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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Haematology, University College
London, and the Department of Haematology, Great Ormond Street
Hospital, London, United Kingdom.
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 ELA2
could 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 ELA2
are not responsible for classical autosomal recessive Kostmann syndrome
but provide further evidence for the role of ELA2 in SCN.
(Blood. 2001;98:2645-2650) Kostmann1 first described severe
congenital neutropenia (SCN) as an autosomal recessive disorder in a
large, intermarried Swedish family in 1956. Subsequently, autosomal
dominant and sporadic forms of the disease have been recognized.
Typically, children are noted in early infancy to have persistent
severe neutropenia of less than 0.2 × 109/L, recurrent
bacterial infections, and maturation arrest at the promyelocyte-myelocyte stage in the bone marrow. Mutations in the
cytoplasmic region of the granulocyte colony-stimulating factor (G-CSF)
receptor have been reported in a proportion of the 10% of children
with SCN in whom acute myeloid leukemia (AML) develops.2 However, such mutations in the cytoplasmic region are an acquired event
and are not the cause of the underlying disease.
Cyclical hematopoiesis is characterized by periodic neutropenia
inter-spaced with normal or near normal neutrophil counts with a
remarkably regular 21-day periodicity.3 In contrast to
SCN, autosomal dominant patterns of inheritance predominate. Recently,
linkage analysis on 13 affected pedigrees identified chromosome 19p13.3
as the likely position for mutations in cyclical hematopoiesis.4 This area contains a family of genes
encoding azurocidin, neutrophil elastase, and proteinase 3. Heterozygous mutations in the ELA2 gene-encoding neutrophil
elastase were detected in all families. Additional work by this group
also demonstrated mutations in ELA2 in patients with
sporadic and autosomal dominant SCN.5 Structural modeling
has suggested that in cyclical hematopoiesis the mutations appear to
cluster around the active site of the enzyme, whereas in
congenital neutropenia the opposite face of the enzyme seems to be
predominantly affected. Neutrophil elastase is synthesized in
promyelocytes and promonocytes and is packaged into the azurophilic
cytoplasmic granules.6 The mature 218-amino acid
glycoprotein is released at sites of inflammation and appears to have a
critical pathophysiological role in a variety of pulmonary diseases.7 To date, neutrophil elastase has no known
function in myelopoiesis.
In the current study, autosomal recessive and autosomal dominant
familial cases of SCN, in addition to sporadic cases, were screened to
investigate whether mutations in the same gene could account for the
pathogenesis of classic, apparently autosomal recessive Kostmann
disease and also to confirm the published results in sporadic SCN.
Patients and clinical details
Eighteen patients were screened. Eight were familial cases from 6 different families, 3 with an apparent autosomal recessive pattern of
inheritance and 3 with an apparent autosomal dominant pattern. Ten were
unrelated sporadic cases. In the first apparently recessive family
(patient 1; Table 1), the parents are second cousins of Sri Lankan
origin. The parents of patient 2 are first cousins of Pakistani origin;
they had 2 other children with SCN who died of infective complications
in early childhood. The parents of patient 3 are also first cousins
from an inter-married Pakistani family, and both are first cousins to a
10-year-old child with SCN. In the apparently autosomal dominant
families, at least 2 members of 2 or more consecutive generations were
affected, but there was no evidence of consanguinity.
Patients 2, 8, and 9 developed acute myeloid leukemia. Patient 2 had a
relapse and died 7 months after undergoing fully engrafted haplo-identical bone marrow transplantation. Patient 8 did not achieve
durable myeloid engraftment from unrelated donor bone marrow
transplantation and died 9 months later of the complications of gut
graft-versus-host disease and recurrent SCN. Patient 9 is alive and
well 4 months after unrelated donor bone marrow transplantation.
Mutational analysis
In the patients in whom no mutation was identified, the 5' promoter region from one member of each kindred was screened using the technique above; primers are detailed in Table 2. The MgCl2 concentration was optimized to 0.6 mM, and the annealing temperature was optimized to 64°C for the initial PCR. RNA was extracted from bone marrow mononuclear cells using Trizol (Gibco BRL, Paisley, United Kingdom). Complementary DNA (cDNA) for the complete ELA2 coding region was produced by reverse transcription and used as a template for PCR with the specified primers (Table 2). Bidirectional sequencing was performed as above. Patients 1, 2, 7, 8, 10, 12, and 17 were only screened using RNA. Confirmatory tests The presence of each mutation was confirmed using restriction endonuclease digestion of the relevant PCR fragment (Table 3). The mutation in patient 14 required the use of a mismatch reverse primer 5'-TGCCCAGAAGGCCCCAGCCCATGGCCAGAC-3') (the mismatch is underlined). For each mutation found, at least 50 healthy controls (and parents, if available) were screened using PCR and restriction endonuclease digestion. Positive screening results were confirmed by bidirectional sequencing using the method detailed above.
Linkage analysis Linkage analysis was performed using 3 polymorphic markers on chromosome 19p13.3: KB9, D19S886, and D19S814. KB9 had the highest peak 2-point lod score of 13.11 in the original study on cyclical hematopoiesis.4 D19S886 and D19S814 are centromeric and telomeric to ELA2, respectively.One primer of each pair (Table 2) was
Mutational analysis No mutations were found in the patients with presumed autosomal recessive disease. In the 5 patients from 3 autosomal dominant families, only one mutation was found. This was a splice donor variant (G4716A) resulting in the deletion of Val161 to Phe170. The G4716A mutation and others leading to this deletion have been described in 7 patients with familial cyclical hematopoiesis, 3 patients with sporadic cyclical hematopoiesis, and 2 patients with SCN.4,5Seven different heterozygous base substitutions were identified in 8 of
10 patients with sporadic SCN (Table 3). They were found in all exons
except exon 1. Six were point substitutions that would lead to the
following amino acid changes: Cys26Tyr, Gly56Glu, Ser97Leu (2 patients), Cys122Ser, Pro176Arg, and Pro228Leu. The remaining mutation
was at a splice acceptor site and would lead to the insertion of 2 amino acids
For the 2 splice variants, cDNA was produced from bone marrow RNA, and the appropriate fragment was amplified by PCR. Wild-type and mutant cDNAs were present in approximately equal quantities; an example is shown in Figure 1B. The 2 bands were separated on low-melting-point agarose. Sequencing confirmed the predicted changes in both. Four of the 7 mutations (seen in patients 9, 10, 14, and 15) and the polymorphism (seen in patient 16) have not been previously described. In the patients in whom no mutation was found in the coding region, the
5' promoter region (starting at Linkage analysis Linkage analysis was performed in the 2 autosomal dominant families without mutations in ELA2 using the 3 polymorphic markers described above. In family A, samples were available from affected relatives spanning 3 generations (Figure 2A). Using the KB9 marker, the results showed that the 2 affected daughters (IIi and IIii) inherited different alleles from their affected mother (Ii), thus excluding linkage to chromosome 19p13.3 in this family. Corroborating evidence was also provided by identical results with the other 2 markers and by data from 3 unaffected half-siblings (data not shown), making recombination most unlikely. Analysis of X chromosome inactivation patterns using the HUMARA gene9 did not show any evidence of skewing in this family, thus excluding an X-linked disorder with extreme lyonization (data not shown).
In family B, the mother (IIi) and all 4 of her male children are affected, her parents are not. Results using KB9, D19S886 (Figure 2B), and D19S814 (data not shown) showed that the mother had passed her paternal chromosome 19 to all her children. Her 61-year-old father had a normal neutrophil count whenever tested and did not experience an excess of infective illnesses. Spontaneous mutation in the mother and linkage to chromosome 19p13.3 cannot, therefore, be excluded in this family. X-linked disease with extreme lyonization in the mother has been excluded by the demonstration of balanced expression of her polymorphic p55 transcripts10 (data not shown). Too few family members were available to perform linkage analysis in the autosomal recessive families.
In the current study, a mutation in the ELA2 gene was detected in 1 of 3 apparently autosomal dominant kindreds with familial SCN. No mutations were identified in the apparently autosomal recessive families. These results are compatible with those published previously showing that mutations were found in 5 of 5 autosomal dominant families,5 but they suggest that not all autosomal dominant SCN is caused by mutations in ELA2. The original disorder described by Kostmann,1 infantile genetic agranulocytosis, was an autosomal recessive disease. The current study is the first report in which mutations in ELA2 have been sought in patients with autosomal recessive SCN; none were found. This is perhaps not surprising in that a single heterozygous mutation that appears to act in a dominant fashion does not fit with an autosomal recessive pattern of inheritance in which, by definition, both copies of a gene are defective. Nevertheless, it is possible that total loss of a putative neutrophil elastase inhibitor (likely to be a recessive event) might lead to a similar disease. Although we only studied 3 families with the autosomal dominant pattern of inheritance, using the KB9 marker linkage to chromosome 19p13.3 could be excluded in 1 of these families, effectively ruling out the involvement of mutations in ELA2 and any immediate promoter-enhancer regions in the pathogenesis of the neutropenia in this kindred. Linkage analysis was unhelpful in the remaining autosomal dominant family. Furthermore, sequencing of the 5' promoter region in all 8 kindreds without a coding region mutation (ie, 3 families with autosomal recessive, 2 families with autosomal dominant, and 3 patients with sporadic disease) only demonstrated a previously identified single nucleotide polymorphism. The results presented in this study confirm the high frequency of heterozygous mutations in the neutrophil elastase gene in sporadic SCN that has been previously reported.5 Four novel mutations and a low-frequency polymorphism were detected. The control group used in this study was predominantly of white United Kingdom origin, whereas the patient with the polymorphism was of mixed race, and the incidence of the polymorphism in the population of origin could be higher than the 1 in 110 found in our study. These data further strengthen the argument for the role of neutrophil elastase mutations in the pathogenesis of SCN. It should be noted that the patients with apparently sporadic disease without ELA2 mutations (patients 16-18) could actually have autosomal recessive disease and that nearly all cases of sporadic SCN may result from de novo heterozygous mutations in ELA2. Figure 3 shows schematically the location
of published mutations and those from the current study in SCN and
cyclical neutropenia. To date, we have only studied 2 cases of
classical cyclical neutropenia; in one there was no mutation and in the
other there was the previously described Ala32Val (coded for by C1900T
in exon 2; data not shown). It can be seen that in SCN the mutations
are scattered throughout the molecule. There is also clear overlap
between the 2 diseases, particularly in the cluster of mutations at the
intron 4 splice donor site, all of which led to the loss of the last 10 residues of exon 4. The latter mutations have now been described in 7 families with cyclical neutropenia, 3 patients with sporadic cyclical
neutropenia, and 3 patients with sporadic congenital
neutropenia.4,5
It remains difficult to explain the different phenotypes and clinical severity of cyclical and severe congenital neutropenia, in particular the different risks for malignant transformation. To date, secondary leukemia has not developed in any patient with cyclical neutropenia (though one patient with a clonal cytogenetic abnormality has recently been described in the literature11), whereas in SCN there is a 1 in 10 risk of developing myelodysplasia or acute myeloid leukemia.2 Furthermore, the presence of a mutant neutrophil elastase does not preclude the development of leukemia (as evidenced by patient 9 in the current study) nor is it an essential prerequisite (patients 2 and 8). Interestingly, there may be variation in phenotype within SCN patients with identical ELA2 mutations. Patient 12 (now aged 5) with the Ser97Leu substitution has continuing severe neutropenia and remains on G-CSF therapy with only a modest response. In contrast, patient 13 (now aged 13) had severe neutropenia (less than 0.1 × 109/L) and recurrent infections until he started G-CSF at the age of 4. He responded well and needed only a small maintenance dose of 2 µg/kg per 24 hours. G-CSF was discontinued when he was 8; he has remained free of major infections and now has a neutrophil count of approximately 0.5 × 109/L. This may reflect the influence of other inherited modifying factors, and it may suggest a more multigenic pathogenesis for SCN. The pathogenicity of mutations in ELA2 remains mysterious. Neutrophil elastase has no known role in myelopoiesis. Interestingly, heterozygous and homozygous ELA2 knockout mice have normal neutrophil counts, and only the latter have a demonstrable increase in susceptibility to infection.12,13 Experiments in which the mutant enzyme has been transfected into cell lines (rat basophilic cells [RBL-1] and murine myeloblasts [32D]) have shown reduced levels of elastase activity in some patients.14 However, other investigators have reported reduced elastase activity as a more general phenomenon in congenital neutropenia15 with or without elastase mutations, implying that this is a general feature of SCN rather than one that is related to ELA2 mutations and disease pathogenesis. Therefore, it appears likely that the pathogenesis is due to a gain of function in the mutant protein, perhaps allowing the enzyme to bind a novel substrate involved in myelopoiesis or changing its binding with an intracellular inhibitor. An alternative explanation may relate to changes in intracellular packaging. During normal myeloid differentiation, the granule matrix immobilizes the digestive enzymes in the azurophilic granules.6 Should the ELA2 mutations lead to significant shape change of the mature protein, this immobilization may no longer be possible and the developing myeloid cells may be killed by autodigestion. Finally, recent evidence shows increased susceptibility to apoptosis of myeloid precursors in SCN,16 but the link between this and mutant neutrophil elastase has not been established.
We thank the families for kindly providing material, we thank the following for their assistance in sample and data collection: Brady Baxter, Great Ormond Street Hospital, London; Peter Arkwright, Department of Child Health, Manchester; Paula Bolton-Maggs, Alder Hey Hospital, Liverpool; Carole Edwards and Sally Kinsey, SCN International Registry, Leeds; Brenda Gibson, Royal Hospital for Sick Children, Glasgow; Diab Haddad and Tanya Bernard, St Peter's Hospital, Chertsey; Judith Marsh, St George's Hospital, London; Janice Simpson and James Nicholson, Addenbrooke's Hospital, Cambridge; and Graham Smith, Royal United Hospital, Bath, United Kingdom.
Submitted February 9, 2001; accepted June 27, 2001.
Supported by the Roald Dahl Foundation, Great Missenden (Bucks, United Kingdom) (P.A.) and by an unrestricted educational grant from Amgen (Cambridge, United Kingdom) (P.A.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Phil Ancliff, Department of Haematology, University College London, 98 Chenies Mews, London, WC1E 6HX, United Kingdom; e-mail: p.ancliff{at}ucl.ac.uk.
1. Kostmann R. Infantile genetic agranulocytosis. Acta Paediatr. 1956;45(suppl 105):1-78[Medline] [Order article via Infotrieve]. 2. Zeidler C, Germeshausen M, Aprikyan A, Touw I, Dale D, Welte K. Mutations of the G-CSF receptor gene in AML secondary to severe congenital neutropenia [abstract]. Blood. 2000;96:501. 3. Palmer SE, Stephens K, Dale DC. Genetics, phenotype and natural history of autosomal dominant cyclical hematopoiesis. Am J Med Genet. 1996;66:413-422[CrossRef][Medline] [Order article via Infotrieve]. 4. Horwitz M, Benson K, Person R, Aprikyan A, Dale D. Mutations in ELA2, encoding for neutrophil elastase, defines a 21-day biological clock in cyclic haematopoiesis. Nat Genet. 1999;23:433-436[CrossRef][Medline] [Order article via Infotrieve].
5.
Dale DC, Person RE, Bolyard AA, et al.
Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.
Blood.
2000;96:2317-2322 6. Gullberg U, Bengtsson N, Bülow E, Garwicz D, Lindmark A, Olsson I. Processing and targeting of granule proteins in human neutrophils. J Immunol Methods. 1999;232:201-210[CrossRef][Medline] [Order article via Infotrieve].
7.
Stockley RA.
New approaches to the management of COPD.
Chest.
2000;117(suppl 2):58S-62S
8.
Srikanth S, Rado TA.
A 30-base pair element is responsible for the myeloid-specific activity of the human neutrophil elastase promoter.
J Biol Chem.
1994;269:32626-32633 9. Gale RE, Mein CA, Linch DC. Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay. Leukemia. 1996;10:362-367[Medline] [Order article via Infotrieve].
10.
Harrison CN, Gale RE, Machin SJ, Linch DC.
A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications.
Blood.
1999;93:417-424
11.
Freedman MH, Bonilla MA, Fier C, et al.
Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy.
Blood.
2000;96:429-436 12. Belaaouaj A, McCarthy R, Baumann M, et al. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med. 1998;4:615-618[CrossRef][Medline] [Order article via Infotrieve]. 13. Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity. 2000;12:201-210[CrossRef][Medline] [Order article via Infotrieve].
14.
Li F-Q, Horwitz M.
Characterization of mutant neutrophil elastase in severe congenital neutropenia.
J Biol Chem.
2001;276:14230-14241 15. Germeshausen M, Schulze H, Ballmaier M, et al. Decreased neutrophil elastase activity in patients with severe congenital neutropenia is not correlated to mutations in ELA2 [abstract]. Blood. 2000;96:609.
16.
Aprikyan AA, Liles WC, Rodger E, Jonas M, Chi EY, Dale DC.
Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia.
Blood.
2001;97:147-153
© 2001 by The American Society of Hematology.
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  N. Berliner Lessons from congenital neutropenia: 50 years of progress in understanding myelopoiesis Blood, June 15, 2008; 111(12): 5427 - 5432. [Abstract] [Full Text] [PDF]
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D. S. Grenda, M. Murakami, J. Ghatak, J. Xia, L. A. Boxer, D. Dale, M. C. Dinauer, and D. C. Link Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis Blood, December 15, 2007; 110(13): 4179 - 4187. [Abstract] [Full Text] [PDF]
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 K. Matsubara, K. Imai, S. Okada, M. Miki, N. Ishikawa, M. Tsumura, T. Kato, O. Ohara, S. Nonoyama, and M. Kobayashi Severe developmental delay and epilepsy in a Japanese patient with severe congenital neutropenia due to HAX1 deficiency Haematologica, December 1, 2007; 92(12): e123 - e125. [Abstract] [Full Text] [PDF]
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M. S. Horwitz, Z. Duan, B. Korkmaz, H.-H. Lee, M. E. Mealiffe, and S. J. Salipante Neutrophil elastase in cyclic and severe congenital neutropenia Blood, March 1, 2007; 109(5): 1817 - 1824. [Abstract] [Full Text] [PDF]
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P. J. Ancliff, M. P. Blundell, G. O. Cory, Y. Calle, A. Worth, H. Kempski, S. Burns, G. E. Jones, J. Sinclair, C. Kinnon, et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia Blood, October 1, 2006; 108(7): 2182 - 2189. [Abstract] [Full Text] [PDF]
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 P. Yang, W. R. Bamlet, Z. Sun, J. O. Ebbert, M.-C. Aubry, W. R. Taylor, R. S. Marks, C. Deschamps, S. J. Swensen, E. D. Wieben, et al. {alpha}1-Antitrypsin and Neutrophil Elastase Imbalance and Lung Cancer Risk Chest, July 1, 2005; 128(1): 445 - 452. [Abstract] [Full Text] [PDF]
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P. Massullo, L. J. Druhan, B. A. Bunnell, M. G. Hunter, J. M. Robinson, C. B. Marsh, and B. R. Avalos Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes Blood, May 1, 2005; 105(9): 3397 - 3404. [Abstract] [Full Text] [PDF]
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D. C. Dale Neutrophil elastase and neutropenia Blood, June 1, 2004; 103(11): 3993 - 3994. [Full Text] [PDF]
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C. Bellanne-Chantelot, S. Clauin, T. Leblanc, B. Cassinat, F. Rodrigues-Lima, S. Beaufils, C. Vaury, M. Barkaoui, O. Fenneteau, M. Maier-Redelsperger, et al. Mutations in the ELA2 gene correlate with more severe expression of neutropenia: a study of 81 patients from the French Neutropenia Register Blood, June 1, 2004; 103(11): 4119 - 4125. [Abstract] [Full Text] [PDF]
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G. Carlsson, A. A. G. Aprikyan, R. Tehranchi, D. C. Dale, A. Porwit, E. Hellstrom-Lindberg, J. Palmblad, J.-I. Henter, and B. Fadeel Kostmann syndrome: severe congenital neutropenia associated with defective expression of Bcl-2, constitutive mitochondrial release of cytochrome c, and excessive apoptosis of myeloid progenitor cells Blood, May 1, 2004; 103(9): 3355 - 3361. [Abstract] [Full Text] [PDF]
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 Z. Duan, F.-Q. Li, J. Wechsler, K. Meade-White, K. Williams, K. F. Benson, and M. Horwitz A Novel Notch Protein, N2N, Targeted by Neutrophil Elastase and Implicated in Hereditary Neutropenia Mol. Cell. Biol., January 1, 2004; 24(1): 58 - 70. [Abstract] [Full Text] [PDF]
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|
 H. Kawaguchi, M. Kobayashi, K. Nakamura, N. Konishi, S.-i. Miyagawa, T. Sato, H. Toyoda, Y. Komada, S. Kojima, Y. Todoroki, et al. Dysregulation of transcriptions in primary granule constituents during myeloid proliferation and differentiation in patients with severe congenital neutropenia J. Leukoc. Biol., February 1, 2003; 73(2): 225 - 234. [Abstract] [Full Text] [PDF]
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D. S. Grenda, S. E. Johnson, J. R. Mayer, M. L. McLemore, K. F. Benson, M. Horwitz, and D. C. Link Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis Blood, October 16, 2002; 100(9): 3221 - 3228. [Abstract] [Full Text] [PDF]
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P. J. Ancliff, R. E. Gale, M. J. Watts, R. Liesner, I. M. Hann, S. Strobel, and D. C. Linch Paternal mosaicism proves the pathogenic nature of mutations in neutrophil elastase in severe congenital neutropenia Blood, June 28, 2002; 100(2): 707 - 709. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-[32P]-ATP
end-labeled, and PCR was performed using the method above with the
following modifications: 0.2 µM concentrations of each primer for
D19S814, annealing temperatures as indicated in Table 
insProGln94. All were confirmed by restriction digestion;
a typical example is shown in Figure 
240 and including all of exon 1) was
sequenced bidirectionally in at least one member of each kindred
(including the 3 patients with sporadic disease). This fragment
includes the critical region between 
