How I manage children with neutropenia

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1 review How I manage children with neutropenia David C. Dale Department of Medicine, University of Washington, Seattle, WA, USA Summary Neutropenia, usually defined as a blood neutrophil count < /l, is a common medical problem for children and adults. There are many causes for neutropenia, and at each stage in life the clinical pattern of causes and consequences differs significantly. I recommend utilizing the age of the child and clinical observations for the preliminary diagnosis and primary management. In premature infants, neutropenia is quite common and contributes to the risk of sepsis with necrotizing enterocolitis. At birth and for the first few months of life, neutropenia is often attributable to isoimmune or alloimmune mechanisms and predisposes to the risk of severe bacterial infections. Thereafterwhenachildisdiscovered to have neutropenia, often associated with relatively minor symptoms, it is usually attributed to autoimmune disorder or viral infection. The congenital neutropenia syndromes are usually recognized when there are recurrent infections, the neutropenia is severe and there are congenital anomalies suggesting a genetic disorder. This review focuses on the key clinical finding and laboratory tests for diagnosis with commentaries on treatment, particularly the use of granulocyte colonystimulating factor to treat childhood neutropenia. Keywords: neutropenia, congenital neutropenia, neonatal neutropenia, childhood neutropenia, granulocyte colony stimulating factor. The diagnosis and treatment of neutropenia in children and adults has been the central focus of my work in haematology for more than 40 years. During this period, great advances have occurred in understanding the basic pathophysiology and the molecular and genetic basis for these disorders. The development of recombinant human granulocyte colony-stimulating factor (G-CSF) in the 1980s provided the first predictably effective therapy for treating neutropenia. G-CSF Correspondence: David C. Dale, Department of Medicine, University of Washington, Box , 1959 NE Pacific St., Rm AA522, Seattle, WA 98195, USA. dcdale@u.washington.edu greatly changed the outlook for children and adults with neutropenia and increased the importance of making a proper diagnosis. This review describes the diagnosis and management of neutropenia in children based on their age and their signs and symptoms. It presents an approach based on published reports, long-term observational studies conducted through the Severe Chronic Neutropenia International Registry (SCNIR) and my personal experience. Neutropenia in children is relatively common. It is particularly common in some ethnic groups, e.g., children of Arabic and African heritage; more than 10% of these children have neutrophil counts < /l (Denic et al, 2016; Ortiz et al, 2016). In these groups, the mild neutropenia appears to be of little clinical consequence, and it persists into adulthood. Neutropenia is also common in premature or small-for-gestational-age newborns; approximately 6% of these infants have absolute neutrophil counts (ANC) less than /l (Schmutz et al, 2008). Beyond a gestational age of 36 weeks, neutropenia is much less common. The ANC measured at 3 10 days after birth ranges from 27 to /l (Schmutz et al, 2008). However, because the ANC is not routinely measured at birth or during the neonatal period, neutropenia is usually first recognized when a newborn becomes severely ill; develops recurrent fevers or fails to gain weight normally. After the neonatal period, blood neutrophil counts soon approximate those of older children and adults (Christensen et al, 2009; Vetter-Laracy et al, 2014). Neutropenia is then usually attributed to viral infections or autoimmune mechanisms (Husain et al, 2012). Recurrent fevers and common infections that are more severe or last longer than usual suggests an underlying genetic cause for neutropenia, particularly if the child has other clinical features suggesting a hereditary disease that is associated with neutropenia (Newburger & Dale, 2013; Walkovich & Boxer, 2013; Maheshwari, 2014; Bartels et al, 2016; Dufour et al, 2016). Overall, congenital or hereditary neutropenia is quite rare, and its true incidence and prevalence of are not known. Swedish and European studies estimate the prevalence is 2 per million inhabitants (Zeidler et al, 2009; Carlsson et al, 2012). Most families with a child with congenital neutropenia have stories about delay in diagnosis and undoubtedly many children do not receive a proper diagnostic work-up. ª 2017 John Wiley & Sons Ltd First published online 17 April 2017 doi: /bjh.14677

2 Neutropenia in premature infants Definitions and pathophysiology Hypertension and pre-eclampsia are strongly associated with prematurity and neonatal neutropenia, possible consequences of restriction of placental blood flow. The result is reduced production of neutrophils by the neonate s bone marrow (Kush et al, 2006; Zook et al, 2009). In some newborns, severe neutropenia is attributable to sepsis; for example, chorioamnionitis develops after rupture of membranes and exposure of the placenta to vaginal bacteria (Funke et al, 2000). In this setting, neutropenia occurs because of a huge demand for neutrophils that exhausts the marrow neutrophil supply; an insult to which premature infants are particularly vulnerable. With modern supportive care, sepsis induced neo-natal neutropenia usually resolves, but the children who develop multisystem dysfunction associated with sepsis often have a slow recovery, and there may be serious neurological and developmental sequelae (Haller et al, 2016). The risk of sepsis in premature infants is multifactorial; it is partly due to the immaturity of innate immunity (Kan et al, 2016). Normally after about 22 weeks of gestation, haematopoiesis occurs primarily in the bone marrow, but the production of neutrophils is probably quite low until the third trimester (Melvan et al, 2010). Blood counts also can fluctuate substantially in premature neonates, ranging from 15 to /l (Melvan et al, 2010). The neutrophil response to infection is also delayed, attributable to a decreased supply of neutrophils in the storage pool in the marrow and a decreased proliferative response of marrow progenitors (al-mulla & Christensen, 1995). A number of studies indicate that neutrophils produced by neonates are qualitatively different from those of older children and adults, but the clinical significance of these abnormalities is difficult to determine (Melvan et al, 2010). A recent study indicates that neonatal neutrophils can engage in the complex process of formation of extracellular traps normally (Byrd et al, 2016). Another study showed that they can be activated with endotoxin to express ITGAM (CD11b), tolllike receptor 4 and produce reactive oxygen intermediate normally (O Hare et al, 2015). A recent review of neonatal intensive care admissions showed that neutrophils are expected to increase to > /l in premature infants by day 7. In this study, neutropenia was independently associated with the risk of necrotizing enterocolitis (Christensen et al, 2015). of hypertension and pre-eclampsia is part of good pre-natal care. Treatment should include adequate rest, good nutrition and anti-hypertensives as necessary. However, there is a lack of evidence that neonatal neutropenia is prevented through prenatal care. Neonatal sepsis may be prevented by meticulous care during delivery and skilful Caesarean deliveries, when necessary. Randomized clinical trials have shown that neutrophil production can be stimulated in neonates by administration of recombinant human G-CSF, but the clinical benefit is controversial (Carr et al, 2003; Kuhn et al, 2009; Lee et al, 2016). A multicentre randomized trial in France in preterm infants with neutropenia (ANC < /l) demonstrated that G-CSF neutrophil counts increased but did not show a benefit in terms of infection-free survival (Kuhn et al, 2009). A randomized controlled trial in Kolkata, India of premature infants with gestational age less than 32 weeks and blood neutrophil counts less than /l, however, showed that administration of G-CSF resulted in significantly improved survival (Chaudhuri et al, 2012). Based on a large experience in managing neonates in intensive care, Christensen (2013) recommended treating neonates with severe neutropenia (ANC < /l) that last for 2 days or more with G-CSF when the cause of severe neutropenia is uncertain. I concur that G-CSF, in addition to judicious antibiotics for suspected sepsis, is indicated, with careful observation for improvement and as short a treatment period as necessary. There is inconclusive evidence for neutrophil transfusions for neonates with confirmed or suspected sepsis, and this approach to management is much more difficult in most locales (Pammi & Brocklehurst, 2011). Intravenous gammaglobulin is also not of proven benefit. Neutropenia in full-term infants Immune-mediated disorders There are three recognized forms of immune-mediated neutropenia in children: alloimmune, isoimmune and autoimmune neutropenia (AIN). They have similar mechanisms, and sometimes it is difficult to distinguish between them. Neonatal alloimmune neutropenia Neonatal alloimmune neutropenia is much less common than neonatal neutropenia of prematurity. It is attributable to fetal-maternal neutrophil antigen incompatibility occurring because of trans-placental transfer of maternal antibodies to paternal antigens expressed on the surface of the neonate s neutrophils. The alleles most commonly involved are those of the human neutrophil antigen (HNA) family, HNA1a, -1b, -1c, and represent polymorphisms of FCGR3B (Fcgamma receptor IIIb, FccRIIIb or CD16b). Incompatibilities of HNA2, -3a, -4a, -4b, and -5a may also cause this form of neutropenia (Agueda et al, 2012; van den Tooren-de Groot et al, 2014; Mraz et al, 2016). The consequences may be quite severe, including severe omphalitis, cellulitis, bacteraemia and meningitis. Normally the alloimmune antibodies 352 ª 2017 John Wiley & Sons Ltd

3 are cleared spontaneously during the first 6 weeks of life, but neutropenia can last as long as 6 months. Neutropenia is attributed to rapid removal of mature, antibody-coated neutrophils by the spleen, liver and lungs. Diagnosis requires knowing about this diagnostic possibility and then testing maternal serum for anti-neutrophil antibodies to the antigens HNA1a, HNA-1b and HNA 1c and others that may be present on the infant and the infant s father (Bux, 2002). A broader panel of neutrophil antigens is used in some reference laboratories. Management is focused on administration of appropriate antibiotics, determined by the prevalence of specific bacteria and their resistance patterns in the environment. The use of myeloid growth factors in this setting is usually not necessary, because the neutropenia is transient. Faced with a difficult clinical situation of severe neutropenia and a very ill child, it would be rational to try to accelerate marrow recovery with G-CSF until there is a clear signal that marrow recovery is well underway. There are no randomized clinical trials and no large observational studies of therapy for this condition. Neonatal iso-immune neutropenia Iso-immune neutropenia is caused by transplacental transfer of pre-existing IgG antibodies from mothers with AIN or who are FccRcRIIIb deficient (Huizinga et al, 1990; Maslanka et al, 2001; Fung et al, 2005; Boxer et al, 2015a). These antibodies can cause severe neutropenia in the infant with a similar risk of severe infections to those observed with alloimmune neutropenia. In most cases, the key to diagnosis is to consider the possibility. The ANC of the newborn should always be determined if the mother has neutropenia. Management is the same as for alloimmune neutropenia. Autoimmune neutropenia Primary AIN is a disorder of unknown cause, which may be associated with development of neutrophil-specific autoantibodies. AIN overlaps with alloimmune and iso-immune neutropenia because the neutrophil-specific antigens are often the same (i.e., HNA1a, HNA-1b and HNA 1c), but in AIN there are no neutrophil-specific antibodies in the maternal serum. It has long been thought that AIN is triggered by environmental antigens or viral infections, e.g., parvovirus, but specificity in these associations has proven to be elusive (Bux et al, 1998; Bruin et al, 1999). Primary AIN usually occurs in the first 2 years of life, with a very high spontaneous remission rate, perhaps greater than 90%. There are similar cases with and without neutrophil-specific autoantibodies. When antibodies cannot be detected the patient is usually given the diagnosis of idiopathic neutropenia. AIN and idiopathic neutropenia are the most common cause of chronic neutropenia in young children. Neutropenia is attributed to shortened survival of circulating neutrophils without a compensatory increase in neutrophil production, but definitive studies to prove this pathophysiology have never been conducted because of the age of the affected patients. The International Granulocyte Immunology Workshops recommends using both the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination test for diagnostic studies (Lucas et al, 2013). Recently the Italian Neutropenia Registry reported a study of 157 paediatric patients with AIN. The median age at onset was 07 years (Farruggia et al, 2015). GIFT, performed in three laboratories, showed a sensitivity of 62% with a single assay; repeat assay showed 82% were positive, when the clinical course of the patients, i.e., spontaneous remission of an acquired neutropenia in early life, was used as the primary feature to define the diagnosis to be AIN. Boxer et al (2015b) also recently reported on antibody test results for children with a clinical diagnosis of AIN or chronic idiopathic neutropenia. In this study outcomes were the same (i.e., spontaneous remissions, response to G-CSF or serious adverse events) for patients with or without positive antibody tests. In general, expert clinicians rely on the patient s history of fever and infections to guide treatment (Farruggia & Dufour, 2015). Treatment with G-CSF is recommended only for patients with ANC < /l and recurrent fever and infections, using the minimal effective dose (usually lg/kg/day, administered on a daily or alternate day basis), a dose sufficient to increase the ANC to about /l. I do not favour prophylactic antibiotics or recommend corticosteroids or intravenous gamma globulin for the treatment of these patients. Congenital causes for childhood neutropenia Congenital or hereditary neutropenia usually presents in the first year of life as recurrent fever attributable to a common type of infection. It is sometimes difficult to know if a child has idiopathic, autoimmune or congenital neutropenia; but usually the congenital disorders are associated with more severe and recurrent infections. The frequent events are fevers, mouth ulcers, gingivitis, sinusitis, pharyngitis, and cellulitis, respiratory and perianal infections. It is particularly important to recognize the severe oral manifestations associated with the congenital disorders (Dale & Welte, 2016). Patients and their families regularly have stories about the long interval between the first disease manifestation and a specific genetic diagnosis, but this situation is changing rapidly with better information sources and the increasing availability of genetic testing. With these rapid changes, it is helpful to use ª 2017 John Wiley & Sons Ltd 353

4 clear, unambiguous names for these genetic disorders. In this review I have used the terms ELANE-neutropenia, HAX1- neutropenia, G6PC3-neutropenia for the conditions for which the genetic cause has been identified, with additional modifiers as necessary. Within the congenital causes for childhood neutropenia there are several distinctive syndromes; the physical examination and laboratory testing can often guide the clinician to the specific genetic test required to confirm the diagnosis. The family history is also very helpful. Mutations in ELANE, the gene for neutrophil elastase, are the most common cause for cyclic and severe congenital neutropenia (SCN), both primarily autosomal dominant diseases (Dale, 2002). Males and females are equally affected, and it is likely that one of the parents of an affected child will also have an ELANE mutation. ELANE is only expressed in myeloid tissues, and therefore mutations only affect neutrophils and monocytes. After gathering the basis clinical data, I find that sequencing of ELANE is the most expeditious course to a genetic diagnosis in the majority of children with congenital neutropenia. By contrast, mutations in numerous other genes affect both haematopoietic and non-haematopoietic tissues and cause a wide diversity of congenital manifestations (Klein, 2011). Many of these conditions are inherited in an autosomal recessive pattern, so the patient is likely to be the only affected family member. For these reasons it is very important to search for clues to a genetic diagnosis through the family history and examine the patient carefully for subtle congenital anomalies. The following sections describe the hereditary neutropenia syndromes presenting in childhood and provide specific recommendations regarding testing and treatment. illness and the mouth ulcers for review at office visits. CyN patients are particularly prone to develop Clostridium sp. infections, attributable to ulcers in the colonic mucosa similar to the ulcers in the mouth. The consistently regular oscillations of the ANC over many years, and even decades, are a remarkable feature of this disorder (Leale, 1910; Rutledge et al, 1930; Bonilla et al, 1994; Palmer et al, 1996). As noted above, in the majority of cases, CyN is attributable to mutations in the gene for the enzyme neutrophil elastase, ELANE (Horwitz et al, 1999; Dale & Link, 2009). I now call this disease ELANE-associated cyclic neutropenia (ELANE-CyN). In these patients, mutant neutrophil elastase is not processed and packaged normally in the cells primary granules. The mutant enzyme impairs survival of myeloid precursors through initiation of programmed cell death (Nustede et al, 2016). Bone marrow aspirates show autophagy of effete myeloid cells and other changes associated with apoptosis of marrow neutrophils. The cellular and molecular mechanisms underlying cycling have puzzled many haematologists and mathematicians. Mackey (1978) was the first to suggest that oscillations occur because of interruptions of the normal feedback loop governing neutrophil production. In Mackey s model, neutrophil production is regulated by longrange stimulators with a built-in delay between stimulation and product because of the time necessary for myeloid cells to differentiate from the progenitor stage to the mature neutrophil (Haurie et al, 1999). Mackey postulated that if there were interruptions in production at an early stage in this process there would be oscillations in the ANC. Consistent with this hypothesis, cycling of the ANC could occur with other genetic or acquired disorders affecting the early stages of myeloid cell proliferation. ELANE associated neutropenia Mutations in ELANE cause both cyclic and SCN. These diseases have overlapping features but are discussed separately because of the difference in severity of infectious complications, prognosis and response to G-CSF. Cyclic neutropenia Cyclic neutropenia (CyN) is a rare disorder characterized by oscillating levels of blood neutrophils and monocytes, usually at 21-day intervals (Wright et al, 1981). At one phase of the cycle, the patient s blood ANC is extremely low, often zero, for 3 5 days and then in a second phase neutrophil counts recover rapidly to about /l. A few days after this peak, the ANC falls again. When the ANC is at its nadir, patients regularly have painful mouth ulcers, respiratory symptoms, and may develop cellulitis, abscesses and severe, even fatal, infections. In considering this diagnosis, it is helpful to have the parent keep a calendar record of signs of CyN usually presents as fever and infection in a child s first year of life. It is diagnosed quickly when a relative is already known to have CyN. Diagnosis is based primarily on analysis of serial ANCs performed at least three times per week for 6 weeks showing typical, approximately 21-day oscillations. Usually there are also oscillations of blood monocytes, and platelets and reticulocytes with the same periodicity, which are probably secondary to the more extreme oscillations in neutrophils and associated recurrent inflammation during period of severe neutropenia. Visual inspection of the counts displayed graphically is often sufficient for diagnosis, but more sophisticated analysis can be made using the Lomb periodogram (Guerry et al, 1973). I recommend sequencing for ELANE mutations, at least in one member of every family. If there is no mutation, it is best to use do a neutropenia gene panel to search for another genetic cause or consult with a haematologist or geneticist specializing in this field. Patients with ELANE-CyN of all ages are at risk for both minor and serious infections, but the risk is greater for 354 ª 2017 John Wiley & Sons Ltd

5 children. Sepsis from bowel perforation and rapidly spreading anaerobic cellulitis in the pelvic region is the most serious risk, a condition that can be rapidly fatal (Langer et al, 1990; Bar-Joseph et al, 1997; Fata et al, 1997; Li et al, 2004). Some patients with even a relatively benign course may have a serious septic event without warning. For this reason, treatment with G-CSF, usually 1 3 lg/kg/day given daily or every other day, is warranted (Hammond et al, 1989; Dale et al, 2003). G-CSF is primarily a preventative therapy. But patients who have a septic episode who are not on G-CSF should be treated with it immediately, in conjunction with antibiotics for both aerobic and anaerobic bacteria. Through the SCNIR we have observed that sepsis and death from sepsis have been eliminated with regularly administered G-CSF. Intermittent G-CSF or G-CSF timed to just treat the neutropenic period is difficult and would probably be less effective. Patients with ELANE-CyN are not at risk or are at extremely low risk for developing myeloid malignancies (Makaryan et al, 2015; Klimiankou et al, 2016). For this reason, I do not recommend annual bone marrow examinations. Severe congenital neutropenia SCN usually presents within the first year of life as fever and infections that do not resolve or resolve only very slowly (Donadieu et al, 2011; Klein, 2011; Boztug & Klein, 2013). The term SCN is used to include all patients with severe neutropenia beginning in early childhood. Some of the specific diseases causing SCN just involve the haematopoietic system. Other diseases causing SCN have additional manifestations in the gastrointestinal, cardiac, neurological, immunological or other organ systems. This is an important division, because patients with only haematopoietic abnormalities are more likely to have mutations in ELANE. On the other hand, congenital anomalies and functional abnormalities in other systems are rare in patients with ELANE-associated neutropenia. Overall, heterozygous mutations in ELANE are the more common cause of SCN, ELANE-SCN (Xia et al, 2009), but about 30 40% of SCN patients are ELANE negative (Dale & Link, 2009). The original family described by Kostmann had autosomal recessive neutropenia due to HAX1 mutations, but one member of this family had both mutations in ELANE and HAX1 (Carlsson et al, 2007). As research on SCN proceeds, we expect to find many additional causes for SCN and we can anticipate that increasing numbers of patients with digenic and multigenic causes for neutropenia will be discovered (Germeshausen et al, 2010). Patients with ELANE-SCN characteristically have a severe marrow defect readily seen with a bone marrow aspirate smear. The marrow shows an abundance of early myeloid precursors but few cells beyond the early myelocyte stage of development, an abnormality descriptively called maturation arrest. In these patients, the ANC is often < /l and blood monocytes are often > /l. Patients have poorly healing pneumonias and deep tissue infections. The marrow defect is attributable to accelerated apoptosis of developing promyelocytes and myelocytes, just at the stage when these cells are producing mutant neutrophil elastase (Nanua et al, 2011; Nayak et al, 2015). There are also associated abnormalities in myeloid-specific transcription factors and decreases in specific cellular proteins (Karlsson et al, 2007; Skokowa & Welte, 2009; Klimenkova et al, 2014). Mutations in the receptor for G-CSF (CSF3R) are early markers and monosomy 7, RAS and RUNX1 mutations are late and very late events in leukaemic evolution (Dong et al, 1995; Germeshausen et al, 2008a; Skokowa et al, 2014; Touw, 2015). The diagnosis of SCN is usually based on a series of ANCs showing very severe neutropenia and a bone marrow aspirate showing maturation arrest. Genetic sequencing (selective, single gene, gene panels or exome sequencing) is helpful for diagnosis and prognosis. In patients with mutations in ELANE, there is now sufficient genotype-phenotype information available to identify mutations which are more likely to be associated with poor response to G-CSF, risk of death from infections and a high risk of developing myelodysplasia (MDS) and acute myeloid leukaemia (AML) (Makaryan et al, 2015). Specifically, ELANE mutations G214R and C151Y as well as frameshift and termination mutations are associated with a poor prognosis (Bellanne-Chantelot et al, 2004; Makaryan et al, 2015). For example, all three known cases of SCN associated with ELANE mutation C223ter have developed AML (Dale et al, 2016). In patients with high-risk mutations, it is prudent to consider haematopoietic stem cell transplantation as early as possible. However, it is not yet possible to state an individual patient s risk of MDS and AML, and when, or if, these complications will occur. Longterm treatment with G-CSF, generally in low doses on a daily or alternate day basis, is highly effective so long as the patient is compliant with the repeated subcutaneous injections and any associated symptoms (Bonilla et al, 1989; Dale et al, 1993, 2003; Borzutzky et al, 2006). The risk of severe infections is virtually eliminated if the patients ANC can be maintained at about /l. Because the overall risk of MDS/AML is approximately 20 30% in these patients (Rosenberg et al, 2006, 2008, 2010), it is prudent to do complete blood counts several times per year and annual surveillance bone marrow aspirates with cytogenetics on an indefinite basis. The indications for haematopoietic stem cell transplantation in patients with ELANE-SCN have gradually changed with introduction of reduced-intensity conditioning and recognition of better outcomes if patients are transplanted before they develop overt leukaemia (Connelly et al, ª 2017 John Wiley & Sons Ltd 355

6 2012; Fioredda et al, 2015; Osone et al, 2016). Patients responding poorly to G-CSF, i.e. requiring doses greater than 8 10 lg/kg/day appear to be a greater risk of evolution to AML as well as patients with monosomy 7 and RUNX1 mutations. CSF3R mutations confer risk, but because these mutations may come and go and there may be many years between their first detection and MDS or AML, authorities differ in their clinical recommendations for individual patients. The decision to transplant also always depends upon the availability of a suitable, well-matched donor. The congenital neutropenias with non-myeloid manifestations The specific causes for chronic neutropenia in children can often be diagnosed from the clinical feature and genetic sequencing. These diseases are briefly described below. If a patient does not have a mutation in ELANE and if the diagnosis cannot be made from the clinical examination and targeted gene sequencing, I usually recommend exome sequencing for a panel of genes associated with neutropenia. Information on testing is available from The Severe Chronic Neutropenia International Registry at ington.edu/registry or re-congenital-neutropenia#diagnosis. therefore annual bone marrow examinations with cytogenetics are recommended. G6PC3-neutropenia This is a rare autosomal recessive condition attributable to mutations in the glucose 6 phosphatase catalytic subunit 3 gene, G6PC3 (Boztug et al, 2009; Desplantes et al, 2014). The gene encodes glucose-6-phosphatase (G6Pase), which catalyses the hydrolysis of G6P to glucose and phosphate. Almost all of the known mutations abolished G6PC3 enzymatic activity. The original report described accelerated apoptosis of developing neutrophils and maturation arrest, but some patients with less severe marrow changes, less severe neutropenia and a variety of other haematological, cardiac and urogenital anomalies have now been described (Arikoglu et al, 2015; Kiykim et al, 2015; Glasser et al, 2016). In the French series of 14 patients, features were prominent veins (n = 12), cardiac malformations (n = 12), and intellectual impairment (n = 7), chronic diarrhoea with steatorrhoea (n = 5) and Crohn disease (n = 3) (Desplantes et al, 2014). They do not have the problem of hypoglycaemia associated with other forms of glycogen storage disease. HAX1-neutropenia Kostmann syndrome, autosomal recessive SCN, is now attributable to mutations in HAX1, HCLS1 Associated Protein X-1, a protein associated with Lyn substrate 1 and with the F-actin-binding protein, cortactin (Klein et al, 2007). Mutations in this gene cause maturation arrest at the promyelocyte stage of neutrophil development, very similar to those seen in patients with mutations in ELANE. HAX1 encodes a protein that plays critical functions in mitochondria, signal transduction and the cyto-skeleton. Mutations in this protein result in a loss of protection from apoptosis of myeloid cells. This mutation causes ineffective neutrophil production and an extremely low output of neutrophils from the bone marrow. Patients may have also developmental delays, cognitive impairments and epilepsy (Boztug et al, 2010; Lebel et al, 2015; Aydogmus et al, 2016), with subtype determined by specific mutations and transcript variants (Germeshausen et al, 2008b; Roques et al, 2014). The diagnosis is based on the clinical history and examination, ANC measurements, bone marrow examination and genetic sequencing. Treatment with G-CSF is effective, usually about 5 lg/kg/day. There is approximately the same risk of MDS and AML as with ELANE-SCN (Lebel et al, 2015); Diagnosis is based on the constellation of clinical features, serial blood counts, bone marrow examination and genetic sequencing. Most patients respond well to G-CSF administered prophylactically on a long-term basis. There are cases of MDS/AML, thus warranting careful long-term surveillance with annual bone marrows (Desplantes et al, 2014). Glycogen storage disease 1b and SLC37A4 (G6PT1)-neutropenia Glycogen storage disease 1b (GSD 1b) is a rare hereditary disorder characterized by recurrent and severe hypoglycaemia, hepatomegaly, enterocolitis, neutropenia and recurrent infections. It is attributable to mutations in the gene for the solute carrier family 37 member 4 (SLC37A4, previously termed glucose 6 phosphate transporter, G6PT1), whose product is responsible for the transport of glucose from the cytoplasm into the endoplasmic reticulum of neutrophils and other cells (Melis et al, 2005; Chou et al, 2010). Before birth, maternal glucose transfer across the placenta prevents hypoglycaemia, but the liver is enlarged at birth and symptoms of hypoglycaemia, unconsciousness or seizures may occur before diagnosis. Chronic neutropenia is present soon after birth, and is attributable to apoptosis of developing and mature neutrophils (Visser et al, 2012). Neutrophil and monocyte 356 ª 2017 John Wiley & Sons Ltd

7 dysfunction add to the risk for recurrent bacterial infections (Visser et al, 2012). Inflammatory bowel disease, very similar to Crohn disease, is another common consequence. GSD 1b is readily diagnosed by the constellation of presenting features and can be confirmed by genetic sequencing. General management includes frequent feeding, often with nocturnal gastric infusions of cornstarch. Treatment with G-CSF corrects neutropenia and usually improves the symptoms of inflammatory bowel disease (Roe et al, 1992; Calderwood et al, 2001; Rake et al, 2002; Alsultan et al, 2010). GSD 1b patients usually have splenomegaly, which may increase markedly with G-CSF; the G-CSF dose should therefore be as low as possible to maintain neutrophils at about /l, usually maintaining the dose at 1 2 lg/kg/day. Liver transplantation corrects the disorder, but it does not resolve chronic neutropenia. AML has been reported in GSD 1b, a patient on long-term treatment with G-CSF (Pinsk et al, 2002). Barth syndrome and TAZ-neutropenia Barth syndrome is a rare X-linked disease attributable to defective phospholipid metabolism primarily affecting mitochondrial membranes (Saric et al, 2016). Its major features are childhood cardiomyopathy, proximal myopathy, delayed motor development, growth delay and neutropenia, resulting in chronic and recurrent infections. The severity of neutropenia varies considerably, and many patients have increased blood monocyte counts. The infections involve both grampositive and gram-negative bacteria; there is no particular predilection to fungal infections. Bone marrow aspirates usually show decreased mature neutrophils with late stage developmental arrest. Blood neutrophil levels may show cyclic oscillations, but this is a rare finding. Diagnosis is usually based on symptoms and physical findings of heart failure in a young male, but some patients present with fever, neutropenia and signs of an acute bacterial infection (Rigaud et al, 2013). Respiratory infections and pneumonia are a particular problem. The diagnosis is suggested by a five-fold or greater increase in urinary 3-methylglutaconic acid and an increased monolysocardiolipin:cardiolipin ratio or DNA sequencing showing a disease-associated variant of TAZ (Saric et al, 2016). Heart transplantation can correct the myocardial abnormality, but does not correct the skeletal problems or neutropenia. Treatment with G-CSF is reserved for patients with neutropenia; observational studies suggest that it is highly effective. Shwachman-Diamond syndrome and SBDSneutropenia Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder attributable to mutations in the SBDS gene. The protein product of this gene is a critical component of ribosomes and the associated defect in ribosomal functions affect multiple organ systems (Shimamura & Alter, 2010; Finch et al, 2011). SDS is usually recognized because of failure to thrive with steatorrhoea, skeletal abnormalities and bacterial infections very early in life (Myers, 2008; Dror et al, 2011; Donadieu et al, 2012). There are numerous other manifestations of this syndrome (Shimamura & Alter, 2010; Dror et al, 2011; Donadieu et al, 2012). Usually neutropenia is the first detected haematological abnormality, but bi-lineage and tri-lineage haematological abnormalities are frequent. MDS and AML are well known complications. The provisional diagnosis of SDS is usually based on evidence of exocrine pancreatic insufficiency, skeletal abnormalities, and neutropenia, but the diversity of clinical presentations are increasingly recognized. Findings of typical mutations in the SBDS gene are diagnostic, but there are patients with typical findings of SDS who lack mutations in SBDS (Myers et al, 2014). Clonal abnormalities in the marrow are relatively common, with the most common molecular changes being abnormalities in 7q and 20q; for this reason surveillance with blood counts and regular bone marrow examinations with cytogenetics are recommended. Management requires a multidisciplinary team: haematology, gastroenterology, neurology and other specialists. Pancreatic insufficiency can usually be managed with supplemental vitamins and replacement of pancreatic enzymes. G-CSF is effective to prevent recurrent infections, together with antibiotics as necessary. There is no convincing evidence to show that G-CSF causes cytogenetic changes or malignant transformation. Serial bone marrow examinations are helpful for the early diagnosis of clonal disorders. Haematopoietic stem cell transplantation is indicated for severe cytopenias, MDS or AML, with a suitable donor. Wiskott-Aldrich syndrome and WASneutropenia Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency disease usually characterized by small platelets, eczema, recurrent infections, autoimmunity and a predilection to malignancies (Massaad et al, 2013). The diversity of manifestations reflects the complexity of the structure and multiple functions of the WAS protein ª 2017 John Wiley & Sons Ltd 357

8 (WASp). X-linked neutropenia is a variant of WAS which may evolve to MDS (Devriendt et al, 2001; Beel et al, 2009). This variant is attributed to activating mutations in the GTPase binding domain of WASp. Devriendt et al (2001) described a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain; a few other mutations have been associated with WAS neutropenia (Ancliff et al, 2006). The pattern of infections is quite variable and may well relate to the severity of the individual patient s marrow defect. Laboratory investigations indicate that the mutant protein is constitutively activated resulting in apoptosis of developing myeloid cells. As with other hereditary myeloid disorders there is an association of apoptosis and the risk of evolution to MDS (Ancliff et al, 2006). The diagnosis of WAS is usually considered in a patient with eczema recurrent infections, small platelets, and evidence for autoimmunity. The diagnosis of WAS-related neutropenia has been missed because a patient lacked these classical features of the syndrome. Thus diagnosis requires consideration of the possibility and genetic sequencing. Management includes a careful assessment of the patient s immune system, surveillance for early detection of infections and antibiotics, as necessary. G-CSF can be used to correct neutropenia and prevent infections. Regular follow-up and periodic bone marrow examinations are necessary for early detection of MDS. Bone marrow transplantation is curative (Massaad et al, 2013). Other causes of congenital neutropenia TCIRG1-neutropenia is an autosomal dominant neutropenia described thus far in a single large US family (Makaryan et al, 2014). All affected members had heterozygous mutations. Homozygous mutations in TCIRG1 cause osteopetrosis. The described mutation affects the hinging region of a protein component of ATPase that controls ph of intracellular organelles. CXCR4-neutropenia: WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome or myelokathexis is an immunodeficiency disorder characterized by severe lymphocytopenia and neutropenia complicated by warts and recurrent bacterial infections (Dotta et al, 2011). This syndrome is caused by mutations in CXCR4, the gene encoding CXCR4, a receptor for CXCL12 (previously called SDF1) (Al Ustwani et al, 2014). CXCR4 plays a critical role in controlling the trafficking of neutrophils, lymphocytes and CD34 + haematopoietic progenitor cells. A novel therapy, a CXCR4 antagonist, has promise as a molecularly targeted treatment for WHIM syndrome (McDermott et al, 2014). JAGN1-neutropenia is a rare disorder characterized by neutrophils with few granules, aberrant glycosylated proteins and increased apoptosis of developing neutrophils. Patients are poorly responsive to G-CSF (Boztug et al, 2014). Cohen syndrome and VPS13B-neutropenia is a rare autosomal recessive disorder with non-progressive psychomotor retardation, microcephaly retinal dystrophy and intermittent neutropenia due to mutations in VPS13B, previously called COH1 (Wang et al, 2006). Neutropenia may be severe and responds to G-CSF. GFI1-neutropenia is attributable to mutations in the gene encoding the transcriptional repressor oncoprotein GFI1. Patients have immunodeficiency and circulating immature myeloid cells (Person et al, 2003). VPS45-neutropenia has occurred in infants from consanguineous families and is characterized by myelofibrosis, defective platelet aggregation and life-threatening neutropenia attributed to the accelerated apoptosis of developing neutrophils. It is reported to be refractory to G-CSF (Stepensky et al, 2013; Vilboux et al, 2013). WDR1-neutropenia syndrome is characterized by recurrent infections, severe stomatitis, variable degrees of neutropenia and impaired wound healing. It is attributable to abnormalities in an actin-interacting protein as a result of a mutation in WDR1 (Kuhns et al, 2016). There are also a number of syndromes involving the formation of lysosomal granules and vesicles within neutrophils and other leucocytes associated with milder forms of neutropenia (Dell Angelica et al, 1999; Bellanne-Chantelot et al, 2007; Gauthier-Vasserot et al, 2017). Undoubtedly, new syndromes will be added to this list with increasing understanding of myeloid cells biology. Drug-and chemotherapy-associated neutropenia in children In adults, idiosyncratic reactions to drugs are probably the most common cause for neutropenia, often occurring in patients receiving multiple drugs. Children are probably at risk for the same type of events, although they have far less exposure to drugs. A recent prospective study in Spain (Medrano- Casique et al, 2016) revealed an incidence of drug-associated neutropenia of 4 per 10 4 hospitalized patients. Antibiotics, particularly beta-lactams, were the most frequent cause. Another recent study showed similar findings (Murphy et al, 2016). Children and adults are at risk of chemotherapy-associated neutropenia. Fortunately for children, the resiliency of their marrow seems to be greater, the recovery from severe neutropenia quicker and the risk of severe infections is less than adults. In most children the period of severe neutropenia is brief and antibiotic treatments are sufficient. When neutropenia is severe and prolonged, as occurs in paediatric AML, G- CSF prophylaxis is beneficial (Sung et al, 2013). General principles for management of neutropenia in children 1 Family history and the pattern of fever and infections in the patient are critical for accurate diagnosis and proper management. 358 ª 2017 John Wiley & Sons Ltd

9 2 It is always good practice to obtain multiple white blood counts with differentials to determine if a patient has neutropenia and its severity. Infections tend to occur when neutrophil counts are the lowest, but patients come to the physician when they are ill and their counts are higher than baseline. 3 The value of genetic testing for the diagnosis of neutropenia is increasing rapidly as the genetically determined disorders of myeloid biology are described more and more precisely. 4 G-CSF is the natural regulator of neutrophil production and deployment. It is widely used to treat neutropenia because of its potency and favourable adverse event profile. In the chronic neutropenias, both congenital and acquired, low dose G-CSF, e.g., <3 lg/kg/day, is often sufficient to raise neutrophils to approximately / l and prevent infections. 5 For most patients with neutropenia, infections occur from surface organisms. 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