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Congenital neutropenia
Congenital neutropenia
Congenital neutropenia Philip James Ancliff Department of Haematology, Great Ormond Street Hospital, London WC1N 3JH, UK
Abstract Congenital neutropenia is strictly defined as neutropenia present at birth. However, it is more generally used to describe neutropenia secondary to inherited genetic mutations. This review will discuss the presentation of such children and the various causes of congenital neutropenia. In particular, it will focus on severe congenital neutropenia (SCN) and the recent discovery of mutations in the gene encoding neutrophil elastase in the majority of cases of SCN. The potential mechanisms of pathogenesis and of transformation to leukaemia will be discussed. Shwachman–Diamond Syndrome and other less common causes of congenital neutropenia will also be reviewed. Finally, an approach to the child with potential congenital neutropenia will be presented. c 2003 Elsevier Ltd. All rights reserved. KEY WORDS: elastase; congenital neutropenia; neutropenia
INTRODUCTION Definition eutropenia is defined as a decrease in the number of circulating neutrophils in the blood. Strictly speaking, congenital is defined as an abnormality present at birth and is usually, but not necessarily, of genetic cause. This review will focus on the genetic causes of neutropenia (in particular severe congenital neutropenia) that usually present in infancy and produce lifelong neutropenia and recurrent infections. Infantile immune neutropenias will also be briefly discussed. The lower limit of normal for a peripheral blood neutrophil count is both age and race dependent. In infants (children 612 months old), the threshold is 1:0 109 l. After infancy, the usual value is 1:5 109 l up to the age of 10 years and the adult threshold of 1:8 109 l is applied thereafter.1 Both total white cell count and neutrophil count are often depressed in people of African descent. Most laboratories regard an absolute neutrophil count above 1:0 109 l as normal in adult Afro-Caribbeans and this is indeed supported by observational data that such individuals with apparently low counts suffer no excess of bacterial infections.2 There is less hard data for normal black infants, but it is clear that the total neutrophil count is less than 1:0 109 l in maybe as many as 20% of such children.3 Neutropenia has been classified as mild (below the lower limit of normal but >1:0 109 l), moderate (between 0.5 and 1:0 109 l), severe (between 0.2 and 0:5 109 l) and very severe ð<0:2 109 lÞ. The lower limits are useful because the risk of severe infections does not increase until the neutrophil count falls below 0:5 109 l and rises rapidly below 0:2 109 l.4
N
Presentation and initial diagnostic difficulties Children typically present in the first few months of life with neutropenia and recurrent bacterial infections. Other nonhaematological features may lead to a specific diagnosis as detailed below. Presentation at birth or in the first months of life raises the possibility of neonatal allo-immune neutropenia (NAIN), often referred to as the myeloid homologue of Rhesus disease of the newborn.5 However, there is one important difference in that, analogous to neonatal allo-immune thrombocytopenia, NAIN can commonly occur in a first pregnancy (Geoff Lucas, National Blood Service, Bristol, personal communication). NAIN is rare (incidence <0.1%) and in contrast to SCN the infections are rarely severe despite the depth of the neutropenia. The diagnosis is confirmed by demonstration of the presence of neutrophil specific antibodies in the sera from the mother and baby against antigens inherited from the father. No specific therapy is usually required and spontaneous recovery occurs in an average of 11 weeks (range 3–28 weeks).6 However, the mother should be warned of the significant risk of recurrence and asked not to become a blood donor in view of the risk of inducing transfusion associated acute lung injury (TRALI) in any recipient with the same antigen as the father. She should also be warned that she could be at a possible risk of TRALI herself if transfused. Passive transfer of antibodies from a mother with auto-immune neutropenia can produce a similar clinical picture in the neonate.6 Primary auto-immune neutropenia (AIN) of childhood is rare, but probably at least 10-fold more common than SCN (incidence of 1 : 100 000 versus 1:1 000 000).7;8 AIN is a relatively benign disorder characterized by neutrophil specific auto-antibodies, often severe neutropenia and a paucity of severe bacterial infections.9 Bone marrow examination is not always necessary if auto-antibodies are identified and there is a lack of serious infections at presentation. Spontaneous recovery occurs in 95% of patients in a median of 17 months.9 Specific treatment is rarely required to elevate the neutrophil count. Indeed, possibly due to endogenous granulocyte colony stimulating factor (G-CSF) release affected children manage to produce sufficient neutrophils to ‘‘overcome’’ the antibody when they have a bacterial infection – the presumed explanation for the usual lack of major sepsis in AIN. G-CSF is now the treatment of choice for the occasional sepsis that necessitates hospital admission or to elevate the neutrophil count prior to surgery. There is no hard evidence to support the role of prophylactic antibiotics in AIN, but their use in AIN with recurrent infections appears sensible.
SEVERE CONGENITAL NEUTROPENIA Presentation Kostmann first described infantile genetic agranulocytosis as an autosomal recessive disease in an intermarried family from northern Sweden in 1956.10 Subsequently, autosomal dominant and sporadic forms have been recognized.11 Typically, children present in infancy with severe neutropenia (often < 0:2 109 l), recurrent bacterial infections and a maturation arrest at the promyelocyte/myelocyte stage in the bone
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Ancliff marrow. There is often a ‘‘compensatory’’ monocytosis and modest eosinophilia. Basic immunological investigations are normal; indeed any abnormalities outside of the haemopoietic system should raise the possibility of an alternative diagnosis. Pathophysiology The cause of such a specific cytopenia as SCN has fascinated and frustrated investigators for a number of years. G-CSF and its receptor (G-CSFR) seemed an obvious starting point. However, levels of G-CSF were shown to be normal or elevated, G-CSFR expression appeared increased (albeit on neutrophils) and at first no abnormalities were detected in the G-CSFR.12–14 Subsequently, point mutations were described in the intra-cytoplasmic region of the G-CSFR in some patients that lead to truncated receptors. Although, initially suggested to be a cause of SCN, it is now clear that these are associated with leukaemic progression in a proportion of patients (see below).15;16 Furthermore, two cases of G-CSF non-responsive SCN have recently been described that have been attributed to mutations in the extra-cellular region of the G-CSFR.17;18 Genetics The surprising discovery of heterozygous mutations in ELA2 encoding neutrophil elastase (NE) in 13 out of 13 families with autosomal dominant cyclical neutropenia, soon led to the same group also reporting NE mutations in sporadic SCN.19;20 A second study confirmed these results for the sporadic form of the disease but not surprisingly did not find any mutations in three autosomal recessive families where a heterozygous pattern of inheritance would not fit the presumed pattern of inheritance.21 At least 50 normal controls and when available, healthy parents were examined for each of the novel mutations and no evidence was found that these substitutions were polymorphisms. One novel polymorphism was described and both groups identified one previously known silent substitution. Nevertheless, despite extremely strong genetic data a convincing pathogenic mechanism has proved elusive, even leading some authors to suggest that SCN may indeed not be caused by a single mutation in the NE gene.22 However, a healthy father who is a somatic mosaic for his daughterÕs elastase mutation has recently been identified.23 Around a quarter of the DNA from his T-cells was shown to contain the mutant whereas less than 5% of his neutrophils were derived from stem cells containing the mutated elastase gene. This elegant experiment of nature is the first direct proof of the pathogenic nature of NE mutations in man. Potential mechanisms of pathogenesis of neutrophil elastase mutations Synthesis of NE is confined to promyelocytes and promonocytes.24 It is packaged into the azurophilic cytoplasmic granules and thus physiologically has been assumed not to have a role in its cell of synthesis.25 The mature 218 amino acid glycoprotein appears to have an important role in both host response to infection and the inflammatory response.26 Following microbial ingestion, NE and the other granule contents are released into the phagocytic vacuole where killing of the ingested microbe occurs.27 Pathophysiologi210
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cally, extracellular release of NE at sites of inflammation appears to have an important role in a variety of conditions including emphysema, cystic fibrosis, adult respiratory distress syndrome and rheumatoid arthritis.28;29 Neutrophil elastase has no known role in myelopoiesis to date. At the outset, haploinsufficiency appears to be an unlikely explanation for the role of NE in SCN, as homozygous and heterozygous NE ‘‘knock-out’’ mice are not neutropenic, although the former do show an increased susceptibility to infections.30;31 Protein modeling suggests that a common feature of all the NE mutations (both in SCN and cyclical neutropenia) is that they can lead to destabilization of the mature protein.32 It is therefore possible to postulate that the highly toxic enzyme is not folded and packaged correctly in the safe environment of the azurophilic granule and instead leaks out of the endoplasmic reticulum and kills the cell. Moreover, very recent work suggests that sub-cellular localization of mutant NE may be abnormal, with mutant enzyme detectable in the cytosol.33 In vitro expression of 14 different types of the mutant enzyme in two haemopoietic cell lines showed that the mutations had a varied effect on proteolytic activity, with most retaining significant activity.34 The mutant enzymes retained vulnerability to inhibition by a-1 antitrypsin and, perhaps surprisingly, were not cytotoxic to their cells of manufacture. One unanticipated finding was that co-expression of mutant and wild-type enzyme led to a reduction in expression of the wild-type enzyme suggesting a potential to interfere with sub-cellular trafficking or post-translational processing.34 Most recently a mouse model has been created containing the V72M NE mutation which has been described in two families with SCN.35 Disappointingly, the mice are haematologically completely normal with no evidence of neutropenia, increased susceptibility to infection or malignant transformation. This suggests that the mechanism may be species specific. An increased susceptibility to apoptosis of myeloid cells in patients with SCN has been suggested and there is some recent intriguing work proposing that the cognate inhibitor of NE, monocyte/neutrophil elastase inhibitor (M/NEI) when complexed with NE may translocate to the nucleus and have a pro-apoptotic role.36;37 However, an augmentation of any such mechanism in SCN remains pure speculation. One alternative explanation involves a possible gain of function with digestion of a novel substrate, or perhaps even loss of function with consequent loss of digestion of an as yet unidentified regulatory molecule. To investigate this concept further, bone marrow CD34+ cells from normal donors and from a SCN patient (with a G56E NE mutation) were cultured in the presence of a pharmacological inhibitor of NEGW311616A (kind gift of GlaxoSmithKline, Stevenage, UK).38 The inhibitor had no effect on the expansion and differentiation of the normal bone marrow stem cells. The reduction in expansion and differentiation seen in in vitro cultured SCN marrow was not improved by the addition of the inhibitor, suggesting that the mechanism of pathogenesis does not involve substrate cleavage by the active site of the mutant NE enzyme.
Congenital neutropenia In summary, although the genetic evidence for NE mutations being the fundamental cause of the majority of cases of SCN is persuasive, the exact pathological mechanism remains to be elucidated. The cause(s) of autosomal recessive SCN and those of sporadic and autosomal dominant disease without NE mutations remain to be discovered. Treatment Prophylactic antibiotics alone are insufficient in SCN, and before the introduction of G-CSF the projected median survival was only 3 years.39 Fortunately, over 90% of patients with SCN respond to pharmacological G-CSF with an elevation in neutrophil counts and a reduction in the number of infections and hospitalizations.11 Dose requirements are relatively large (a median of 11:5 lg=kg=day in the phase III trial) and treatment probably needs to be maintained for life.40 Prognosis Although the vast majority of children can lead a reasonably normal life, patients with SCN run a very significant lifetime risk of transformation into myelodysplasia or acute myeloid leukaemia, with the crude rate now reported at 12% overall or up to a 2% annual risk.41 Bone marrow transplantation from a sibling is a relatively safe process if performed prior to transformation, although very difficult afterwards with only a handful of successful cases being reported.42 Therefore, from a clinical point of view, the most important piece of information that the laboratory could provide would be an estimate of the risk of transformation – sadly there is no current means of predicting the likelihood of this event. Possible causes of leukaemic transformation No clear role has been established for NE mutations in leukaemic transformation, with both involved and un-involved cases being reported to transform.20;43 However, three other genetic abnormalities have been described in association with transformation in SCN. Firstly, monosomy 7 is the most frequent cytogenetic abnormality associated with transformation in SCN and other bone marrow failure syndromes.44 Secondly, activating mutations in the oncogene ras have been described in 5 out of 10 patients in one small series.45 Finally, G-CSFR mutations have been associated with transformation in SCN, although cases have recently been reported where such mutations have been present for many years without overt transformation taking place.11;43 The criticality of all of these steps is this subject of much debate. Not all patients have chromosome 7 abnormalities at transformation and in a case recently reported this abnormality had disappeared at relapse,43 implying that it is a late and clearly non-essential event. Similarly, it is clear that not all transformed cases have activating ras mutations. However, the role of G-CSFR mutations remains the most controversial subject. Initially suggested to be the cause of SCN,15 these are now generally accepted as being associated with leukaemic progression.16 Since this acceptance, debate has raged as to whether such mutations are a critical point on the pathway to leukaemia or merely a marker of clonal haemopoiesis.46 Support for the first view point comes from the fact that the mutations all remove the negative regulatory C-terminal do-
main of the mature G-CSFR protein and when knock-in mice were engineered, an excess of neutrophil proliferation was noted in the presence of exogenous G-CSF.47;48 However, despite administration of large amounts of G-CSF for prolonged periods, not one mouse has been reported to develop any form of leukaemic condition. Moreover, a case has been recently reported where a very small amount of mutant G-CSF receptor DNA was been detectable for over 8 years, without any obvious signs of progression.43 In addition, Jeha et al.49 reported spontaneous remission of a secondary leukaemia in an SCN patient following withdrawal of exogenous G-CSF on two occasions. The authors implied that the child had a G-CSFR mutation that was still present in spontaneous complete remission and thus did not provide a significant degree of proliferative stimulus. This fits with the suggestion that such mutations may only be a ‘‘bystander phenomenon’’ and simply a marker of clonal haemopoiesis.46 In summary, transformation in SCN is likely to be a multistep process occurring on the background of a haemopoietically stressed bone marrow. The most important genetic event(s) in transformation remain(s) to be elucidated.
CYCLICAL NEUTROPENIA (CyN) Presentation Although also present from birth, the presentation of cyclical neutropenia tends to be later than that of SCN as the infections are generally less severe. CyN is characterized by periodic neutropenia (< 0:2 109 l for 3–5 days) inter-spaced with normal or near normal neutrophil counts with a remarkably regular 21-day periodicity.50 Children typically present with a history of recurrent fever, mouth ulcers and an excess of typical childhood upper respiratory tract and ear infections. More severe cases also have episodes of peri-anal cellulitis, more typical of SCN. The recommendation from the Severe Chronic Neutropenia International Registry (SCNIR) of thrice weekly blood counts for 6 weeks to establish the diagnosis is often difficult to achieve in children, and in view of the recent genetic data may no longer be regarded as an essential investigation. Pathogenesis As noted above, it is now clear that the majority of cases (probably nearly all with a classical 21-day cycle) of CyN are caused by heterozygous mutations in ELA2 encoding NE.19 It was initially thought that the mutations that cause the CyN and SCN were distinct,20 but as further cases have been described it is clear that there is overlap at both the genetic and protein level with identical DNA mutations being reported in both diseases and no obvious clustering of mutations on protein models. This overlap, and the fact that CyN patients do not have an excess risk of AML, remain two of the many difficulties yet to be resolved in the pathogenic mechanism of NE mutations.44 Treatment and outcome G-CSF treatment increases the amplitude of neutrophil oscillations, shortens both the cycle length and duration of neutropenia and leads to marked clinical improvement.
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Ancliff Smaller doses (typically 1–5 lg=kg=day) are required than in SCN.50 Interestingly, none of 132 patients followed by the SCNIR has developed AML.44
IDIOPATHIC NEUTROPENIA This diagnosis of exclusion is used to cover those patients who present with neutropenia in childhood or adult life without any evidence of myelodysplasia or other identifiable cause of neutropenia. Although most likely of heterogeneous cause, clinically these patients have a good prognosis, typically with few infections, good response to G-CSF when required and no apparent risk of malignant transformation.44;51
SHWACHMAN–DIAMOND SYNDROME (SDS) Presentation SDS is a rare autosomal recessive disease characterized by exocrine pancreatic dysfunction, neutropenia (often intermittent) and skeletal abnormalities.52 It presents in infancy with failure to thrive and an excess of bacterial infections. A severe eczematous-like skin condition is also common at presentation and improves with treatment of the pancreatic insufficiency. Other clinical features include short stature, metaphyseal dysostosis, epiphyseal dysplasia, liver function abnormalities, renal tubular defects and possibly psychomotor retardation. Pathogenesis In contrast to SCN and CyN, SDS is quite clearly a multi-system disorder and there is significant heterogeneity in both the number of systems affected and the severity. Observational studies on the haemopoietic system have demonstrated reduced bone marrow progenitor colony forming potential, bone marrow stromal abnormalities, increased bone marrow cell apoptosis and defects of neutrophil chemotaxis.53–55 Very recently, the likely causative gene for SDS has been identified (SBDS, from Shwachman–Bodian–Diamond Syndrome).56 A pseudogene (SBDSP) with 97% nucleotide sequence identity resides in a locally duplicated 305 kb segment. Recurring mutations resulting from gene conversion (recombination events occurring between SBDS and SBDSP) were found in 89% of unrelated individuals with SDS (141 of 158 cases), with 60% carrying two converted alleles. Point mutations, an insertion and two deletions accounted for the 15 other mutated alleles in this study. Clearly, although every case of SDS did not have two mutated alleles, there is little doubt that mutations in SBDS are responsible for the majority, if not all cases of SDS. Genotype/phenotype studies may yet prove very interesting in this disease. SBDS was a previously uncharacterized gene and is a member of a highly conserved protein family that includes RNA processing genes, suggesting that SDS may be due to a defect in RNA metabolism essential for the development of the exocrine pancreas, haematopoiesis and chrondrogenesis. Treatment and outcome The mainstay of treatment is the replacement of exocrine pancreatic enzymes. This clearly has to be performed in 212
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conjunction with a gastroenterologist and often leads to a remarkable improvement in a previously very lethargic child. Curiously, around half of all children show an improvement in pancreatic function with increasing age, sufficient to stop enzyme supplementation.57 The treatment of the haematological abnormalities is less straightforward. Probably, the majority of children require no regular treatment or only prophylactic antibiotics. Although, SDS children do respond to G-CSF (with an increase in neutrophil number), the risk of malignant transformation and the functional defects of neutrophils in SDS make haematologists less enthusiastic to prescribe it than in SCN. Transformation to MDS/AML probably occurs in around 15% of SDS patients,58 although it is very difficult to give an exact figure as far fewer patients with SDS than SCN have been registered with the SCNIR and patients with milder disease may consult only a gastroenterologist. It should be noted that cytogenetic abnormalities in the absence of MDS/ AML, particularly isochrome 7q, do not indicate imminent transformation in SDS and indeed may occasionally regress spontaneously.59;60
GLYCOGEN STORAGE DISEASE TYPE 1b (GSD1b) GSD1b results from deficiency of the glucose-6-phosphate translocase enzyme. This enzyme transports glucose-6-phosphate into the endoplasmic reticulum for conversion to glucose and phosphate by the enzyme glucose-6-phosphatase. Absence of the enzyme leads to an inability to produce glucose by either gluconeogenesis or glycogenolysis. Patients are thus dependent on dietary carbohydrate to maintain blood glucose levels and prevent hypoglycemia and lactic acidosis. GSD1b patients (in contrast to GSD1a patients) also have a marked neutropenia and neutrophil function defects.61 The mechanism of neutropenia remains uncertain, but presumably directly involves the haemopoietic system as orthotopic liver transplantation in two patients failed to correct the neutropenia.62 G-CSF treatment leads to both an increase in neutrophil count and reduction in infections.63 There is also now increasing evidence that the often severe chronic inflammatory bowel disease that these patients are affected by is significantly ameliorated by G-CSF therapy.64 Until recently, there appeared to be no risk of malignant myeloid transformation in GSD1b, but of concern is the recent publication of a case of AML in a GSD1b patient who had received long-term G-CSF support.65
CONGENITAL NEUTROPENIA AS A COMPONENT OF CONGENITAL IMMUNODEFICIENCY Neutropenia is a feature of many immunodeficiency syndromes and for a detailed discussion readers are referred to the recent review by Cham et al.66 In particular neutropenia has been reported in a significant number of patients with CD40-ligand (CD40L) deficiency (hyper IgM syndrome), X-linked agammaglobulinaemia, cartilage hair hypoplasia
Congenital neutropenia and reticular dysgenesis. The mechanism of neutropenia is often thought to be auto-immune although hard evidence for this is difficult to find. For example, in one study 68% of patients with CD40L deficiency were reported to be neutropenic, but anti-neutrophil antibodies could not be demonstrated.67 A recent interesting report suggests that CD40L stimulates bone marrow stromal cells to release the cytokines flt3-ligand and thrombopoietin that are necessary for myelopoiesis,68 this fact may thus explain the neutropenia in patients with CD40L deficiency. It should be noted that the bone marrow examination in patients with CD40L deficiency can show a striking maturation arrest with vacuolated promyelocytes, indistinguishable from that seen in SCN. This serves to illustrate the importance of a brief immunological work-up in new patients presenting with congenital neutropenia.
EXTREMELY RARE CAUSES OF CONGENITAL NEUTROPENIA Activating mutations in the Wiskott Aldrich syndrome protein (WASp) Recently, a large kindred in which only the males suffered from congenital neutropenia has been described.69 Unusually, affected individuals had a monocytopenia (most SCN patients have a monocytosis) and a variable lymphopenia with particularly low CD4+ cell count. Two further cases have since been identified, one with striking myelodysplasia.70 Different mutations have been described in the three kindreds, but all are predicted to lead to a constitutively active WASp. It is at present uncertain why this leads to neutropenia. Myelokathexis Myelokathexis is an autosomal dominant moderate to severe neutropenia with unusual morphology in that most of the patientÕs neutrophils have cytoplasmic vacuoles and abnormal nuclei with hypersegmentation and only very thin filaments connecting the nuclear lobes. The mechanism of neutropenia may relate to decreased expression of bcl-x and accelerated apoptosis of neutrophil precursors within the bone marrow.71 Most patients do not suffer from serious infections, but G-CSF has been reported to be effective in a few individuals.71 Pearson’s syndrome PearsonÕs syndrome comes into the differential diagnosis of a child with suspected SDS but it is usually relatively easy to identify the characteristic bone marrow appearance. The disease manifests as refractory sideroblastic anaemia with vacuolization of bone marrow precursors, variable degrees of neutropenia and thrombocytopenia and exocrine pancreatic failure.72 It is caused by mitochondrial DNA deletions.73
sepsis.74 A limited number of these children have been reported to respond well to G-CSF and it seems sensible to have a lower threshold for using G-CSF in this condition.75 The disease is caused by mutations in the X-linked G4.5 gene which encodes a protein involved in cardiolipin biosynthesis,76;77 again the pathogenesis of the neutropenia remains to be elucidated.
APPROACH TO THE CHILD WITH SUSPECTED CONGENITAL NEUTROPENIA The age of the child at presentation, the depth of the neutropenia, severity of infections and presence/absence of non-haematological signs and symptoms guides the initial investigation of a child with suspected congenital neutropenia. Presentation in the first few weeks of life with an excess of bacterial infections (atypical infections suggest a congenital immunodeficiency) suggests either NAIN or SCN. Clearly, previous history of an affected child will help with the diagnosis of NAIN and in such instances a bone marrow examination can be deferred whilst the anti-neutrophil antibody results are awaited. The absence of such a history and the presence of life-threatening infections together with a profound neutropenia mandate an early bone marrow aspirate. In a child older than 6 months who had been well prior to the event that precipitated referral and has not had any life-threatening infections, the diagnosis is almost always auto-immune neutropenia and a bone marrow aspirate is not necessary if anti-neutrophil antibodies are detected. It is recommended to perform a basic lymphocyte subset analysis and immunoglobulin levels in all patients without clear isolated auto-immune neutropenia. The stool should be analysed for elastase (of pancreatic not neutrophil origin) in children with symptoms suggestive of malabsorption to exclude SDS.
CONCLUSIONS The last few years have seen the description of the gene responsible for the majority of cases of congenital neutropenia and more recently the likely causative gene for SDS has been identified. Clinically, the treatment of SCN has been revolutionized in the last decade by the introduction of G-CSF. The challenge for the coming years is to translate some of the important basic science research into clinical benefit for the patients. Genotype/phenotype studies on the NE mutations are a priority to attempt to establish whether or not there is a sub-group of SCN children who are at a higher risk of malignant transformation that may benefit from early bone marrow transplantation.
Acknowledgements
Barth syndrome Barth syndrome is characterized by the unfortunate combination of neutropenia and cardiomyopathy, many patients succumb to cardiac failure during an episode of neutropenic
PA was funded jointly by the Roald Dahl Foundation, Great Missenden, Bucks, UK; REACH, London, UK and Amgen Ltd (unrestricted educational grant), Cambridge, UK. I am indebted to Rosemary Gale and David Linch, UCL, London, UK
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Ancliff for their furtherance of my research and critical review of this manuscript. 16.
Correspondence to: Dr. P.J. Ancliff, MA, MB, BChir, MRCP, MRCPath, Department of Haematology, Great Ormond Street Hospital, London WC1N 3JH, UK. Tel.: +44-20-7405-9200; Fax: +44-20-7813-8410;
17.
E-mail: [email protected]
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myeloid leukemia preceded by severe congenital neutropenia. N Engl J Med 1995; 333: 487–493. Tidow N, Pilz C, Teichmann B et al. Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia. Blood 1997; 89: 2369–2375. Ward AC, van Aesch YM, Gits J et al. Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment. J Exp Med 1999; 190: 497–507. Sinha S, Watkins S, Corey SJ. Genetic and biochemical characterization of a deletional mutation of the extra-cellular domain of the human G-CSF receptor in a child with severe congenital neutropenia unresponsive to neupogen [abstract]. Blood 2001; 98: 440a. 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. 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. Ancliff PJ, Gale RE, Liesner R, Hann IM, Linch DC. 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. Blood 2001; 98: 2645–2650. Germeshausen M, Schulze H, Ballmaier M, Zeidler C, Welte K. Mutations in the gene encoding neutrophil elastase (ELA2) are not sufficient to cause the phenotype of congenital neutropenia. Br J Haematol 2001; 115: 222–224. Ancliff PJ, Gale RE, Watts MJ et al. Paternal mosaicism proves the pathogenic nature of mutations in neutrophil elastase in severe congenital neutropenia. Blood 2002; 100: 707–709. Fouret P, du Bois RM, Bernaudin JF et al. Expression of the neutrophil elastase gene during human bone marrow cell differentiation. J Exp Med 1989; 169: 833–845. Gullberg U, Bengtsson N, Bulow E et al. Processing and targeting of granule proteins in human neutrophils. J Immunol Methods 1999; 232: 201–210. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89: 3503–3521. Lehrer RI, Ganz T. Antimicrobial polypeptides of human neutrophils. Blood 1990; 76: 2169–2181. Lehr HA, Arfors KE. Mechanisms of tissue damage by leukocytes. Curr Opin Hematol 1994; 1: 92–99. Stockley RA. Neutrophils and the pathogenesis of COPD. Chest 2002; 121: 151S–155S. Tkalcevic J, Novelli M, Phylactides M et al. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 2000; 12: 201–210. 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. Morris EC, Carrell RW, Coughlin PB. Intracellular serpins in haemopoietic and peripheral blood cells. Br J Haematol 2001; 115: 758–766.
Congenital neutropenia 33. Aprikyan A, Oganesian A, Dale D. Aberrant subcellular localization of neutrophil elastase in cyclic neutropenia (abstract. Blood 2003; 100: 80a–81a. 34. Li FQ, Horwitz M. Characterization of mutant neutrophil elastase in severe congenital neutropenia. J Biol Chem 2001; 276: 14230–14241. 35. Grenda DS, Johnson SE, Mayer JR et al. Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis. Blood 2002; 100: 3221–3228. 36. Aprikyan AA, Liles WC, Rodger E et al. Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia. Blood 2001; 97: 147–153. 37. Belmokhtar CA, Torriglia A, Counis MF et al. Nuclear translocation of a leukocyte elastase inhibitor/elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity. Exp Cell Res 2000; 254: 99–109. 38. Ancliff PJ, Gale RE, Somervaille TC et al. Complete inhibition of neutrophil elastase fails to correct the in vitro phenotype of severe congenital neutropenia (abstract). Blood 2002; 100: 243. 39. Alter BP, Young NS. The bone marrow failure syndromes. In: Nathan DG, Orkin SH, eds. Nathan and OskiÕs Hematology of Infancy and Childhood. Philadelphia: WB Saunders Company; 1998: 306. 40. Dale DC, Bonilla MA, Davis MW et al. A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. Blood 1993; 81: 2496–2502. 41. Freedman MH, Alter BP. Risk of myelodysplastic syndrome and acute myeloid leukemia in congenital neutropenias. Semin Hematol 2002; 39: 128–133. 42. Zeidler C, Welte K, Barak Y et al. Stem cell transplantation in patients with severe congenital neutropenia without evidence of leukemic transformation. Blood 2000; 95: 1195–1198. 43. Ancliff PJ, Gale RE, Liesner R, Hann IM, Linch DC. Long-term follow-up of G-CSF receptor mutations in patients with severe congenital neutropenia: implications for leukaemogenesis and therapy. Br J Haematol 2003; 120: 685–690. 44. 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. 45. Kalra R, Dale D, Freedman M et al. Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia. Blood 1995; 86: 4579–4586. 46. Bernard T, Gale RE, Evans JP, Linch DC. Mutations of the granulocyte-colony stimulating factor receptor in patients with severe congenital neutropenia are not required for transformation to acute myeloid leukaemia and may be a bystander phenomenon. Br J Haematol 1998; 101: 141–149. 47. McLemore ML, Poursine-Laurent J, Link DC. Increased granulocyte colony-stimulating factor responsiveness but normal resting granulopoiesis in mice carrying a targeted granulocyte colony-stimulating factor receptor mutation derived from a patient with severe congenital neutropenia. J Clin Invest 1998; 102: 483–492. 48. Hermans MH, Antonissen C, Ward AC et al. Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene. J Exp Med 1999; 189: 683–692.
49. Jeha S, Chan KW, Aprikyan AG et al. Spontaneous remission of granulocyte colony-stimulating factor-associated leukemia in a child with severe congenital neutropenia. Blood 2000; 96: 3647–3649. 50. Dale DC, Bolyard AA, Aprikyan A. Cyclic neutropenia. Semin Hematol 2002; 39: 89–94. 51. Palmblad JE, dem Borne AE. Idiopathic, immune, infectious, and idiosyncratic neutropenias. Semin Hematol 2002; 39: 113–120. 52. Dror Y, Freedman MH. Shwachman–Diamond syndrome. Br J Haematol 2002; 118: 701–713. 53. Dror Y, Freedman MH. Shwachman–Diamond syndrome: an inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty marrow microenvironment. Blood 1999; 94: 3048–3054. 54. Dror Y, Freedman MH. Shwachman–Diamond syndrome marrow cells show abnormally increased apoptosis mediated through the Fas pathway. Blood 2001; 97: 3011–3016. 55. Dror Y, Ginzberg H, Dalal I et al. Immune function in patients with Shwachman–Diamond syndrome. Br J Haematol 2001; 114: 712–717. 56. Boocock GR, Morrison JA, Popovic M et al. Mutations in SBDS are associated with Shwachman–Diamond syndrome. Nat Genet 2003; 33: 97–101. 57. Mack DR, Forstner GG, Wilschanski M, Freedman MH, Durie PR. Shwachman syndrome: exocrine pancreatic dysfunction and variable phenotypic expression. Gastroenterology 1996; 111: 1593–1602. 58. Ginzberg H, Shin J, Ellis L et al. Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar. J Pediatr 1999; 135: 81–88. 59. Dror Y, Durie P, Ginzberg H et al. Clonal evolution in marrows of patients with Shwachman–Diamond syndrome: a prospective 5-year follow-up study. Exp Hematol 2002; 30: 659–669. 60. Cunningham J, Sales M, Pearce A et al. Does isochromosome 7q mandate bone marrow transplant in children with Shwachman–Diamond syndrome. Br J Haematol 2002; 119: 1062–1069. 61. Kannourakis G. Glycogen storage disease. Semin Hematol 2002; 39: 103–106. 62. Matern D, Starzl TE, Arnaout W et al. Liver transplantation for glycogen storage disease types I, III, and IV. Eur J Pediatr 1999; 158(suppl 2): S43–S48. 63. Calderwood S, Kilpatrick L, Douglas SD et al. Recombinant human granulocyte colony-stimulating factor therapy for patients with neutropenia and/or neutrophil dysfunction secondary to glycogen storage disease type 1b. Blood 2001; 97: 376–382. 64. Dieckgraefe BK, Korzenik JR, Husain A, Dieruf L. Association of glycogen storage disease 1b and Crohn disease: results of a North American survey. Eur J Pediatr 2002; 161(suppl 2): S88–S92. 65. Pinsk M, Burzynski J, Yhap M et al. Acute myelogenous leukemia and glycogen storage disease 1b. J Pediatr Hematol Oncol 2002; 24: 756–758. 66. Cham B, Bonilla MA, Winkelstein J. Neutropenia associated with primary immunodeficiency syndromes. Semin Hematol 2002; 39: 107–112. 67. Levy J, Espanol-Boren T, Thomas C et al. Clinical spectrum of X-linked hyper-IgM syndrome. J Pediatr 1997; 131: 47–54. 68. Solanilla A, Dechanet J, El Andaloussi A et al. CD40-ligand stimulates myelopoiesis by regulating flt3-ligand and thrombopoietin
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production in bone marrow stromal cells. Blood 2000; 95: 3758–3764. Devriendt K, Kim AS, Mathijs G et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat Genet 2001; 27: 313–317. Ancliff PJ, Blundell MP, Gale RE et al. Activating mutations in the Wiskott Aldrich syndrome protein may define a sub-group of severe congenital neutropenia with specific and unusual laboratory features [abstract]. Blood 2001; 98: 439. Aprikyan AA, Liles WC, Park JR et al. Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression of bcl-x in neutrophil precursors. Blood 2000; 95: 320–327. Pearson HA, Lobel JS, Kocoshis SA et al. A new syndrome of refractory sideroblastic anemia with vacuolization of marrow
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precursors and exocrine pancreatic dysfunction. J Pediatr 1979; 95: 976–984. Rotig A, Cormier V, Blanche S et al. PearsonÕs marrow-pancreas syndrome. A multisystem mitochondrial disorder in infancy. J Clin Invest 1990; 86: 1601–1608. Barth PG, Scholte HR, Berden JA et al. An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes. J Neurol Sci 1983; 62: 327–355. Zeidler C, Barth PG, Bonilla MA et al. Neutropenia in Barth syndrome: clinical course and treatment of neutropenia [abstract]. Blood 2001; 98: 300. Bione S, DÕAdamo P, Maestrini E et al. A novel X-linked gene, G4.5. is responsible for Barth syndrome. Nat Genet 1996; 12: 385–389. McMillin JB, Dowhan W. Cardiolipin and apoptosis. Biochim Biophys Acta 2002; 1585: 97–107.
Congenital neutropenia Philip James Ancliff Department of Haematology, Great Ormond Street Hospital, London WC1N 3JH, UK
Abstract Congenital neutropenia is strictly defined as neutropenia present at birth. However, it is more generally used to describe neutropenia secondary to inherited genetic mutations. This review will discuss the presentation of such children and the various causes of congenital neutropenia. In particular, it will focus on severe congenital neutropenia (SCN) and the recent discovery of mutations in the gene encoding neutrophil elastase in the majority of cases of SCN. The potential mechanisms of pathogenesis and of transformation to leukaemia will be discussed. Shwachman–Diamond Syndrome and other less common causes of congenital neutropenia will also be reviewed. Finally, an approach to the child with potential congenital neutropenia will be presented. c 2003 Elsevier Ltd. All rights reserved. KEY WORDS: elastase; congenital neutropenia; neutropenia
INTRODUCTION Definition eutropenia is defined as a decrease in the number of circulating neutrophils in the blood. Strictly speaking, congenital is defined as an abnormality present at birth and is usually, but not necessarily, of genetic cause. This review will focus on the genetic causes of neutropenia (in particular severe congenital neutropenia) that usually present in infancy and produce lifelong neutropenia and recurrent infections. Infantile immune neutropenias will also be briefly discussed. The lower limit of normal for a peripheral blood neutrophil count is both age and race dependent. In infants (children 612 months old), the threshold is 1:0 109 l. After infancy, the usual value is 1:5 109 l up to the age of 10 years and the adult threshold of 1:8 109 l is applied thereafter.1 Both total white cell count and neutrophil count are often depressed in people of African descent. Most laboratories regard an absolute neutrophil count above 1:0 109 l as normal in adult Afro-Caribbeans and this is indeed supported by observational data that such individuals with apparently low counts suffer no excess of bacterial infections.2 There is less hard data for normal black infants, but it is clear that the total neutrophil count is less than 1:0 109 l in maybe as many as 20% of such children.3 Neutropenia has been classified as mild (below the lower limit of normal but >1:0 109 l), moderate (between 0.5 and 1:0 109 l), severe (between 0.2 and 0:5 109 l) and very severe ð<0:2 109 lÞ. The lower limits are useful because the risk of severe infections does not increase until the neutrophil count falls below 0:5 109 l and rises rapidly below 0:2 109 l.4
N
Presentation and initial diagnostic difficulties Children typically present in the first few months of life with neutropenia and recurrent bacterial infections. Other nonhaematological features may lead to a specific diagnosis as detailed below. Presentation at birth or in the first months of life raises the possibility of neonatal allo-immune neutropenia (NAIN), often referred to as the myeloid homologue of Rhesus disease of the newborn.5 However, there is one important difference in that, analogous to neonatal allo-immune thrombocytopenia, NAIN can commonly occur in a first pregnancy (Geoff Lucas, National Blood Service, Bristol, personal communication). NAIN is rare (incidence <0.1%) and in contrast to SCN the infections are rarely severe despite the depth of the neutropenia. The diagnosis is confirmed by demonstration of the presence of neutrophil specific antibodies in the sera from the mother and baby against antigens inherited from the father. No specific therapy is usually required and spontaneous recovery occurs in an average of 11 weeks (range 3–28 weeks).6 However, the mother should be warned of the significant risk of recurrence and asked not to become a blood donor in view of the risk of inducing transfusion associated acute lung injury (TRALI) in any recipient with the same antigen as the father. She should also be warned that she could be at a possible risk of TRALI herself if transfused. Passive transfer of antibodies from a mother with auto-immune neutropenia can produce a similar clinical picture in the neonate.6 Primary auto-immune neutropenia (AIN) of childhood is rare, but probably at least 10-fold more common than SCN (incidence of 1 : 100 000 versus 1:1 000 000).7;8 AIN is a relatively benign disorder characterized by neutrophil specific auto-antibodies, often severe neutropenia and a paucity of severe bacterial infections.9 Bone marrow examination is not always necessary if auto-antibodies are identified and there is a lack of serious infections at presentation. Spontaneous recovery occurs in 95% of patients in a median of 17 months.9 Specific treatment is rarely required to elevate the neutrophil count. Indeed, possibly due to endogenous granulocyte colony stimulating factor (G-CSF) release affected children manage to produce sufficient neutrophils to ‘‘overcome’’ the antibody when they have a bacterial infection – the presumed explanation for the usual lack of major sepsis in AIN. G-CSF is now the treatment of choice for the occasional sepsis that necessitates hospital admission or to elevate the neutrophil count prior to surgery. There is no hard evidence to support the role of prophylactic antibiotics in AIN, but their use in AIN with recurrent infections appears sensible.
SEVERE CONGENITAL NEUTROPENIA Presentation Kostmann first described infantile genetic agranulocytosis as an autosomal recessive disease in an intermarried family from northern Sweden in 1956.10 Subsequently, autosomal dominant and sporadic forms have been recognized.11 Typically, children present in infancy with severe neutropenia (often < 0:2 109 l), recurrent bacterial infections and a maturation arrest at the promyelocyte/myelocyte stage in the bone
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Ancliff marrow. There is often a ‘‘compensatory’’ monocytosis and modest eosinophilia. Basic immunological investigations are normal; indeed any abnormalities outside of the haemopoietic system should raise the possibility of an alternative diagnosis. Pathophysiology The cause of such a specific cytopenia as SCN has fascinated and frustrated investigators for a number of years. G-CSF and its receptor (G-CSFR) seemed an obvious starting point. However, levels of G-CSF were shown to be normal or elevated, G-CSFR expression appeared increased (albeit on neutrophils) and at first no abnormalities were detected in the G-CSFR.12–14 Subsequently, point mutations were described in the intra-cytoplasmic region of the G-CSFR in some patients that lead to truncated receptors. Although, initially suggested to be a cause of SCN, it is now clear that these are associated with leukaemic progression in a proportion of patients (see below).15;16 Furthermore, two cases of G-CSF non-responsive SCN have recently been described that have been attributed to mutations in the extra-cellular region of the G-CSFR.17;18 Genetics The surprising discovery of heterozygous mutations in ELA2 encoding neutrophil elastase (NE) in 13 out of 13 families with autosomal dominant cyclical neutropenia, soon led to the same group also reporting NE mutations in sporadic SCN.19;20 A second study confirmed these results for the sporadic form of the disease but not surprisingly did not find any mutations in three autosomal recessive families where a heterozygous pattern of inheritance would not fit the presumed pattern of inheritance.21 At least 50 normal controls and when available, healthy parents were examined for each of the novel mutations and no evidence was found that these substitutions were polymorphisms. One novel polymorphism was described and both groups identified one previously known silent substitution. Nevertheless, despite extremely strong genetic data a convincing pathogenic mechanism has proved elusive, even leading some authors to suggest that SCN may indeed not be caused by a single mutation in the NE gene.22 However, a healthy father who is a somatic mosaic for his daughterÕs elastase mutation has recently been identified.23 Around a quarter of the DNA from his T-cells was shown to contain the mutant whereas less than 5% of his neutrophils were derived from stem cells containing the mutated elastase gene. This elegant experiment of nature is the first direct proof of the pathogenic nature of NE mutations in man. Potential mechanisms of pathogenesis of neutrophil elastase mutations Synthesis of NE is confined to promyelocytes and promonocytes.24 It is packaged into the azurophilic cytoplasmic granules and thus physiologically has been assumed not to have a role in its cell of synthesis.25 The mature 218 amino acid glycoprotein appears to have an important role in both host response to infection and the inflammatory response.26 Following microbial ingestion, NE and the other granule contents are released into the phagocytic vacuole where killing of the ingested microbe occurs.27 Pathophysiologi210
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cally, extracellular release of NE at sites of inflammation appears to have an important role in a variety of conditions including emphysema, cystic fibrosis, adult respiratory distress syndrome and rheumatoid arthritis.28;29 Neutrophil elastase has no known role in myelopoiesis to date. At the outset, haploinsufficiency appears to be an unlikely explanation for the role of NE in SCN, as homozygous and heterozygous NE ‘‘knock-out’’ mice are not neutropenic, although the former do show an increased susceptibility to infections.30;31 Protein modeling suggests that a common feature of all the NE mutations (both in SCN and cyclical neutropenia) is that they can lead to destabilization of the mature protein.32 It is therefore possible to postulate that the highly toxic enzyme is not folded and packaged correctly in the safe environment of the azurophilic granule and instead leaks out of the endoplasmic reticulum and kills the cell. Moreover, very recent work suggests that sub-cellular localization of mutant NE may be abnormal, with mutant enzyme detectable in the cytosol.33 In vitro expression of 14 different types of the mutant enzyme in two haemopoietic cell lines showed that the mutations had a varied effect on proteolytic activity, with most retaining significant activity.34 The mutant enzymes retained vulnerability to inhibition by a-1 antitrypsin and, perhaps surprisingly, were not cytotoxic to their cells of manufacture. One unanticipated finding was that co-expression of mutant and wild-type enzyme led to a reduction in expression of the wild-type enzyme suggesting a potential to interfere with sub-cellular trafficking or post-translational processing.34 Most recently a mouse model has been created containing the V72M NE mutation which has been described in two families with SCN.35 Disappointingly, the mice are haematologically completely normal with no evidence of neutropenia, increased susceptibility to infection or malignant transformation. This suggests that the mechanism may be species specific. An increased susceptibility to apoptosis of myeloid cells in patients with SCN has been suggested and there is some recent intriguing work proposing that the cognate inhibitor of NE, monocyte/neutrophil elastase inhibitor (M/NEI) when complexed with NE may translocate to the nucleus and have a pro-apoptotic role.36;37 However, an augmentation of any such mechanism in SCN remains pure speculation. One alternative explanation involves a possible gain of function with digestion of a novel substrate, or perhaps even loss of function with consequent loss of digestion of an as yet unidentified regulatory molecule. To investigate this concept further, bone marrow CD34+ cells from normal donors and from a SCN patient (with a G56E NE mutation) were cultured in the presence of a pharmacological inhibitor of NEGW311616A (kind gift of GlaxoSmithKline, Stevenage, UK).38 The inhibitor had no effect on the expansion and differentiation of the normal bone marrow stem cells. The reduction in expansion and differentiation seen in in vitro cultured SCN marrow was not improved by the addition of the inhibitor, suggesting that the mechanism of pathogenesis does not involve substrate cleavage by the active site of the mutant NE enzyme.
Congenital neutropenia In summary, although the genetic evidence for NE mutations being the fundamental cause of the majority of cases of SCN is persuasive, the exact pathological mechanism remains to be elucidated. The cause(s) of autosomal recessive SCN and those of sporadic and autosomal dominant disease without NE mutations remain to be discovered. Treatment Prophylactic antibiotics alone are insufficient in SCN, and before the introduction of G-CSF the projected median survival was only 3 years.39 Fortunately, over 90% of patients with SCN respond to pharmacological G-CSF with an elevation in neutrophil counts and a reduction in the number of infections and hospitalizations.11 Dose requirements are relatively large (a median of 11:5 lg=kg=day in the phase III trial) and treatment probably needs to be maintained for life.40 Prognosis Although the vast majority of children can lead a reasonably normal life, patients with SCN run a very significant lifetime risk of transformation into myelodysplasia or acute myeloid leukaemia, with the crude rate now reported at 12% overall or up to a 2% annual risk.41 Bone marrow transplantation from a sibling is a relatively safe process if performed prior to transformation, although very difficult afterwards with only a handful of successful cases being reported.42 Therefore, from a clinical point of view, the most important piece of information that the laboratory could provide would be an estimate of the risk of transformation – sadly there is no current means of predicting the likelihood of this event. Possible causes of leukaemic transformation No clear role has been established for NE mutations in leukaemic transformation, with both involved and un-involved cases being reported to transform.20;43 However, three other genetic abnormalities have been described in association with transformation in SCN. Firstly, monosomy 7 is the most frequent cytogenetic abnormality associated with transformation in SCN and other bone marrow failure syndromes.44 Secondly, activating mutations in the oncogene ras have been described in 5 out of 10 patients in one small series.45 Finally, G-CSFR mutations have been associated with transformation in SCN, although cases have recently been reported where such mutations have been present for many years without overt transformation taking place.11;43 The criticality of all of these steps is this subject of much debate. Not all patients have chromosome 7 abnormalities at transformation and in a case recently reported this abnormality had disappeared at relapse,43 implying that it is a late and clearly non-essential event. Similarly, it is clear that not all transformed cases have activating ras mutations. However, the role of G-CSFR mutations remains the most controversial subject. Initially suggested to be the cause of SCN,15 these are now generally accepted as being associated with leukaemic progression.16 Since this acceptance, debate has raged as to whether such mutations are a critical point on the pathway to leukaemia or merely a marker of clonal haemopoiesis.46 Support for the first view point comes from the fact that the mutations all remove the negative regulatory C-terminal do-
main of the mature G-CSFR protein and when knock-in mice were engineered, an excess of neutrophil proliferation was noted in the presence of exogenous G-CSF.47;48 However, despite administration of large amounts of G-CSF for prolonged periods, not one mouse has been reported to develop any form of leukaemic condition. Moreover, a case has been recently reported where a very small amount of mutant G-CSF receptor DNA was been detectable for over 8 years, without any obvious signs of progression.43 In addition, Jeha et al.49 reported spontaneous remission of a secondary leukaemia in an SCN patient following withdrawal of exogenous G-CSF on two occasions. The authors implied that the child had a G-CSFR mutation that was still present in spontaneous complete remission and thus did not provide a significant degree of proliferative stimulus. This fits with the suggestion that such mutations may only be a ‘‘bystander phenomenon’’ and simply a marker of clonal haemopoiesis.46 In summary, transformation in SCN is likely to be a multistep process occurring on the background of a haemopoietically stressed bone marrow. The most important genetic event(s) in transformation remain(s) to be elucidated.
CYCLICAL NEUTROPENIA (CyN) Presentation Although also present from birth, the presentation of cyclical neutropenia tends to be later than that of SCN as the infections are generally less severe. CyN is characterized by periodic neutropenia (< 0:2 109 l for 3–5 days) inter-spaced with normal or near normal neutrophil counts with a remarkably regular 21-day periodicity.50 Children typically present with a history of recurrent fever, mouth ulcers and an excess of typical childhood upper respiratory tract and ear infections. More severe cases also have episodes of peri-anal cellulitis, more typical of SCN. The recommendation from the Severe Chronic Neutropenia International Registry (SCNIR) of thrice weekly blood counts for 6 weeks to establish the diagnosis is often difficult to achieve in children, and in view of the recent genetic data may no longer be regarded as an essential investigation. Pathogenesis As noted above, it is now clear that the majority of cases (probably nearly all with a classical 21-day cycle) of CyN are caused by heterozygous mutations in ELA2 encoding NE.19 It was initially thought that the mutations that cause the CyN and SCN were distinct,20 but as further cases have been described it is clear that there is overlap at both the genetic and protein level with identical DNA mutations being reported in both diseases and no obvious clustering of mutations on protein models. This overlap, and the fact that CyN patients do not have an excess risk of AML, remain two of the many difficulties yet to be resolved in the pathogenic mechanism of NE mutations.44 Treatment and outcome G-CSF treatment increases the amplitude of neutrophil oscillations, shortens both the cycle length and duration of neutropenia and leads to marked clinical improvement.
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Ancliff Smaller doses (typically 1–5 lg=kg=day) are required than in SCN.50 Interestingly, none of 132 patients followed by the SCNIR has developed AML.44
IDIOPATHIC NEUTROPENIA This diagnosis of exclusion is used to cover those patients who present with neutropenia in childhood or adult life without any evidence of myelodysplasia or other identifiable cause of neutropenia. Although most likely of heterogeneous cause, clinically these patients have a good prognosis, typically with few infections, good response to G-CSF when required and no apparent risk of malignant transformation.44;51
SHWACHMAN–DIAMOND SYNDROME (SDS) Presentation SDS is a rare autosomal recessive disease characterized by exocrine pancreatic dysfunction, neutropenia (often intermittent) and skeletal abnormalities.52 It presents in infancy with failure to thrive and an excess of bacterial infections. A severe eczematous-like skin condition is also common at presentation and improves with treatment of the pancreatic insufficiency. Other clinical features include short stature, metaphyseal dysostosis, epiphyseal dysplasia, liver function abnormalities, renal tubular defects and possibly psychomotor retardation. Pathogenesis In contrast to SCN and CyN, SDS is quite clearly a multi-system disorder and there is significant heterogeneity in both the number of systems affected and the severity. Observational studies on the haemopoietic system have demonstrated reduced bone marrow progenitor colony forming potential, bone marrow stromal abnormalities, increased bone marrow cell apoptosis and defects of neutrophil chemotaxis.53–55 Very recently, the likely causative gene for SDS has been identified (SBDS, from Shwachman–Bodian–Diamond Syndrome).56 A pseudogene (SBDSP) with 97% nucleotide sequence identity resides in a locally duplicated 305 kb segment. Recurring mutations resulting from gene conversion (recombination events occurring between SBDS and SBDSP) were found in 89% of unrelated individuals with SDS (141 of 158 cases), with 60% carrying two converted alleles. Point mutations, an insertion and two deletions accounted for the 15 other mutated alleles in this study. Clearly, although every case of SDS did not have two mutated alleles, there is little doubt that mutations in SBDS are responsible for the majority, if not all cases of SDS. Genotype/phenotype studies may yet prove very interesting in this disease. SBDS was a previously uncharacterized gene and is a member of a highly conserved protein family that includes RNA processing genes, suggesting that SDS may be due to a defect in RNA metabolism essential for the development of the exocrine pancreas, haematopoiesis and chrondrogenesis. Treatment and outcome The mainstay of treatment is the replacement of exocrine pancreatic enzymes. This clearly has to be performed in 212
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conjunction with a gastroenterologist and often leads to a remarkable improvement in a previously very lethargic child. Curiously, around half of all children show an improvement in pancreatic function with increasing age, sufficient to stop enzyme supplementation.57 The treatment of the haematological abnormalities is less straightforward. Probably, the majority of children require no regular treatment or only prophylactic antibiotics. Although, SDS children do respond to G-CSF (with an increase in neutrophil number), the risk of malignant transformation and the functional defects of neutrophils in SDS make haematologists less enthusiastic to prescribe it than in SCN. Transformation to MDS/AML probably occurs in around 15% of SDS patients,58 although it is very difficult to give an exact figure as far fewer patients with SDS than SCN have been registered with the SCNIR and patients with milder disease may consult only a gastroenterologist. It should be noted that cytogenetic abnormalities in the absence of MDS/ AML, particularly isochrome 7q, do not indicate imminent transformation in SDS and indeed may occasionally regress spontaneously.59;60
GLYCOGEN STORAGE DISEASE TYPE 1b (GSD1b) GSD1b results from deficiency of the glucose-6-phosphate translocase enzyme. This enzyme transports glucose-6-phosphate into the endoplasmic reticulum for conversion to glucose and phosphate by the enzyme glucose-6-phosphatase. Absence of the enzyme leads to an inability to produce glucose by either gluconeogenesis or glycogenolysis. Patients are thus dependent on dietary carbohydrate to maintain blood glucose levels and prevent hypoglycemia and lactic acidosis. GSD1b patients (in contrast to GSD1a patients) also have a marked neutropenia and neutrophil function defects.61 The mechanism of neutropenia remains uncertain, but presumably directly involves the haemopoietic system as orthotopic liver transplantation in two patients failed to correct the neutropenia.62 G-CSF treatment leads to both an increase in neutrophil count and reduction in infections.63 There is also now increasing evidence that the often severe chronic inflammatory bowel disease that these patients are affected by is significantly ameliorated by G-CSF therapy.64 Until recently, there appeared to be no risk of malignant myeloid transformation in GSD1b, but of concern is the recent publication of a case of AML in a GSD1b patient who had received long-term G-CSF support.65
CONGENITAL NEUTROPENIA AS A COMPONENT OF CONGENITAL IMMUNODEFICIENCY Neutropenia is a feature of many immunodeficiency syndromes and for a detailed discussion readers are referred to the recent review by Cham et al.66 In particular neutropenia has been reported in a significant number of patients with CD40-ligand (CD40L) deficiency (hyper IgM syndrome), X-linked agammaglobulinaemia, cartilage hair hypoplasia
Congenital neutropenia and reticular dysgenesis. The mechanism of neutropenia is often thought to be auto-immune although hard evidence for this is difficult to find. For example, in one study 68% of patients with CD40L deficiency were reported to be neutropenic, but anti-neutrophil antibodies could not be demonstrated.67 A recent interesting report suggests that CD40L stimulates bone marrow stromal cells to release the cytokines flt3-ligand and thrombopoietin that are necessary for myelopoiesis,68 this fact may thus explain the neutropenia in patients with CD40L deficiency. It should be noted that the bone marrow examination in patients with CD40L deficiency can show a striking maturation arrest with vacuolated promyelocytes, indistinguishable from that seen in SCN. This serves to illustrate the importance of a brief immunological work-up in new patients presenting with congenital neutropenia.
EXTREMELY RARE CAUSES OF CONGENITAL NEUTROPENIA Activating mutations in the Wiskott Aldrich syndrome protein (WASp) Recently, a large kindred in which only the males suffered from congenital neutropenia has been described.69 Unusually, affected individuals had a monocytopenia (most SCN patients have a monocytosis) and a variable lymphopenia with particularly low CD4+ cell count. Two further cases have since been identified, one with striking myelodysplasia.70 Different mutations have been described in the three kindreds, but all are predicted to lead to a constitutively active WASp. It is at present uncertain why this leads to neutropenia. Myelokathexis Myelokathexis is an autosomal dominant moderate to severe neutropenia with unusual morphology in that most of the patientÕs neutrophils have cytoplasmic vacuoles and abnormal nuclei with hypersegmentation and only very thin filaments connecting the nuclear lobes. The mechanism of neutropenia may relate to decreased expression of bcl-x and accelerated apoptosis of neutrophil precursors within the bone marrow.71 Most patients do not suffer from serious infections, but G-CSF has been reported to be effective in a few individuals.71 Pearson’s syndrome PearsonÕs syndrome comes into the differential diagnosis of a child with suspected SDS but it is usually relatively easy to identify the characteristic bone marrow appearance. The disease manifests as refractory sideroblastic anaemia with vacuolization of bone marrow precursors, variable degrees of neutropenia and thrombocytopenia and exocrine pancreatic failure.72 It is caused by mitochondrial DNA deletions.73
sepsis.74 A limited number of these children have been reported to respond well to G-CSF and it seems sensible to have a lower threshold for using G-CSF in this condition.75 The disease is caused by mutations in the X-linked G4.5 gene which encodes a protein involved in cardiolipin biosynthesis,76;77 again the pathogenesis of the neutropenia remains to be elucidated.
APPROACH TO THE CHILD WITH SUSPECTED CONGENITAL NEUTROPENIA The age of the child at presentation, the depth of the neutropenia, severity of infections and presence/absence of non-haematological signs and symptoms guides the initial investigation of a child with suspected congenital neutropenia. Presentation in the first few weeks of life with an excess of bacterial infections (atypical infections suggest a congenital immunodeficiency) suggests either NAIN or SCN. Clearly, previous history of an affected child will help with the diagnosis of NAIN and in such instances a bone marrow examination can be deferred whilst the anti-neutrophil antibody results are awaited. The absence of such a history and the presence of life-threatening infections together with a profound neutropenia mandate an early bone marrow aspirate. In a child older than 6 months who had been well prior to the event that precipitated referral and has not had any life-threatening infections, the diagnosis is almost always auto-immune neutropenia and a bone marrow aspirate is not necessary if anti-neutrophil antibodies are detected. It is recommended to perform a basic lymphocyte subset analysis and immunoglobulin levels in all patients without clear isolated auto-immune neutropenia. The stool should be analysed for elastase (of pancreatic not neutrophil origin) in children with symptoms suggestive of malabsorption to exclude SDS.
CONCLUSIONS The last few years have seen the description of the gene responsible for the majority of cases of congenital neutropenia and more recently the likely causative gene for SDS has been identified. Clinically, the treatment of SCN has been revolutionized in the last decade by the introduction of G-CSF. The challenge for the coming years is to translate some of the important basic science research into clinical benefit for the patients. Genotype/phenotype studies on the NE mutations are a priority to attempt to establish whether or not there is a sub-group of SCN children who are at a higher risk of malignant transformation that may benefit from early bone marrow transplantation.
Acknowledgements
Barth syndrome Barth syndrome is characterized by the unfortunate combination of neutropenia and cardiomyopathy, many patients succumb to cardiac failure during an episode of neutropenic
PA was funded jointly by the Roald Dahl Foundation, Great Missenden, Bucks, UK; REACH, London, UK and Amgen Ltd (unrestricted educational grant), Cambridge, UK. I am indebted to Rosemary Gale and David Linch, UCL, London, UK
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Ancliff for their furtherance of my research and critical review of this manuscript. 16.
Correspondence to: Dr. P.J. Ancliff, MA, MB, BChir, MRCP, MRCPath, Department of Haematology, Great Ormond Street Hospital, London WC1N 3JH, UK. Tel.: +44-20-7405-9200; Fax: +44-20-7813-8410;
17.
E-mail: [email protected]
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