Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins. IntroductionSevere congenital neutropenia (SCN) is a rare disorder characterized by severe neutropenia present at birth, an arrest of granulocytic differentiation at the promyelocyte or myelocyte stage, and a marked propensity to develop acute myeloid leukemia and myelodysplasia. 1,2 Cyclic neutropenia (CN) is a related disorder of granulopoiesis, characterized by periodic oscillations in the numbers of circulating neutrophils and other peripheral blood cells. 3 Mutations of the ELA2 gene encoding neutrophil elastase (NE) have been identified in nearly all patients with CN and in approximately 50% of cases of SCN. [4][5][6][7][8][9] To date, more than 50 distinct mutations of the ELA2 gene have been reported in patients with CN or SCN. [5][6][7]10 Most of the mutations (ϳ80%) are missense mutations, although mutations that lead to splicing defects (ϳ10%) and premature stop codons (ϳ10%) also have been observed. With a few exceptions, specific ELA2 mutations are associated with SCN or CN, but not both, suggesting a genotypephenotype correlation.The molecular mechanisms by which ELA2 mutations disrupt granulopoiesis are unclear. Genetic studies provide 2 important clues. First, in all cases, the ELA2 mutations are heterozygous, [5][6][7] suggesting a dominant mechanism of action. Second, a case report of paternal mosaicism for an ELA2 mutation provides evidence that expression of mutant NE inhibits granulopoiesis in a cell intrinsic fashion, since no toxic paracrine effects of mutant NE protein on wild-type granulocytic cells in this mosaic individual were observed. 11 NE is a serine protease expressed at extremely high levels at the promyelocyte stage of granulocytic differentiation. 12 However, an extensive in vitro biochemical characterization of a l...
Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNAlike ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane.
Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of their Ela2 gene ("V72M") reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established.
Extracorporeal membrane oxygenation (ECMO) is an established therapy in the management of patients with refractory cardiogenic shock or acute respiratory failure. In this report, we describe the rapid development and implementation of an organized ECMO program at a facility that previously provided ad hoc support. The program provides care for patients within the Emory Healthcare system and throughout the Southeastern United States. From September 2014 to February 2015, 16 patients were treated with either venovenous or venoarterial ECMO with a survival to decannulation of 53.3% and survival to intensive care unit discharge of 40%. Of the 16 patients, 10 were transfers from outside facilities of which 2 were remotely cannulated and initiated on ECMO support by our ECMO transport team. Complications included intracerebral hemorrhage, bleeding from other sites, and limb ischemia. The results suggest that a rapidly developed ECMO program can provide safe transport services and provide outcomes similar to those in the existing literature. Key components appear to be an institutional commitment, a physician champion, multidisciplinary leadership, and organized training. Further study is required to determine whether outcomes will continue to improve.
Extracorporeal membrane oxygenation (ECMO) has become an increasingly utilized modality for the support of patients with severe cardiac or pulmonary dysfunction. Unfortunately, the costs and expertise required to maintain a formal ECMO program preclude the vast majority of hospitals from employing such technology routinely. These barriers to implementation of an effective ECMO program highlight the importance of the safe transport of patients in need of extracorporeal support. While many centers with extensive expertise in the management of patients on extracorporeal support have demonstrated their ability to transport those same patients, the ability of new ECMO programs to provide such transportation remains poorly studied. We established an ECMO program at our institution and immediately provided equipment and personnel to transport patients in need of or receiving extracorporeal support to our institution. Overall, we found that 13 out of 28 patients transported to our institution on ECMO or for consideration of ECMO support during the first 15 months of the program survived to hospital discharge. During that period, four incidents associated with patient transport occurred but none were related to ECMO support or adversely affected patient outcome. These observations demonstrate that new ECMO programs can safely and reliably transport patients on or in need of extracorporeal support.
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