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Keywords:

  • hereditary thrombocytosis;
  • thrombopoietin;
  • thrombopoietin-receptor;
  • THPO mutations;
  • MPL mutations

Summary

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

Familial thrombocytosis can be divided into two broad categories. The first includes inherited syndromes that affect only the megakaryocytic lineage with Mendelian inheritance, high penetrance and polyclonal haematopoiesis. The second category includes inherited predisposition to true Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by low penetrance, clonal haematopoiesis and presence of somatic mutations such as JAK2 V617F. It must be underlined that these two categories represent two well separate entities, with different patterns of proliferation and different transmission modalities. This review will focus on the molecular pathogenesis of hereditary thrombocytosis, underlining those clinical pictures that are specifically associated with mutations in the genes of thrombopoietin or in its receptor. Moreover, we propose an approach for the diagnosis and therapy of these syndromes.

The platelet count normally ranges from 100 to 400 × 109/l, only occasionally reaching values between 400 and 450 × 109/l. This range of normality is generally accepted for both adults and children (Dame & Sutor, 2005; Tefferi et al, 2005; Cazzola, 2008; Kaushansky, 2009). In clinical practice, the term ‘thrombocytosis’ refers to platelet counts above 450 × 109/l. Thrombocytosis may be classified into mild (platelet counts: 450–700 × 109/l), moderate (700–900 × 109/l) or severe (>900 × 109/l) (Dame & Sutor, 2005), while ‘extreme thrombocytosis’ refers to platelet counts above 1500 × 109/l (Schafer, 2001).

Megakaryocytopoiesis is supported by multiple growth factors, the most important of which is thrombopoietin; both megakaryocytes and their platelet progeny have the thrombopoietin receptor, also known as ‘myeloproliferative leukaemia’ (MPL). Thrombocytosis can be considered as primary, if sustained by a defect intrinsic to the haematopoietic progenitors, or as secondary (or reactive), if caused by a disease that persistently stimulates, by means of growth factors, the otherwise normal megakaryocytopoiesis (Schafer, 2004; Dame & Sutor, 2005;Cazzola, 2008; Skoda, 2009). Secondary thrombocytoses constitute by far the most frequent forms in both adults (Griesshammer et al, 1999; Aydogan et al, 2006) and children (Dame & Sutor, 2005) and are mainly caused by acute or chronic infective and inflammatory states, iron deficiency, acute blood loss, hyposplenism, haemolysis or malignancies (Schafer, 2004). In a recent study reviewing the medical records of 2000 patients with thrombocytosis, an underlying disease was documented in 96·7% of cases, with infections being the most common (50·1% of patients) (Aydogan et al, 2006). In contrast, primary thrombocytoses arise from a primitive myeloproliferation caused by a molecular defect targeting the haematopoietic progenitors. Primary thrombocytosis includes both acquired and hereditary forms, and primitive genetic abnormalities sustaining both forms are so far only partly understood. In acquired thrombocytosis these abnormalities are detectable exclusively in cells belonging to the haematopoietic lineage, whilst in hereditary thrombocytosis the underlying defects can be detected in both somatic and germ line cells and are transmitted as a hereditary character. Acquired primary thrombocytosis is observed in the course of several chronic myeloproliferative neoplasms (MPN), both BCR/ABL1- positive (Chronic Myeloid Leukaemia) and BCR/ABL1-negative, namely Essential Thrombocythaemia (ET), Polycythaemia Vera (PV) and Primary Myelofibrosis (PMF); moreover, thrombocytosis is also observed in the course of certain myelodysplastic syndromes [i.e. refractory anaemia with ring sideroblasts and del(5q) syndrome]. In all these conditions thrombocytosis develops within a wider haematopoietic proliferation, affecting also other lineages, and derives from the clonal expansion of the pathological progenitor cells that have gained a growth advantage over normal cells. The molecular hallmark of Ph-negative MPN is the constitutive activation of the tyrosine kinase JAK2, mainly resulting from the acquired mutation in its pseudo-kinase domain (JAK2V617F mutation) (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005; Zhao et al, 2005). Other mutations have been documented in a minority of MPN patients, either involving exon 12 of JAK2 (Scott et al, 2007) or activating the trans-membrane domain of MPL (MPL-W515L; MPL-W515K) (Pardanani et al, 2006; Pikman et al, 2006) or affecting the inhibitory adaptor protein gene SH2B3 (LNK) (Oh et al, 2010). Interestingly, familial clustering has been reported in about 8% of patients with Ph-negative MPN (Skoda & Prchal, 2005; Rumi, 2008; Percy & Rumi, 2009). In one third of the affected families, different phenotype of MPN (PV or ET or PMF) or different molecular defects (i.e. JAK2V617F and exon 12 JAK2 mutations) may recur in the same pedigree. In addition, first-degree relatives of patients with MPN may have a one- to seven- fold higher risk of developing MPN (Landgren et al, 2008). Moreover, it has been recently demonstrated that the JAK2 haplotype plays a pivotal role in favouring the acquisition of JAK2V617F mutation, leading to the development of an overt MPN (Jones et al, 2009; Olcaydu et al, 2009).

Undoubtedly, all of these discoveries provided valuable insight into the pathogenesis of MPN, and in the meanwhile it has somewhat blurred the boundary between hereditary and acquired forms (Bellanné-Chantelot et al, 2006; Higgs et al, 2008). Nevertheless, it must be underlined that familial MPN with thrombocytosis and hereditary thrombocytosis represent two well-separated entities, with different patterns of proliferation (the former is often clonal and shows multilineage-involvement; the latter is polyclonal and selectively affects the megakaryocytic lineage) and different transmission modalities (complex and on multi-genetic basis in the first and mainly autosomal dominant in the latter). Lastly, although familial MPN have the potential to acquire JAK2 or MPL mutations, these are not the primary pathogenetic event (Percy & Rumi, 2009), whilst in hereditary thrombocytosis mutations of thrombopoietin (THPO) or MPL genes are de facto disease-causing defects. In the past, hereditary thrombocytoses have often been indicated as ‘familial thrombocythaemia’ or ‘hereditary thrombocythaemia’ (Dror & Blanchette, 1999; Schafer, 2001; Skoda & Prchal, 2005). Nevertheless, in order to prevent confusion between acquired familial MPN and thrombocytosis caused by hereditary genetic defects, we prefer refer to these latter exclusively as ‘hereditary thrombocytosis’ avoiding the term ‘familial’.

This review aims to summmarize the state of the art regarding the molecular pathogenesis of hereditary thrombocytosis, to depict clinical pictures common to all molecular defects and, at the same time, to underline the different peculiarities specific of each defect. Finally, we suggest a diagnostic and therapeutic approach specific for this cohort of patients.

Thrombopoietin and MPL

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

Megakaryocytes are derived from the haematopoietic stem cell through successive lineage commitment steps, and they undergo a unique maturation process that includes polyploidization, development of an extensive internal demarcation membrane system, and finally formation of pro-platelet processes. Platelets are shed from these processes into vascular sinusoids within the bone marrow. The most important determinants for platelet production are thrombopoietin and MPL (Kaushansky, 2005; Deutsch & Torner, 2006; Geddis, 2010).

Thrombopoietin is encoded by the THPO gene, located on chromosome 3q27, which was independently cloned in 1994 by five groups (Kaushansky et al, 1994; Kuter et al, 1994; de Sauvage et al, 1994; Sohma et al, 1994; Wendling et al, 1994). The precursor protein is a glycoprotein of 353 amino acids; the mature molecule, composed of 332 amino acids, is acidic and heavily glycosylated. Thrombopoietin is involved in nearly every step of megakaryocyte development, preventing apoptosis, inducing mobilization of stem cells, stimulating megakaryocyte proliferation and lineage differentiation. THPO- and MPL-deficient mice show a marked decrease in the number of megakaryocytes and platelets, accompanied by a significant reduction of all-lineage progenitors (Gurney et al, 1994; Carver-Moore et al, 1996). Interestingly, these animals are the experimental counterpart of congenital amegakaryocytic thrombocytopenia (CAMT), a paediatric disease resulting from the loss of function of the MPL gene, in which newborns display severe thrombocytopenia and later develop aplastic anaemia (Ballmaier & Germeshausen, 2009). The principal site of thrombopoietin production in the steady state condition is the liver (Qian et al, 1998), with minor contributions from bone marrow stromal cells and kidney (Sungaran et al, 1997). Thrombopoietin is removed from the circulation by receptor-mediated uptake and destruction by the mature cells that bear its receptor (Stoffel et al, 1996) and thrombopoietin blood levels are inversely related to the platelet count (Kuter & Rosenberg, 1995). Thus, when platelet levels are high, a larger quantity of thrombopoietin is removed from the blood, leading to a lower concentration of the hormone. Conversely, when platelet levels are low, smaller amounts of thrombopoietin are removed and its blood level rises, resulting in increased platelet production. The surprising finding of high thrombopoietin levels in patients with ET (Griesshammer et al, 2000) can be explained through the impaired expression of MPL (due to its impaired glycosylation) by neoplastic megakaryocytes, resulting in inefficient thrombopoietin clearance (Moliterno et al, 1998; Teofili et al, 2002). Accordingly, paradoxical pronounced thrombocytosis is observed in transgenic mice expressing reduced levels of thrombopoietin-receptor in platelets and in terminally differentiated megakaryocytes (Tiedt et al, 2009). A similar mechanism probably occurs also in hereditary thrombocytosis sustained by MPL mutations producing receptors with low binding affinity to thrombopoietin (cf. below). Interestingly, thrombopoietin production by liver is constitutive and is inducible mainly by inflammatory mediators (especially interleukin-6) (Kaser et al, 2001), whilst it is scarcely influenced by thrombocytopenia. In contrast, the low platelet level exerts its feedback on thrombopoietin production through the regulation of THPO expression in bone marrow stromal cells (Sungaran et al, 2000). In reactive thrombocytosis or during platelet recovery, the rise of thrombopoietin production is obtained by increasing the THPO mRNA translation into protein (Ghilardi et al, 1998; Cazzola & Skoda, 2000). Actually, the translation of normal full length THPO mRNA is physiologically very inefficient, as it is almost completely inhibited by the presence in the 5′-untranslated region (5′-UTR) of seven AUG codons, which define seven short upstream open reading frames (uORF) (Ghilardi et al, 1998). These uORFs prevent the ribosome from reaching the physiological start codon (codon AUG 8). In particular, uORF 7 is a potent inhibitor of translation, most likely because of its extension beyond the physiological start site, whilst uORF 5 and 6 are much weaker inhibitors (Ghilardi et al, 1998). As a result, mutations occurring in the 5′-UTR of THPO cause thrombocytosis through the loss of the normal physiological inhibition of THPO mRNA translation (Cazzola & Skoda, 2000).

The thrombopoietin receptor is encoded by the MPL gene, which consists of 12 exons and is located on chromosome 1p34. MPL is a member of the haematopoietic cytokine receptor family (which includes the receptors for erythropoietin, interleukin-3, granulocyte colony-stimulating factor and several other cytokines) and it was identified as a result of its homology to the murine oncogene Mpl, the transforming factor of the murine myeloproliferative leukaemia virus (Souyri et al, 1990; Vigon et al, 1992). The MPL gene encodes a 635-amino acids protein, constituted by two cytokine receptor motifs (CRMs, e approximately 200 amino acids each), a 22 residues trans-membrane domain (amino acid 492–513) and an intracellular domain containing two conserved motifs termed Box 1 and Box 2 (Geddis, 2010). The incomplete glycosylation of MPL results in its impaired expression on the cell surface (Moliterno et al, 1998). Unlike other cytokine receptors, MPL contains an amphipathic RWQFP motif at the junction between the membrane and the cytoplasmic domains: both the amphipathic and the trans-membrane domains are encoded by the exon 10 and are crucial in the structure of MPL because they prevent the autonomous receptor activation (Onishi et al, 1996; Staerk et al, 2006). In addition, the membrane distal CRM appears to have an inhibitory effect on signalling, as its deletion results in the constitutive activation of the receptor (Sabath et al, 1999). MPL exists as a homodimer that has no intrinsic kinase activity but associates with the cytoplasmic tyrosine kinase JAK2 through its Box 1 domain. Upon ligand binding, the receptor conformation shifts and the closer juxtaposition of the two JAK2 molecules allows their cross-activation. Indeed, thrombopoietin stimulation results in phosphorylation of MPL-bound JAKs and the subsequent activation of several downstream pathways, including the signal transducer and activator of transcription (STAT), Ras/mitogen-activated protein kinase (RAS/MAPK), and phosphatidylinositol 3-kinase (PI3K)/AKT. This cascade drives cell survival and proliferation. In parallel, ligand binding activates several downstream pathways that limit cell signalling, including phosphatases and suppressors of cytokine signalling (SOCS). An alternative mechanism of negative feedback regulation occurs through the binding of SH2B3 (LNK) adaptor protein, which binds phospho-tyrosine residues on JAK2 (Tong & Lodish, 2004). After thrombopoietin stimulation, MPL is degraded by both the lysosomal and proteasomal pathways via ubiquitination of intracellular lysine residues (K553 and K573); the substitution of these residues confer hyperproliferation to the expressing cells (Saur et al, 2010). About 3–15% of patients with ET or PMF show gain-of-function MPL mutations. These mutations often involve the amphipatic iuxta-membrane motif and consist of the substitution of tryptophan at 515 position by leucine (MPL-W515L, the first described) (Pikman et al, 2006) or by lysine (MPL-W515K) or by alanine (MPL-W515A) (Pardanani et al, 2006). As previously stated, the integrity of the RWQFP amphipatic motif at the junction between the trans-membrane and the cytoplasmic domain is required for maintaining the receptor in a signalling-inactive conformation and, importantly, for imparting specific signalling to ligand-activated receptors (Staerk et al, 2006). Actually, all the above described MPL-W515L/K/A mutations induce autonomous cell proliferation, tumorigenesis in nude mice, spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and promote a G1/S-phase transition (Chalignéet al, 2008). Moreover, the presence of the tyrosine at position 112 of the intra-cytoplasmatic domain appears necessary to mediate their pathological effects (Pecquet et al, 2010). In addition, in order to phosphorylate appended JAK2, MPL-W515L mutant requires membrane localization (Marty et al, 2009). Much more rarely, somatic mutations affect the trans-membrane domain (MPL-A506T, MPL- L510P, MPL-S505N) (Chalignéet al, 2008). Among them, the MPL-S505N – detected in two patients (one with ET and one with PMF) (Beer et al, 2008) – was the first germ line MPL mutation described (Ding et al, 2004).

Hereditary THPO gene mutations

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

Four different mutations have been described so far: the specific mechanisms responsible for the increased thrombopoietin production and the associated clinical pictures are reported in Table I. The first mutation was discovered by Wiestner et al (1998) in 11 members (spanning four generations) of a previously described Dutch family (Schlemper et al, 1994): it was the first example of a human disease caused by the increased efficiency of mRNA translation. The authors found a G to C mutation in the splice donor site of exon 3, causing the exon skipping and the deletion of the uORF 7. As a consequence, the THPO coding sequence was shifted and THPO translation was anticipated from the eighth to the fifth and sixth AUG codons. Interestingly, in all five patients with evaluable serum samples, the thrombopoietin level appeared increased above the normal range (Wiestner et al, 1998). More recently, Liu et al (2008) identified the identical mutation in 11 members of a Polish family, confirming that the thrombopoietin serum levels in these patients were significantly higher in comparisons with unaffected relatives. Interestingly, the authors demonstrated by haplotype analysis that the two families exhibited differences in genetic polymorphisms flanking the mutation, indicating that the mutation was not related to a founder effect but rather that it arose de novo in the Polish families (Liu et al, 2008).

Table I.   Molecular, haematological and clinical findings in hereditary thrombocytosis. Abbreviations as in the text.
Involved gene (inheritance)Molecular alterationFunctionAssociated findingsMain references
THPO (autosomal dominant)G to C mutation in the splice donor site of intron 3Deletion of the uORF 7, exon skipping and increased thrombopoietin translationThrombocytosis High thrombopoietin level Microcirculation disturbance and thrombosis Mild splenomegalySchlemper et al (1994) Wiestner et al (1998) Liu et al (2008)
THPO (autosomal dominant)Deletion of a single G nucleotide in 5′UTRuORF 7 in frame with THPO coding sequence and translation initiation at uORF 7Thrombocytosis High thrombopoietin level Skeletal abnormalitiesKondo et al (1998) Ghilardi and Skoda (1999) Graziano et al (2009)
THP0 (autosomal dominant)G to T substitution in the 516 position in 5′UTRShortening the uORF 7 and translational reinitiationThrombocytosis High thrombopoietin levelKikuchi et al (1995) Ghilardi et al (1999)
THPO (autosomal dominant)A to G mutation in the intron 3Not yet characterizedThrombocytosis High thrombopoietin level Mild splenomegalyJorgensen et al (1998)
MPL (autosomal dominant)S to N substitution in the position 505 of the trans-membrane domainConstitutive receptor activationThrombocytosis Arterial and vein thrombosis Mild to severe splenomegaly and bone marrow fibrosis with ageingDing et al (2004) Teofili et al (2007a) Liu et al (2009) Teofili et al (2010)
MPL (autosomal dominant with incomplete penetrance)K to N substitution in the position 39 of the CRM (MPL Baltimore polymorphism)Reduced binding affinity to thrombopoietinThrombocytosis (heterozygote carriers) Reduced MPL expression 7% of African-American individualsMoliterno et al (2004)
MPL (autosomal recessive)P to L substitution in position 106 of the CRMReduced binding affinity to thrombopoietinThrombocytosis (homozygote carriers) Reduced MPL expression High thrombopoietin levels 6% of Arab individualsEl-Harith et al (2009)

The second mutation was discovered in five members of a Japanese family in 1998 (Kondo et al, 1998). This mutation involves the deletion of a guanine located 47 bases upstream of the physiological initiation AUG codon. Also in this case, the unaffected relatives manifested normal levels of thrombopoietin, whereas the affected members presented thrombopoietin levels circa 6–20 times higher than unaffected relatives. Immediately after, Ghilardi and Skoda (1999) demonstrated that the deletion caused the frameshift in the 5′-UTR of the THPO mRNA, which places the uORF 7 in frame with the THPO coding sequence, thus eliminating the strong inhibitory effect normally exerted by uORF 7.

A novel mutation in the 5′-UTR THPO gene was further reported by Ghilardi et al (1999) in four members of a second Japanese family (unrelated to the abovementioned one) that had been previously described by Kikuchi et al (1995). The mutation consisted of a G to T substitution in the 516 position of the 5′-UTR and created a novel stop codon, which shortened the uORF 7 and generated a gap of 31 nucleotides between uORF 7 and the physiological THPO start codon, allowing translational reinitiation and thus enhancing the translational efficiency. Finally, an additional A to G mutation in the intron 3 of the 5′-UTR codon associated with thrombopoietin overproduction has been reported, four bases downstream of the first one described (Jorgensen et al, 1998). To our knowledge this mutation has not been functionally characterized, but it is expected to cause the same aberrant splicing as the Dutch mutation. All the four described mutations share the inheritance model of autosomal dominant transmission.

As shown, all the currently described THPO mutations do not alter the sequence of the mature thrombopoietin protein, promoting the secretion of a biologically active molecule (Cazzola & Skoda, 2000) (Fig 1B). In reality, the THPO gene contains sites responsive to platelet protein-mediated transcriptional repression that are not yet well defined; these elements appear to be located in an approximately 1·9 kb region between 250 bp upstream of the transcriptional initiation site, within the second intron (McIntosh & Kaushansky, 2008). Nevertheless, although these regulatory elements contribute to platelet homeostasis under steady state conditions, they seem unable to detect increased platelet levels and to down-regulate thrombopoietin production in patients with hereditary thrombocytosis.

image

Figure 1.  Schematic representation of platelet production by megakaryocytes upon MPL activation by thrombopoietin. (A) In normal conditions, upon thrombopoietin binding, MPL conformation shifts, allowing cross-activation with JAK2 molecules and subsequent activation of several downstream pathways, including the signal transducer and activator of transcription (STAT). The receptor-mediated uptake leads to the thrombopoietin removal from the circulation. (B) All the THPO mutations so far described determine increased translational efficiency of THPO m-RNA, with over-production of normal thrombopoietin molecules and exaggerated megakaryocyte stimulation (C) Mutation of MPL in the trans-membrane region (MPL-S505N) causes the constitutive activation of the receptor, independently of the binding of thrombopoietin. (D) Mutations of MPL involving the cytokine receptor motifs of the extracellular portion (MPL-K39N and MPL-P106L) determine low binding affinity of MPL to thrombopoietin, resulting in high thrombopoietin levels. Moreover, due to a glycosylation defect, MPL expression is impaired and the thrombopoietin clearance is reduced in these hereditary thrombocytoses.

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Hereditary MPL gene mutations

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

Three germ line MPL mutations have been reported so far: the MPL-S505N (Ding et al, 2004), the MPL-K39N (Moliterno et al, 2004) and the MPL-P106L (El-Harith et al, 2009) (Table I).

MPL-S505N involves the trans-membrane domain of MPL; it was first reported in eight members of a Japanese family (Ding et al, 2004) and subsequently in 24 patients belonging to eight Italian families (Teofili et al, 2007a, 2010). In all these cases the thrombocytosis was inherited in an autosomal-dominant manner. Ding et al (2004) demonstrated that the presence of the mutation was associated with the autonomous activation in MPL downstream signalling pathways, both in vitro (cell transfected with the mutant) and in vivo (platelets obtained from affected individuals). More recently, the same group showed that MPL-S505N transduces the signal through the autonomous dimerization of the MPL protein caused by strong amino acid polarity (Ding et al, 2009) (Fig 1C). Indeed, this mutation appears functionally similar to MPL-W515K/L acquired mutations reported in patients with MPN; actually, they involve the trans-membrane or the iuxta-membrane intracellular domains, which play pivotal roles in arranging the spatial conformation of MPL in order to prevent the JAK2 auto-phosphorylation and the spontaneous receptor activation. A haplotype analysis recently performed in all the nine families carrying the MPL-S505N mutation (eight Italian and one Japanese) documented that the Italian families exhibited a de novo MPL-S505N mutation, unrelated to that reported in the Japanese family (Liu et al, 2009). Moreover it has been highlighted that the clustering of familial cases reported in Italy is due to a founder effect (Liu et al, 2009) (Fig 2).

image

Figure 2.  Haplotype analysis of the MPL locus in the eight Italian HT families and one Japanese family with the MPL-S505N mutation. The chromosomal region containing the MPL gene is shown. Arrows indicate the distances of microsatellite markers from the location of the MPL mutation. Names of microsatellite markers are shown below the locus, and numbers below the markers indicate the sizes in nucleotides of the PCR products of the co-segregating mutated alleles in each of the nine families. Families in which the marker was non-informative (n.i.) are marked. The two different haplotypes in the Italian and Japanese families are highlighted in grey. Reprinted by permission from Liu et al (2009). ©Ferrata Storti Foundation, Pavia, Italy.

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The MPL-K39N – also known as MPL Baltimore – results from the substitution of lysine with asparagine at amino acid 39 and was first identified in three African-American patients with suspected MPN. By a wide subsequent screening of patients and healthy individuals, it was elucidated that MPL- K39N is a polymorphism restricted to African Americans and that about 7% of African Americans are heterozygous for this mutation (Moliterno et al, 2004). This polymorphism is autosomal and appears to conform to a pattern of autosomal dominance with incomplete penetrance, considering that some heterozygotes have normal platelet counts whereas others have sustained elevations. The mutation in the homozygous state is associated with severe thrombocytosis. In the individuals carrying the MPL-K39N mutation, the high platelet count is associated with a reduced expression by platelets of MPL, due to a defective translation of m-RNA (Fig 1D).

The MPL- P106L mutation was first described in a study of a consanguineous Arabic family, with two members showing severe thrombocytosis and three more relatives showing mild thrombocytosis (El-Harith et al, 2009). It resulted from the substitution of proline with leucine in position 106. The frequency of the mutation is about 6% among Arabic individuals, whilst it has not been detected among Caucasian individuals. The homozygous state was associated with mild or severe thrombocytosis, while heterozygotes had mostly normal platelet count and only few individuals had mild thrombocytosis. Indeed, the inheritance mode can be regarded as autosomal-recessive. As reported for MPL Baltimore, homozygotes for the MPL- P106L polymorphism also have low or absent MPL expression on platelets. Moreover, thrombopoietin levels in homozygotes are markedly elevated in comparison to both wild type and heterozygous subjects. Both MPL- K39N and MPL- P106L mutations involve the CRM1 of the extracellular domain of MPL. Thus, it is conceivable that these defects both affect the receptor’s ability to bind thrombopoietin, resulting in reduced thrombopoietin serum clearance and in the over-stimulation of megakaryocytopoiesis (Fig 1D).

Clinical correlates of THPO and MPL mutations

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

The first THPO and MPL gene mutations were discovered in 1998 (Wiestner et al, 1998) and 2004 (Ding et al, 2004), respectively, but – due to their rarity – their clinical correlates have been better elucidated only over the last 2 years. Before the description of THPO mutations, several cases of ‘familial thrombocytosis’ or ‘familial thrombocythaemia’ were reported and patients were generally referred to as asymptomatic (Williams & Shahidi, 1991; Kikuchi et al, 1995; Cohen et al, 1997) or, at most, as suffering from minor haemorrhages (Schlemper et al, 1994). Thereafter, studies designed to expand into the molecular pathogenesis of hereditary thrombocytosis, investigated very small numbers of patients and did not detail neither clinical findings nor follow up data of affected members and of unaffected relatives. Subjects with hereditary thrombocytosis were mostly referred to haematologists for suspected MPN and– whenever investigated – the bone marrow histology was seemingly indistinguishable from that typical for ET. Nevertheless, in contrast to patients with ET, who have frequent vascular complications and possible evolution in fibrosis or acute leukaemia, patients with hereditary forms due to THPO mutations were regarded as asymptomatic (Kikuchi et al, 1995; Jorgensen et al, 1998; Kondo et al, 1998) and vascular complications were not reported in family members carrying the MPL-S505N mutation (Ding et al, 2004). As a result, the notion that hereditary thrombocytosis are benign diseases became widely accepted, having a vascular risk significantly lower than ET and rarely associated with mild splenomegaly (Dror & Blanchette, 1999; Skoda & Prchal, 2005). Overall, it appeared evident that subjects with hereditary thrombocytosis were significantly younger at diagnosis than patients with ET (Fernandez-Robles et al, 1990; Dror & Blanchette, 1999), thus demanding a specific diagnostic approach (Dame & Sutor, 2005; Teofili et al, 2007b, 2008). Liu et al (2008) identified in a Polish family with hereditary thrombocytosis the identical mutation previously described in a Dutch family (Wiestner et al, 1998). Overall, the authors evaluated 23 affected members belonging to the Polish and to the Dutch families carrying the same kind of THPO mutation, and compared them to a cohort of 107 patients with sporadic ET. This was the first study on hereditary thrombocytosis where haematological and clinical data were recorded on a sizeable number of patients. Surprisingly, all complications investigated, including venous thrombotic events, major vasomotor events, arterio-vascular events and haemorrhage occurred at comparable rates in both patients with THPO mutation and with ET. Stroke was the cause of death in two patients with THPO mutation aged over 70 years, but transient ischaemic attack (two patients, one on hydroxycarbamide treatment), deep venous thrombosis (one case) and miscarriage (one case) were reported also in patients aged less than 50 years. Furthermore, the authors reported that symptoms due to the impaired microcirculation, such as Raynaud phenomenon and erythromelalgia, responded well to aspirin and did not require cytoreductive treatment. Moreover, as investigated according to standard criteria for grading of marrow fibrosis (Thiele et al, 2005), no progression to myelofibrosis was observed in patients without cytoreductive treatment. In particular, the bone marrow histology of these patients is reminiscent of a chronic myeloproliferative disorder, with the increase and clustering of megakaryocytes, marrow hypercellularity and occasional mild increase in reticulin fibres. In addition, minimal or mild splenomegaly was documented in about one third of patients. Indeed, this study highlighted for the first time that persons with THPO mutation have an increased thrombotic risk, and, importantly, raised the issue of an appropriate therapeutic approach. Actually, the absence of JAK2 mutation in about half of the cases of ET leads to the fact that many patients with hereditary thrombocytosis are diagnosed and treated as having ET. This issue appears even more important if we consider that patients with hereditary thrombocytosis often undergo haematological investigation at a very young age. By investigating the presence of molecular markers of MPN in a series of 29 children with ET, we found the MPL-S505N mutation in 9 of 11 children with familial recurrence of thrombocytosis (Teofili et al, 2007a). The patients belonged to four unrelated families, suggesting a high frequency of the defect in the general Italian population. This finding prompted us to extended the search for this mutation to all patients with ET who had familial background of thrombocytosis or ET. We identified four additional families carrying the MPL-S505N mutation and, overall, detected the mutation in 21 patients: among them, 6 had received therapy with hydroxycarbamide or interferon for a pre-existing diagnosis of ET (Teofili et al, 2008). Haematological findings and clinical follow up of these patients and of 20 relatives with referred thrombocytosis were recorded. A high incidence of major thrombosis was documented (15 episodes; 9 casualties, including stroke in four cases, myocardial infarction in seven cases, fetal loss in two cases, deep vein thrombosis of the leg in one case and Budd-Chiari syndrome in a young female patient). Interestingly, except for two patients aged 76 and 80 years who experienced stroke during the follow up (both were on therapy with aspirin and hydroxycarbamide), all thromboses occurred before the diagnosis, in the absence of any antiplatelet therapy. During follow-up, many patients exhibited an increase of spleen size and a progressive bone marrow fibrosis, as evaluated by the standardized scoring system (Thiele et al, 2005). The histological picture was characterized in young patients by hypercellular bone marrow and in adult and elderly patients by overt bone marrow fibrosis, paralleled by the progressive decrease of platelet count and of haemoglobin level. Overall, we showed that the life expectancy of family members with thrombocytosis was significantly shorter than that of relatives without thrombocytosis, because they had an increased thrombotic risk and developed, with ageing, a clinical picture very similar to primary myelofibrosis. This observation is not so surprising if we consider that, in the field of MPN, ET patients positive for MPL-W515L/K exhibit bone marrow fibrosis and anaemia more frequently than those with wild type MPL (Beer et al, 2008; Vannucchi et al, 2008). However, Eyster et al (1986) had already described a family (five members over two generations) with elevated platelet counts; interestingly, two of the affected adults had suffered myocardial infarctions when aged 52 and 57 years, respectively, while two children were asymptomatic. Furthermore, the blood smear examination documented teardrop poikilocytosis in all three adult family members but not in children, suggesting that haematological findings could be affected by age (Eyster et al, 1986). Indeed, this description is highly evocative for an underlying MPL-S505N mutation. In respect to MPL-K39N and MPL-P106L polymorphisms, no thrombotic accidents were reported among the heterozygous carriers (Moliterno et al, 2004; El-Harith et al, 2009). Nevertheless, only analysis on larger samples of individuals belonging to specific ethnic groups will clarify if they might have a clinical impact in homozygotes or in heterozygous individuals exposed to thrombosis predisposing conditions.

Finally, it must be emphasized that, to date, progression to acute leukaemia has never been recorded among patients with hereditary thrombocytosis, either from THPO or MPL mutations (Liu et al, 2008; Teofili et al, 2010).

Recently, an interesting report described a family in which three out of four subjects with hereditary thrombocytosis due to a G516 T mutation in the THPO gene (Wiestner et al, 1998) also presented congenital transverse limb defects (Graziano et al, 2009). Considering that inherited unilateral limb defects are extremely rare and that some sporadic cases may have a disruptive vascular pathogenesis (due to intrauterine vascular accident or to ischaemic process), this study argues in favour of a possible involvement of thrombopoietin in vasculogenesis, suggesting that vascular disruptions might be, in these cases, secondary to specific gene derangements. In line with this hypothesis is another recent report of two patients (mother and child) with severe thrombocytosis and similar skeletal abnormalities in the mother and the child (Robins & Niazi, 2008). These patients were not investigated for germ-line THPO mutation, nevertheless, the presence of high thrombopoietin levels and the absence of JAK2 or MPL mutations strongly suggest that THPO could be involved.

Therapeutic approach

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

Hereditary thrombocytosis are a heterogeneous group of disease and their rarity undoubtedly hampered the precise definition of their clinical correlates and did not allow specific guide lines to be designed for proper therapies. In addition, symptomatic patients with hereditary thrombocytosis are often diagnosed as affected by ET and receive both antiplatelet and cytoreductive therapy. Recent papers exploring a fairly high number of affected individuals highlighted that, in contrast to previous beliefs, hereditary thrombocytosis due to splice donor THPO mutation and to MPL-S505N mutation are associated with an increased thrombotic risk (Liu et al, 2008; Teofili et al, 2010) and this risk appears even more pronounced in the second type of defect (Teofili et al, 2010). Three important aspects emerged from these studies, which should be taken in account in deciding how to prevent thromboses in patients with hereditary thrombocytosis: (i) these patients are often very young at diagnosis, (ii) vascular complications are much more frequent among patients not receiving antiplatelet drugs and (iii) no cases of evolution to acute leukaemia have been so far described. Liu et al (2008) reported that patients with THPO mutation suffering from microcirculation disturbance are mostly responsive to low dose aspirin, while they are resistant to hydroxycarbamide (Liu et al, 2008). Moreover, it was observed that hydroxycarbamide failed in preventing thrombosis in two patients with MPL-S505N mutation (Teofili et al, 2010) and in one patient with THPO mutation (Liu et al, 2008), suggesting that different pathogenetic mechanisms could underlie the vascular complications in MPN and hereditary thrombocytosis. Indeed, we suggest that the prevention of thrombosis and of microcirculation disturbances in these latter patients should be primarily carried out by antiplatelet therapies, while only those rare patients with recurrent thrombosis or refractory symptoms of microcirculation dysfunction should be treated with an antiproliferative drug. Similarly, low dose aspirin was found to be safe and efficacious in pregnancy, in order to prevent miscarriage (Teofili et al, 2010). Finally, among our patients with MPL-S505N mutation, we treated two young sisters with α-interferon in order to control the discomfort due to splenomegaly: therapy was interrupted after few weeks in one of them, while it actually reduced the spleen size in the other.

On the whole, it appears to be very important to distinguish hereditary thrombocytosis from ET, where thrombosis history or age over 60 years constitute well-established indications to cytoreductive treatment (Finazzi & Barbui, 2008). To this purpose, a specific diagnostic approach is provided in Fig 3. This is an important topic if we consider the young age of affected individuals, the potential oncogenic risk associated with some antiproliferative drugs, and the absence of an intrinsic leukaemic potential of hereditary thrombocytosis. For instance, in one of the described families with hereditary thrombocytosis, two patients receiving cytostatic treatment for a long time showed an abnormal chromosome 7, which is frequently observed in association with myelodysplastic disorders (Slee et al, 1981). Finally, whenever available, alternative drugs inhibiting the MPL downstream signalling pathway could reveal some efficacy.

image

Figure 3.  Proposed diagnostic approach to patients with hereditary thrombocytosis.

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Concluding remarks

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

We would conclude this review by emphasizing some quite obvious but peculiar aspects in the diagnosis and management of hereditary thrombocytosis. The first one is the importance of the accurate collection of familial history when young patients present with thrombocytosis: in the presence of a well defined familial background, they should be tested for all possible inheritable defects of THPO and MPL genes and should not be given cytoreductive treatment (Fig 3). The second aspect is that all patients, who are often at a young age, should be aware of their diagnosis and of the possibility that their progeny could inherit the disease (these defects are mostly transmitted in an autosomal dominant manner). Moreover, although the exact prevalence of MPL-S505N mutation in our country it is not note, clustering of this defect in central Italy leads into thinking that individuals who are homozygous for this defect might be generated. Finally, clinical data on hereditary thrombocytosis enable us to recommend a careful consideration in the use of two new thrombopoietin mimetic drugs, Eltrombopag and Romiplostim, recently approved for the treatment of resistant Immune Thrombocytopenia (ITP) (Kuter, 2007). Romiplostim is a thrombopoietin mimetic peptide that binds to MPL in the extra-membrane region, in competition with thrombopoietin. Conversely, Eltrombopag is a non-peptide thrombopoietin mimetic and binds to MPL in its trans-membrane region, directly activating the signal transduction downstream pathway. Indeed, Romiplostim reproduces increased MPL stimulation by thrombopoietin over-production, while Eltrombopag mimics the constitutive activation of MPL. As these drugs have to be administered lifelong, it will be very interestingly to compare their potential effect in inducing bone marrow fibrosis and splenomegaly in non-splenectomized ITP patients.

In conclusion, hereditary thrombocytoses are rare but possibly serious diseases. Any future cooperative efforts should be encouraged in order to learn more about their clinical course and treatment and to characterize those families in which the disease-causing mutation is still unknown (Wiestner et al, 2000; Tecuceanu et al, 2006).

During the editing process of this review, Posthuma et al. (Blood 2010, 116: 3375-3376) reported the occurrence of acute myeloid leukemia in one affected member of the Dutch family with hereditary thrombocytosis due to the G to C transversion in the splice donor of intron 3 of THPO. The patient was treated with acetyl salicylic acid and never received cytoreductive treatment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References

We are indebted with Dr M. Martini and Dr T. Cenci for their invaluable work in carrying out molecular analysis. Moreover, we are deeply grateful to Prof R. Foà and to Dr F. Giona for their precious contribution of childhood patients. Finally, we tank Prof G. Leone for his unfailing and expert assistance. This work was supported by Prin 2008, Ministero Università e Ricerca Scientifica (Rome, Italy) and by Fondi d’Ateneo, Progetti D1 2008–2009, Università Cattolica (Rome, Italy).

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  3. Thrombopoietin and MPL
  4. Hereditary THPO gene mutations
  5. Hereditary MPL gene mutations
  6. Clinical correlates of THPO and MPL mutations
  7. Therapeutic approach
  8. Concluding remarks
  9. Acknowledgements
  10. References
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