Hereditary thrombocytosis: the genetic landscape

The normal range for platelets in humans is quoted as 150–450 × 109/l although the quoted normal range may vary in various laboratories and may also vary between ethnic groups. Platelets are derived from megakaryocytes in the bone marrow and released into the circulation, with a circulating lifespan of around 10 days. Platelet production is driven by the primary humoral regulator thrombopoietin (TPO). TPO binds to the thrombopoietin receptor on the platelet (MPL) and activates JAK and STAT signal transduction pathways, stimulating megakaryocyte growth and platelet production. TPO production is maintained at a constant rate in the liver and the levels of TPO are inversely proportional to the rate of platelet production.1 In coordination with endothelial cells and coagulation proteins, platelets act as the principal mediator of vascular homeostasis and thrombosis.2 Thrombocytopenia, defined as a platelet count below <150 × 109/l, may be associated with spontaneous bruising, purpura and bleeding. In contrast, thrombocytosis with a platelet count above 450 × 109/l may be associated with thrombotic complications and rarely at very high platelet levels with bleeding. Platelet elevation or thrombocytosis can be classified into primary or secondary with numerous aetiologies. Causes of primary thrombocytosis consist of myeloproliferative neoplasms including chronic myeloid leukaemia (CML), polycythaemia vera (PV), primary myelofibrosis (PMF), myelodysplastic syndromes (MDS) or very rarely hereditary thrombocytosis (HT). The term familial is also used in association with this condition but this term is preferably reserved for any myeloproliferative neoplasm with a familial element with a variety of genetic linkages and hereditary reserved for a thrombocytosis with a clear inherited pattern. Causes of secondary or reactive thrombocytosis include reactive responses to systematic infection, chronic inflammatory states, hyposplenism, malignancy, iron deficiency, haemorrhage, surgery, and trauma. Spurious elevation of platelet counts can also result when other cellular factors, including microspherocytes, schistocytes or infectious organisms, are mistakenly counted as platelets. Essential thrombocythaemia is a myeloproliferative neoplasm, in which an acquired clone drives excess cell production, predominantly of platelets. Current World Health Organisation (WHO) diagnostic criteria for essential thrombocythaemia requires fulfilment of all four major criteria or the first three major criteria and one minor criterion. Major criteria consist of: (i) sustained platelet count ≥450 × 109/l, (ii) bone marrow biopsy exhibiting morphological abnormalities of proliferation of the megakaryocyte lineage with increased numbers of mature, enlarged megakaryocytes with hyperlobulated nuclei and no significant increase or left-shift in neutrophil granulopoiesis, (iii) not meeting WHO criteria for another myeloproliferative neoplasm (BCR-ABL1 CML, PV, PMF, MDS or other myeloid neoplasms), and (iv) the presence of an acquired mutation of JAK2, CALR or MPL. Minor criteria include the presence of a clonal marker or absence of evidence for reactive thrombocytosis.3 Patients with unexplained thrombocytosis are referred to a haematologist for further investigation and management. While the majority of cases are attributed to secondary reactive causes or fulfil the WHO diagnostic criteria of essential thrombocytosis, rare cases may be hereditary or familial thrombocytosis. Over many years, rare families exhibiting an inherited genetic abnormality leading to a thrombocytosis have been described. HT is a heterogeneous disorder that is usually transmitted in an autosomal dominant pattern with variable penetrance. HT is an inherited myeloproliferative disorder in which the clinical features resemble sporadic essential thrombocythaemia.4 In light of progress in molecular genetics, clusters of families with inherited thrombocytosis have been extensively studied and four particular genes demonstrated association with HT as shown in Fig 1. This review will focus on each of the genes and families in which mutations have been discovered and describe the scientific findings and any clinical consequences. Following the isolation of the human thrombopoietin (THPO) gene in 1994, a number of THPO genetic alterations have been identified among autosomal dominant HT cases in Japan, Italy, The Netherlands and Poland.5 The human THPO gene consists of seven exons and six introns with the locus spanning over 6 kb. THPO gene encodes for a humoral growth factor (332 amino acids with a molecular mass of ˜70 kDa) which exerts profound stimulatory effects on megakaryopoiesis and thrombopoiesis.6 Genetic sequencing of a Dutch family harbouring 11 cases of HT which exhibited autosomal dominant inheritance revealed a common genetic alteration in the THPO gene amongst affected individuals. Through extensive nucleotide sequencing, Wiestner et al. identified a G to C transversion in the splice donor site of intron 3 within the THPO gene (Table I).7 The point mutation resulted in exon 3 skipping and shortened the 5′-untranslated regions (UTR) of TPO mRNA. Though the loss of AUG codons in the 5′-UTR of the TPO mRNA normally represses translation, this splice donor mutation subsequently enhanced the translation of THPO transcripts and increased the synthesis of TPO.7 A similar mutation was also identified in a Polish family with 23 cases of HT.8 Distal limb defects 9, 10 Progression to acute leukaemia and myelofibrosis 7 Vascular events in over 40 years old patients 29, 30 c.2265T>A c.2813G>A p.(S755R) p.(R938Q) Family history of cerebral infarction. 34 Genetic analysis of a Japanese kindred with HT exhibiting elevated TPO serum levels revealed a distinct gain-of-function mutation within the THPO gene. The authors discovered that G to T transversion at position 516 of exon 3 led to increased translation of TPO mRNA and TPO production. The mutation removed the inhibitory effect of upstream open reading frame 7 (uORF7) within 5′-UTR via generation of a premature stop codon, which subsequently stimulated TPO mRNA production.9 Furthermore, Graziano et al. have identified a G to T transversion at position 185 in the 5′-UTR of exon 2 in a family with HT with associated limb defects (absence of critical bones in hands and feet).10 Three out of four members of this kindred exhibited a THPO G185T mutation along with transverse limb defects, suggesting potential TPO involvement in embryonic vasculogenesis.10 In addition, Zhang discovered a T to C point mutation at the splice donor of intron 2 in the 5′-UTR of the THPO gene within a Filipino family with HT.11 Kondo et al. also identified a one-base deletion of G nucleotide in the 5′-UTR of the THPO gene in the affected Japanese family.12 Overall, the afore-mentioned mutations within the THPO gene strongly suggest the importance of the 5′-UTR region of the THPO gene in maintaining platelet homeostasis.12 As a principal regulator of megakaryopoiesis, the cytokine thrombopoietin (TPO) exerts its signalling via its receptor, TPO-R (MPL). Myeloproliferative leukaemia virus oncogene (MPL) protein consists of 635 amino acids and is predominantly expressed on the surfaces of megakaryocytes, platelets and haematopoietic stem cells.13 Binding of TPO to the extracellular portion of partially pre-dimerized cell surface MPL triggers a conformational change of MPL into a homodimeric receptor complex and phosphorylates two tethered Janus kinases (JAK2 and TYK2). Phosphorylation of Janus kinases activates the downstream substrates including the MPL itself, transcriptional factors including STAT3 and STAT5, adaptor proteins including SHC and SHP2 and signalling intermediates such as MAPK, protein kinase C and PI3K.14 Approximately 5–10% of essential thrombocythaemia and primary myelofibrosis exhibit gain-of-function mutations in codon 515 of MPL, implicating its potential role in megakaryopoiesis.15 Molecular analysis of a Japanese family with HT over three generations revealed a G>A nucleotide substitution at position 1073 in exon 10 of the MPL gene. The point mutation resulted in a single amino acid exchange from serine to asparagine (S505N) within the transmembrane domain of the MPL gene. Using methylthiotetrazole (MTT) assays comparing the cell clones transfected with wild-type or mutant MPL illustrated that mutant MPL permitted IL-3-independent survival capacity. In contrast to wild-type cells that undergo apoptosis immediately after the withdrawal of IL-3, cells expressing MPL S505N demonstrated prolonged survival even in the absence of IL-3 via autonomous phosphorylation of the downstream signalling molecules including MEK1/2 and STAT5b.16 The same mutation was identified in eight Italian families with HT. Fifteen out of 41 affected members underwent major thrombotic complications ranging from cerebrovascular accidents, myocardial infarction, deep vein thrombosis, Budd–Chiari syndrome, and fetal loss along with splenomegaly, progression into bone marrow fibrosis and overall reduced life expectancy.17 The MPL S505N mutation identified in the eight Italian families with HT is likely to have arisen due to a founder effect.18 A novel MPL germline mutation in two siblings in an Arab family was identified with a further six patients from three other Arab families with HT. In contrast to the aforementioned mutations, the following genetic alteration exhibited an autosomal-recessive inheritance pattern. Extensive sequence analysis illustrated a single base C to T substitution at nucleotide 317 (c.317 C>T), which subsequently resulted in a MPL missense mutation MPL P106L. Homozygous patients demonstrated severe thrombocytosis, whereas heterozygotes exhibited a mild degree of thrombocytosis or normal platelet counts. The MPL S505N allele was observed in approximately 3·3% of Arabs, whereas in 0% of a control group of different ethnicity. The high rate of consanguinity within the Arab population may contribute to the high prevalence of the allele.19 A large series of 115 Arab patients with the MPL P106L mutation was presented recently. Homozygosity was associated with higher platelets counts and the risk of thrombosis appeared low.20 A further study of 64 patients with MPL mutations in familial thrombocytosis in the Saudi population is presented in this journal.21 Of the group, 41% were <14 years at presentation. Although 26 tribes were represented in the group of 65, 48% came from only two tribes. Four different MPL mutations were seen but 60 (92%) had the c.317C>T as reported previously of which 40 (61%) were homozygous and 19 (31%) heterozygous. A c.117G>T was present in six (9%) with two homozygotes and four heterozygotes. Two patients were heterozygotes for c.358C>T and a single patient was heterozygous for c.509G>A. Three patients had two mutations. Two were compound heterozygotes for c.317C>T and c.117G>T and one for c.317C>T and c.358C>T. Sequencing of MPL patients revealed a single nucleotide change leading to the alternation at amino acid 39 from lysine to asparagine (MPL K39N) (referred to as MPL Baltimore).22 In-vitro analysis of this MPL variant demonstrated reduced MPL expression on platelets, which paradoxically results in a predisposition to supraphysiological megakaryocytopoiesis via reduced TPO serum clearance and low binding affinity of MPL to TPO.22, 23 The MPL K39N was restricted to African-American populations in which approximately 7% of African-Americans were heterozygotes. Such individuals had significantly higher platelet counts than controls. The authors identified an autosomal dominant inheritance pattern with incomplete penetrance, as some heterozygotes exhibited normal platelet counts. Homozygous cases displayed severe thrombocytosis but MPL Baltimore has not been reported to increase the risk of thrombotic complications.22 Direct sequencing of genomic DNA of a father and daughter presenting with isolated thrombocytosis illustrated a germline MPL W515R mutation in exon 10 of MPL. Affected patients neither reported thrombotic events nor displayed evidence of myeloproliferative neoplasm and hepatosplenomegaly.24 Sequencing analysis of one family presenting with two cases of mild thrombocytosis has revealed germline a MPL R102P heterozygous mutation in the proband and his daughter. Interestingly, homozygous MPL R102P mutation was first identified in the congenital amegakaroycytic thrombocytopenia attributed to the loss-of-function effect. Nevertheless, the same mutation in its heterozygous state has shown to stimulate megakaryopoiesis due to reduced expression of MPL on the cell surface of platelets that results in reduced TPO clearance and supraphysiological megakaryocytopoiesis.25 In total, these variations in the MPL gene can account for some cases of HT. The Janus kinase (JAK) family comprises JAK1, JAK2, JAK3 and TYK2 and serves a vital role as the intracellular signalling transducers of downstream cytokine activation. Upon the binding of its cognate ligand, JAK receptor subunits undergo reorientation or oligomerization into active states with phosphorylated loop residues within the kinase domain. Activated JAK subsequently phosphorylates tyrosine within the receptor cytoplasmic domain, which permits recruitment and phosphorylation of the downstream effector STATs.26 Studies have illustrated the presence of activating mutations in JAK2, most commonly JAK2 V617F occurring at exon 14 of JAK2, in myeloproliferative neoplasms. Guanine to thymine substitution results in a valine to phenylalanine substitution at position 617 within the pseudokinase JH2 domain of JAK2. The mutation was observed in 95% of patients with PV, 50–60% of patients with essential thrombocythemia (ET) and PMF.27 The JAK2 V617F mutation exhibits a gene dosage effect with heterozygosity associated with ET and homozygosity associated with PV.28 Mutations in the JAK2 gene have been discovered associated with some cases of HT. Pyrosequencing of a 53-year-old proband who presented with an ischaemic cerebrovascular event and thrombocytosis revealed a heterozygous V617I mutation. Further screening over three generations identified five additional cases of JAK2 V617I mutations with thrombocytosis. Bone marrow analysis displayed megakaryocyte hyperplasia without myelofibrosis. Three of the positive cases above 40 years of age underwent vascular events including ischaemic heart disease or ischaemic cerebrovascular events but did not have any evidence of splenomegaly or leukaemic transformation.29 Transcriptional assays illustrated weaker constitutive activation in JAK2 V617I compared to JAK2 V617F.30 These findings highlight the need to screen for non-JAK2 V617F mutations to exclude HT in patients presenting with thrombocytosis. Sequence analysis of a six-year-old boy with prolonged thrombocytosis (platelet count 800–1300 × 109/l) revealed the first description of a germline mutation in JAK2 located in a residue other than V617. This novel mutation involved a single nucleotide substitution in exon 13 of the JAK2 gene c.1691G>A, which resulted in an arginine to glutamine substitution at position 564 (JAK2 R564Q). Evaluation of the sister and mother of the proband who also had thrombocytosis (500–600 × 109/l) revealed the same genetic mutation, whereas the father with a normal platelet count did not harbour the mutation. Despite similar levels of increased kinase activity, JAK2 R564Q displayed milder growth-promoting effects compared to JAK2 V617F. Moreover, JAK2 R564Q expressing cells were far more sensitive to the commercially available JAK inhibitor ruxolitinib with a significant reduction in viable cell number at 1 000-fold lower concentrations. Administration of low doses of ruxolitinib may be effective in treating patients with familial myeloproliferative neoplasm (MPN) associated with alternate JAK2 mutations.31 Other heterozygous JAK2 germline mutations were discovered in two families with HT compatible with an autosomal dominant inheritance. One family carried a single point mutation in the JAK2 kinase domain at R867Q and the other family displayed two mutations in the same JAK2 allele within the pseudokinase (S755R) and kinase (R938Q) domains. Thrombotic complications were rare in the affected family members. Expression of JAK R867Q and JAK2 S755R/R938Q in BA/F3-thrombopoietin receptor (MPL) cell lines induced constitutive signalling via spontaneous phosphorylation of the downstream signalling molecules (STAT 1, STAT3, STAT5, ERK and AKT), but to a lesser extent than JAK2 V617F. Constitutive signalling was not evident in Ba/F3-human receptor of erythropoietin cells (EPOR) cells, suggesting these mutations promote thrombocytosis, but not erythrocytosis. In addition, BA/F3-MPL cells expressing JAK R867Q and JAK2 S755R/R938Q mutations displayed longer half-lives, increased binding to the heat shock protein 90 (HSP90) chaperone, and higher MPL cell-surface expression. The novel mutations displayed reduced sensitivity to current JAK2 and HSP90 inhibitors compared to JAK2 V617F mutants.32 Next-generation sequencing (NGS) of a cohort of 61 MPN families identified a novel germline JAK2 mutation in kindred with suspected familial ET. The proband carried a heterozygous C to A missense mutation in the JAK2 gene, resulting in a substitution of histidine to asparagine at position 608 (JAK2 H608N). The mutation was identified in other affected family members. Patients harbouring the JAK2 H608N mutation exhibited isolated thrombocytosis in the absence of splenomegaly, bone marrow fibrosis and thrombotic complications. Expression of JAK2 H608N induced a higher level of phosphorylation of STAT5. These findings implicate the JAK2 H608N mutation aetiology of HT.33 Whole-exome sequencing of a 46-year-old Japanese patient with ET revealed a non-canonical mutation in exon 18 of the JAK2 gene. The mutation was characterized by a single nucleotide C to A substitution, resulting in an amino acid substitution of threonine to asparagine at position 875 (JAK2 T875N). A family history of suspected ET with a father with thrombocytosis and cerebral infarction at the age of 51 years was reported. The patient had a history of placental abruption, hypertension and bilateral glaucoma but had no thrombotic or haemorrhagic complications and no splenomegaly or hepatomegaly. Cells transfected with JAK2 cDNA with the T875N mutation displayed increased levels of phosphorylated JAK2 and Erk along with enhanced STAT3 and STAT5 signalling activity. Ba/F3 cells transduced with JAK2 T875N demonstrated enhanced cellular growth without IL-3 stimulation. In contrast to previously described JAK2 R867Q or JAK2 S775R/R938Q mutations, JAK2 T875N-expressing cells were more sensitive to commercial ruxolitinib treatment. The following data suggests the need to determine optimal dosage of ruxolitinib based on the mutation site for patients with MPNs with JAK2 mutations other than JAK2 V617F.34 Gelsolin is a calcium-regulated actin-binding protein of about 15 kDa that is involved in actin cytoskeleton assembly and filament remodelling. Since its discovery in 1979, there is increasing evidence that gelsolin is a multifunctional regulator of cell metabolism.35 Witke et al. illustrated morphological disruption in the platelets, impaired platelet function, and prolonged bleeding times in gelsolin knockout mice (GSN−/−), suggesting the potential role of gelsolin in platelet homeostasis.36 DNA sequencing of a HT pedigree with 21 affected family members has revealed C>T transversion, resulting in a glycine to cysteine amino acid substitution within the gelsolin protein. The mutation was present in 15 out of 21 affected family members over five generations. Platelet biogenesis in-vitro assay of the cell line transfected with mutant gelsolin has displayed increased release of platelet-like particles. Moreover, transgenic mice expressing the mutant gelsolin gene developed thrombocytosis and increased megakaryocytes in the bone marrow. Although the mechanism explaining the pathogenicity of the mutation has not been elucidated, data suggest a role of gelsolin in the development of thrombocytosis.37 A number of families with HT with specific genetic mutations have been described as listed in Table I However, the clinical picture is only known from these described cases and no systematic study of the clinical picture in HT is available. The questions which arise in those with a germline driven platelet count elevation are the risk of thromboembolic events and whether the genetic lesions result in increased risk of progression to myelofibrosis or leukaemia. In many of the families, there is no association with thromboembolic events or it is stated to be rare.32 However, the JAK2V617I mutation in a family is associated with three affected individuals with vascular events occurring under the age of 40 years29 and the JAK2 T875N mutation is associated with a family history of cerebral infarction in presumed affected individuals.34 The MPL mutation S505N in an Italian family was associated with a high rate of thrombosis.17 However, in the large retrospectively studied group with MPL mutations presented in this issue of the journal.21 62 (97%) had no significant physical abnormalities. The vast majority (98%) did not have any thrombotic or haemorrhagic events and the one patient who did have a thrombosis was post liver transplant at the time (liver transplant having been carried out for another pathology, Wilson's disease). The average follow-up in this study was 3·9 years. Other genetic defects such as distal limb defects are seen with a TPO mutation.10 It is of note that some mutations are associated with development of complications such as acute myeloid leukaemia, as seen in one affected family member with a TPO mutation.38 In families in which the bone marrow has been examined systematically, expected megakaryocyte hyperplasia is seen.29 There are however some reports of progression to myelofibrosis associated with specific mutations.17 Specific complications and progression can be associated with individual mutations in HT. Patients presenting with an elevated platelet count warrant careful investigation to exclude classical MPN, reactive thrombocytosis and HT. Clinicians should obtain a comprehensive history from the patient that must include a detailed family history of MPN, thrombocytosis and thrombotic events. Physical examination to check for hepatosplenomegaly is also necessary. A confirmatory full blood count analysis to confirm a sustained elevation in platelet count is required. Hereditary thrombocytosis is suspected in young patients and/or those with two or more family members with thrombocytosis. Patients with a classical MPN with driver mutations and a family history would not be considered for further investigation for HT as in these cases the familial element is associated with all the classical acquired MPNs, not HT. Likewise the issue arises should all triple-negative ET be investigated for HT but this would generate a lot of redundant investigation and some further index of suspicion of HT is required to investigate for HT such as a younger patient. A bone marrow biopsy with morphological examination and cytogenetics is the next step undertaken in a patient with thrombocytosis and this is helpful in looking for the patterns consistent with classical MPN but will not confirm a diagnosis of HT. When the bone marrow is examined in HT hypercellular patterns with prominent megakaryocytes are seen with perhaps more regular shapes than in ET.8 Patients with a sustained thrombocytosis in whom a HT is suspected in the absence of reactive causes should then undergo full genetic analysis of the relevant genes using a targeted NGS panel or full sequencing of THPO, MPL, JAK2 and gelsolin (see diagnostic algorithm, Fig 2).38 To aid the distinction between germline and somatic variants the variant allele frequency (VAF) is helpful as heterozygous loci should be near 50% VAF, but homozygous loci should be near 100%. This should be used in conjugation with VAF databases such as the GnomAD,39 Exome Aggregation Consortium (ExAC)40 or dbSNP.41 The classification of gene variants uses a combination of functional data, population frequency (GnomAD), disease phenotype, familial pedigree, and biological evidence to assign the level of clinical significance.42 The model is based on a five-tier system that defines gene variants as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign using a combination of databases, e.g. GnomAD, as well as in silico tools such as SIFT,43 PolyPhen,44 and splice prediction tools. A measurement of the TPO level which would be expected to be elevated is another helpful investigation. Due to its rarity and distinctive mutations observed in different kindreds, there is no evidence to guide management. In view of the lack of this, the first line of management would be observation only. However, low-dose aspirin may be considered as prophylaxis of thromboembolic events in those with no contraindication to aspirin. This is extrapolated from the benefit in low-dose aspirin in ET,45 which is an acquired neoplasm. However, in another retrospective study of patients with ET and CALR mutations, low-dose aspirin was associated with an increased risk of bleeding46 so even in ET the benefit of aspirin prophylaxis is not clear. Thromboembolic events should be managed by the accepted protocols, but secondary prophylaxis should be continued life-long as the underlying associated cause is germline. In a patient with recurrent thromboembolic events, one would consider if reducing the platelet could would be of benefit. Cytoreductive therapy to reduce the platelet count would generally be contraindicated in a germline disorder. However, there are recorded instances of the administration of hydrocarbamide either before the diagnosis30 was clear or in an attempt to reduce the platelet count.17, 20, 45 There is no information on efficacy. In a patient with recurrent thrombotic events there would be an argument to try interferon to reduce the platelet count. This has been attempted on some occasions17 but there is no information on effectiveness. In in-vitro studies ruxolitinib has been shown to ameliorate the up- regulation of the JAK/STAT pathway associated with some JAK mutations in a dose-dependent manner. Therefore, ruxolitinib might be a useful therapy in the management of some HT-associated JAK mutations. The effective dose depends on the mutation as some respond to lower than conventional doses.31, 32, 34 Investigation and discovery of mutations causing HT needs to be carefully communicated with patients and their families with full consent. They will want to know implications of any diagnosis and with such rare disorders it may be difficult to give comprehensive information. If available, involvement of medical genetics and genetic counselling services may be beneficial. Various attempts have been made to collate and maintain registers of these types of rare mutation47 but there is an ongoing need for international collation and maintenance of molecular and clinical data with this and other rare disorders so that knowledge of outcomes can be progressed. Extensive sequencing analysis of HT kindreds has revealed key mutations involving the THPO, JAK2, MPL and gelsolin genes. Although the majority of HT is benign, specific mutations have been associated with complications including congenital limb defects, thromboembolic events, and myeloproliferative neoplasms. In patients presenting with an elevated platelet count, genetic analysis of the four genes should be conducted to diagnose HT. Specific genetic mutations should then be used to guide prognosis, prophylactic treatment, follow-up regimen and genetic counselling. EYH wrote the manuscript and edited the manuscript. MC wrote the manuscript and edited the manuscript. MFM wrote the manuscript, edited the manuscript and provided supervision. MFM has participated in the advisory boards and received honorarium from Novartis. EYH and MC have no conflicts of interest to declare.