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Karl Welte - One of the best experts on this subject based on the ideXlab platform.
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a zebrafish model for HAX1 associated congenital neutropenia
Haematologica, 2020Co-Authors: Larissa Doll, Narges Aghaallaei, Julia Skokowa, Karl Welte, Advaita M Dick, Baubak BajoghliAbstract:Severe congenital neutropenia (CN) is a rare heterogeneous group of diseases, characterized by a granulocytic maturation arrest. Autosomal recessive mutations in the HAX1 gene are frequently detected in affected individuals. However, the precise role of HAX1 during neutrophil differentiation is poorly understood. To date, no reliable animal model has been established to study HAX1-associated CN. Here we show that knockdown of zebrafish HAX1 impairs neutrophil development without affecting other myeloid cells and erythrocytes. Furthermore, we have found that interference with the HAX1 function decreases the expression level of key target genes of the granulocyte-colony stimulating factor (G-CSF) signaling pathway. The reduced neutrophil numbers in the morphants could be reversed by G-CSF, which is also the main therapeutic intervention for patients who have CN. Our results demonstrate that zebrafish is a suitable model for HAX1-associated neutropenia. We anticipate that this model will serve as an in vivo platform to identify new avenues for developing tailored therapeutic strategies for CN patients, particularly for those individuals that do not respond to the G-CSF treatment.
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gene correction of HAX1 reversed kostmann disease phenotype in patient specific induced pluripotent stem cells
Blood Advances, 2017Co-Authors: Erik Pittermann, Karl Welte, Nico Lachmann, Glenn A Maclean, Stephan Emmrich, Mania Ackermann, Gudrun Gohring, Brigitte Schlegelberger, Axel SchambachAbstract:Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 (HAX1) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1-centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1W44X-iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.
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Impaired DNA Damage Repair in Severe Congenital Neutropenia Patients
Blood, 2016Co-Authors: Maksim Klimiankou, Cornelia Zeidler, Karl Welte, Benjamin Dannenmann, Klaus Schulze-osthoff, Lothar Kanz, Julia SkokowaAbstract:Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1 (mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation. We first investigated differences in DNA damage susceptibility of CD34 + and CD33 + bone marrow cells from CN- ELANE (n = 3) and CN- HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair ( BRCA1 and RAD51 ), mismatch repair ( MSH2 and PCNA ) as well ascell cycle regulation ( CHEK2 and CDKN2C ) in CD33 + of both CN groups as compared to healthy individuals. Interestingly, CN- ELANE and CN- HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B , RAD50 and ATR expression in the CN- HAX1 group only and of MRE11A in the CN- ELANE group only. Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation. Disclosures No relevant conflicts of interest to declare.
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Optimization of CSF3R Mutation Detection in Severe Congenital Neutropenia and Cyclic Neutropenia Patients for Routine Diagnostics Using Next Generation Sequencing
Blood, 2016Co-Authors: Maksim Klimiankou, Cornelia Zeidler, Karl Welte, Sabine Mellor-heineke, Ingeborg Steiert, Karin Haehnel, Julia SkokowaAbstract:Severe congenital neutropenia (CN) is a group of bone marrow failure syndromes characterized by absolute neutrophil counts below 0.5x109/L, susceptibility to bacterial infections and frequently associated with maturation arrest at promyelocyte stage in the bone marrow (BM). There is a high incidence of malignant transformation among CN patients with a cumulative rate of MDS/AML 22 % after 15 years of G-CSF treatment. The acquisition of G-CSFR truncating mutations is a risk factor for leukemic transformation in CN patients. Therefore, annual monitoring of CSF3Rmutations by means of next generation sequencing (NGS) is required for identification of CN patients with high risk of MDS/AML development. Since CSF3R mutations usually occur at low frequency without additional clinical features, it is important to carefully select suitable clinical sample type and methods for mutation detection. Next, it remains to be evaluated which CN genetic subgroups should be considered for annual screening of CSF3Rmutations. We performed CSF3R mutational screening in DNA and/or cDNA in 101 patients (ELANE, n = 42; HAX1, n = 16; G6PC3, n = 7; JAGN1, n = 2; WASP, n = 1; digenic ELANE, HAX1, n =1; digenic HAX1 and G6PC3, n =1; inherited mutations in CSF3R, n = 2; genetically unclassified CN, n = 9; cyclic neutropenia (CyN), n = 20) from the European Branch of the Severe Congenital Neutropenia Registry (SCNIR). Using DNA deep sequencing we screened 63 of 81 CN-patients and 20 CyN patients. Using this method, we identified CSF3R mutations in 22.2% (14/63) of CN patients and 10% (2/20) of CyN patients. The frequency of CSF3R mutations in CN patients with known inherited mutations was 20% (11/55): 30 % (3/10) in CN-HAX1 patients, and 22.9 % (8/35) in CN-ELANE patients. Interestingly, 3/8 (37.5 %) patients harbouring CSF3R mutations were observed in genetically unclassified CN. We did not detect any acquired CSF3R mutations in the small groups of CN patients (n=10) harbouring inherited G6PC3, JAGN1, CSF3Ror digenic mutations. In order to increase the sensitivity of mutation detection we performed cDNA deep sequencing of the critical region of G-CSFR. We sequenced 38 CN patients (ELANE, n = 15; HAX1, n = 11; JAGN1, n = 2; G6PC3, n = 2; WASP, n = 1; germline CSF3R, n = 1; genetically unclassified, n = 6). We found 13% (2/15) CN-ELANE, 27% (3/11) CN-HAX1 and 33% (2/6) genetically unclassified CN patients to be positive for acquired mutations in the critical region of G-CSFR. One CN patient with WASP mutation also acquired CSF3R mutation. Based on our sequencing data we would suggest CSF3Rmutation sequencing in all studied groups of patients regardless of mutations in ELANE and HAX1 genes. Intriguingly, 3 out of 5 CN patients with CSF3R mutations detected by cDNA deep sequencing were negative based on results of previous DNA deep sequencing. All of them were found to acquire low frequency CSF3R mutant clones (ELANE pos. patient with 0.3% of p.Q739* clone; genetically unclassified CN patient with 2% of p.Q749*clone; HAX1pos. patient with 0.9% of p.Q749* clone) in cDNA deep sequencing. In 2 patients (one CyN-ELANE and one CN-HAX1) with multiple acquired CSF3R mutations we compared mutant clone enrichment in different cell types (BM MNC; BM PMN; PB MNC and PB PMN) by means of cDNA and DNA deep sequencing. In the CyN-ELANE patient with 2 CSF3R mutant clones, the highest mutant allele frequency (MAF) was detected in the cDNA sample of PB PMN (11% of p.Q749* clone and 0.44% of p.Q739* clone), whereas in the PB MNC cDNA sample clone p.Q749* had only 2.5% MAF and clone P.Q739 was not detectable. Similar to that, in the CN-HAX1 patient the highest MAFs for all 3 CSF3R mutant clones were in PB PMN cDNA and the lowest in PB MNC DNA sample. Frequency of mutated CSF3Rclones in BM PMNs of both patients was comparable to PB PMN samples. Taken together, sequencing of cDNA extracted from peripheral blood or bone marrow PMN samples may provide better results than from MNC in terms of frequency of CSF3R mutation detection in CN and CyN patients. Sequencing of cDNA extracted from BM or PB samples allows enrichment of G-CSFR expressing mutant cells, but due to intrinsic low fidelity of reverse transcriptase the threshold level for positive calls could not be improved significantly (current threshold for candidate calls is 0.2-0.5%). We would suggest CSF3R mutation screening using deep-sequencing of cDNA from peripheral blood PMN in all patient groups (CN and CyN) for routine diagnostics. Disclosures No relevant conflicts of interest to declare.
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update on the risk of secondary leukemia in genetic subgroups elane HAX1 was g6pc3 p14 of congenital neutropenia in europe
Blood, 2011Co-Authors: Cornelia Zeidler, Peter Vandenberghe, Martin Zimmermann, Manuela Germeshausen, Irina Schafer, Karl WelteAbstract:Abstract 1106 Congenital neutropenia (CN) is well known as one of the premalignant bone marrow failure syndromes with an overall incidence of secondary leukemia of more than 10 percent. With the identification of new causative gene mutations the number of genetic subgroups is still increasing. Due to the limited patient numbers for each subgroup we re-assessed the incidence and potential risk factors of leukemic transformation in CN patients with known gene mutations in ELANE, HAX1, G6PT, G6PC3, WAS, SBDS, TAZ1 and p14 or no identified mutation, respectively, by analyzing all available data from the European Branch of the Severe Chronic Neutropenia Registry (SCNIR). For comparison we also analyzed patients with cyclic neutropenia (CyN) with or without ELANE mutations. Results from genetic testing were available for 255 of 336 CN patients. Of the 255 CN patients 71 patients revealed ELANE mutations, 31 HAX1 mutations, 47 SBDS , 18 WAS , 21 G6PT , 9 G6PC3 , 4 p14 and 5 TAZ1 mutations. In addition, in 36 patients neither ELANE nor HAX1 mutation was detectable. 81 patients were not tested to date, but further genetic evaluation is not yet completed. Results from genetic testing were also available in 28 of 66 patients with CyN of whom 22 revealed ELANE mutations and 6 were negative for ELANE mutations. Secondary malignancies occurred in 36 of the 336 CN patients and 1 of the 66 patients with CyN. Leukemia distribution is unequal in genetic subtypes of CN: Patients with ELANE-CN and HAX1-CN and ELANEneg/HAX1neg-CN have the highest risks followed by patients with WAS-CN and Shwachman-Diamond syndrome. Progression to MDS or leukemia has so far not been reported in G6PT, G6PC3, TAZ1 or p14 CN cases in our registry. All subgroups benefit from G-CSF treatment. However, patients requiring higher maintenance doses of G-CSF are at greater risk of leukemic transformation (Rosenberg, Zeidler et al., 2010). G-CSF receptor mutations usually occur without additional clinical features, e.g. change in response to G-CSF treatment in different CN subtypes. Mutations can be used as early indicators for the process of malignant transformation. Conclusion: The incidence of secondary leukemia reflects the heterogeneity of congenital neutropenia ranging between no leukemia and more than 10 percent in patients with an underlying mutations in ELANE, HAX1, WAS or ELANE/HAX1 negative patients. However, patient numbers within each genetic subgroup are still limited. Despite mutations in the ELANE gene patients with cyclic neutropenia exhibit no increased risk for malignant transformation. Mutational analysis is helpful to identify the genetic cause of severe congenital or cyclic neutropenia but with limited numbers in genetic subgroups still does not serve to identify patients at risk of leukemic transformation. Detection of G-CSF receptor mutations should result in close observation of the patient with consideration of stem cell transplantation as soon as additional aberrations occur. Disclosures: No relevant conflicts of interest to declare.
Christoph Klein - One of the best experts on this subject based on the ideXlab platform.
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Machine Learning Unveils Proteotypic Mimicry in Genetically Defined SCN Variants
Blood, 2019Co-Authors: Sebastian Hesse, Christoph Klein, Nima Rezaei, Rappsilber Juri, Yoko Mizoguchi, Monika Linder, Piotr Grabowski, Zahra Alizadeh, Zahra Pourpak, Sorin IurianAbstract:Background Novel computational algorithms for multi-omics analysis bear great potential to highlight pathomechanisms of monogenic diseases. We recently defined the in-depth proteome of primary human neutrophil granulocytes (PMID 30630937). Here, we ask the question whether proteotypic patterns differ between defined genetic subtypes associated with severe congenital neutropenia (SCN). We focus on two novel genetic variants in constituents of the signal recognition particle (SRPRA and SRP19) and previously reported SCN genotypes SRP54, HAX1, and ELANE. Methods We analyzed proteomes of highly purified neutrophil granulocytes from a total of 26 SCN patients, including 5 with homozygous splice site mutations in SRP19, one patient with a de-novo heterozygous missense mutation in SRPRA (using 5 biological replicates collected months apart) as well as 6 patients with SRP54, 8 with HAX1 and 6 with ELANE mutations. Samples of 70 healthy donors (HD) served as controls. Whole cell proteome analysis was based on data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer. Data analysis was performed in R and Cytoscape, machine learning approaches included lasso regression and random forest. Results Differential expression analysis in comparison to HD showed in all genotypes overexpression of ribosomes, the translational apparatus, mitochondria, cell-substrate junctions and response to unfolded proteins. Underexpressed proteins showed genotype specific enrichment for granule subsets. Whereas ELANE showed deficiency of primary and secretory granules, HAX1 showed deficiency of specific, tertiary and secretory granules. All SRP genotypes showed markedly reduced abundance of proteins in all granule subsets. Principal component analysis showed clear separation of healthy and diseased proteotypes on the first component, whereas the separation of patient genotypes became clear only using five dimensions. We derived genotype specific proteome signatures by lasso regression, consisting of 26 (minimal specific set) to 128 (comprehensive signature) proteins, and a signature of 48 proteins when joining the SRP genotypes as one group. This signatures allow for perfect separation of the genotypes, demonstrating a clear genotype specific effect on protein abundance levels. We asked the question if the genotypes SRP19 and SRPRA show more similar proteomic profile to SRP54 than the other genotypes (ELANE, HAX1) by training a random forest model on proteome data from SRP54, HAX1, ELANE and HD and subsequently testing if the other SRP samples get classified as SRP54. We observe only few misclassifications using either all proteins (7/10) or using the lasso derived genotype defining proteins (8/10). This strongly supports our hypothesis that mutations in different subunits of the same complex lead to similar proteotype changes, a phenomenon we propose to call "proteotypic mimicry". For a systems biology perspective we selected proteins that were exclusively regulated in the SRP genotypes and restricted a network of interactions to these proteins together with their direct interactors (based on APID level 1). The resulting network contained 464 proteins and 3587 interactions. MCODE analysis identified 16 clusters that were consequently annotated using BINGO enrichment analysis. The SRP specific network shows features of the translational apparatus, the proteasome, the septin complex, splicing and cell-metabolic processes. Further studies to dissect specific pathomechanisms are under way. Conclusion Here we provide for the first time evidence for the correlation between SCN causing genotypes and their corresponding neutrophil proteotypes. In particular, we demonstrate significant overlap of all SRP related proteotypes, indicating a phenomenon we propose to be called "proteotypic mimicry". Studies on similarities and disparities of neutrophil proteotypes will help to raise new hypothesis on distinct cellular dysfunction in defined genetic defects of neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.
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Kostmann’s Disease and HCLS1-Associated Protein X-1 (HAX1)
Journal of Clinical Immunology, 2017Co-Authors: Christoph KleinAbstract:Severe congenital neutropenia (SCN), originally described by the Swedish pediatrician Rolf Kostmann, constitutes a heterogeneous disorder associated with a dramatic decrease of peripheral neutrophil granulocytes. Patients suffer from life-threatening bacterial infections unless treated by recombinant human granulocyte colony stimulating factor (G-CSF) or allogeneic hematopoietic stem cells. This review is focused on the SCN variant caused by mutations in HCLS1 Associated Protein X-1 ( HAX1 ) (SCN3, “Kostmann Disease”). HAX1 is a ubiquitously expressed protein with pleotropic functions, including control of cellular viability, migration, and cancer progression. Even though scientific evidence on the molecular mechanisms regarding HAX1 accumulates, no unified picture has emerged. This review highlights historical milestones and our current understanding of SCN related to mutations in HAX1 .
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kostmann s disease and hcls1 associated protein x 1 HAX1
Journal of Clinical Immunology, 2017Co-Authors: Christoph KleinAbstract:Severe congenital neutropenia (SCN), originally described by the Swedish pediatrician Rolf Kostmann, constitutes a heterogeneous disorder associated with a dramatic decrease of peripheral neutrophil granulocytes. Patients suffer from life-threatening bacterial infections unless treated by recombinant human granulocyte colony stimulating factor (G-CSF) or allogeneic hematopoietic stem cells. This review is focused on the SCN variant caused by mutations in HCLS1 Associated Protein X-1 (HAX1) (SCN3, “Kostmann Disease”). HAX1 is a ubiquitously expressed protein with pleotropic functions, including control of cellular viability, migration, and cancer progression. Even though scientific evidence on the molecular mechanisms regarding HAX1 accumulates, no unified picture has emerged. This review highlights historical milestones and our current understanding of SCN related to mutations in HAX1.
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Genetic etiologies of severe congenital neutropenia
Current Opinion in Pediatrics, 2011Co-Authors: Kaan Boztug, Christoph KleinAbstract:To review recent advances in severe congenital neutropenia (SCN) syndromes. The majority of patients with SCN bear monoallelic mutations in the neutrophil elastase (ELANE) gene. Biallelic mutations in the antiapoptotic gene HAX1 were identified in patients with autosomal recessive SCN. G6PC3 deficiency is a syndromic variant of SCN associating congenital neutropenia with various developmental defects including cardiac or urogenital malformations. The pathophysiology of these distinct genetic variants of SCN is complex. Increased apoptosis of neutrophil granulocytes may be caused by various molecular mechanisms including destabilization of the mitochondrial membrane potential and/or activation of the so-called ‘unfolded protein response’. SCN represents a heterogenous group of disorders that may be caused by genetic defects in ELANE, GFI1, HAX1, G6PC3 or activating mutations in the Wiskott–Aldrich syndrome (WAS) gene. Ongoing research will uncover additional genetic defects in SCN patients.
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Genetic Insights into Congenital Neutropenia
Clinical Reviews in Allergy & Immunology, 2010Co-Authors: Christoph Klein, Karl WelteAbstract:Congenital neutropenia syndromes comprise a heterogeneous group of disorders leading to increased susceptibility to bacterial infections. Recent work has elucidated the molecular basis of several congenital neutropenia syndromes such as mutations in ELA2 , HAX1 , GF11 , and WAS . In addition, a number of complex clinical syndromes associating congenital neutropenia have been recognized and elucidated on a genetic level, e.g. p14-deficiency or G6PC3-deficiency. The clinical and genetic findings of various neutropenia syndromes are being discussed.
Ewa Grzybowska - One of the best experts on this subject based on the ideXlab platform.
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HAX1 impact on collective cell migration cell adhesion and cell shape is linked to the regulation of actomyosin contractility
Molecular Biology of the Cell, 2019Co-Authors: Anna Balcerak, Ryszard Konopinski, Alicja Trebinskastryjewska, Maciej Wakula, Mateusz Chmielarczyk, Urszula Smietanka, Tymon Rubel, Ewelina Macechklicka, Ewa GrzybowskaAbstract:HAX1 protein is involved in the regulation of apoptosis, cell motility and calcium homeostasis. Its overexpression was reported in several tumors, including breast cancer. This study demonstrates that HAX1 has an impact on collective, but not single-cell migration, thus indicating the importance of cell-cell contacts for the HAX1-mediated effect. Accordingly, it was shown that HAX1 knockdown affects cell-cell junctions, substrate adhesion, and epithelial cell layer integrity. As demonstrated here, these effects can be attributed to the modulation of actomyosin contractility through changes in RhoA and septin signaling. Additionally, it was shown that HAX1 does not influence invasive potential in the breast cancer cell line, suggesting that its role in breast cancer progression may be linked instead to collective invasion of the epithelial cells but not single-cell dissemination.
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cytoplasmic HAX1 is an independent risk factor for breast cancer metastasis
Journal of Oncology, 2019Co-Authors: Alicja Trebinskastryjewska, L Szafron, A Rembiszewska, Maciej Wakula, Sylwia Tabor, Renata Sienkiewicz, Joanna Owczarek, Anna Balcerak, Anna Felisiakgolabek, Ewa GrzybowskaAbstract:HAX1 is an antiapoptotic factor involved in the regulation of cell migration and calcium homeostasis, overexpressed in several cancers, including breast cancer. It has been suggested that HAX1 is also implicated in metastasis. Herein we report the results of meta-analysis of HAX1 expression, based on publicly available data, which confirms its significant overexpression in breast cancer and demonstrates copy number gain and prognostic value of HAX1 overexpression for metastatic relapse in ER
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comment on HAX1 augments cell proliferation migration adhesion and invasion induced by urokinase type plasminogen activator receptor
Journal of Oncology, 2013Co-Authors: Alicja Trebinska, Ryszard Konopinski, Ewa GrzybowskaAbstract:Multifunctional HAX-1 protein is involved in the regulation of several key processes like calcium homeostasis, apoptosis, and cell migration. Clarification of its role in these or other processes and its molecular mechanisms of function remains to be established. Mekkawy et al. [1] report HAX-1 influence on cell proliferation, migration, adhesion, and invasion induced by the urokinase-type plasminogen activator receptor (uPAR), as a followup of the previous study, in which the authors demonstrated uPAR-HAX-1 interaction [2]. We would like to report our serious concerns about the HAX-1 expression system used in the study of Mekkawy et al., 2012 [1]. The authors use either pGEM-3Zf(+)HAX1 or pGEM-3Zf(+) as an empty vector to transfect human cell lines. These vectors are intended for in vitro transcription and propagation in bacteria; they are not mammalian expression vectors and do not possess the appropriate promoter sequences. pGEM-HAX-1 used in this study was obtained from Maria Olsson and is described in the work of Dufva et al. [3] as a vector generated for in vitro transcription by cloning HAX-1 coding region into pGEM-3Zf(+). Unless the vector was modified, we do not see the possibility of HAX-1 expression, other than endogenous, in the studied cell lines. Furthermore, no evidence of HAX-1 overexpression was provided (e.g., Western blot). Regarding these concerns, we consider the results indicating HAX-1 influence meaningless.
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Comment on “HAX1 Augments Cell Proliferation, Migration, Adhesion, and Invasion Induced by Urokinase-Type Plasminogen Activator Receptor”
Journal of Oncology, 2013Co-Authors: Alicja Trebinska, Ryszard Konopinski, Ewa GrzybowskaAbstract:Multifunctional HAX-1 protein is involved in the regulation of several key processes like calcium homeostasis, apoptosis, and cell migration. Clarification of its role in these or other processes and its molecular mechanisms of function remains to be established. Mekkawy et al. [1] report HAX-1 influence on cell proliferation, migration, adhesion, and invasion induced by the urokinase-type plasminogen activator receptor (uPAR), as a followup of the previous study, in which the authors demonstrated uPAR-HAX-1 interaction [2]. We would like to report our serious concerns about the HAX-1 expression system used in the study of Mekkawy et al., 2012 [1]. The authors use either pGEM-3Zf(+)HAX1 or pGEM-3Zf(+) as an empty vector to transfect human cell lines. These vectors are intended for in vitro transcription and propagation in bacteria; they are not mammalian expression vectors and do not possess the appropriate promoter sequences. pGEM-HAX-1 used in this study was obtained from Maria Olsson and is described in the work of Dufva et al. [3] as a vector generated for in vitro transcription by cloning HAX-1 coding region into pGEM-3Zf(+). Unless the vector was modified, we do not see the possibility of HAX-1 expression, other than endogenous, in the studied cell lines. Furthermore, no evidence of HAX-1 overexpression was provided (e.g., Western blot). Regarding these concerns, we consider the results indicating HAX-1 influence meaningless.
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HAX‐1 is a nucleocytoplasmic shuttling protein with a possible role in mRNA processing
FEBS Journal, 2012Co-Authors: Ewa Grzybowska, Valery Zayat, Ryszard Konopinski, Alicja Trebinska, Maria M. Szwarc, Elżbieta Sarnowska, Ewelina Macech, Jarosław Korczyński, A. Knapp, Janusz A. SiedleckiAbstract:HAX-1 is a multi-functional protein that is involved in the regulation of apoptosis, cell motility and calcium homeostasis. It is also reported to bind RNA: it associates with structural motifs present in the 3′ untranslated regions of at least two transcripts, but the functional significance of this binding remains unknown. Although HAX-1 has been detected in various cellular compartments, it is predominantly cytoplasmic. Our detailed localization studies of HAX-1 isoforms revealed partial nuclear localization, the extent of which depends on the protein isoform. Further studies demonstrated that HAX-1 is in fact a nucleocytoplasmic shuttling protein, dependent on the exportin 1 nuclear export receptor. Systematic mutagenesis allowed identification of the two nuclear export signals in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after inhibition of nuclear export by leptomycin B, but also after specific cellular stress. The biological role of HAX-1 nuclear localization and shuttling remains to be established, but the HAX-1 transcript-binding properties suggest that it may be connected to mRNA processing and surveillance. In this study, HAX-1 status was shown to influence mRNA levels of DNA polymerase β, one of the HAX-1 mRNA targets, although this effect becomes pronounced only after specific stress is applied. Moreover, HAX-1 tethering to the reporter transcript caused a significant decrease in its expression. Additionally, the HAX-1 co-localization with P-body markers, reported here, implies a role in mRNA processing. These results suggest that HAX-1 may be involved in the regulation of expression of bound transcripts, possibly as part of the stress response. Structured digital abstract HAX1 and DCP1A, colocalize by fluorescence microscopy (View Interaction: 1, 2) HAX1 physically interacts with XPO1 by anti tagcoimmunoprecipitation (View interaction)
Cornelia Zeidler - One of the best experts on this subject based on the ideXlab platform.
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Optimization of CSF3R Mutation Detection in Severe Congenital Neutropenia and Cyclic Neutropenia Patients for Routine Diagnostics Using Next Generation Sequencing
Blood, 2016Co-Authors: Maksim Klimiankou, Cornelia Zeidler, Karl Welte, Sabine Mellor-heineke, Ingeborg Steiert, Karin Haehnel, Julia SkokowaAbstract:Severe congenital neutropenia (CN) is a group of bone marrow failure syndromes characterized by absolute neutrophil counts below 0.5x109/L, susceptibility to bacterial infections and frequently associated with maturation arrest at promyelocyte stage in the bone marrow (BM). There is a high incidence of malignant transformation among CN patients with a cumulative rate of MDS/AML 22 % after 15 years of G-CSF treatment. The acquisition of G-CSFR truncating mutations is a risk factor for leukemic transformation in CN patients. Therefore, annual monitoring of CSF3Rmutations by means of next generation sequencing (NGS) is required for identification of CN patients with high risk of MDS/AML development. Since CSF3R mutations usually occur at low frequency without additional clinical features, it is important to carefully select suitable clinical sample type and methods for mutation detection. Next, it remains to be evaluated which CN genetic subgroups should be considered for annual screening of CSF3Rmutations. We performed CSF3R mutational screening in DNA and/or cDNA in 101 patients (ELANE, n = 42; HAX1, n = 16; G6PC3, n = 7; JAGN1, n = 2; WASP, n = 1; digenic ELANE, HAX1, n =1; digenic HAX1 and G6PC3, n =1; inherited mutations in CSF3R, n = 2; genetically unclassified CN, n = 9; cyclic neutropenia (CyN), n = 20) from the European Branch of the Severe Congenital Neutropenia Registry (SCNIR). Using DNA deep sequencing we screened 63 of 81 CN-patients and 20 CyN patients. Using this method, we identified CSF3R mutations in 22.2% (14/63) of CN patients and 10% (2/20) of CyN patients. The frequency of CSF3R mutations in CN patients with known inherited mutations was 20% (11/55): 30 % (3/10) in CN-HAX1 patients, and 22.9 % (8/35) in CN-ELANE patients. Interestingly, 3/8 (37.5 %) patients harbouring CSF3R mutations were observed in genetically unclassified CN. We did not detect any acquired CSF3R mutations in the small groups of CN patients (n=10) harbouring inherited G6PC3, JAGN1, CSF3Ror digenic mutations. In order to increase the sensitivity of mutation detection we performed cDNA deep sequencing of the critical region of G-CSFR. We sequenced 38 CN patients (ELANE, n = 15; HAX1, n = 11; JAGN1, n = 2; G6PC3, n = 2; WASP, n = 1; germline CSF3R, n = 1; genetically unclassified, n = 6). We found 13% (2/15) CN-ELANE, 27% (3/11) CN-HAX1 and 33% (2/6) genetically unclassified CN patients to be positive for acquired mutations in the critical region of G-CSFR. One CN patient with WASP mutation also acquired CSF3R mutation. Based on our sequencing data we would suggest CSF3Rmutation sequencing in all studied groups of patients regardless of mutations in ELANE and HAX1 genes. Intriguingly, 3 out of 5 CN patients with CSF3R mutations detected by cDNA deep sequencing were negative based on results of previous DNA deep sequencing. All of them were found to acquire low frequency CSF3R mutant clones (ELANE pos. patient with 0.3% of p.Q739* clone; genetically unclassified CN patient with 2% of p.Q749*clone; HAX1pos. patient with 0.9% of p.Q749* clone) in cDNA deep sequencing. In 2 patients (one CyN-ELANE and one CN-HAX1) with multiple acquired CSF3R mutations we compared mutant clone enrichment in different cell types (BM MNC; BM PMN; PB MNC and PB PMN) by means of cDNA and DNA deep sequencing. In the CyN-ELANE patient with 2 CSF3R mutant clones, the highest mutant allele frequency (MAF) was detected in the cDNA sample of PB PMN (11% of p.Q749* clone and 0.44% of p.Q739* clone), whereas in the PB MNC cDNA sample clone p.Q749* had only 2.5% MAF and clone P.Q739 was not detectable. Similar to that, in the CN-HAX1 patient the highest MAFs for all 3 CSF3R mutant clones were in PB PMN cDNA and the lowest in PB MNC DNA sample. Frequency of mutated CSF3Rclones in BM PMNs of both patients was comparable to PB PMN samples. Taken together, sequencing of cDNA extracted from peripheral blood or bone marrow PMN samples may provide better results than from MNC in terms of frequency of CSF3R mutation detection in CN and CyN patients. Sequencing of cDNA extracted from BM or PB samples allows enrichment of G-CSFR expressing mutant cells, but due to intrinsic low fidelity of reverse transcriptase the threshold level for positive calls could not be improved significantly (current threshold for candidate calls is 0.2-0.5%). We would suggest CSF3R mutation screening using deep-sequencing of cDNA from peripheral blood PMN in all patient groups (CN and CyN) for routine diagnostics. Disclosures No relevant conflicts of interest to declare.
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Impaired DNA Damage Repair in Severe Congenital Neutropenia Patients
Blood, 2016Co-Authors: Maksim Klimiankou, Cornelia Zeidler, Karl Welte, Benjamin Dannenmann, Klaus Schulze-osthoff, Lothar Kanz, Julia SkokowaAbstract:Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1 (mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation. We first investigated differences in DNA damage susceptibility of CD34 + and CD33 + bone marrow cells from CN- ELANE (n = 3) and CN- HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair ( BRCA1 and RAD51 ), mismatch repair ( MSH2 and PCNA ) as well ascell cycle regulation ( CHEK2 and CDKN2C ) in CD33 + of both CN groups as compared to healthy individuals. Interestingly, CN- ELANE and CN- HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B , RAD50 and ATR expression in the CN- HAX1 group only and of MRE11A in the CN- ELANE group only. Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation. Disclosures No relevant conflicts of interest to declare.
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update on the risk of secondary leukemia in genetic subgroups elane HAX1 was g6pc3 p14 of congenital neutropenia in europe
Blood, 2011Co-Authors: Cornelia Zeidler, Peter Vandenberghe, Martin Zimmermann, Manuela Germeshausen, Irina Schafer, Karl WelteAbstract:Abstract 1106 Congenital neutropenia (CN) is well known as one of the premalignant bone marrow failure syndromes with an overall incidence of secondary leukemia of more than 10 percent. With the identification of new causative gene mutations the number of genetic subgroups is still increasing. Due to the limited patient numbers for each subgroup we re-assessed the incidence and potential risk factors of leukemic transformation in CN patients with known gene mutations in ELANE, HAX1, G6PT, G6PC3, WAS, SBDS, TAZ1 and p14 or no identified mutation, respectively, by analyzing all available data from the European Branch of the Severe Chronic Neutropenia Registry (SCNIR). For comparison we also analyzed patients with cyclic neutropenia (CyN) with or without ELANE mutations. Results from genetic testing were available for 255 of 336 CN patients. Of the 255 CN patients 71 patients revealed ELANE mutations, 31 HAX1 mutations, 47 SBDS , 18 WAS , 21 G6PT , 9 G6PC3 , 4 p14 and 5 TAZ1 mutations. In addition, in 36 patients neither ELANE nor HAX1 mutation was detectable. 81 patients were not tested to date, but further genetic evaluation is not yet completed. Results from genetic testing were also available in 28 of 66 patients with CyN of whom 22 revealed ELANE mutations and 6 were negative for ELANE mutations. Secondary malignancies occurred in 36 of the 336 CN patients and 1 of the 66 patients with CyN. Leukemia distribution is unequal in genetic subtypes of CN: Patients with ELANE-CN and HAX1-CN and ELANEneg/HAX1neg-CN have the highest risks followed by patients with WAS-CN and Shwachman-Diamond syndrome. Progression to MDS or leukemia has so far not been reported in G6PT, G6PC3, TAZ1 or p14 CN cases in our registry. All subgroups benefit from G-CSF treatment. However, patients requiring higher maintenance doses of G-CSF are at greater risk of leukemic transformation (Rosenberg, Zeidler et al., 2010). G-CSF receptor mutations usually occur without additional clinical features, e.g. change in response to G-CSF treatment in different CN subtypes. Mutations can be used as early indicators for the process of malignant transformation. Conclusion: The incidence of secondary leukemia reflects the heterogeneity of congenital neutropenia ranging between no leukemia and more than 10 percent in patients with an underlying mutations in ELANE, HAX1, WAS or ELANE/HAX1 negative patients. However, patient numbers within each genetic subgroup are still limited. Despite mutations in the ELANE gene patients with cyclic neutropenia exhibit no increased risk for malignant transformation. Mutational analysis is helpful to identify the genetic cause of severe congenital or cyclic neutropenia but with limited numbers in genetic subgroups still does not serve to identify patients at risk of leukemic transformation. Detection of G-CSF receptor mutations should result in close observation of the patient with consideration of stem cell transplantation as soon as additional aberrations occur. Disclosures: No relevant conflicts of interest to declare.
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Digenic mutations in severe congenital neutropenia
Haematologica, 2010Co-Authors: Manuela Germeshausen, Cornelia Zeidler, Manfred Stuhrmann, Marina Lanciotti, Matthias Ballmaier, Karl WelteAbstract:Severe congenital neutropenia a clinically and genetically heterogeneous disorder. Mutations in different genes have been described as causative for severe neutropenia, e.g. ELANE, HAX1 and G6PC3. Although congenital neutropenia is considered to be a group of monogenic disorders, the phenotypic heterogeneity even within the yet defined genetic subtypes points to additional genetic and/or epigenetic influences on the disease phenotype. We describe congenital neutropenia patients with mutations in two candidate genes each, including 6 novel mutations. Two of them had a heterozygous ELANE mutation combined with a homozygous mutation in G6PC3 or HAX1, respectively. The other 2 patients combined homozygous or compound heterozygous mutations in G6PC3 or HAX1 with a heterozygous mutation in the respective other gene. Our results suggest that digenicity may underlie this disorder of myelopoiesis at least in some congenital neutropenia patients.
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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
Blood, 2009Co-Authors: Cornelia Zeidler, Jean Donadieu, Audrey Anna Bolyard, Peter Vandenberghe, Gusal Pracht, Blandine Beaupain, Martin Zimmermann, Christine Bellannechantelot, Daniel C Link, Christoph KleinAbstract:Abstract 3597 Poster Board III-534 An 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: 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: Conclusion Patients 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.
Manuela Germeshausen - One of the best experts on this subject based on the ideXlab platform.
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update on the risk of secondary leukemia in genetic subgroups elane HAX1 was g6pc3 p14 of congenital neutropenia in europe
Blood, 2011Co-Authors: Cornelia Zeidler, Peter Vandenberghe, Martin Zimmermann, Manuela Germeshausen, Irina Schafer, Karl WelteAbstract:Abstract 1106 Congenital neutropenia (CN) is well known as one of the premalignant bone marrow failure syndromes with an overall incidence of secondary leukemia of more than 10 percent. With the identification of new causative gene mutations the number of genetic subgroups is still increasing. Due to the limited patient numbers for each subgroup we re-assessed the incidence and potential risk factors of leukemic transformation in CN patients with known gene mutations in ELANE, HAX1, G6PT, G6PC3, WAS, SBDS, TAZ1 and p14 or no identified mutation, respectively, by analyzing all available data from the European Branch of the Severe Chronic Neutropenia Registry (SCNIR). For comparison we also analyzed patients with cyclic neutropenia (CyN) with or without ELANE mutations. Results from genetic testing were available for 255 of 336 CN patients. Of the 255 CN patients 71 patients revealed ELANE mutations, 31 HAX1 mutations, 47 SBDS , 18 WAS , 21 G6PT , 9 G6PC3 , 4 p14 and 5 TAZ1 mutations. In addition, in 36 patients neither ELANE nor HAX1 mutation was detectable. 81 patients were not tested to date, but further genetic evaluation is not yet completed. Results from genetic testing were also available in 28 of 66 patients with CyN of whom 22 revealed ELANE mutations and 6 were negative for ELANE mutations. Secondary malignancies occurred in 36 of the 336 CN patients and 1 of the 66 patients with CyN. Leukemia distribution is unequal in genetic subtypes of CN: Patients with ELANE-CN and HAX1-CN and ELANEneg/HAX1neg-CN have the highest risks followed by patients with WAS-CN and Shwachman-Diamond syndrome. Progression to MDS or leukemia has so far not been reported in G6PT, G6PC3, TAZ1 or p14 CN cases in our registry. All subgroups benefit from G-CSF treatment. However, patients requiring higher maintenance doses of G-CSF are at greater risk of leukemic transformation (Rosenberg, Zeidler et al., 2010). G-CSF receptor mutations usually occur without additional clinical features, e.g. change in response to G-CSF treatment in different CN subtypes. Mutations can be used as early indicators for the process of malignant transformation. Conclusion: The incidence of secondary leukemia reflects the heterogeneity of congenital neutropenia ranging between no leukemia and more than 10 percent in patients with an underlying mutations in ELANE, HAX1, WAS or ELANE/HAX1 negative patients. However, patient numbers within each genetic subgroup are still limited. Despite mutations in the ELANE gene patients with cyclic neutropenia exhibit no increased risk for malignant transformation. Mutational analysis is helpful to identify the genetic cause of severe congenital or cyclic neutropenia but with limited numbers in genetic subgroups still does not serve to identify patients at risk of leukemic transformation. Detection of G-CSF receptor mutations should result in close observation of the patient with consideration of stem cell transplantation as soon as additional aberrations occur. Disclosures: No relevant conflicts of interest to declare.
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Digenic mutations in severe congenital neutropenia
Haematologica, 2010Co-Authors: Manuela Germeshausen, Cornelia Zeidler, Manfred Stuhrmann, Marina Lanciotti, Matthias Ballmaier, Karl WelteAbstract:Severe congenital neutropenia a clinically and genetically heterogeneous disorder. Mutations in different genes have been described as causative for severe neutropenia, e.g. ELANE, HAX1 and G6PC3. Although congenital neutropenia is considered to be a group of monogenic disorders, the phenotypic heterogeneity even within the yet defined genetic subtypes points to additional genetic and/or epigenetic influences on the disease phenotype. We describe congenital neutropenia patients with mutations in two candidate genes each, including 6 novel mutations. Two of them had a heterozygous ELANE mutation combined with a homozygous mutation in G6PC3 or HAX1, respectively. The other 2 patients combined homozygous or compound heterozygous mutations in G6PC3 or HAX1 with a heterozygous mutation in the respective other gene. Our results suggest that digenicity may underlie this disorder of myelopoiesis at least in some congenital neutropenia patients.
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clinical implications of ela2 HAX1 and g csf receptor csf3r mutations in severe congenital neutropenia
British Journal of Haematology, 2009Co-Authors: Cornelia Zeidler, Christoph Klein, Manuela Germeshausen, Karl WelteAbstract:Summary Congenital Neutropenia (CN) is a heterogeneous bone marrow failure syndrome characterized by a maturation arrest of myelopoiesis at the level of the promyelocyte/myelocyte stage with peripheral blood absolute neutrophil counts below 0AE5 · 10 9 /l. There are two major subtypes of CN as judged by inheritance: an autosomal dominant subtype, e.g. defined by neutrophil elastase mutations (approximately 60% of patients) and an autosomal recessive subtype (approximately 30% of patients), both presenting with the same clinical and morphological phenotype. Different mutations have been described (e.g. HAX1, p14 etc) in autosomal recessive CN, with HAX1 mutations in the majority of these patients. CN in common is considered as a preleukemic syndrome, since the cumulative incidence for leukemia is more than 25% after 20 years of observation. Leukemias occur in both, the autosomal dominant and recessive subtypes of CN. The individual risk for each genetic subtype needs to be further evaluated. Numbers of patients tested for the underlying genetic defect are still limited. Acquired G-CSFR (CSF3R) mutations are detected in approximately 80% of CN patients who developed acute myeloid leukemia independent of the ELA2 or HAX1 genetic subtype, suggesting that these mutations are involved in leukemogenesis. As the majority of patients benefit from G-CSF administration, HSCT should be restricted to nonresponders and patients with leukaemic transformation.
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Clinical implications of ELA2‐, HAX1‐, and G‐CSF‐receptor (CSF3R) mutations in severe congenital neutropenia
British Journal of Haematology, 2008Co-Authors: Cornelia Zeidler, Christoph Klein, Manuela Germeshausen, Karl WelteAbstract:Summary Congenital Neutropenia (CN) is a heterogeneous bone marrow failure syndrome characterized by a maturation arrest of myelopoiesis at the level of the promyelocyte/myelocyte stage with peripheral blood absolute neutrophil counts below 0AE5 · 10 9 /l. There are two major subtypes of CN as judged by inheritance: an autosomal dominant subtype, e.g. defined by neutrophil elastase mutations (approximately 60% of patients) and an autosomal recessive subtype (approximately 30% of patients), both presenting with the same clinical and morphological phenotype. Different mutations have been described (e.g. HAX1, p14 etc) in autosomal recessive CN, with HAX1 mutations in the majority of these patients. CN in common is considered as a preleukemic syndrome, since the cumulative incidence for leukemia is more than 25% after 20 years of observation. Leukemias occur in both, the autosomal dominant and recessive subtypes of CN. The individual risk for each genetic subtype needs to be further evaluated. Numbers of patients tested for the underlying genetic defect are still limited. Acquired G-CSFR (CSF3R) mutations are detected in approximately 80% of CN patients who developed acute myeloid leukemia independent of the ELA2 or HAX1 genetic subtype, suggesting that these mutations are involved in leukemogenesis. As the majority of patients benefit from G-CSF administration, HSCT should be restricted to nonresponders and patients with leukaemic transformation.
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risk of leukemia in genetic subgroups of congenital neutropenia cn comparison of patients with mutations in HAX1 or ela2
Blood, 2008Co-Authors: Cornelia Zeidler, Jean Donadieu, Gusal Pracht, Blandine Beaupain, Martin Zimmermann, Christine Bellannechantelot, Christoph Klein, Manuela Germeshausen, Karl WelteAbstract:An increased risk for malignant transformation (myelodysplastic syndrome (MDS) or leukemia) is well documented in patients with congenital neutropenia (CN). However, the risk of defined genetic subgroups of CN remains unknown. We therefore assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with ELA2 and HAX1 mutations, respectively. The overall cumulative incidence of secondary leukemias in CN, as documented by long term follow up data from the European Severe Chronic Neutropenia Registry (SCNER) and the French Neutropenia Registry (FR) is 39 out of 343 patients (11.4 %). Up to now genetic testing has been performed in 177 of the 343 CN patients revealing ELA2 mutations in 99 (56%) and HAX1 mutations in 19 (11%) of tested CN patients. In 107patients no ELA2 mutation was detectable. Out of these 107 ELA2 negative patients 59 were also negative for HAX1 mutations (33% ELA2/HAX1 negative patients). In both, patients with ELA2-CN and HAX1-CN, the clinical phenotype is characterized by a maturation arrest of myelopoiesis and severe neutropenia and cannot be discriminated morphologically. There is also no difference in the gender distribution. Both subgroups benefit from G-CSF treatment and respond well to similar G-CSF doses (median G-CSF dose in ELA2-CN is 8,5 μg/kg/day versus 6 μg/kg/day in HAX1-CN. Secondary malignancies occurred in 17 (5 MDS, .11. AML, 1 bi-phenotypic leukemia) out of 99 (17%) ELA2-CN patients, 4 (2 MDS, 1 AML, 1 bi-phenotypic leukemia) out of 19 (21%) HAX1-CN patients, and 5 (1 MDS, 3 AML, 1 ALL) out of 59 (8 %) double negative patients. Mutations in the G-CSF receptor gene have been detectable in both groups of CN patients. The frequency of mutations increases over time with a significant higher frequency of G-CSFR mutations in the CN patients who developed leukemia indicative for an involvement of G-CSFR mutations in the process of leukemic transformation. Interestingly, G-CSFR mutations are detectable in 5 of 8 ELA2-CN leukemic patients tested and 1 of 2 HAX1-CN leukemic patients tested, but also in 1 out of 4 ELA2/HAX1 negative leukemic patients. Independent of the underlying genetic defect the dose response to G-CSF treatment seems to indicate the risk for leukemia. Patients being treated with less than 5 μg/kg/day had an leukemia incidence of 14% versus 26% in patients who received 5 μg/kg/day or more (p=0.027). Within the group of patients being treated with less than 5 μg/kg/day, 14% developed leukemia, whereas in the group of patients receiving = 5 μg/kg/day 26% developed leukemia (p=0.027), suggesting that those patients requiring higher doses of GCSF may be at higher risk for malignant transformation. Leukemia outcome is dependent on the success of hematopoietic stem cell transplantation. The outcome could be improved dramatically since SCT was initiated immediately after confirmation of the leukemia diagnosis. It would be interesting to further discuss why different genetic backgrounds (ELA2 and HAX1) reveal the same clinical phenotype in terms of morphology, response to G-CSF treatment, acquisition of G-CSFR mutations and the development of leukemia.
