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Maria A Chavez - One of the best experts on this subject based on the ideXlab platform.
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purification and partial characterization of human Neutrophil Elastase inhibitors from the marine snail cenchritis muricatus mollusca
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2007Co-Authors: Yamile Gonzalez, Maday Alonso Del Rivero, Maria Luiza Vilela Oliva, Izaura Y Hirata, Aparecida S Tanaka, Mariana S. Araujo, Maria A ChavezAbstract:Abstract Human Neutrophil Elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human Neutrophil Elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human Neutrophil Elastase strongly, with equilibrium dissociation constant ( K i ) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic Elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human Neutrophil Elastase inhibitors described in a mollusk.
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purification and partial characterization of human Neutrophil Elastase inhibitors from the marine snail cenchritis muricatus mollusca
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2007Co-Authors: Yamile Gonzalez, Maday Alonso Del Rivero, Maria Luiza Vilela Oliva, Izaura Y Hirata, Aparecida S Tanaka, Mariana S. Araujo, Maria A ChavezAbstract:Abstract Human Neutrophil Elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human Neutrophil Elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human Neutrophil Elastase strongly, with equilibrium dissociation constant ( K i ) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic Elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human Neutrophil Elastase inhibitors described in a mollusk.
David C Dale - One of the best experts on this subject based on the ideXlab platform.
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mutations in the Neutrophil Elastase gene in cyclic and congenital neutropenia
Current Opinion in Immunology, 2001Co-Authors: Andrew A G Aprikyan, David C DaleAbstract:Abstract Severe neutropenia disorders are characterized by extremely low levels of peripheral blood Neutrophils, a maturation block of bone marrow progenitor cells and recurring severe bacterial and fungal infections. Recent reports indicated that severe neutropenia is a consequence of an impaired survival and abnormal cell cycle progression of myeloid progenitor cells in both cyclic and severe congenital neutropenia. Mutations in the Neutrophil Elastase gene were identified in all patients with cyclic neutropenia and most of the patients with severe congenital neutropenia. We hypothesize that expression of mutant Neutrophil Elastase protein results in deregulation of intracellular activity and premature cell death of myeloid-committed progenitor cells in these disorders, resulting in the lack of peripheral blood Neutrophils. The potential molecular mechanisms of mutant-protein-mediated neutropenia is discussed.
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clinical implications of mutations of Neutrophil Elastase in congenital and cyclic neutropenia
Journal of Pediatric Hematology Oncology, 2001Co-Authors: David C Dale, W. Conrad Liles, Daniel Garwicz, Andrew A G AprikyanAbstract:Clinical implications of mutations of Neutrophil Elastase in congenital and cyclic neutropenia
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mutations in the gene encoding Neutrophil Elastase in congenital and cyclic neutropenia
Blood, 2000Co-Authors: David C Dale, Audrey Anna Bolyard, Mary Ann Bonilla, George Kannourakis, Richard E Person, Andrew A G Aprikyan, Cornelia Zeidler, Karl Welte, Laurence A. Boxer, Kathleen F BensonAbstract:Congenital neutropenia and cyclic neutropenia are disorders of Neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding Neutrophil Elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of Neutrophil Elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the Neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding Neutrophil Elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.
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mutations in ela2 encoding Neutrophil Elastase define a 21 day biological clock in cyclic haematopoiesis
Nature Genetics, 1999Co-Authors: Marshall Horwitz, Andrew A G Aprikyan, Richard E Person, Kathleen F Benson, David C DaleAbstract:Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity1,2. Circulating Neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection3. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding Neutrophil Elastase (EC 3.4.21.37, also known as leukocyte Elastase, Elastase 2 and medullasin), a serine protease of Neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil Elastase (a 240-aa mature protein predominantly found in Neutrophil granules4) is the target for protease inhibition by α1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between Neutrophil Elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.
Kathleen F Benson - One of the best experts on this subject based on the ideXlab platform.
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contributions to neutropenia from pfaap5 n4bp2l2 a novel protein mediating transcriptional repressor cooperation between gfi1 and Neutrophil Elastase
Molecular and Cellular Biology, 2009Co-Authors: Stephen J. Salipante, Brice Korkmaz, Zhijun Duan, Jeremy Wechsler, Richard E Person, Kathleen F Benson, Meghan E B Rojas, Leighton H Grimes, Marshall S. HorwitzAbstract:“Neutropenia” refers to deficient numbers of Neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which Neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease Neutrophil Elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating Neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and Neutrophil Elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows Neutrophil Elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of Neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which Neutrophil Elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature Neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.
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Neutropenia-Associated Mutations of PFAAP5, a Novel Protein Mediating Transcriptional Repressor Interaction between Gfi1 and Neutrophil Elastase.
Blood, 2006Co-Authors: Stephen J. Salipante, Kathleen F Benson, Richard Person, Raffaele Badolato, Marshall S. HorwitzAbstract:Mutations in ELA2, encoding the protease Neutrophil Elastase, cause cyclic neutropenia and are also the most common cause of the Kostmann syndrome of congenital neutropenia. Mutations in the transcriptional repressor and intrinsic regulator of hematopoiesis, Gfi1, are another rare cause of congenital neutropenia. ELA2 is a target of Gfi1; consequently, mutation of Gfi1 leads to over-expression of Neutrophil Elastase. Defects in a predicted feedback circuit offer a possible explanation for the periodic oscillations of cyclic neutropenia, but how Neutrophil Elastase and Gfi1 regulate hematopoiesis with respect to each other, and what additional factors may be involved, has remained undefined. To identify other components of a potential hematopoietic feedback circuit, we performed yeast two-hybrid screens with Gfi1 and Neutrophil Elastase using bone marrow cDNA libraries. We found 25 proteins that interacted with Gfi1. Only two proteins, however, interacted with the isoform of Neutrophil Elastase containing an intact carboxyl terminal extension (deletion of which is the most frequently occurring type of mutation in congenital neutropenia). Remarkably, one of the two, the previously uncharacterized protein PFAAP5, was among the 25 found to also interact with Gfi1. PFAAP5 may therefore play a unique role in mediating interactions between Neutrophil Elastase and Gfi1. We further studied PFAAP5. Immunofluorescent localization with an epitope-tagged construct transfected into cultured cells demonstrates its localization in the nucleus. To determine if PFAAP5 influences Gfi1’s activities as a transcription factor in the presence or absence of Neutrophil Elastase, we performed transient transfection assays with a Gfi1 responsive reporter and PFAAP5 siRNA knockdown in NIH3T3 cells, which express PFAAP5, but not Gfi1 or Neutrophil Elastase, endogenously. Not surprisingly, transfected Neutrophil Elastase demonstrated no effect as a transcriptional repressor by itself. Unexpectedly, however, transcriptional repression by transfected Gfi1 increased with co-expression of Neutrophil Elastase. Knockdown of PFAAP5 had no effect on transfected Gfi1 repressor activity alone, but knockdown of PFAAP5 blocked the ability of co-expressed Neutrophil Elastase to increase Gfi1 repressor activity. Therefore, Neutrophil Elastase can increase Gfi1 repressor function, but only in the presence of PFAAP5. Finally, we evaluated PFAAP5 as a candidate gene in cases of congenital neutropenia in which ELA2 or Gfi1 mutations are absent. One homozygous and two different heterozygous coding sequence substitutions were detected in four of 230 unrelated patients, yet absent among double the number of controls. The mutations prevent PFAAP5 from interacting with either Neutrophil Elastase or Gfi1 in the yeast two-hybrid assay. Thus, mutations of Neutrophil Elastase, Gfi1, or PFAAP5 may each similarly disrupt a common circuit in which Gfi1 represses ELA2 and Neutrophil Elastase feeds back to cooperate with Gfi1 in regulating its own transcription.
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mutations associated with neutropenia in dogs and humans disrupt intracellular transport of Neutrophil Elastase
Nature Genetics, 2003Co-Authors: Kathleen F Benson, Fengqian Li, Zhijun Duan, Jeremy Wechsler, Dalila Albani, Richard E Person, Kimberly Meadewhite, Kayleen WilliamsAbstract:Cyclic hematopoiesis is a stem cell disease in which the number of Neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease Neutrophil Elastase1, found in lysosome-like granules, cause cyclic hematopoiesis2 and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of Neutrophil Elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity4. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis5, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of Neutrophil Elastase exposes an AP3 interaction signal responsible for redirecting Neutrophil Elastase trafficking from membranes to granules. Disruption of either Neutrophil Elastase or AP3 perturbs the intracellular trafficking of Neutrophil Elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of Neutrophil Elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.
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mutations in the gene encoding Neutrophil Elastase in congenital and cyclic neutropenia
Blood, 2000Co-Authors: David C Dale, Audrey Anna Bolyard, Mary Ann Bonilla, George Kannourakis, Richard E Person, Andrew A G Aprikyan, Cornelia Zeidler, Karl Welte, Laurence A. Boxer, Kathleen F BensonAbstract:Congenital neutropenia and cyclic neutropenia are disorders of Neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding Neutrophil Elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of Neutrophil Elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the Neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding Neutrophil Elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.
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mutations in ela2 encoding Neutrophil Elastase define a 21 day biological clock in cyclic haematopoiesis
Nature Genetics, 1999Co-Authors: Marshall Horwitz, Andrew A G Aprikyan, Richard E Person, Kathleen F Benson, David C DaleAbstract:Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity1,2. Circulating Neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection3. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding Neutrophil Elastase (EC 3.4.21.37, also known as leukocyte Elastase, Elastase 2 and medullasin), a serine protease of Neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil Elastase (a 240-aa mature protein predominantly found in Neutrophil granules4) is the target for protease inhibition by α1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between Neutrophil Elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.
Yamile Gonzalez - One of the best experts on this subject based on the ideXlab platform.
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purification and partial characterization of human Neutrophil Elastase inhibitors from the marine snail cenchritis muricatus mollusca
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2007Co-Authors: Yamile Gonzalez, Maday Alonso Del Rivero, Maria Luiza Vilela Oliva, Izaura Y Hirata, Aparecida S Tanaka, Mariana S. Araujo, Maria A ChavezAbstract:Abstract Human Neutrophil Elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human Neutrophil Elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human Neutrophil Elastase strongly, with equilibrium dissociation constant ( K i ) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic Elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human Neutrophil Elastase inhibitors described in a mollusk.
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purification and partial characterization of human Neutrophil Elastase inhibitors from the marine snail cenchritis muricatus mollusca
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2007Co-Authors: Yamile Gonzalez, Maday Alonso Del Rivero, Maria Luiza Vilela Oliva, Izaura Y Hirata, Aparecida S Tanaka, Mariana S. Araujo, Maria A ChavezAbstract:Abstract Human Neutrophil Elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human Neutrophil Elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human Neutrophil Elastase strongly, with equilibrium dissociation constant ( K i ) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic Elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human Neutrophil Elastase inhibitors described in a mollusk.
Andrew A G Aprikyan - One of the best experts on this subject based on the ideXlab platform.
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Neutrophil Elastase mutations and risk of leukaemia in severe congenital neutropenia
British Journal of Haematology, 2007Co-Authors: Philip S Rosenberg, Audrey Anna Bolyard, Mary Ann Bonilla, Yigal Dror, Blanche P Alter, Andrew A G Aprikyan, Daniel C Link, Steven Stein, Elin Rodger, George KannourakisAbstract:Severe congenital neutropenia (SCN) is a heterogeneous bone marrow failure syndrome predisposing to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). We studied 82 North American and Australian SCN patients enrolled in the Severe Chronic Neutropenia International Registry who were on long-term treatment with granulocyte colony-stimulating factor and for whom the Neutrophil Elastase (ELA2) gene was sequenced. There was no significant difference in the risk of MDS/AML in patients with mutant versus wild-type ELA2: the respective cumulative incidences at 15 years were 36% and 25% (P = 0·96). Patients with either mutant or wild-type ELA2 should be followed closely for leukaemic transformation.
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mutations in the Neutrophil Elastase gene in cyclic and congenital neutropenia
Current Opinion in Immunology, 2001Co-Authors: Andrew A G Aprikyan, David C DaleAbstract:Abstract Severe neutropenia disorders are characterized by extremely low levels of peripheral blood Neutrophils, a maturation block of bone marrow progenitor cells and recurring severe bacterial and fungal infections. Recent reports indicated that severe neutropenia is a consequence of an impaired survival and abnormal cell cycle progression of myeloid progenitor cells in both cyclic and severe congenital neutropenia. Mutations in the Neutrophil Elastase gene were identified in all patients with cyclic neutropenia and most of the patients with severe congenital neutropenia. We hypothesize that expression of mutant Neutrophil Elastase protein results in deregulation of intracellular activity and premature cell death of myeloid-committed progenitor cells in these disorders, resulting in the lack of peripheral blood Neutrophils. The potential molecular mechanisms of mutant-protein-mediated neutropenia is discussed.
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clinical implications of mutations of Neutrophil Elastase in congenital and cyclic neutropenia
Journal of Pediatric Hematology Oncology, 2001Co-Authors: David C Dale, W. Conrad Liles, Daniel Garwicz, Andrew A G AprikyanAbstract:Clinical implications of mutations of Neutrophil Elastase in congenital and cyclic neutropenia
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mutations in the gene encoding Neutrophil Elastase in congenital and cyclic neutropenia
Blood, 2000Co-Authors: David C Dale, Audrey Anna Bolyard, Mary Ann Bonilla, George Kannourakis, Richard E Person, Andrew A G Aprikyan, Cornelia Zeidler, Karl Welte, Laurence A. Boxer, Kathleen F BensonAbstract:Congenital neutropenia and cyclic neutropenia are disorders of Neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding Neutrophil Elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of Neutrophil Elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the Neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding Neutrophil Elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.
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mutations in ela2 encoding Neutrophil Elastase define a 21 day biological clock in cyclic haematopoiesis
Nature Genetics, 1999Co-Authors: Marshall Horwitz, Andrew A G Aprikyan, Richard E Person, Kathleen F Benson, David C DaleAbstract:Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity1,2. Circulating Neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection3. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding Neutrophil Elastase (EC 3.4.21.37, also known as leukocyte Elastase, Elastase 2 and medullasin), a serine protease of Neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil Elastase (a 240-aa mature protein predominantly found in Neutrophil granules4) is the target for protease inhibition by α1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between Neutrophil Elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.