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

Abstract Severe 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.

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Mutations in the Gene for Neutrophil Elastase in Severe Congenital Neutropenia Are Heterozygous and of Male Origin.

Blood ◽

10.1182/blood.v104.11.1455.1455 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1455-1455

Author(s):

Steven Stein ◽

Marcia Paddock ◽

Audrey Anna Bolyard ◽

Chris Raymond ◽

Maynard Olson ◽

...

Keyword(s):

Neutrophil Elastase ◽

Autosomal Recessive ◽

Autosomal Dominant ◽

Paternal Allele ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Informative Marker ◽

Affected Child ◽

Sporadic Cases ◽

Parent Of Origin

Abstract Severe congenital neutropenia (SCN) occurs sporadically and as an autosomal-dominant or autosomal-recessive inherited disorder. Most sporadic and autosomal dominant cases are caused by mutations in the gene for neutrophil elastase (NE or ELA2). The gene for this potent serine protease is located on chromosome 19p13.3 and has 5 exons. In collaboration with the Severe Chronic Neutropenia International Registry (SCNIR), we have sequenced the NE gene from blood, bone marrow or other tissues samples for more than 300 patients with the clinical diagnosis of SCN using Big Dye Terminator, version 3.1 and ABI 3700. These studies have included 21 families with 2 or more affected members and 15 families with sporadically occurring SCN, i.e., two healthy parents with normal blood counts having an affected child. In 15 families with sporadic cases of SCN attributable to 13 different mutations in the NE gene, we sought to determine the parent of origin for the NE mutation. The 5 exons of the NE gene were sequenced twice in the parents and the affected child using Big Dye Terminator, version 3.1 and ABI 3700. After confirming the mutation in each patient and the absence of mutations in each parent, PCR was used to scan the adjacent region to the NE gene in the parents and the child to identify polymorphisms that could be used to distinguish between maternally and paternally inherited chromosomes. A fosmid library of 2-4X coverage for each patient’s DNA sample was made which successfully separated the patient’s genome into its constituent’s haplotypes. The fosmids containing the NE gene were identified by PCR and genotyped at the site of the mutant allele and the parent-informative marker. In all cases, the new NE gene mutation in the affected child was on the paternal allele.

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Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia

Blood ◽

10.1182/blood.v96.7.2317 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2317-2322 ◽

Cited By ~ 378

Author(s):

David C. Dale ◽

Richard E. Person ◽

Audrey Anna Bolyard ◽

Andrew G. Aprikyan ◽

Cindy Bos ◽

...

Keyword(s):

Neutrophil Elastase ◽

Rna Splicing ◽

Bacterial Infections ◽

Autosomal Dominant ◽

Mononuclear Cells ◽

Mutational Analysis ◽

Congenital Neutropenia ◽

Gene Encoding ◽

Cyclic Neutropenia ◽

Shwachman Diamond Syndrome

Abstract 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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Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis

Blood ◽

10.1182/blood-2002-05-1372 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3221-3228 ◽

Cited By ~ 49

Author(s):

David S. Grenda ◽

Sonja E. Johnson ◽

Jill R. Mayer ◽

Morgan L. McLemore ◽

Kathleen F. Benson ◽

...

Keyword(s):

Neutrophil Elastase ◽

Blood Analysis ◽

Severe Congenital Neutropenia ◽

Normal Numbers ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Gene Encoding ◽

Cyclic Neutropenia ◽

Related Disorder ◽

Increased Risk

Abstract 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 theirEla2 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. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.

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Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia

Blood ◽

10.1182/blood.v96.7.2317.h8002317_2317_2322 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2317-2322 ◽

Cited By ~ 4

Author(s):

David C. Dale ◽

Richard E. Person ◽

Audrey Anna Bolyard ◽

Andrew G. Aprikyan ◽

Cindy Bos ◽

...

Keyword(s):

Neutrophil Elastase ◽

Rna Splicing ◽

Bacterial Infections ◽

Autosomal Dominant ◽

Mononuclear Cells ◽

Mutational Analysis ◽

Congenital Neutropenia ◽

Gene Encoding ◽

Cyclic Neutropenia ◽

Shwachman Diamond Syndrome

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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Unique Evidence for Autosomal Dominant Inheritance of Severe Congenital Neutropenia.

Blood ◽

10.1182/blood.v104.11.1457.1457 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1457-1457

Author(s):

Laurence A. Boxer ◽

Steven Stein ◽

Danielle Buckley ◽

Sandhya Subramanian ◽

Audrey Anna Bolyard ◽

...

Keyword(s):

Neutrophil Elastase ◽

Autosomal Dominant ◽

Autosomal Dominant Inheritance ◽

Paternal Allele ◽

Severe Congenital Neutropenia ◽

Dominant Inheritance ◽

Congenital Neutropenia ◽

Sperm Donor ◽

The Mean ◽

Single Child

Abstract Severe congenital neutropenia was originally described as an autosomal recessive disorder characterized by severe neutropenia and recurrent bacterial infections from birth, but other reports indicate that autosomal dominant inheritance occurs. Several report now indicate that most, but not all, patients with severe congential neutropenia have heterozygous mutation in the gene for neutrophil elastase (NE or ELA2). We recently investigated an unusual series of cases of severe congenital neutropenia occurring when the same sperm donor was utilitized by four different families to impregnate mothers by either artificial insemination or in vitro fertilization. Three sets of twins were conceived and a single child in the four families. Collectively one set of twins and one child each from the other two families conceiving twins and the single child were all found to have severe congenital neutropenia. The mean for their median blood neutrophils (ANC) at diagnosis was 84 +/−32 x109/L (range 0–166 x 109 /L); and their other blood counts were remarkably similar. On treatment with G-CSF (mean dose 6.7+/− 1.3 SEM mcg/kg/day) the mean for the median ANC increased to 3290 +/−1410 x109/L( range 420–8600 x 109 /L ) To determine whether the sperm donor was responsible for transmitting severe congenital neutropenia, geonomic DNA from peripheral blood leukocytes was used to sequence the gene for neutrophil elastase in the affected children and their mothers using standard techniques. None of the mothers had a mutation in their NE gene but all five affected children had the same mutation effecting the fourth exon at site S97L. Since it was not possible to perform genetic analysis on biological samples from the donor, more detailed genetic linkage analysis was performed on the affected children and their mothers. Linkage mapping analysis of 22 microsatellite markers on chromosome 14 and 19 confirmed that all affected children possessed one of the two paternal alleles. For an 11 centiMorgan region immediately adjacent to the NE gene on chromosome 19, all affected children shared a single paternal allele, and 4 of 5 affected children shared a single paternal allele for a 20 centiMorgan region adjacent to the NE gene. The studies indicate that the father of the affected children provided consistent haplotypes that led to the expression of severe congenital neutropenia in all of the affected children. This investigation strongly implicates that autosomal dominant inheritance for severe congenital neutropenia. It also raises important questions for the evaluation of subjects in sperm donor programs.

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Dispensable Role of Neutrophil Elastase in Survival and Differentiation of Human Myeloid Progenitor Cells

Blood ◽

10.1182/blood.v112.11.3561.3561 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3561-3561

Author(s):

Andrew A Aprikyan ◽

Vahagn Makaryan ◽

Qian Si ◽

Kelly Treonze ◽

Nara Markosyan ◽

...

Keyword(s):

Cell Survival ◽

Progenitor Cells ◽

Neutrophil Elastase ◽

Autosomal Dominant ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Abstract Severe congenital neutropenia (SCN) is a rare autosomal dominant or recessive disorder with a characteristic “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow and extremely low level of neutrophils in peripheral circulation. SCN patients may evolve to develop myelodysplastic syndrome and acute myeloid leukemia (MDS/AML) with ~30% cumulative incidence of leukemia. Heterozygous mutations in the neutrophil elastase (NE, ELA2) gene have been identified in most of SCN patients with acquired and autosomal dominant inheritance (Dale et al., Blood 2000). It has been reported that accelerated apoptosis of bone marrow myeloid progenitor cells is the cellular mechanism of severe neutropenia in SCN. We and others also reported that expression of mutant elastase triggers impaired cell survival in human myeloid progenitor cells (Aprikyan et al., Exp Hem 2003; Massullo et al, Blood 2005; Kollner et al, Blood 2006; Grenda et al, Blood 2007). However, it remains unclear whether the abnormal cell survival and impaired myeloid differentiation in SCN is due to the gain-of-function or dominant negative effect of mutant NE. To answer these questions, we established tet-off HL-60 human myeloid progenitor cell lines with inducible expression of mutant or normal forms of NE and examined their survival and differentiation characteristics. Induced expression of del.145–152 mutant NE resulted in a significantly increased apoptosis of myeloid cells and a characteristic block of myeloid differentiation (p<0.05, n=4), thus closely recapitulating the human SCN phenotype. Importantly, induced overexpression of wild type NE in the myeloid progenitor cells did not alter the cell survival characteristics as determined by flow cytometry analyses of annexin V or DIOC6 stained cells (p>0.05, n=4). In addition, the overexpression of wild type NE had no effect on differentiation capacity of myeloid progenitor cells induced into granulocytic differentiation with retinoic acid compared with control cells expressing physiological levels of endogenous NE as determined by morphological evaluation of stained cells. Furthermore, nearly complete inhibition of NE proteolytic activity in control human myeloid progenitor cells expressing endogenous elastase with a cell-penetrant small molecule inhibitor of NE (Merck, NJ) also had no significant effect and impaired neither the cell survival nor their differentiation characteristics compared with control untreated cells (p>0.05, n=3). Thus, these data demonstrate that neutrophil elastase appears to be dispensable for the formation and differentiation of HL-60 human myeloid progenitor cells and accelerated apoptosis and impaired myeloid differentiation in SCN is attributable to a gain-of-function effect of pro-apoptotic mutant elastase. These data also suggest that small molecule inhibitors of NE may represent a promising therapy in severe congenital neutropenia.

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Novel HAX1 Gene Mutation in a Vietnamese Boy with Severe Congenital Neutropenia

Case Reports in Pediatrics ◽

10.1155/2018/2798621 ◽

2018 ◽

Vol 2018 ◽

pp. 1-4

Author(s):

Tham Thi Tran ◽

Quang Van Vu ◽

Taizo Wada ◽

Akihiro Yachie ◽

Huong Le Thi Minh ◽

...

Keyword(s):

Early Onset ◽

Bacterial Infections ◽

Complete Blood Count ◽

Hereditary Diseases ◽

Severe Neutropenia ◽

Sequencing Analysis ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Direct Dna Sequencing ◽

Life Threatening

Severe congenital neutropenia (SCN) is a rare disease that involves a heterogeneous group of hereditary diseases. Mutations in the HAX1 gene can cause an autosomal recessive form of SCN-characterized low blood neutrophil count from birth, increased susceptibility to recurrent and life-threatening infections, and preleukemia predisposition. A 7-year-old boy was admitted due to life-threatening infections, mental retardation, and severe neutropenia. He had early-onset bacterial infections, and his serial complete blood count showed persistent severe neutropenia. One older sister and one older brother of the patient died at the age of 6 months and 5 months, respectively, because of severe infection. Bone marrow analysis revealed a maturation arrest at the promyelocyte/myelocyte stage with few mature neutrophils. In direct DNA sequencing analysis, we found a novel homozygous frameshift mutation (c.423_424insG, p.Gly143fs) in the HAX1 gene, confirming the diagnosis of SCN. The patient was successfully treated with granulocyte colony-stimulating factor (G-CSF) and antibiotics. A child with early-onset recurrent infections and neutropenia should be considered to be affected with SCN. Genetic analysis is useful to confirm diagnosis. Timely diagnosis and suitable treatment with G-CSF and antibiotics are important to prevent further complication.

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