blood mononuclear cells from patients with severe congenital neutropenia are capable of producing granulocyte colony-stimulating factor

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1234-1237 ◽  
Author(s):  
T Pietsch ◽  
C Buhrer ◽  
K Mempel ◽  
T Menzel ◽  
U Steffens ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Leukemia Cell Line ◽  
Severe Neutropenia ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Blood Mononuclear Cells ◽  
Stimulating Factor

Abstract Severe congenital neutropenia (SCN) is a disorder of myelopoiesis characterized by severe neutropenia or absence of blood neutrophils secondary to a maturational arrest at the level of promyelocytes. We examined peripheral blood mononuclear cells (PBMC) of SCN patients who demonstrated normalization of their blood neutrophil counts in a phase II clinical study with recombinant human granulocyte colony-stimulating factor (rhG-CSF). When stimulated in vitro with bacterial lipopolysaccharides (LPS), PBMC of those SCN patients produced G-CSF activity, as judged by proliferation induction of the murine leukemia cell line, NFS-60. Western and Northern blot analysis showed G-CSF protein and G-CSF-mRNA indistinguishable in size from those of normal controls. We conclude that PBMC of the SCN patients tested are capable of synthesizing and secreting biologically active G-CSF in vitro.

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

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

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

Clinical and Genetic Characteristics of Patients with Severe Congenital Neutropenia in Japan,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3213-3213
Author(s):  
Yoko Mizoguchi ◽  
Kazuhiro Nakamura ◽  
Shuhei Karakawa ◽  
Satoshi Okada ◽  
Hiroshi Kawaguchi ◽  
...  
Keyword(s):  
Psychomotor Retardation ◽  
Severe Neutropenia ◽  
Case Report Form ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
Genetic Characteristics ◽  
The Mean ◽  
Malignant Transformations ◽  
Chronic Neutropenia

Abstract Abstract 3213 Severe congenital neutropenia (SCN) includes a variety of hematologic disorders characterized by severe neutropenia, with absolute neutrophil counts (ANC) below 0.5 × 109/L, and associated with severe systemic bacterial infections from early infancy. Mutations in ELANE, HAX1, G6PC3, WAS and GFI1 have been so far identified in patients with SCN. The Severe Chronic Neutropenia International Registry (SCNIR) has collected data to monitor the clinical course, treatments, and disease outcomes for SCN patients. In this study, we analyzed the clinical and genetic characteristics of patients with SCN based on the Japanese cohort study collecting the data on chronic neutropenia using a standardized case report form. This study is approved by the ethics committees of the Hiroshima University School of Medicine. Forty-six patients with SCN in Japan were enrolled in this study. Mean present age of patients was 13.6 years old ranged from 1 to 39. The Mean age at diagnosis was 4.6 months ranged from 0 to 24 months. Approximately 90% of patients were diagnosed before 12 months. Twenty-three patients were female. As initial clinical presentation, subcutaneous abscess and cutaneous cellulitis were dominated (37%), followed by unknown fever (17%), stomatitis (13%) and lymphadenitis (13%). On the other hand, infections which patients had experienced after diagnosis were bacterial pneumonia (50%), cutaneous infections (50%) and oral infections such as stomatitis (48%) and gingivitis (48 %). Total 8 patients (17%) including 4 patients with mutations in HAX1 suffered from psychomotor retardation that was higher than that in general Japanese population (about 1%), suggesting that psychomotor retardation may be one of considerable complications in patients with SCN. Thirty-three out of 46 patients with SCN were preformed gene analysis and 29 patients (approximately 88 %) were identified mutations; 25 patients had heterozygous mutations in ELANE, 4 patients had homozygous mutations in HAX1. The proportion of mutations in ELANE (76%) was higher and that in HAX1 (12%) was relatively lower in Japan compared with those in Western counties. Thirty-six patients (78%) were treated with G-CSF, including regular use in 26 patients (56%) and on demand use during infections in 10 patients (22%). In 26 patients treated with regular G-CSF therapy, the mean cumulative duration was 6.6 years, ranged from 0.4 to 19. Two patients showed no response to G-CSF therapy. Four patients among total 46 patients developed MDS/AML and 3 patients were alive after receiving hematopoietic stem cell transplantation (HSCT). Total 12 out of 46 patients (26%) with SCN were underwent HSCT before malignant transformations due to recurrent infections and/or the long-term use of G-CSF. Reduced intensity conditionings (RIC) were used in part of patients. All patients were alive after HSCT. In Japan, high proportion of patients was treated with HSCT using RIC before malignant transformations. The accumulation of cases is necessary to establish suitable conditioning regimen and appropriate indication of HSCT in patients with SCN. Disclosures: No relevant conflicts of interest to declare.

Successful Hematopoietic Stem Cell Transplantation Using an Immunosuppressive Conditioning Regimen in Ten Patients with Severe Congenital Neutropenia: A Single-Institute Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3688-3688
Author(s):  
Yoko Mizoguchi ◽  
Mizuka Miki ◽  
Aya Furue ◽  
Shiho Nishimura ◽  
Maiko Shimomura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Acute Gvhd ◽  
Conditioning Regimen ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
Engraftment Failure

Abstract Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity. Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.

Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2182-2189 ◽  
Author(s):  
Phil J. Ancliff ◽  
Michael P. Blundell ◽  
Giles O. Cory ◽  
Yolanda Calle ◽  
Austen Worth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bacterial Infections ◽  
Killer Cell ◽  
Significant Proportion ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Wiskott Aldrich Syndrome ◽  
Immunologic Function ◽  
Syndrome Protein

Abstract Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.

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