ELA2 Mutations Research Articles

Acute lymphoblastic leukemia in a patient with congenital neutropenia without G-CSF-R and ELA2 mutations

Acute lymphoblastic leukemia in a patient with congenital neutropenia without G-CSF-R and ELA2 mutations

Autosomal-dominant primary immunodeficiencies

The vast majority of known primary immunodeficiencies (PIDs) are autosomal or X-linked recessive Mendelian traits. Only four classical primary immunodeficiencies are thought to be autosomal-dominant, three of which still lack a well-defined genetic etiology: isolated congenital asplenia, isolated chronic mucocutaneous candidiasis, and hyper IgE syndrome. The large deletions on chromosome 22q11.2 associated with Di George syndrome suggest that this disease may be dominant but not Mendelian, possibly involving several genes. The clinical and genetic features of six novel autosomal-dominant primary immunodeficiencies have however been described in recent years. These primary immunodeficiencies are caused by germline mutations in seven genes: ELA2, encoding a neutrophil elastase, and GFI1, encoding a regulator of ELA2 (mutations associated with severe congenital neutropenia); CXCR4, encoding a chemokine receptor (warts, hypogammaglobulinemia, infections and myelokathexis syndrome); LCRR8, encoding a key protein for B-cell development (agammaglobulinemia); IFNGR1, encoding the ligand-binding chain of the interferon-gamma receptor; STAT1, encoding the signal transducer and activator of transcription 1 downstream from interferon-gammaR1 (Mendelian susceptibility to mycobacterial diseases); and IKBA, encoding IkappaBalpha, the inhibitor alpha of NF-kappaB (anhidrotic ectodermal dysplasia with immunodeficiency). These recent data suggest that many more autosomal-dominant PIDs are likely to be identified in the near future.

Can mutations in ELA2, neutrophil elastase expression or differential cell toxicity explain sulphasalazine-induced agranulocytosis?

BackgroundDrug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene (ELA2), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in ELA2 or that sulphasalazine may affect human neutrophil elastase activity and protein expression.MethodsWe screened for genetic differences in ELA2 in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity.ResultsNone of the mutations in ELA2, which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line.ConclusionNeutrophil elastase expression and ELA2 mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.

Acquired Mutation and Reversion of the Neutrophil Elastase Gene, ELA2, in Kostmann Syndrome and Cyclic Neutropenia Suggest that Some Mutations Cause Neutropenia while Others Paradoxically Suppress It.

Heterozygous mutations in the gene, ELA2, encoding neutrophil elastase, cause cyclic neutropenia and are the commonest cause of severe congenital neutropenia, a genetically heterogeneous leukemia-predisposing condition also known as Kostmann syndrome. The cataloged mutations have divergent effects on the enzyme, defying singular explanations for how mutations cause disease. Until now, all mutations have appeared to be constitutional and heritable. We describe two exceptional patients with Kostmann syndrome and cyclic neutropenia who each respectively demonstrate somatic mosaicism in the bone marrow of multiple different ELA2 sequences representing acquired mutations of constitutionally wild type alleles or, conversely, acquired reversion of constitutionally mutant alleles. The first patient has Kostmann syndrome, lacks a family history of neutropenia, and comes from a consanguineous population where recessive inheritance is anticipated. Sequencing of PCR-amplified genomic DNA from peripheral blood revealed two novel, apparently heterozygous ELA2 mutations, V69L and V72L, absent in both parents. The mother did possess an adjacent intronic polymorphism inherited by the patient, and we employed this polymorphism to determine the parent of origin of the mutation by sequencing genomic PCR fragments subcloned in bacteria. Remarkably, either or both mutation, as well as a third (V72M), appeared on either maternal or paternal alleles. The mutations have thus arisen independently in multiple different cells of the bone marrow. We propose that Kostmann syndrome here results from inheritance of an unidentified second gene and that the resulting neutropenia paradoxically leads to the selection in the bone marrow of counteracting, “neutrophilic” ELA2 mutations. The second patient has cyclic neutropenia lessening in severity since birth. Sequencing PCR-amplified genomic DNA from peripheral blood revealed two different, apparently heterozygous ELA2 mutations, IVS4+1 G>A (ΔV161-F170), the commonest ELA2 mutation observed in cyclic neutropenia, and T99I, not previously reported. Both mutations were absent in the unaffected parents but present in the patient's buccal cells, demonstrating their de novo constitutional origins. Although the parents lacked distinguishing polymorphisms, the presence of two mutations allowed us to assess for somatic mosaicism. We subcloned genomic PCR fragments in bacteria and found that, in addition to wild type and both mutations in cis, some alleles contained just one or the other mutation, indicating that the mutations have undergone stepwise reversion in multiple, independent cells in the bone marrow. In colony formation assays of circulating stem cells, reversion to wild type occurred exclusively in CFU-GM, but not more primitive CFU-GEMM, suggesting selection in myeloid progenitors. Selection and counter-selection in these two complementary patients imply that there are two opposing categories of ELA2 mutations: some cause neutropenia and others suppress it. This may be the first description of an unexpected genetic phenomenon, where different mutations in a single gene can either cause or ameliorate the same disease, and may explain the divergent effect of various mutations.

Mutations in the Gene of Neutrophil Elastase Associated with Congenital Neutropenia Lead to Alteration of Intracellular Trafficking and Proteolytic Function of NE Protein.

Severe congenital (SCN) and cyclic neutropenia (CyN) are characterized by early-stage maturation arrest of myelopoiesis and often are found associated with different heterozygous mutations in the gene encoding neutrophil elastase (ELA2). Neutrophil elastase (NE) is a serin protease stored mainly in azurophilic granules. However, the consequences of the mutations of this protein up to date are not clear. Recent studies suggest a correlation with incorrect intracellular transport leading to mis-localization of the mutant protein. In the present study, we have generated an ELA2-inducible expression system based on the monoblast-like cell line U937. This system allows controlled expression of the transgene to study functional correlations with specific mutations of ELA2. Applying this system, we have studied intracellular processing and proteolytic activities of ten different mutations of ELA2 that are typically detected in patients suffering from SCN or CyN. We detected two clusters within the neutrophil elastase protein, in which mutations caused alteration of protein glycosylation, associated with prevention of membrane localization of the mutant protein. Instead, the mutant protein was incorrectly directed to the nuclear region. Proteolytic activities of NE protein were measured using different substrates. NE activities were suppressed in most but not all mutations tested. To extend our localization studies, we analyzed the intracellular distribution of NE in granulocytes of patients suffering from CyN or SCN. Again, NE protein of patients was mis-localized to the nucleus correlated with loss of detection in granules and membranes as compared with healthy donors without or with rh-G-CSF treatment. In summary, our data suggest that intracellular processing and localization is altered due to specific mutations of ELA2, which may be crucial for the molecular mechanisms resulting in the neutropenic phenotype.

Mutations in the ELA2 gene correlate with more severe expression of neutropenia: a study of 81 patients from the French Neutropenia Register

AbstractHeterozygous mutations of the gene encoding neutrophil elastase (ELA2) have been associated with cyclic neutropenia (CN) and severe congenital neutropenia (SCN). To date, 30 different mutations have been reported, but no correlation has been found with the degree of neutropenia. To address this issue, we analyzed the clinical, hematologic, and molecular characteristics of 81 unrelated patients with SCN (n = 54) or CN (n = 27). We identified mutations in 31 patients, two thirds of whom had sporadic forms. Familial cases were consistent with dominant inheritance. Seventeen novel mutations were identified, showing that the mutational spectrum encompasses not only the region encoding the mature enzyme but also the prodomains and promoter region. Genotype-phenotype analysis strongly suggested that ELA2 mutations correlate with more severe expression of neutropenia, specifically in patients diagnosed with SCN. This study underlines the importance of ELA2 molecular screening to identify patients who may be at particular risk of severe bacterial infections and/or acute myeloid leukemia/myelodysplasia. By phenotypic analysis of affected relatives and carriers of the same ELA2 mutations, we showed that the expression of neutropenia in CN and SCN may be either homogeneous or variable according to the type of mutations, suggesting different pathogenetic mechanisms.

A novel notch protein, N2N, targeted by neutrophil elastase and implicated in hereditary neutropenia.

Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes-terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.

Congenital and Acquired Neutropenia

Our understanding of the pathogenesis of congenital and acquired neutropenia is rapidly evolving. New ground-breaking observations have identified the genes responsible for many of the congenital neutropenia syndromes and are also providing new insights into normal neutrophil commitment and differentiation. Acquired neutropenia remains a poorly understood syndrome, although new insights into its pathogenesis are also emerging, especially with regard to subsets of immune neutropenia. In Section I, Dr. Marshall Horwitz reviews the current understanding of the genetic basis, molecular pathology, and approaches to treatment of congenital neutropenia and cyclic hematopoiesis. Mutations in the ELA2 gene, which encodes for neutrophil elastase, cause cyclic hematopoiesis. ELA2 mutations are also the most common cause of congenital neutropenia, where their presence may equate with a more severe clinical course and higher frequency of leukemic progression. Emerging evidence indicates interrelatedness with Hermansky Pudlak syndrome and other disorders of neutrophil and platelet granules. In Section II, Dr. Nancy Berliner presents an overview of the clinical approach to the evaluation and treatment of acquired neutropenia. This includes a review of the pathogenesis of primary and secondary immune neutropenia, drug-induced neutropenia, and non-immune chronic idiopathic neutropenia of adults. Studies used to evaluate patients for potential immune neutropenia are reviewed. Management issues, especially the use of granulocyte colony-stimulating factor (G-CSF), are discussed. In Section III, Dr. Thomas Loughran, Jr., reviews the pathogenesis and clinical manifestations of large granular lymphocyte (LGL) leukemia. Possible mechanisms of neutropenia are discussed. In particular, discussion focuses on the relationship between LGL leukemia, rheumatoid disease, and Felty's syndrome, and the complex interplay of defects in neutrophil production, distribution, destruction, and apoptosis that underly the development of neutropenia in those syndromes.

Lymphoid Enhancer Factor-1 Links Two Hereditary Leukemia Syndromes through Core-binding Factor α Regulation of ELA2

Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor alpha (CBFalpha). In mice, CBFalpha regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFalpha regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/beta-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFalpha binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFalpha co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFalpha. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFalpha activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFalpha to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.

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 elastase, found in lysosome-like granules, cause cyclic hematopoiesis 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 activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, 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) beta-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.

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Leukemia in Severe Congenital Neutropenia: Defective Proteolysis Suggests New Pathways to Malignancy and Opportunities for Therapy

Severe congenital neutropenia (SCN), a heterogeneous disorder that includes Kostmann syndrome, predisposes to myelodysplasia and acute myelogenous leukemia. Recently identified heterozygous mutations in the gene ELA2, encoding neutrophil elastase on human chromosome 19pter, account for the majority of autosomal dominant cases of SCN, including those demonstrating neoplastic progression. The involvement of the serine protease neutrophil elastase, localized to the granules of neutrophils and monocytes, implies an unexpected role for proteolytic regulation of hematopoiesis. Continued elucidation of the clinical features, molecular genetics, and biochemistry is likely to provide insight into novel pathways of leukemia induction with attendant prospects for new avenues of therapy.

Role of neutrophil elastase in bone marrow failure syndromes: molecular genetic revival of the chalone hypothesis.

Two forms of inherited deficiency of neutrophil numbers are cyclic hematopoiesis and severe congenital neutropenia. In cyclic hematopoiesis, neutrophil counts oscillate opposite monocytes in a 3-week cycle. Severe congenital neutropenia consists of static neutropenia and a predisposition to myelodysplasia and acute myelogenous leukemia. All cases of cyclic neutropenia and most cases of severe congenital neutropenia result from heterozygous germline mutations in the gene encoding neutrophil elastase, ela2. Recent work extends the list of neutropenia genes to include WASp, Gfi-1, adaptin, and tafazzin. Studies of mosaic patients suggest that ela2 mutations act in a cell-autonomous fashion. A hypothetical feedback circuit potentially interconnects these genes. Genetic dissection of signaling in model organisms along with experimental hematology implicate C/EPBepsilon, RUNX1/AML1, Notch family members, LEF1, and Cdc42 as additional nodes in this pathway. The authors propose that neutrophil elastase acts as an inhibitor of myelopoiesis, substantiating a chalone hypothesis proposed many years ago.

Possibility of somatic mosaicism of ELA2 mutation overlooked in an asymptomatic father transmitting severe congenital neutropenia to two offspring

The article by Germeshausen et al (2001) describes a pedigree in which severe congenital neutropenia (CN) has been transmitted by a father to two children. Sequence analysis of the neutrophil elastase gene (ELA2) identified heterozygous changes at amino acid 122 in both the affected children as well as the father. The authors interpret the lack of neutropenia in the father to be evidence that the mutation in ELA2 is not sufficient to cause CN. CN is known to be primarily a sporadic disease and as such the authors have overlooked the most likely interpretation of their results which is that the father is mosaic for the C122Y mutation. Somatic and germline mosaicism is a common occurrence in severe genetic diseases in which reduced genetic fitness mandates that de novo mutations account for a substantial proportion of new cases. These include Duchenne's muscular dystrophy, achondroplasia, tuberous sclerosis, Hunter's disease, osteogenesis imperfecta, retinoblastoma, neurofibromatosis type 1 and haemophilia A (Leuer et al, 2001 and references within). Most recently, Ancliff et al (2001a) have demonstrated somatic mosaicism of a C42R ELA2 mutation in the father of a girl presenting with CN. Further analysis determined that, while about half the father's lymphocytes and other cells contained the C42R ELA2 mutation found in his daughter, < 5% of his neutrophils contained a mutant allele, unequivocally demonstrating the pathogenic nature of ELA2 mutation on neutrophil production and CN genesis. The authors go on to suggest that their one case proves that mutations in the ELA2 gene are not the single cause of CN; however, it has never been stated that there is a single cause of CN. An elegant study by Ancliff et al (2001b) has shown that mutations in ELA2 account for approximately 75% of sporadic cases of CN, a figure similar to that obtained by Dale et al (2000). ELA2 mutations are less common in familial cases of the disease and linkage analysis and the exclusion of the 19p13.3 region in one pedigree with autosomal dominant CN suggests that at least one other gene is involved (Ancliff et al, 2001b). The possibility that this other gene or genes may function in a common pathway or actually directly regulate neutrophil elastase expression is supported by the authors themselves who have pointed out that CN patients with undetectable mutations in the ELA2 gene nonetheless demonstrate reduced elastase activity (Germeshausen et al, 2000). In closing, the demonstration of somatic mosaicism in the case of CN presented by Germeshausen et al (2001), which we believe to be a likely scenario that has not been addressed, would lead to the opposite conclusion as that proffered by the authors: mutations in ELA2 are causative of CN and sufficient.

Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease

AbstractSevere congenital neutropenia (SCN) was originally described as an autosomal recessive disorder. Subsequently, autosomal dominant and sporadic forms of the disease have been recognized. All forms are manifest by persistent severe neutropenia and recurrent bacterial infection. In contrast, cyclical hematopoiesis is characterized by periodic neutropenia inter-spaced with (near) normal neutrophil counts. Recently, linkage analysis on 13 affected pedigrees identified chromosome 19p13.3 as the likely position for mutations in cyclical hematopoiesis. Heterozygous mutations in the ELA2 gene encoding neutrophil elastase were detected in all families studied. Further work also demonstrated mutations in ELA2 in sporadic and autosomal dominant SCN. However, all mutations described to date are heterozygous and thus appear to act in a dominant fashion, which is inconsistent with an autosomal recessive disease. Therefore, the current study investigated whether mutations in ELA2could account for the disease phenotype in classical autosomal recessive SCN and in the sporadic and autosomal dominant types. All 5 exons of ELA2 and their flanking introns were studied in 18 patients (3 autosomal recessive, 5 autosomal dominant [from 3 kindreds], and 10 sporadic) using direct automated sequencing. No mutations were found in the autosomal recessive families. A point mutation was identified in 1 of 3 autosomal dominant families, and a base substitution was identified in 8 of 10 patients with the sporadic form, though 1 was subsequently shown to be a low-frequency polymorphism. These results suggest that mutations in ELA2are not responsible for classical autosomal recessive Kostmann syndrome but provide further evidence for the role of ELA2 in SCN.

Mutations in the gene encoding neutrophil elastase (ELA2) are not sufficient to cause the phenotype of congenital neutropenia

Mutations in the ELA2 gene encoding human neutrophil elastase have been reported recently to be involved in the aetiology of both, cyclic (CyN) and congenital neutropenia (CN). We analysed the correlation between the occurrence of ELA2 mutations and the neutropenic phenotype in a family with two children affected with CN. The two children harboured the same heterozygous mutation in the ELA2 gene that was inherited from their unaffected father. We conclude that ELA2 mutations are not the single cause of CN although they might be a necessary prerequisite for the expression of the neutropenic phenotype in a subgroup of CN patients.

Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Severe congenital neutropenia is a heritable human disorder characterized by neutropenia and acute myelogenous leukemia. We recently determined that the majority of cases result from de novo or autosomal dominantly inherited heterozygous mutations in ELA2, encoding neutrophil elastase. Neutrophil elastase is a chymotryptic serine protease localized in granules of neutrophils and monocytes and is the major target of inhibition of the serpin alpha(1)-antitrypsin. The mutations causing severe congenital neutropenia consist of amino acid missense substitutions, in-frame deletion, splice donor mutation producing a deletion, splice acceptor mutation causing insertion of novel residues, and protein truncating mutations of the carboxyl terminus resulting from nonsense substitutions and deletions leading to frameshifts. We have expressed 14 mutant forms of neutrophil elastase in vitro and have characterized their biochemical properties. The mutations have variable effects on proteolytic activity, eliminating the possibility that the disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by alpha(1)-antitrypsin, but demonstrate variable avidity for interaction with this serpin. Somewhat surprisingly, the mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal subcellular trafficking or post-translational processing.

Neutrophil elastase mutations in cyclic hematopoiesis, congenital neutropenia, and leukemia alter proteolytic specificity

We have previously identified mutations in the ELA2 gene, encoding neutrophil elastase, a serine protease of neutrophil granules, as the hereditary cause of cyclic hematopoiesis (CH) and severe congenital neutropenia (SCN). CH is an autosomal dominant human disease in which neutrophils and monocytes sinusoidally oscillate in opposite phase with 21 day frequency. SCN is a genetically heterogeneous human disorder typified by a promyelocytic arrest in the bone marrow, with severe peripheral neutropenia. Both diseases lead to opportunistic infection and an increased risk for acute myelogenous leukemia. Genetic linkage analysis was used to map the locus for CH to chromosome 19pter and heterozygous mutations were identified in ELA2 by positional cloning. As a candidate gene, mutations in ELA2 were also identified in most cases of SCN. From a total of 24 unique mutations in ELA2 detected among 41 different patients (7 mutations causing CH, 15 associated with SCN, 2 found in both patient populations, and 5 mutations occurring in individuals developing leukemia), we have now investigated the biochemical properties and intracellular processing of the mutant elastase protein through in vitro expression in cultured hematopoietic cells. We find that the mutations appear to be normally processed and routed to granules, collectively diminish activity toward the native substrate, and demonstrate a proclivity for alternate peptide substrates. We conclude that change of specificity mutations in a granule protease may be the basis for the spectrum of hereditary neutropenic and leukemic disorders in the “ela-opathies”.

Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis

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 periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. 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 granules) is the target for protease inhibition by alpha1-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.