Hereditary thrombocytosis: the genetic landscape

Abstract

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 with a significant in cell number at of low of may be in patients with familial myeloproliferative neoplasm associated with JAK2 heterozygous JAK2 germline mutations were discovered in two families with HT with an autosomal dominant family a single point mutation in the JAK2 kinase domain at and the other family displayed two mutations in the same JAK2 allele within the pseudokinase and kinase complications were rare in the affected family of JAK and JAK2 in receptor (MPL) cell constitutive signalling via spontaneous phosphorylation of the downstream signalling molecules STAT5, and but to a than JAK2 V617F. signalling was not in receptor of cells suggesting these mutations thrombocytosis, but not In addition, cells expressing JAK and JAK2 mutations displayed increased binding to the protein and higher MPL The novel mutations displayed reduced to JAK2 and compared to JAK2 V617F sequencing of a of families identified a novel germline JAK2 mutation in kindred with familial The proband a heterozygous C to A missense mutation in the JAK2 in a substitution of to asparagine at position (JAK2 The mutation was identified in other affected family Patients harbouring the JAK2 mutation exhibited isolated thrombocytosis in the absence of splenomegaly, bone marrow fibrosis and thrombotic of JAK2 a higher of phosphorylation of These findings the JAK2 mutation of sequencing of a Japanese patient with ET revealed a mutation in exon of the JAK2 gene. The mutation was by a single nucleotide C to A in an amino acid substitution of to asparagine at position (JAK2 A family history of ET with a father with thrombocytosis and cerebral at the age of years was The patient had a history of and but had no thrombotic or complications and no splenomegaly or transfected with JAK2 with the mutation displayed increased levels of phosphorylated JAK2 and along with enhanced STAT3 and signalling cells with JAK2 demonstrated enhanced cellular growth without IL-3 In contrast to previously JAK2 or JAK2 mutations, JAK2 cells were more sensitive to The following the need to dosage of on the mutation site for patients with with JAK2 mutations other than JAK2 is a protein of that is involved in and its in is evidence that is a regulator of cell et al. illustrated morphological in the platelets, platelet and prolonged in suggesting the potential role of in platelet DNA sequencing of a HT with affected family members has revealed in a to amino acid substitution within the The mutation was present in out of affected family members over five Platelet of the cell transfected with mutant has displayed increased of Moreover, expressing the mutant gene thrombocytosis and increased megakaryocytes in the bone Although the the of the mutation has not been suggest a role of in the of A number of families with HT with genetic mutations have been as in the clinical is only from these cases and no systematic study of the clinical in HT is The which in with a germline driven platelet count elevation are the risk of events and the genetic result in increased risk of progression to myelofibrosis or In many of the is no association with events or is to be the mutation in a family is associated with three affected individuals with vascular events occurring the age of 40 and the JAK2 mutation is associated with a family history of cerebral in affected The MPL mutation S505N in an Italian family was associated with a high rate of in the large studied group with MPL mutations presented in this of the journal.21 had no significant The majority did not have any thrombotic or events and the one patient who did have a thrombosis was liver at the been out for another The in this study was genetic defects such as limb defects are seen with a TPO is of that some mutations are associated with of complications such as acute myeloid as seen in one affected family with a TPO In families in which the bone marrow has been megakaryocyte hyperplasia is are some of progression to myelofibrosis associated with complications and progression can be associated with mutations in HT. Patients presenting with an elevated platelet count investigation to exclude reactive thrombocytosis and HT. a history from the patient that include a family history of thrombocytosis and thrombotic to for is also A count analysis to a sustained elevation in platelet count is thrombocytosis is in patients with two or more family members with thrombocytosis. Patients with a with mutations and a family history not be for further investigation for HT as in these cases the familial element is associated with all the acquired not HT. the all ET be for HT but this a of investigation and some further of of HT is to for HT such as a A bone marrow biopsy with morphological and is the in a patient with thrombocytosis and this is in for the with but will not a of HT. the bone marrow is in HT with megakaryocytes are seen with more than in Patients with a sustained thrombocytosis in a HT is in the absence of reactive causes undergo genetic analysis of the genes a or sequencing of MPL, JAK2 and diagnostic Fig the between germline and the variant allele is as heterozygous be but homozygous be This be used in with such as the or The of gene a of population disease familial and evidence to the of clinical The is on a that gene as likely variant of likely or a of as as in such as and splice A of the TPO which be to be elevated is another to its and mutations observed in different is no evidence to management. In of the of the first of be may be as of events in with no to This is from the in in which is an acquired in another study of patients with ET and CALR mutations, was associated with an increased risk of even in ET the of is not events be by the but secondary be as the associated is In a patient with one the platelet be of to the platelet count be in a germline are of the of the was clear or in an to the platelet is no on In a patient with thrombotic events be an to to the platelet This has been on some but is no on In has been shown to the of the associated with some JAK mutations in a be a in the of some JAK mutations. The on the mutation as some to than 34 and of mutations HT to be with patients and families with will to of any and with such rare may be to involvement of and genetic may be have been to and of these of rare but is an need for and of molecular and clinical with this and other rare that of can be Extensive sequencing analysis of HT has revealed mutations the JAK2, MPL and Although the majority of HT is mutations have been associated with complications including congenital limb defects, and myeloproliferative neoplasms. In patients presenting with an elevated platelet genetic analysis of the four genes be to HT. genetic mutations be used to and genetic the and the the and the the the and has in the and from and have no of to

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