Hereditary erythrocytosis, thrombocytosis and neutrophilia

Best Practice & Research Clinical Haematology 27 (2014) 95e106

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Hereditary erythrocytosis, thrombocytosis and neutrophilia Wan-Jen Hong, M.D., Adjunct Clinical Assistant Professor, Jason Gotlib, M.D., M.S., Associate Professor of Medicine (Hematology) * Division of Hematology, Stanford Cancer Institute, Stanford University School of Medicine, 875 Blake Wilbur Drive, Stanford, CA 94305, USA

Keywords: hereditary erythrocytosis familial polycythaemia thrombocytosis neutrophilia JAK2 MPL TPO

Hereditary erythrocytosis, thrombocytosis, and neutrophilia are rare inherited syndromes which exhibit Mendelian inheritance. Some patients with primary hereditary erythrocytosis exhibit a mutation in the erythropoietin receptor (EPOR) which is associated with low serum erythropoietin (EPO) levels. Secondary congenital erythrocytosis may be characterized by normal or high serum EPO levels, and is related to high oxygen affinity haemoglobin variants, mutation of the enzyme biphosphoglycerate mutase (BPGM), or defects in components of the oxygen-sensing pathway. Hereditary thrombocytosis was first linked to mutations in genes encoding thrombopoietin (THPO) or the thrombopoietin receptor, MPL. More recently, germline mutations in JAK2, distinct from JAK2 V617F, and mutation of the gelsolin gene, were uncovered in several pedigrees of hereditary thrombocytosis. Hereditary neutrophilia has been described in one family with an activating germline mutation in CSF3R. The mutational basis for most hereditary myeloproliferative disorders has yet to be identified. © 2014 Published by Elsevier Ltd.

Introduction Hereditary (or congenital) myeloproliferative disorders (e.g. erythrocytosis, thrombocytosis, and leucocytosis/neutrophilia) are uncommon inherited syndromes that typically affect a single blood * Corresponding author. Stanford Cancer Institute, Stanford University School of Medicine, 875 Blake Wilbur Drive, Room 2324, Stanford, CA 94305-5821, USA. Tel.: þ1 650 736 1253; Fax: þ1 650 724 5203. E-mail addresses: [email protected] (W.-J. Hong), [email protected] (J. Gotlib).

http://dx.doi.org/10.1016/j.beha.2014.07.002 1521-6926/© 2014 Published by Elsevier Ltd.

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lineage, and exhibit Mendelian transmission with high penetrance and polyclonal haematopoiesis. Ruling out secondary causes of elevated blood counts, and obtaining a thorough family history is critical to unmasking the presence of these disorders as well as their mode of genetic transmission. These diseases should be distinguished from acquired myeloproliferative neoplasms (MPNs), which are clonal disorders derived from one or more somatic mutations (e.g. JAK2 V617F, MPL W515L/K) that usually arise in a haematopoietic stem cell (HSC) or early pluripotent progenitor blood cell. MPNs are characterized by the proliferation of one or more of the myeloid lineages and can transform into acute myeloid leukaemia (AML). Hereditary myeloproliferative disorders should also be distinguished from familial MPNs which are exemplified by pedigrees with an increase in the number of MPN cases compared to the expected frequency in the general population. In such families, autosomal dominant or autosomal recessive patterns of inheritance have been observed. In some family members, JAK2 V617F or other characteristic MPN-associated somatic mutations may be found, but in others family members with an overt MPN, no acquired mutations or shared germline mutation(s) have been identified. The increased predisposition to MPNs in these pedigrees can manifest as several family members with the same or different MPN (e.g. polycythaemia vera, essential thrombocythaemia, primary myelofibrosis, chronic myelogenous leukaemia, or systemic mastocytosis). The basis for the increased genetic susceptibility to MPNs within certain families remains an active area of investigation, and several inherited polymorphisms have been identified which contribute to this higher risk of MPN development (e.g. JAK2 46/1 or GGCC haplotype, TERT, SH2B3, etc) [1e6]. The focus of this review is hereditary myeloproliferative disorders. Primary erythrocytosis is caused by a gain of function mutation in the erythropoietin receptor (EPOR). Patients with secondary congenital erythrocytosis may carry mutations in haemoglobin chains or biphosphoglycerate mutase (BPGM) that result in high oxygen affinity haemoglobin variants. Mutations in genes encoding components of the oxygen-sensing pathway, such as von Hippel-Lindau (VHL), hypoxia inducing factor-2a (HIF2A; EPAS1) and prolyl hydroxylase 2 (PHD2; EGLN1) have also been identified as causes of secondary congenital erythrocytosis. Mutations in the genes for thrombopoietin (THPO), and the receptor for thrombopoietin (MPL), have been discovered in pedigrees with hereditary thrombocytosis (HT). More recently, germline mutations in JAK2, distinct from the V617F mutation, have also been identified in families with hereditary thrombocytosis. Patients with leucocyte adhesion deficiencies (LAD) exhibit germline mutations and have recurrent infections as well as neutrophilia. An activating mutation in CSF3R, the receptor for granulocyte colony stimulating factor (G-CSF), was found in one family with hereditary neutrophilia, and is the same acquired variant uncovered in patients with atypical chronic myelogenous leukaemia [7,8]. Hereditary erythrocytosis Hereditary or congenital erythrocytosis (CE) can be classified into primary or secondary disorders (Table 1). Patients with primary erythrocytosis (or primary familial congenital polycythaemia (PFCP)) have a defect in haematopoietic progenitors and exhibit low serum erythropoietin (EPO) levels. Secondary congenital erythrocytosis is characterized by inappropriately normal or high serum EPO levels Table 1 Online Mendelian Inheritance in Man (OMIM) classification, gene, chromosome location and proteins associated with hereditary/congenital erythrocytosis [9,23]. Disease group

OMIM number

Gene

Chromosome location

Protein

Inheritance

ECYT1 ECYT2 ECYT3

133100 263400 609820

EPOR VHL EGLN1

19p13.2 3p25.3 1q42.1

Autosomal dominant Autosomal recessive Autosomal dominant

ECYT4 High O2 affinity Hb variant BPGM

611783

EPAS1 HBB, HBA

222880

BPGM

2p21 11p15.3 16p13.3 7q33

Epo Receptor (EPOR) Von Hippel Landau (VHL) Prolyl hydroxylase domain-containing protein 2 (PHD2) Hypoxia inducible factor (HIF-2a) Haemoglobin (Hb) Bisphosphoglycerate mutase (BPGM)

Autosomal recessive

Autosomal dominant Autosomal dominant

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due to high oxygen affinity haemoglobin variants or defects in the oxygen-sensing pathway. A diagnostic algorithm for the work-up of patients with suspected congenital erythrocytosis is shown in Fig. 1. Over 160 mutations associated with congenital erythrocytosis have been described. However, this accounts for only approximately 30% of these patients. The molecular defects in the majority of CE patients have yet to be identified [9]. The European Congenital Erythrocytosis Consortium (ECE-C) established by MPN & MPNr-EuroNet manages a registry of CE patients. The consortium website is available at www.erythrocytosis.org and displays a list of published mutations as well as laboratories that provide relevant genetic testing. Primary erythrocytosis/primary familial congenital polycythaemia (PFCP) EPO is the primary regulator of erythropoiesis and is involved in the maturation and differentiation of erythroid progenitor cells. EPO's effects are mediated through binding to the EPO receptor (EPOR). PFCP is an autosomal dominant disorder caused by mutations in EPOR. The first mutation in EPOR was reported in a large Finnish family with 29 affected family members with erythrocytosis [10]. Since the initial report, more than 22 heterozygous mutations have been identified. All of these mutations are located in exon 8 and the majority of these mutations lead to the truncation of the C-terminal negative regulatory domain [9]. These mutations induce a gain of function phenotype resulting in the increase in sensitivity of erythroid progenitors to EPO. Although a slight increase in vascular complications have been observed in affected individuals [11], there are otherwise no major health effects in these patients, including no increased risk for transformation to acute leukaemia [12]. High oxygen (O2) affinity haemoglobin variants Haemoglobin consists of a tetramer formed by a1b1 and a2b2 subunits. There are two conformations of haemoglobin: the R or “relaxed” conformation is a high O2 affinity state and the T or “tense” conformation is a low O2 affinity state. The first high affinity O2 haemoglobin variant as a cause of erythrocytosis was first reported in 1966 by Cherache et al. when a patient with erythrocytosis was found to have an abnormal haemoglobin band on electrophoresis and also a left shifted oxygen dissociation curve. The abnormal haemoglobin was isolated and structural analysis revealed an a-chain variant, now called haemoglobin (Hb) Chesapeake [13]. High O2 affinity haemoglobin variants are typically autosomal dominant and carry substitutions that are involved in the regulation of oxygen

Fig. 1. A diagnostic algorithm for isolated erythrocytosis after acquired causes is ruled out. EPO, erythropoietin; PFCP, primary familial and congenital polycythaemia; p50, partial pressure of oxygen in the blood at which the haemoglobin is 50% saturated; Hb, haemoglobin, 2,3-BPG, 2,3-bisphosphoglycerate; WT, wild-type [12,16].

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transport and stimulate the erythropoietic drive [14]. These variants are caused by substitutions in aor b-globin chains that stabilize the high O2 affinity state relative to the low O2 affinity state. Some of the substitutions involve the a1b2 interface, where T to R state transitions are mediated, or the Cterminal end of the b-chain which impairs the release of oxygen [15]. Normally, 2,3bisphosphoglycerate (2,3-BPG) binds globin chains and stimulates the release of oxygen. Substitutions in the 2,3-BPG binding site inhibit the interaction with globin chains and reduce the release of oxygen [15]. Patients with high O2 affinity haemoglobin variants have increased oxygen affinity of the haemoglobin molecule, resulting in low tissue O2 tension and a left shift of the oxygen dissociation curve to the left with a reduction of p50 which in turn increases the erythropoietic drive [16]. Almost 100 high O2 affinity haemoglobin variants have been now identified [14]. A complete and updated listing is available on the Globin Gene Server in the Hb Var database at (http://globin.cse.psu. edu/). BPGM Deficiency in bisphosphoglycerate mutase (BPGM) is a rare cause of congenital secondary erythrocytosis. BPGM controls the level of 2,3-BPG, previously known as 2,3-diphosphoglycerate (DPG), and is involved in the regulation of oxygen delivery by haemoglobin. When 2,3-BPG binds to haemoglobin, haemoglobin is allosterically converted to a low oxygen affinity state and shifts the oxygen dissociation curve to the right. Mutations in BPGM result in the deficiency of BPGM and reduction in 2,3-BPG which leads to a left shift in the oxygen dissociation curve. Haemoglobin remains in a high oxygen affinity state, causing tissue hypoxia and compensatory erythrocytosis. The first patient with complete BPGM deficiency was described in 1978. This patient was found to have minimal mutase activity [17]. This abnormal enzyme was caused by a mutation leading to the amino acid substitution R89C and is called BPGM Creteil [18]. Additional testing in the family revealed that his three sisters had the same mutation and phenotype, while his two children had an intermediate phenotype. The phenotype of the proband and sisters was discovered to be caused by a compound heterozygote with one allele coding for an inactive enzyme (BPGM Creteil I) and the other with a frameshift mutation (BPGM Creteil II) [19]. A second family of Meshadi heritage with complete BPGM deficiency was found to have a homozygous R61Q substitution [20]. Methaemoglobinemia Methaemoglobinemia is another condition that leads to impaired oxygen delivery from haemoglobin. Methaemoglobin is formed through the oxidation of ferrous iron (Fe2þ) in haemoglobin to the ferric state (Fe3þ). Under normal physiologic conditions, methaemoglobin is efficiently reduced by methaemoglobin reductase enzymes, such cytochrome b5 reductase, and accounts for only 1% of the total haemoglobin [21]. Because the ferric iron in methaemoglobin does not bind oxygen, an increase in methaemoglobin leads to reduction in oxygen supply to tissues, resulting in cyanosis and also secondary erythrocytosis [21e23]. Hereditary methaemoglobinemia can occur in patients with a Hb M variant, deficiency of cytochrome b5 reductase, or cytochrome b5 deficiency. Most patients with haemoglobin M have variants in which the proximal or distal histidine in either the a or b subunit is replaced by a tyrosine residue. The presence of the tyrosine residue in the heme pocket results in the stabilization of the oxidized ferric state [22]. Hb M is inherited in an autosomal dominant pattern and patients are cyanotic, but are largely asymptomatic. Recessive hereditary methaemoglobinemia is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (cytb5r). Two types of cytb5r deficiency are recognized. Type I is characterized by cyanosis and results from the deficiency in the red cell soluble cytb5r isoform. Patients with Type II are cyanotic and also have severe mental retardation and neurologic impairment resulting deficiency of both the soluble and membrane-bound forms of cytb5r [24]. Oxygen sensing pathway variants In normoxic conditions, proline hydroxylase (PHD) hydroxylates hypoxia induced factor (HIF-a) which provides a recognition site for von Hippel-Lindau (VHL). The VHL protein is part of an E3

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ubiquitin ligase complex that targets HIF-2a for degradation. Under hypoxic conditions, HIF-2a is not hydroxylated and therefore not degraded by VHL allowing stabilization of HIF-2a and activating translation of genes such as EPO (Fig. 2). VHL. Chuvash polycythaemia (CP) is an autosomal recessive disorder endemic to the Chuvash autonomous republic in Russia caused by the homozygous VHL mutation 598C > T (R200W) [25,26]. This loss-of-function mutation in VHL impairs the interaction of VHL with HIF-a, leading to the reduction in the rate of HIFa degradation and increased levels of HIF1, and other target genes such as EPO, solute carrier family 2 (SLC2A1), transferrin (TF), transferrin receptor (TFRC) and vascular endothelial growth factor (VEGF) [27]. CP patients have increased serum HIF-1a and EPO levels and erythrocytosis despite tissue normoxia. Patients have an increased incidence in vascular abnormalities, such as vertebral haemangiomas, varicose veins and lower blood pressures. The median age of death from a cerebral vascular event in CP patients was 42 years, compared to 70 years in a control population. Classical VHL disease is a typically a rare autosomal dominant syndrome which predisposes patients to the development of highly vascularized tumours. Mutations are distributed throughout the coding sequence except in the first 53 codons and the phenotype depends on the levels of HIF [28]. Unlike patients with the typical VHL syndrome, CP patients do not have an increased risk of developing malignancies, such as renal cell carcinomas, pheochromocytomas, pancreatic neuroendocrine tumours, CNS haemangioblastomas and other vascular tumours [29]. The VHL R200W mutation was subsequently identified in several non-Chuvash families around the world, including in Asia and Western Europe, which most likely originated from a single founder event [30e33].

Fig. 2. Oxygen-sensing pathway involving hypoxia-inducible factor (HIF). In normoxic conditions, prolyl hydoxylase protein (PHD) hydroxylates HIF-a on specific prolyl residues and factor inhibiting HIF (FIH) hydroxylates HIF-a on a specific asparaginyl residue. Von Hippel Lindau (VHL) protein, a component of E3 ubiquitin ligase complex, recognizes the hydroxylated prolyl, leading to the ubiquitination and degradation of HIF-a. Under hypoxic conditions, the prolyl residues are not hydroxylated which stabilizes HIF-a and dimerization with HIF-b. p300 can bind HIF-a under these conditions and the complex can bind the hypoxia response element (HRE), activating transcription of genes such as EPO [23].

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Additional mutations in VHL as the cause of secondary erythrocytosis have also been identified. In Croatia, VHL 571C > G (H191D) was identified in two families which arose from a common ancestor. Patients with VHL H191D have higher erythropoietin levels than VHL R200W patients and their erythroid progenitors are not sensitive to erythropoietin [32,34]. A total of 16 other mutations in VHL have been described which have been associated with secondary congenital erythrocytosis [9]. EGLN1 (PHD2). Three prolyl hydroxylase domain proteins (PHD) have been identified, PHD1, PHD2, and PHD3. PHD2 is encoded by the gene egg-laying defective nine protein 1 (EGLN1) and is the key enzyme in catalyzing the prolyl hydroxylation of HIF-a and uses oxygen as a co-substrate [35]. A loss-offunction mutation in PHD2 (EGLN1) was first reported in a family with erythrocytosis. A heterozygous 950C > G transversion was identified, resulting in a P317R substitution. The PHD2 P317R mutation results in a decrease in PHD2 enzyme activity. EPO levels were inappropriately normal given the elevated haemoglobin levels in the affected patients [35]. Multiple other mutations in PHD2 have since been identified [36,37]. A PHD2 H374R mutation was identified in a patient who presented with isolated erythrocytosis who subsequently developed a paraganglioma [38]. A different mutation resulting in N203K was identified in a patient who also had a JAK2 mutation in exon 12. This patient had a low EPO level suggesting that this patient's disease was likely driven by the somatic JAK2 mutation [39]. EPAS1 (HIF2A). Hypoxia-inducible factor (HIF) is composed of two subunits, a constitutively expressed b-subunit and a labile a-subunit [40]. HIF-a has three isoforms, HIF-1a, HIF-2a and HIF-3a. HIF-2a is the primary transcription factor that induces hepatic EPO expression [41,42]. A missense 1609G > T (G537W) gain-of-function mutation in exon 12 of EPAS1, which encodes HIF-2a, was described in a family with a history of erythrocytosis. In vitro assays showed decrease in binding of PHD2 to the HIF2a G537W mutant compared to wild type as well as decrease in hydroxylation of the mutant HIF-2a [43]. Additional missense mutations in EPAS1, including I533V, P534L, M535V, M535T, M535I, G537R, N539Q, and F540L have been identified in patients with hereditary erythrocytosis [33e38]. These residues are near the proline 531 residue which is the primary site of hydroxylation and results in the impaired degradation and aberrant stabilization of HIF-2a [44]. A novel germline mutation in EPAS1 F374Y in exon 9 was recently described in a patient who developed a pheochromcytoma/paraganglioma. The EPAS1 F374Y substitution leads to the stabilization of mutant HIF-2a, exhibiting decreased binding to VHL compared to wild type HIF-2a [45].

Hereditary thrombocytosis Hereditary thrombocytosis results from mutations in thrombopoietin (THPO) or the gene encoding the receptor for thrombopoietin (Myeloproliferative Leukaemia Virus Oncogene (MPL)). Thrombopoietin (TPO) is a growth factor that binds to receptors on megakaryocytes and platelets and is the principal hormone involved in megakaryocyte development, proliferation and differentiation. MPL is a type I cytokine receptor that has no intrinsic kinase activity. Upon stimulation by TPO, MPL is bound by JAK family members, including JAK2. Phosphorylation of several downstream signalling pathways ensues, including activation of the JAK-STAT axis, which in turn leads to cellular proliferation [46]. Mutations of THPO or MPL have been excluded in some families with hereditary thrombocytosis [47,48], implicating other molecular abnormalities as a basis for HT. Recently, novel germline mutations have been identified in JAK2 as well as the gene encoding gelsolin. A summary of mutations that mediate HT is summarized in Table 2. THPO The first mutation reported in THPO was reported in a Dutch family with 11 members over four generations with thrombocytosis [49,50]. In these patients, serum TPO concentrations were elevated and haemoglobin and leucocyte counts were normal. All affected family members exhibited a G to C transversion in the splice donor site of intron 3 of the THPO gene. This mutation leads to an mRNA transcript with a shortened 50 -untranslated region (UTR) that is more efficiently translated. Some of these patients experienced haemorrhagic complications as well as vaso-occlusive symptoms that

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Table 2 Genes involved in hereditary thrombocytosis [46]. Gene

Protein

Mutation

Inheritance

Reference

THPO

Thrombopoietin (TPO)

G > C in the splice donor site of intron 3 Deletion of single G in 5’ UTR G > T substitution in 5’ UTR A > G mutation in intron 3 T > C mutation in intron 2

Autosomal dominant

Schlemper et al. (1994) [49]

Autosomal dominant

Kondo et al. (1998) [52]

Autosomal dominant

Kikuchi et al. (1995) [54]

Autosomal dominant

Jorgensen et al. (1998) [83]

Autosomal dominant

Zhang et al. (2011) [56]

S505N

Autosomal dominant

P106L W515R K39N

Autosomal recessive Autosomal dominant Autosomal dominant with incomplete penetrance

Ding et al. (2004) [61] Teofili et al. (2010) [63] El-Harith et al. (2008) [65] Vilaine et al. (2012) [66] Moliterno et al. (2004) [67]

MPL

Thrombopoietin receptor, MPL

JAK2

Janus kinase 2

V617I R564Q H608N R867Q S755R/R938Q

Autosomal Autosomal Autosomal Autosomal Autosomal

dominant dominant dominant dominant dominant

GSN

Gelsolin

C > T transversion

Autosomal dominant

Mead et al. (2012, 2013) [69,70] Etheridge et al. (2014) [71] Rumi et al. (2014) [72] Marty et al. (2014) [73] Marty et al. (2014) [73] Pianta et al. (2013) [76]

responded to treatment with aspirin. The same mutation was also identified in a Polish family with 11 affected members [51]; this mutation arose independently in these two families. Clinically, these patients displayed microvasculatory disturbances manifested by episodes of lightheadedness and syncope which improved with aspirin. A deletion of a single G nucleotide in the 50 -UTR of THPO was identified in a Japanese family. The affected individuals had higher TPO levels compared to unaffected relatives [52]. Similar to the aforementioned cases, the mutation in the 50 -UTR region causes a frameshift and eliminates the inhibitory effect of upstream open reading frame 7 (uORF7) which leads to increase in mRNA translation [53]. A G to T transversion in position 516 of the 50 -UTR region was found in a different Japanese family. The G516T transversion shortens uORF7, which creates a premature stop codon and relieves the translational inhibition of uORF7 [54,55]. Another novel point mutation was identified at the T to C transition at the splice donor of intron 2 in the 50 -UTR in a Filipino family [56]. The parents of the proband did not carry the mutation or exhibit thrombocytosis, suggesting that this was a sporadic mutation that developed in utero. The affected mother (proband) and two children had a significant increase in serum TPO levels compared to unaffected members. Phospho-STAT5 levels in myeloid progenitors were also significantly higher in the affected patients. A single base pair deletion (DG) in the 5’-UTR in THPO was also described in a family with unilateral limb defects [57]. Other limb defects were also observed in a family with a 185G > T mutation in the 50 UTR region of THPO [58]. These findings suggest that excess TPO may affect development of the embryonic vasculature. MPL Somatic gain-of-function mutations in codon 515 of MPL (e.g. W515K/L/A) have been reported in approximately 5e10% of patients with essential thrombocythaemia and primary myelofibrosis [59,60]. MPL S505N is an activating germline mutation that involves the transmembrane domain of protein, and has been identified in patients with HT. The mutation induces autonomous dimerization of MPL and signal activation in the absence of its ligand TPO. This mutation was first reported in eight members in a Japanese family [61,62]. Eight Italian families were subsequently found to carry the same mutation. These patients exhibit a high risk of thrombosis and develop splenomegaly and bone

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marrow fibrosis over time. However, these patients do not appear to develop cytopenias or an increased risk of progression to AML [63]. Analysis of microsatellite markers and single nucleotide polymorphisms found that the mutation in the eight Italian families is due to a founder effect [64]. The MPL P106L mutation was first identified in two siblings in an Arab family. An additional six patients from three other Arab families were also found to carry the same mutation. This mutation is autosomal recessive and is present in approximately 6e7% of Arabs. Similar to patients with MPL Baltimore (see below), patients homozygous for the mutation exhibit severe thrombocytosis, whereas heterozygotes demonstrate mild thrombocytosis [65]. A MPL W515R mutation was identified in a father and daughter who had isolated thrombocytosis. The affected members did not have any evidence of an MPN, no hepatosplenomegaly nor history of thrombosis. In vitro, MPL W515R is an activating mutation which increases MPL activity, but to a lesser effect than the aforementioned somatic mutations [66]. A single nucleotide substitution 1238 G > T resulting in a K39N amino acid substitution (referred to as MPL Baltimore) was identified in three African-American women evaluated by investigators at Johns Hopkins University. This polymorphism is only found in African-Americans, of whom 7% are heterozygous for this variant. In vitro studies revealed that this mutation is associated with incomplete processing and a reduction in MPL protein [67]. Another patient was reported in an Ethiopian Jewish boy who presented with thrombocytosis and was found to be homozygous for MPL Baltimore [68]. This polymorphism has not been reported to have an increase risk in thrombotic complications. JAK2 JAK2 V617F is a somatic mutation that is found in 95e98% of patients with polycythaemia vera (PV) and 50e60% of patients with ET and PMF. Recently, a germline JAK2 V617I mutation was identified in six family members over three generations. Affected patients older than 40 years old suffered vascular events such as ischaemic heart disease or ischaemic cerebrovascular event(s), but did not have any evidence of polycythaemia, leucocytosis, splenomegaly or transformation to myelofibrosis or leukaemia. There was lack of cytokine-independent colony formation with JAK2 V617I, unlike colonies derived from JAK2 V617F which are cytokine-independent. JAK2 V617I only has weak constitutive signalling compared to JAK2 V617F due to reduction in the threshold for cytokine-induced activation [69,70]. A germline mutation in JAK2 in a residue other than V617 was identified in three family members in two generations. This novel mutation is a single nucleotide substitution resulting in a R564Q amino acid change in exon 13. JAK2 R564Q exhibits similar levels of increased kinase activity compared to JAK2 V617F, but less growth-promoting effects. Cells expressing JAK2 V564Q are more sensitive to ruxolitinib, a JAK1/JAK2 inhibitor, compared to cells expressing JAK2 V617F [71]. Another mutation in JAK2 was discovered in three family members with isolated thrombocytosis, a missense C > A variant resulting in a H608N substitution. Affected patients did not demonstrate any thrombotic events, splenomegaly or bone marrow fibrosis. When expressed in Ba/F3 cells, the JAK2 H608N mutation resulted in more STAT5 phosphorylation compared to wild type JAK2. H608 is located in the JH2 (pseudokinase) domain which normally inhibits the JH1 kinase domain. Mutations in this region may abrogate the function of JH2, resulting in increased kinase activity [72]. In a recent report, two families with three heterozygous JAK2 germline mutations were described [73]. One family had a single point mutation in the JAK2 kinase domain at R867Q and the other family had two mutations on the same JAK2 allele in the pseudokinase (S755R) and kinase (R948Q) domains. Ba/F3-MPL cells expressing the mutants exhibit longer half-lives compared to JAK2 V617F, increased binding to HSP90, and higher MPL expression on the cell surface. These mutants were also less sensitive to both JAK2 and HSP90 inhibitors compared to JAK2 V617F mutants. Gelsolin Gelsolin is an actin binding protein that is involved in actin filament assembly and disassembly [74]. Gelsolin knockout mice (GSN/) have impaired platelet function leading to prolonged bleeding times and also delayed neutrophil migration [75]. In 2013, a C > T transversion, resulting in a glycine to cysteine amino acid change in the gelsolin (GSN) gene, was identified in 15 affected family members in

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five generations with HT. In vitro assays show that this mutation increases the release of platelet-like particles. In an in vivo model, transgenic mice developed thrombocytosis as well as increased megakaryocytes in the bone marrow [76]. The mechanism of how this mutation in GSN causes thrombocytosis is currently unknown. Hereditary neutrophilia CSF3R Colony stimulating factor 3 receptor (CSF3R) encodes for the granulocyte colony-stimulating factor (G-CSF) receptor which is involved in the differentiation and proliferation of granulocytes. Acquired mutations in CSF3R have been previously described to cause Kostmann Syndrome, also known as severe congenital neutropenia. Patients with Kostmann Syndrome have an increased risk for transformation to leukaemia [77]. Several novel mutations (two clusters of proximal membrane and carboxy-terminal truncation mutations) in CSF3R were recently identified in patients with chronic neutrophilic leukaemia and atypical chronic myeloid leukaemia [7,8,78]. To date, the only CSF3R germline mutation identified was in twelve affected individuals across three generations of a family with chronic neutrophilia. CSF3R T617N (this mutation is equivalent to T640N in current gene nomenclature) results in constitutive activation of the G-CSF receptor, and hypersensitivity to G-CSF in vitro. When immunodeficient mice were injected with CD34þ cells from affected patients, there was an increase in the number of engrafted progenitors and skewed differentiation of HSCs into the myeloid lineage. One of twelve patients progressed to myelodysplastic syndrome (MDS) [79]. Leucocyte adhesion deficiencies (LAD) Leucocyte adhesion deficiency (LAD) is a group of autosomal recessive disorders characterized by immunodeficiency and neutrophilia. LAD is caused by a defect in the adhesion of leukocytes to the vascular wall [80]. There are three subtypes of LAD, LAD-I, LAD-II and LAD-III. The most common subtype, LAD-1, is caused by a mutation in integrin b2 (ITGB2). This mutation leads to decreased expression or functioning of CD18, the b2 subunit of the leucocyte b2 integrin. Clinical characteristics of patients with LAD-1 include delayed separation of the umbilical cord, leucocytosis and severe bacterial infections in the first year of life [81]. LAD-II is caused be a deficiency in a Golgi GDP-fucose transport protein (GFTP) which is encoded by SLC35C1, solute carrier family 35 member C1 or FUCT1 (GDP-fucose transporter 1). Deficiency of GFTP results in decreased leucocyte extravasation and recruitment to the site of infection. Patients with LAD-II have less severe infectious complications compared to patient with LAD-I because of an intact b2 integrin. These patients also have dysmorphic facial features, short stature and severe mental retardation [82]. Patients with LAD-III, also known as LAD-1/variant, have mutations in ferritin family homologue 3 (FERMT3) which encodes kindlin-3. Similar to patients with LAD-1, patients with LAD-III have recurrent infections as well as a severe Glanzmann thrombasthenia-like bleeding disorder [80]. Summary Hereditary myeloproliferative disorders are rare syndromes wherein the undertaking of a thorough family history is critical to their identification and characterization of mode of inheritance. For affected families, defining the molecular basis for hereditary erythrocytosis or thrombocytosis is particularly relevant for genetic counselling of proband(s) to gauge the potential for morbid complications or premature death. Cooperative efforts and shared registries are needed to better understand the genotypeephenotype correlations of these conditions. In addition, next generation sequencing techniques are expected to quickly elucidate the molecular underpinnings of those disorders which remain uncharacterized. Role of funding source No funding source to disclose.

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Conflict of interest statement Authors do not have any financial or personal conflict of interests to disclose.

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