Essential thrombocythaemia (ET) is a chronic myeloproliferative syndrome due to sustained proliferation of megakaryocytes, which results in elevated numbers of circulating platelets, thrombotic or haemorrhagic episodes and occasional leukaemic transformation. The cause of ET is unknown. Hereditary thrombocythaemia (HT) with autosomal-dominant transmission has been described with manifestations similar to those of sporadic ET. As the thrombopoietin gene (THPO) encodes a lineage-restricted growth factor with profound stimulatory effects on megakaryopoiesis and platelet production, we tested the hypothesis that HT results from a mutation in the human THPO gene. In a Dutch family with eleven affected individuals, the thrombopoietin protein (TPO) concentrations in serum were consistently elevated in individuals with HT. We derived an intragenic CA marker for the human THPO gene and performed linkage analysis in fourteen informative meioses in this family. This resulted in a lod score of 3.5 at theta=0. A G-->C transversion was found in the splice donor site of intron 3 of the THPO gene in all affected family members. This mutation leads to THPO mRNAs with shortened 5'-untranslated regions (UTR) that are more efficiently translated than the normal THPO transcripts. We conclude that a splice donor mutation in THPO leads to systemic overproduction of TPO and causes thrombocythaemia.

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription- polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.

Summary. Hereditary thrombocythaemia (HT) with clinical features very similar to essential thrombocythaemia (ET) has been found to be transmitted as an autosomal dominant trait in several families. Here we studied the pathogenesis of HT in a previously described Japanese kindred. We found markedly elevated thrombopoietin (TPO) serum levels in all affected individuals and identi®ed a novel point mutation in the TPO gene, a G ! T transversion at position 516 of the TPO mRNA (G516T) that co-segregated with the HT phenotype in all affected family members. This mutation is located in the 5 H -untranslated region (5 H -UTR) of the TPO mRNA and when assayed in reticulocyte lysates, improved translational ef®ciency of in vitro transcribed TPO mRNA. Cell lines transfected with the mutant TPO cDNA secreted up to 8-fold more TPO protein than cells transfected with the normal cDNA. We provide a molecular model of how the mutation partially disables the physiologic repression of TPO translation and thereby causes thrombocytosis. This is the third family in which HT has been caused by the loss of translational inhibition of TPO mRNA.

We generated mice expressing a fulllength Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl Ϫ/Ϫ background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wildtype controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpltransgenic Mpl ؊/؊ mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count. IntroductionThrombopoietin (Tpo) and its receptor Mpl are the principal regulators of megakaryopoiesis. 1,2 Mice deficient in Tpo or Mpl continue to produce functional platelets, albeit at much lower levels, 3,4 suggesting that Mpl mainly controls quantitative aspects of thrombopoiesis. Tpo serum levels are controlled by the platelet mass through Mpl-mediated Tpo uptake and degradation. 5,6 Consequently, Mpl Ϫ/Ϫ mice show increased Tpo levels. 3 Although an important function of Mpl is to regulate platelet numbers, it is also expressed on hematopoietic stem cells (HSCs) and early progenitors. 7,8 Consistently, Mpl-deficient mice show markedly decreased numbers of hematopoietic progenitors, and competitive repopulation assays indicate that the numbers of murine HSCs is reduced by 7-to 8-fold. 7,8 In humans, loss-of-function mutations in Mpl lead to congenital amegakaryocytic thrombocytopenia, a disorder that frequently leads to bone marrow failure. [9][10][11] The reason for the more severe phenotype in humans remains unknown.Mutant versions of Mpl can lead to uncontrolled proliferation and survival signals as exemplified by the retroviral fusion oncogene v-Mpl, which can immortalize hematopoietic progenitors. 12 An autosomal-dominant point mutation in the transmembrane domain of Mpl (S505N) was identified as the cause of thrombocytosis in families with hereditary thrombocytosis. 13,14 Recently, point mutations in the cytoplasmic domain of Mpl (W515L, W515K) were identified in patients with primary myelofibrosis and essential thrombocythemia, and W515L was shown in mouse models to elicit myeloproliferative disease (MPD) with marked thromboc...

Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine that regulates megakaryopoiesis and platelet production. The major site of TPO biosynthesis is the liver. Despite easily detectable levels of liver TPO mRNA, the circulating TPO serum levels are very low. We have observed that translation of TPO mRNA is inhibited by the presence of inhibitory elements in the 5′-untranslated region (5′-UTR). Alternative promoter usage and differential splicing generate at least three TPO mRNA isoforms that differ in the composition of their 5′-UTR. Using mutational analysis we show that physiologically the translation of these TPO mRNA isoforms is strongly inhibited by the presence of AUG codons, which define several short open reading frames (ORFs) in the 5′-UTR and suppress efficient initiation at the physiologic start site. The two regularly spliced isoforms, which account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an efficient mechanism to prevent overproduction of this highly potent cytokine.