ELA2 Mutations Research Articles

ELANE Neutropenia Beyond the Classification into Cyclic Neutropenia and Severe Congenital Neutropenia: The Inconstant Time Clock

▪Introduction: ELANE neutropenia represents the cause of 25-30% of the cases of congenital neutropenia. Classically, its appears in the literature as in OMIM, under two distinct entities: Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN). The delineation between the 2 entities is the “cyclicity” i.e. the periodic variation of the absolute neutrophils count (ANC), also called a 21-day time clock 1. However, it is extremely difficult to obtain enough sequential complete blood counts (CBC) at the onset of the disease, during an enough length period, while the patient is not experiencing a severe infection or receiving GCSF therapy. The purpose of our study is to analyze the ANC periodicity at the onset of the disease, prior to the initiation of GCSF in a cohort of patients with ELANE neutropenia.Methods : Available data from the patients, with ELANE class 4 and 5 variants, enrolled in the French Severe Chronic Neutropenia Registry, were analysed. The final diagnostic of CyN and SCN was performed considering all the follow up period (median 16.7 years) and based on the presence of recurrent periodic variation of ANC in the absence of GCSF therapy. CyN was defined as multiple documented ANCs >500 cells/mm 3 , with intermittent ANC variation (n=49), while SCN is defined as ANC persistently <500 cells/mm 3 (n=94). In case of irregularity (i.e. not a regular periodic pattern during all the follow up), the classification takes in consideration the majority of the follow up. A comprehensive analysis of the infectious profile is available elsewhere 2. We were focused here on the diagnostic period (roughly the 2 first months since the diagnosis). We have analysed the initial blood count of the patients and cast the patients by categories if at least 4 ANCs can be evaluated. ANC oscillations defined 4 groups: Group 1: oscillation of ANC values above and below 500 ANC/mm 3 for at least 2 cycles lead to consider the patient as Cyclic; Group 2: clear oscillation of ANC values above and below 200 ANC/mm 3 (but ever<500 ANC/mm 3) for at least 2 cycles; Group 3: no oscillation of ANC values whose level are ever below 500 ANC/mm 3; Group 4: Early GCSF treatment.Results: Among the 143 patients enrolled in this study (Table 1), 137 have at least 4 CBC evaluable during the diagnosis period, including 30 who have been treated almost front line after diagnosis of neutropenia by GCSF hampering evaluation of periodicity. Such patients were all initially considered as SCN. Among the 67 finally classified as SCN, 28 (27.38%) showed an oscillation pattern (group 1), 14 (15.48%) showed minor oscillations (group 2), while 25 (30.95%) had a persistent and severe neutropenia (group 3). Among the 40 CyN, 1 have showed minor oscillations (group 2) below 500/mm 3, while 3 had a persistent and severe neutropenia (group 3). Globally, 32 /107 patients were miss- classified at diagnosis compare to the final diagnosis. Additional data shows that many health indicators could not be deducted from the initial classification like the infections rate, the use of GCSF, the death rate, the sequels rate.Conclusions:Periodic variation of ANC despite being the criteria to define the sub type of ELANE neutropenia is difficult to evaluate at the initial presentation of the disease. In addition, cyclicity is not a permanent feature in ELANE neutropenia, some patients being cyclic only for a certain time in their life span. It results a high rate of miss classification if we compare the initial diagnostic period and all the medical history of the patients. Noteworthy, the rate of several severe complications is not so clearly different between diagnosis sub categories. We propose to consider ELANE neutropenia as a unique disease characterized by a clinical spectrum ranging from more severe forms (corresponding to SCN) to milder forms, the latter often characterized at onset by ANC fluctuations. In addition, some intermediate severity forms could be characterized by minor oscillations.References1 Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat.Genet. 1999;23:433-436. 2 Rotulo GA, Plat G, Beaupain B et al. Recurrent bacterial infections, but not fungal infections, characterise patients with ELANE-related neutropenia: a French Severe Chronic Neutropenia Registry study. Br J Haematol. 2021 doi: 10.1111/bjh.17695 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Turkish National Severe Congenital Neutropenia Registry

Introduction: Severe congenital nötropenia (SCN) is a very rare disease. Genetic mutation in neutrophil elastase gene (ELANE) is the most frequent mutation in European and North American registries. However, differences could be expected in the countries where consanguineous marriages are common. It is important to find out whether the approach for genetic typing shall be the same in western Europe, eastern Europe and middle East. We aimed to establish a national neutropenia registry in Turkey, a country with an extraordinary mixed population of Caucasian and Asian decent and the proportion of consanguineous families being higher than in most other parts of Europe.Patients and Methods: In this study, establishment of a national registry for severe congenital neutropenia (SCNR) and national bone marrow failure (BMFR) was aimed. Clinical and laboratory findings of 699 patients with BMF including 223 patients (31.9%) with SCN were entered into Turkish National BMFR.Results: The median age of the children with SCN (male/ female: 0.96) was 11.7 years (range 1 month to 35 years). The median follow up period for patients were 7.7 years (range 3 months to 34 years). Consanguinity between the parents of SCN patients was 36.3 %. HAX1 mutation was the most frequently seen mutation among the patients entered into national registry (n=51, 22.8%). The same mutation, homozygote c130-131insA, p.W44X, was detected in 47 of 51 patients (92.1%) with HAX-1 mutation. ELA2 mutation was detected in 16 patients (7.2%) and it was the second most common mutation for SCN. In this series 7 patients (3.1%) had a mutation in G6PC3 gene. CSF3R and JAGN1 mutations were seen in 4 (1.8%) and 2 (1.5%) patients respectively.No mutation was found in 66 (29.5%) tested patients (All tested for ELANE and HAX1 and 22 of them were also tested for G6PC3). Fifty two patients (22.4%) were not tested for SCN. Fifty eight percent of the patients were given GCSF. The median dose was 5 mcg/ kg for 3 days a week. Two patients died with infectious complications and six developed MDS/ AML.Conclusion: In Turkey SCN mostly resulted from the p W44X mutation in HAX1 gene. Therefore in Turkey mutation analysis should be started with HAX1 and if it is negative ELANE and G6PC3 should be checked. Rare mutations should be tested in mutation negative patients because of very high percentage of consanguineous marriage.This study was supported by TUBITAK and Turkish Pediatric Hematology Association. DisclosuresYilmaz Karapinar:TUBITAK: Research Funding.

Intermittent Neutropenia as an Early Feature of Mild Mevalonate Kinase Deficiency

A 15-month-old boy, born to Iranian consanguineous parents presented with intermittent neutropenia interspersed with episodes of fever and leukocytosis since early infancy. No ELA2 mutations were found and the bone marrow study was normal. At age 4years he progressed to more typical attacks of periodic attacks of fever, abdominal pain, oral aphthous ulcers, cutaneous rash and leukocytosis. The clinical and laboratory features were compatible with the mild form of mevalonate kinase deficiency, usually named "Hyper-IgD and periodic fever syndrome" (HIDS). Genomic sequencing of the mevalonate kinase (MVK) gene revealed homozygous missense mutation (p.Val377Ile). On demand dexamethasone resulted in a rapid amelioration of febrile episodes. The presentation of intermittent neutropenia has not been reported in HIDS and deserves more attention in large patient cohorts.

The neutrophil elastase mutant affects viability and differentiation of the human monocytic THP-1 cell

Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP-1, to carry the human wild-type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild-type THP-1 cells. Exogenous wild-type ELA2 markedly increased THP-1 differentiation, whereas G185R ELA2 was incompetent to promote THP-1 differentiation in response to all-trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up-regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival.

IL-1 blockade as a novel approach to treatment of hyperzincemia and hypercalprotectinemia, a possible new autoinflammatory syndrome

Methods We describe a patient initially evaluated at 10 months of age for gross developmental delay, failure to thrive, splenomegaly, and microcytic anemia. Physical exam was significant for weight <3rd percentile, splenomegaly (down 5cm), and head lag. Initial laboratories revealed microcytic anemia (hemoglobin 8.7 g/dL); elevated platelets 499 K/uL, C-reactive protein 23.2 mg/dL (<0.2) and erythrocyte sedimentation rate 120 mm/h (0-10). Radiographs showed Erlenmeyer flask deformities of the femurs. Workup for lysosomal storage disorders, oncologic processes, and infections were negative. Genetic testing of known mutations in FAS, CIAS1, ELA2, LPIN2, MVK, PSTPIP1, and TNFRSF1A were negative. Immunoglobulins were elevated with normal T and B cells. On further workup for failure to thrive, a plasma zinc level was found elevated at 532 mcg/dL (60-120). Due to his persistently inflamed state, there was concern for hyperzincemia related to hypercalprotectinemia. Calprotectin level, measured by enzyme-linked immunosorbent assay, was elevated: 428,300 ng/mL (<420). On the basis of these findings the diagnosis of hyperzincemia associated with hypercalprotectinemia was made (OMIM 194470). Additionally, the patient’s workup has revealed the following mutations with unknown clinical significance: a heterozygous variant in the MEFV gene (T577A), a hemizygous duplication of exons 61-79 of the dystrophin gene (CPK normal), and a maternally inherited microduplication at Xp21.2. Colchicine was started due to the MEFV gene mutation with little to no improvement. Based on calprotectin’s role in inflammation, and possible link to IL-1, anakinra was initiated.

Suppressed Neutrophil Development in Hematopoiesis of Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with ELA2 Mutation

Abstract 730Severe congenital neutropenia (SCN) is a rare disorder characterized by severe neutropenia present at birth, an arrest of neutrophilic differentiation at the promyelocyte or myelocyte stage, and a propensity to develop acute myeloid leukemia and myelodysplasia. Mutations of the ELA2 gene encoding neutrophil elastase have been identified in majority cases of SCN. To date, more than 50 distinct mutations of the ELA2 gene have been reported in patients with SCN. However, the mechanisms by which these mutations disrupt neutrophil development are unclear.To understand the roles of ELA2 mutation in SCN, we established three human induced pluripotent stem (iPS) cell clones (SPN0101, SPN0102 and SPN0103) from bone marrow stromal cells of a patient having heterozygous mutation in ELA2 gene by transfection with retrovirus vector whichexpressed human OCT3/4, SOX2, KLF4, and c-MYC (SCN-iPS cells). The silencing of exogenous genes and the capability to differentiate into three germ lines by teratoma formation were confirmed in the three SCN-iPS cell clones.We first examined the hematopoietic differentiation of SCN-iPS and control iPS cells which were generated from healthy donors by the same method to SCN-iPS cells, using coculture system with murine embryonic aorta-gonad-mesonephros region-drived stromal cell line (AGM-3 cells) (Ma F. et al., PNAS, 2009). The cocultured cells were harvested at day 12 and analyzed for the hematopoietic colony formation by 10,000 CD34+ cells which were separated using magnetic beads system. There was no difference between SCN-iPS and control iPS cells in the number and size of erythroid and mixed-lineage colonies (erythroid colonies; 11.5±3.7 in SCN-iPS cells and 10.3±4.5 in control and mixed-lineage colonies; 25.5±8.0 in SCN-iPS cells and 18.7±4.2 in control). However, the number of myeloid colonies derived from SCN-iPS cells was significantly smaller than that in control (48.0±21.3 in SCN-iPS cells and 206.7±37.6 in control, p<0.01), and their size was tiny as compared to that in control. The myeloid colonies derived from SCN-iPS cells consisted of monocytic cells and a few immature myeloid cells whereas those from control iPS cells contained a substantial number of mature neutrophils. Furthermore, since SCN patients need higher dose of granulocyte colony-stimulating factor (G-CSF) treatment to increase neutrophil numbers and avoid recurrent life-threatening bacterial or fungal infections, we compared the sensitivity of myeloid colony formation to G-CSF between SCN-iPS and control iPS cells. The number and size of myeloid colonies derived from control iPS cells reached a plateau at a concentration of 1 ng/mL G-CSF, while those from SCN-iPS cells were enhanced in association with the escalated concentrations of G-CSF up to 1,000 ng/mL although the number of the colonies was fewer, and their size was smaller than control even at 1,000 ng/mL of G-CSF. The large colonies containing more than 100 cells were formed from control iPS cells at 1 ng/mL of G-CSF, but SCN-iPS cells at higher doses of 100 ng/mL.We next examined the neutrophil development in more detail in suspension culture. 10,000 CD34+ cells derived from SCN-iPS and control iPS cells were cocultured with AGM-3 stromal cells in the presence of various cytokines including 10 ng/mL of G-CSF, but not erythropoietin to exclude the growth of erythroid cells. The cells from control iPS cell increased 23.3-fold for 2 weeks, and most of the cultured cells were neutrophils. By contrast, the cells from SCN-iPS cells gradually decreased, and contained few neutrophils, but mainly monocytic cells.These results indicated thatthe development of neutrophils was selectively impaired in hematopoiesis derived from SCN-iPS cells, although other lineages of cells, such as erythroid and monocytic cells, were not affected, and that the stimulation of high concentration of G-CSF compensated the impaired neutrophil development to some extent in SCN-iPS cells. The present study demonstrated that hematopoiesis derived from SCN-iPS cells reflected the pathophysiological status of SCN patients including neutrocytopenia and its improvement by G-CSF treatment, and established SCN-iPS cells could contribute to understand the pathophysiology and leukemogenesis in SCN. Further researches are under investigation to clarify the relationship between ELA2 mutation and clinical status in SCN including leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Neutrophil Extracellular Traps (NETs) in Patients with Congenital Neutropenia

Abstract 15Activated neutrophils kill microbes by phagocytosis and extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of decondensed chromatin and granular proteins such as neutrophil elastase (NE) and cathepsin G. Enzymatic generation of reactive oxygen species (ROS) by NADPH oxidase and the release of serine proteases such as NE have been shown to be essential factors for NET formation. Patients with chronic granulmatous disease (CGD), who lack a normal generation of ROS, show a defective NET formation.Since myeloid cells of patients with severe congenital neutropenia (CN) show an aberrant expression pattern of granular proteases such as neutrophil elastase (NE) or myeloperoxidase, we aimed to analyse NET formation in activated neutrophils of these patients. CN is a heterogeneous hematological disorder, characterized by peripheral blood neutrophil counts below 0,2×109/l and a maturation arrest of myelopoiesis at the promyelocytic/myelocytic stage. 60% of CN patients harbor autosomal dominant mutations within the ELA2 gene encoding for NE, but also mutations in other genes (e.g. HAX1, G6PC3, WAS, GFI1, p14) have been found to be disease-causing. Previously, we described severely diminished levels of NE in myeloid cells of CN patients. Here, we aimed to explore NET formation in neutrophils of CN patients. Moreover, we intended to analyze the effects of a reduced ELA2 expression and gene mutations as seen in CN patients on NET formation in vitro.Granulocytes of CN patients undergoing G-CSF therapy were extracted by density centrifugation, stimulated with phorbol myristate acetate (PMA, 50 nM, up to 240 min) and then tested for NET formation. NETs were stained with an extracellular DNA dye or DAPI. Our analyses showed normal NET formation in peripheral blood granulocytes of two patients with HAX1-related neutropenia, whereas there was a significantly lower amount of NETs in two patients with ELA2 mutations. One further patient out of three CN patients with unknown mutations showed a reduced amount of NETs in bone marrow PMNs.To further evaluate the possible effect of downregulated ELA2 expression on NET formation, we transduced primary human CD34+ cells with a lentiviral-based shRNA construct downregulating the expression of NE. Subsequently, these cells were differentiated into granulocytes with a cytokine cocktail containing G-CSF and tested for their ability to form NETs. We found an almost completely abolished NET formation in cells transduced with ELA2 shRNA as compared to control cells.Hitherto, CGD is the only immunodeficiency with a clearly defective NET formation. Our results point to an impaired formation of NETs also in CN patients carrying ELA2 mutations. This supports the recent finding of a central role for NE in NET formation. Two patients with HAX1 related CN showed a normal ability to form NETs. Our further work will aim to better define the subgroup of CN patients defective in NET formation. Disclosures:No relevant conflicts of interest to declare.

Heterozygous M1V variant of ELA‐2 gene mutation associated with G‐CSF refractory severe congenital neutropenia

Severe congenital neutropenia is an autosomal recessive disorder characterized by maturation arrest at the promyelocyte/myelocyte phase in the bone marrow, absolute neutrophil count <0.5 × 10(9) /L and recurrent bacterial infections. Homozygous mutations of either HAX-1 or ELA-2 have been described. We report the case of a premature male infant with congenital neutropenia, associated with multiple infections, refractory to treatment with granulocyte colony stimulating factor who subsequently underwent matched sibling donor stem-cell transplant. He was found to be heterozygous for the M1V variant of the ELA-2 gene that we postulate to be causative for his severe neutropenia

Secondary acute myeloid leukemia following severe congenital neutropenia: Case presentation.

e20001 Background: Therapy with granulocyte colony stimulating factor (G-CSF) is standard of care to prevent sepsis in severe congenital neutropenia (SCN). This successful approach has unmasked the predisposition to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). This appears greatest in patients who require higher doses of G-CSF to achieve an adequate neutrophil response. Methods: 9 year old boy started severe infections since early infancy, neutropenia consistently below 500. Severe congenital neutropenia (SCN) was diagnosed at age 3 months, after marrow assessment. On high dose G-CSF since then, 10 to 15 mcg/Kg/d, to keep adequate ANC. Asymptomatic since then, normal development. No physical stigmata. Blood smears always dysplastic on myeloid line. At age 8, on check-up, had mild bicytopenia (anemia and thrombocytopenia). Was followed with bone marrow aspirates and biopsies. Normocellular, myeloid blasts 10 to 12%, by morphology and immunophenotype. Normal karyotype. Peripheral cytopenias advanced, until becoming transfusion-dependent for RBC and later platelets. WBC remained stable after discontinuing G-CSF. Viral and other infections were rulled out. Samples were analyzed for ELA-2 mutation, which was positive. G-CSF receptor mutation was negative. Course was progressive, transfusions given frequently. Splenomegaly stable at 3cm. Bone marrow performed monthly. Started iron chelation after 15 RBC transfusions and ferritin over 1000mcg/dl. No sibling available for HSCT. Results: Transformed to overt AML, with a rapid disease progression, despite giving AML chemotherapy. He ultimately died of refractory disease, after 2 blocks of chemotherapy. Conclusions: It is imperative that all patients with severe congenital neutropenia be closely monitored for leukaemic transformation. Risks and benefits of early haematopoietic stem cell transplantation should be evaluated for patients who respond poorly to a high dose of G-CSF.

Correspondence: Cyclic neutropenia associated with T cell immunity to granulocyte proteases and a double de novo mutation in GFI1, a transcriptional regulator of ELANE

correspondence: Cyclic neutropenia associated with T cell immunity to granulocyte proteases and a double <i>de novo</i> mutation in <i>GFI1</i>, a transcriptional regulator of <i>ELANE</i>

Resolving a genetic paradox throughout preimplantation genetic diagnosis for autosomal dominant severe congenital neutropenia

Severe congenital neutropenia is an inherited disease characterized by low peripheral blood neutrophils, amenable to bone marrow transplantation. Genetic analysis in the family here described detected a ELA2 splice-site mutation in the affected child and also in his asymptomatic father. The parents requested preimplantation genetic diagnosis (PGD), coupled with HLA matching, to obtain a suitable bone marrow donor for the affected child. A PGD protocol was developed, based on multiplex nested PCR for direct analysis of the ELA2 mutation, flanking polymorphic markers and HLA typing. The amplification efficiency of the mutation was > 90% in single leukocytes from the affected child but only 67% in the father. Analysis of single haploid sperm cells from the father demonstrated three different sperm-cell populations: (1) sperm cells harboring the ELA2 mutation on the 'affected' haplotype, (2) sperm cells without the ELA2 mutation on the 'normal' haplotype, and (3) sperm cells without the ELA2 mutation on the 'affected' haplotype. These data demonstrate that the ELA2 mutation in the father occurred de novo during his embryonic development, resulting in somatic as well as germ-line mosaicism. This conclusion was also taken into consideration when PGD was performed.

Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Abstract 551Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures:No relevant conflicts of interest to declare.

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).

Abstract 3597Poster Board III-534An 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:CN subtype by gene mutationTotal patient number (n)MDS/Leukemia (n/%)ELA2-CN20931 (14. 8%)HAX1-CN204 (20%)ELA2 neg + HAX1 neg576 (10.5%)ELA2 neg/ HAX1 not tested915 (5.5%)WAS184 (22.2%)G6PC380p1440Total40750 (12.3%)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:CN subtype by gene mutationWithout leukemia (n)Median G-CSF dose (μg/kg/d)With leukemia (n)Median G-CSF dose (μg/kg/d)ELA2-CN755.01515HAX1-CN155.0413.4ELA2-/HAX1-367.2410.5WAS152.643.0G6PC383.90nap1445.10na ConclusionPatients 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.

Severe Chronic Neutropenia in Korean Children.

Abstract 4513▪ IntroductionSevere chronic neutropenia (SCN) is a rare hematologic disorder defined by an absolute neutrophil count less than 0.5×109/L for several months or years. They usually suffer from recurrent infections. Principal subtypes of SCN are congenital, cyclic, idiopathic and primary autoimmune neutropenia (AIN). Patients and MethodsMedical records collected from a national survey were retrospectively analyzed on newly diagnosed SCN patients in Korea between January, 1999 and December, 2008 in respect to the diagnosis, clinical manifestations, treatments and prognosis of the patients. ResultsThere were 64 patients (Male, 20; Female 44) reported from 16 hospitals: congenital, 19; cyclic, 16; idiopathic, 25; and immune in origin, 4. The main clinical manifestation was various types of bacterial infections. Two cases (1 congenital, 1 cyclic) were diagnosed by family histories. The median age at diagnosis was 12 months (11 days-158 months). A bone marrow examination was done in 45 patients (70.3 %) at the median age of 26 months (1 day-158 months), with the interval between the initial CBC and BM study being 7.3 months (9 days-138 months). The ELA2 mutation, done in 6 patients, was not detected. Only one patient with congenital SCN evolved to AML at 54 months after diagnosis, who is under chemotherapy. Most patients were treated with G-CSF (5-10 μg/kg/day) during infection episodes. The median follow up duration was 23 months (11 days – 176 months). Two patients of congenital SCN died of infection (pneumonia, meningitis) and 8 patients were lost to follow up, and the remaining are alive. ConclusionsSCN is a rare hematologic disease with inherent vulnerability to infections, thus early detection with proper management should be important for survival of SCN patients. We propose a nation-wide, prospective study to delineate the prevalence, molecular diagnosis, natural history, the optimal use of G-CSF, and prognostic factors in Korean patients with SCN. Disclosures:No relevant conflicts of interest to declare.

Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

AbstractSevere congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis which follows an autosomal dominant or autosomal recessive pattern of inheritance. Genetic analyses indicate mutations in the ELA2 gene in most patients. We have identified LEF-1 as a decisive transcription factor in granulopoiesis controlling proliferation and granulocytic differentiation by direct activation of its target gene, C/EBPα. In patients with CN, the expression of LEF-1 and C/EBPα was abrogated in myeloid progenitors leading to maturation arrest of granulopoiesis. In the present study we demonstrated that ELA2 mRNA expression in myeloid progenitors and plasma protein levels of neutrophil elastase (NE) were markedly reduced in patients with CN harboring mutations in either ELA2 or HAX-1 genes. The ELA2 gene promoter is positively regulated by the direct binding of LEF-1 or C/EBPα, documenting the role of LEF1 in the diminished ELA2 expression. We found that transduction of hematopoietic cells with LEF-1 cDNA resulted in the up-regulation of ELA2/NE synthesis, whereas inhibition of LEF-1 by shRNA led to a marked reduction in the levels of ELA2/NE. LEF-1 rescue of CD34+ cells isolated from 2 patients with CN resulted in granulocytic differentiation of the cells which was in line with increased levels of functionally active ELA2/NE.

Contributions to Neutropenia from PFAAP5 (N4BP2L2), a Novel Protein Mediating Transcriptional Repressor Cooperation between Gfi1 and Neutrophil Elastase

"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.

Genetic Insights into Congenital Neutropenia

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.

Ela2 Mutations and Clinical Manifestations in Familial Congenital Neutropenia

Three familial cases of each of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for neutrophil elastase (Ela2) gene mutation. The contents of the neutrophil-specific granule proteins cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were also analyzed in SCN. Genomic DNA was extracted from the patients' peripheral blood or bone marrow, and the coding sequence of the Ela2 gene was amplified by polymerase chain reaction and subjected to direct sequencing. The contents of antimicrobial peptides were analyzed by flow cytometry. Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN (W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case was a novel mutation of Ela2. Prophylactic treatment for growth factors or antibiotic prophylaxis against bacterial infection was useful for lowering the frequency of infectious episodes. Adult patients tended to have less frequent infections compared with minors in the same family. The contents of both cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were significantly reduced in SCN compared with healthy controls. Prophylaxis by growth factor or antibiotics is useful for decreasing risks of bacterial infections in SCN and CN. Adults were likely to have less frequent infections than children in familial cases of SCN and CN with the same mutation of Ela2.

Progenitor cell self‐renewal and cyclic neutropenia

Cyclic neutropenia (CN) is a rare genetic disorder where patients experience regular cycling of numbers of neutrophils and various other haematopoietic lineages. The nadir in neutrophil count is the main source of problems due to risk of life-threatening infections. Patients with CN benefit from granulocyte colony stimulating factor therapy, although cycling persists. Mutations in neutrophil elastase gene (ELA2) have been found in more than half of patients with CN. However, neither connection between phenotypic expression of ELA2 and CN nor the mechanism of cycling is known. Recently, a multicompartment model of haematopoiesis that couples stem cell replication with marrow output has been proposed. In the following, we couple this model of haematopoiesis with a linear feedback mechanism via G-CSF. We propose that the phenotypic effect of ELA2 mutations leads to reduction in self-renewal of granulocytic progenitors. The body responds by overall relative increase of G-CSF and increasing progenitor cell self-renewal, leading to cell count cycling. The model is compatible with available experimental data and makes testable predictions.

Congenital neutropenia

Congenital neutropenia comprises a variety of genetically heterogeneous phenotypic traits. Molecular elucidation of the underlying genetic defects has yielded important insights into the physiology of neutrophil differentiation and function. Non-syndromic variants of congenital neutropenia are caused by mutations in ELA2, HAX1, GFI1, or WAS. Syndromic variants of congenital neutropenia may be due to mutations in genes controlling glucose metabolism (SLC37A4, G6PC3) or lysosomal function (LYST, RAB27A, ROBLD3/p14, AP3B1, VPS13B). Furthermore, defects in genes encoding ribosomal proteins (SBDS, RMRP) and mitochondrial proteins (AK2, TAZ) are associated with congenital neutropenia syndromes. Despite remarkable progress in the field, many patients with congenital neutropenia cannot yet definitively be classified by genetic terms. This review addresses diagnostic and therapeutic aspects of congenital neutropenia and covers recent molecular and pathophysiological insights of selected congenital neutropenia syndromes.