Thrombopoietin Gene Research Articles

Familial dominant thrombocytopenia: clinical, biologic, and molecular studies.

Inherited thrombocytopenias are a heterogenous group of disorders. Different criteria have been suggested to classify the forms, such as the inheritance mechanism and the platelet volume as well as the number and morphology of megakaryocytes. However, the classification is often descriptive, and the precise mechanism of thrombocytopenia still remains unknown. We describe the clinical, biologic, and molecular findings of an autosomal dominant thrombocytopenia in a large family. The 17 patients had normocellular bone marrow and normal platelet volume. Platelets also showed a normal aggregation test and normal response to ADP and thrombopoietin (TPO). In the affected subjects, the mean +/- SD levels of platelet count and plasma TPO were 62+/-25 and 258+/-151, respectively. Comparative analysis showed that the patients with platelet count <70000 had higher plasma TPO concentration. The data are consistent with a mild clinical form of the disease associated with only a few episodes of bleeding. To exclude the possible role of TPO and its receptor c-mpl in the etiology of this condition, linkage analysis was performed using microsatellite markers close to the TPO and c-mpl genes on chromosomes 3q26.3-q27 and 1p34, respectively. The absence of cosegregation within the affected family indicated that these genes, as well as two other candidate loci on chromosomes 11 and 21, are not responsible for this hereditary dominant form of thrombocytopenia. A genome-wide search and subsequent identification of the gene will provide new insight into the pathogenesis of this disorder.

A Single-Base Deletion in the Thrombopoietin (TPO) Gene Causes Familial Essential Thrombocythemia Through a Mechanism of More Efficient Translation of TPO mRNA

To the Editor: Deletion of a single G nucleotide (ΔG) in exon 3 of the thrombopoietin (TPO) gene was found to cosegregate with thrombocytosis and elevated TPO serum levels in a Japanese kindred with thrombocythemia.[1][1] The ΔG mutation affects the 5′-untranslated region (5′-UTR) of the TPO

Genomic organisation of the human chordin gene and mutation screening of candidate Cornelia de Lange syndrome genes.

We have determined the genomic organisation of the human chordin gene, CHRD, and have shown that it maps within a gene cluster at 3q27 containing THPO (thrombopoietin), CLCN2 (a voltage-gated chloride-channel gene) and EIF4G1 (a eukaryotic translation-initiation-factor-gamma gene). The CHRD and THPO genes are very close neighbours and are transcribed from opposing DNA strands from promoters that are spaced less than 2 kb apart. We considered that the CHRD gene and the chordin-regulating GSC (goosecoid) gene could be candidate genes for Cornelia de Lange syndrome (CDLS), a developmental malformation syndrome which is primarily characterised by mental handicap, growth retardation, distinctive facial features and limb-reduction defects. CDLS patients typically occur as sporadic cases, but several reports have suggested dominant inheritance. The candidacy of the CHRD and GSC genes was supported by several lines of evidence: prior evidence for a CDLS gene at 3q26.3-q27; a report suggesting a significant association between CDLS and thrombocytopenia; suspected genetic heterogeneity in CDLS; location of the GSC gene in close proximity to a 14q32 breakpoint detected in a CDLS patient with a balanced de novo translocation; known regulation of chordin expression by goosecoid; and the pattern of embryonic expression of the mouse GSC gene. Another candidate gene at 3q27, SOX2, was also considered because of its suspected role as a transcription factor in early development and because of known examples of SOX genes that are loci for dominantly inherited developmental disorders. However, mutation screening failed to identify CDLS patient-specific mutations in CHRD, GSC or SOX2.

Expression of the Thrombopoietin Gene in Human Fetal and Neonatal Tissues

Thrombopoietin (TPO) regulates megakaryopoiesis and platelet production. In the adult, TPO is mainly produced by the liver and the kidneys. This study focuses on fetal and neonatal TPO mRNA expression. In 26 human fetuses and preterm neonates, samples from liver, kidney, spleen, lung, and bone marrow were extracted for total RNA. We measured platelet counts, TPO serum concentrations by enzyme-linked immunosorbent assay, and TPO mRNA contents by reverse transcription/competitive polymerase chain reaction. TPO mRNA concentrations per microgram total RNA were similar in liver, spleen, and bone marrow, slightly lower in kidney, and significantly lower in lung. When related to gram tissue, TPO mRNA levels were highest in the liver. Considering the total amount of TPO mRNA produced in liver, kidney, and spleen, the liver accounted for 95.3%. No correlations between TPO mRNA expression and serum TPO concentration, blood platelet count, or gestational age were observed. In conclusion, the liver is the primary site of TPO gene expression in human fetuses and neonates. The spleen may contribute to TPO production during fetal life. Like in the adult, TPO mRNA is expressed in fetal bone marrow.

Hepatic thrombopoietin mRNA levels in acute and chronic liver failure of childhood.

The liver is the main production site of the hormone thrombopoietin (TPO), the major regulator of megakaryopoiesis. To investigate the role of an impaired TPO gene expression in the pathogenesis of thrombocytopenia in pediatric patients suffering from liver failure, we measured hepatic TPO mRNA in children with acute or chronic end-stage liver disease undergoing orthotopic liver transplantation. Tissue samples for RNA extraction were obtained from 12 children with compensated cirrhosis (CC), 22 children with decompensated cirrhosis (DC), and 9 children with acute liver failure (ALF). TPO mRNA was quantitated by competitive polymerase chain reaction (PCR), following reverse transcription (RT). Furthermore, in 9 children with ALF, serum TPO levels were measured by enzyme-linked immunosorbent assay before and 10 to 14 days after liver transplantation. The hepatic TPO mRNA concentration was highest in children with CC (median, 50.9 amol/micrograms RNA). This value was significantly reduced in children with DC (30.2 amol/micrograms RNA) or ALF (13.8 amol/micrograms RNA). Children with ALF (139 cells/nL) or DC (200 cells/nL) had lower platelet counts than children with CC (368 cells/nL). The serum TPO concentration increased from a median of 156 pg/mL in patients with ALF to 547 pg/mL after liver transplantation. These results show that the thrombocytopenia in children with liver failure is associated with reduced hepatic TPO mRNA levels. It remains to be investigated whether the serum TPO level and platelet counts are markers for the severity of liver damage that may serve as a prognostic indicator.

Thrombopoietin stimulation of hematopoietic stem/progenitor cells.

The recent cloning of the thrombopoietin gene, and the production of recombinant protein, have allowed studies on both its biological actions and clinical utility. Thrombopoietin not only affects the cells of the megakaryocytic lineage, but has a diverse set of cellular targets. In particular, it stimulates the ex vivo expansion of hematopoietic stem/progenitor cells suggesting that it may play a role in transplantation studies. Pre-clinical but limited clinical studies indicate that under defined conditions, thrombopoietin may accelerate white blood cell count and platelet recoveries following myelosuppression or radiotherapy.

Functional Roles of Thrombopoietin-c-mpl System in Essential Thrombocythemia

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and throm-bopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl-proto-oncogene). In addition to its physiologic role, the TPO-c-mpl system has been suggested to participate in the pathophysiology of essential thrombocythemia (ET) which is a clonal disorder characterized by a sustained elevation of the circulafing platelet count and bone-marrow hyperplasia with excessive proliferation of megakaryocytes. Recent studies have demonstrated that serum TPO levels are slightly elevated or within normal range in ET patients, whereas serum TPO levels tend to be inversely correlated with platelet mass. Flow cytometric, Western blot, and Northern blot analyses have revealed that the expression of platelet c-Mpl is strikingly reduced in all of patients with ET, possibly due to the decreased expression of c-mpl mRNA. These results suggest that normal or slightly elevated levels of serum TPO in ET patients may be attributable to the impaired uptake and catabolism of TPO owing to the low c-Mpl expression. Furthermore, immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TPO-induced protein-tyrosine phosphorylation, including c-Mpl-tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl-mediated signaling pathway was not constitutively activated in platelets of ET patients. Although activating mutation in the TPO gene, which leads to overexpression of TPO mRNA, has been reported in familial thrombocythemia, these results suggest that TPO-c-Mpl system may not be directly linked to pathogenesis of sporadic ET.

Thrombopoietin-Induced Signal Transduction and Potentiation of Platelet Activation

IntroductionThe presence of thrombopoietin, a humoral regulator of megakaryopoiesis and thrombopoiesis, had been suggested for years,1 however, some investigators were skeptical about the existence of such a factor. Modern studies of thrombopoietin begin with the cloning of c-mpl (the cellular counterpart of the v-mpl oncogene) and its cognate ligand genes. Following the cloning of the c-mpl proto-oncogene,2 Methia et al3 found that, in the presence of oligonucleotides antisense to the gene, CD34+ cells developed into megakaryocytic precursors less efficiently. As the development into other lineage was nearly intact, it was reasonably argued that the c-mpl protein may be the receptor for a thrombopoietin-like factor.3 This study inspired numerous investigations, which culminated in the cloning of the thrombopoietin gene in 1994.1, 4-8

The activating splice mutation in intron 3 of the thrombopoietin gene is not found in patients with non‐familial essential thrombocythaemia

Essential thrombocythaemia (ET) is a condition of unknown aetiology characterized by sustained thrombocytosis in the absence of a detectable systemic cause. Although usually considered a clonal disease affecting myeloid cells, recent data indicate that a significant proportion of patients have polyclonal haemopoiesis. In some patients the thrombopoietin (TPO) levels are normal or raised. Recently a mutation has been described in the TPO gene in familial thrombocythaemia that results in elevated TPO levels. We have therefore screened 51 patients diagnosed with non-familial ET for the presence of this mutation, but it was not detected in any patient. The constitutional presence of this mutation is therefore unlikely to contribute to the pathogenesis of ET.

Familial Essential Thrombocythemia Associated With One-Base Deletion in the 5′-Untranslated Region of theThrombopoietin Gene

Familial essential thrombocythemia (ET) is inherited in an autosomal-dominant manner. This finding implies that familial ET may arise as a consequence of a mutation(s) that activates platelet production. In 1994, the thrombopoietin (TPO) gene was isolated and cloned. The TPO-TPO receptor, encoded for by thec-mpl gene, are essential regulators of thrombopoiesis. Alterations of TPO or c-Mpl thus may constitute a pathogenic event leading to familial ET. In a case of familial ET presented in our institute, serum TPO levels were significantly elevated in affected members of the family as compared with nonaffected members. Moreover, we identified a one-base deletion in the 5′-untranslated region of theTPO gene in affected but not in nonaffected family members. In vitro experiments showed that the identified mutation increased TPO production. Based on our findings, we propose that this region of theTPO gene may play a crucial role in regulating TPO expression. Our results strongly suggest that the identified mutation leads to familial ET. © 1998 by The American Society of Hematology.

The activating splice mutation in intron 3 of the thrombopoietin gene is not found in patients with non-familial essential thrombocythaemia

The activating splice mutation in intron 3 of the thrombopoietin gene is not found in patients with non-familial essential thrombocythaemia

An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia.

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.

Elemental study of gene therapy with thrombopoietin

Thrombopoietin (TPO) is also referred to as the c-mpl ligand or megakaryocyte growth and development factor, inducing thrombopoiesis by proliferation of megakaryocyte progenitors and promoting endoreplicatton of mature megakaryocytes, t The identification of TPO, and the demonstration that administration to normal experimental animals with recombinant TPO results in a marked increase in platelets count, 2 has opened the possibility of using TPO to rescue patients from life-threatening thrombocytopenia with high dose chemotherapy and radiotherapy. But recombinant TPO is a foreign protein and has adverse effects on patients to different extent. This study is to establish a new TPO administration by gene therapeutic methods, i.e. that after transferred into animals, the TPO gene expresses functional TPO in vivo and maintains high platelet level.

Expression in Escherichia coli and purification of human thrombopoietin.

Human thrombopoietin (TPO) has been successfully overexpressed in Escherichia coli, with an expression level of about 12% of total cellular protein. The full-length TPO gene was subcloned into the prokaryotic expression vector pKK233-2 under the control of the inducible tac promoter. The recombinant protein was produced mainly in the form of inclusion body. By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity. The specific activity and yield of recombinant TPO can reach 2 x 10(4) units/mg and 2 mg/g of wet E. coli cells respectively.

Characterization of the Human Thrombopoietin Gene Promoter: A POSSIBLE ROLE OF AN Ets TRANSCRIPTION FACTOR, E4TF1

Thrombopoietin (TPO), the ligand for c-Mpl, is a cytokine that regulates megakaryocyte growth and development. We have cloned the 5'-flanking region of the human TPO gene and analyzed its promoter activity. The human TPO gene promoter lacks a TATA box and directs transcription initiation at multiple sites over a 50-nucleotide region. Transient expression in a human liver cell line (PLC) of promoter fragment-luciferase reporter gene constructs containing a series of 5'-truncated sequences or site-directed mutations identified a sequence 5'-ACTTCCG-3' from -69 to -63 as a positive cis-acting element for high level expression of TPO gene. This sequence contains a core motif (C/A)GGA(A/T) for Ets family proteins in the noncoding strand. Gel mobility shift assays performed with nuclear protein from PLC cells identified a DNA binding protein(s) specific for the element. Anti-E4TF1-60(GABPalpha) or anti-E4TF1-53/47(GABPbeta) antibodies supershifted the complex in gel shift assay. Furthermore, co-expression of E4TF1-60 and E4TF1-53/47 squelched TPO gene expression in PLC and HepG2 cells. It is concluded that Ets family transcription factor E4TF1(GABPalpha/beta), an ubiquitously expressed protein, is required for high level expression of the TPO gene in liver.

Dysregulated Mpl-Ligand production by hemopoietic cellsinduces a fatal myeloproliferative syndrome in mice

To evaluate the effects of long-term high-dose exposure to Mpl-ligand also called thrombopoietin (TPO), C57BL/6J murine marrow cells were infected with a retrovirus carrying the murine TPO gene. Mice were treated 4 days by 5-FU and marrow cells were then infected by coculture using a MPZen vector containing the murine TPO cDNA. Non adherent marrow cells were transplanted into lethally irradiated recipients. A majority of hematopoietic cells in the marrow, spleen, thymus and blood was transduced by the retroviral vector, one and three months after reconstitution. Plasma TPO activity in transplanted mice was extremely high (104 U/ml). A disease with two distinct steps was observed. During the two first months after transplantation, platelet (plt) and white blood cell (WBC) counts increased 4- and 10-fold, respectively. Abnormal platelet size and granules were observed. Spleen weight increased 4-fold and marrow cellularity decreased 5-fold. Histology revealed hyperplasia of the megakaryocytic and myeloid cells. Total numbers of CFU-MK and CFU-GM increased. In contrast, the hematocrit progressively fell accompanied by a decrease in the erythroblasts and CFU-E numbers. Beginning two months after transplantation, plt and WBC numbers also declined. Thrombocytopenia was noted 5 months after transplantation. The Hcts continued to decrease. Few cells were isolated from the marrow cavities and spleens. Histology revealed fibrosis of the marrow and spleen and significant osteosclerosis of the marrow. An extramedullary hematopoiesis was observed in numerous organs such as the liver or the kidney. Total numbers of progenitors were very low in hematopoietic organs. Mice died 7 months after transplantation with severe pancytopenia. Two early deaths were observed with a marked increase in blast cells. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described [Yan et al (1995) Blood 86: 4025]. In conclusion, dysregulated TPO production by hemopoietic cells in mice results in a fatal myeloproliferative disease which mimics the clinical evolution of idiopathic myelofibrosis observed in man.

トロンボポエチンのクローニングと臨床応用の可能性

Recently, we purified rat thrombopoietin (TPO) from the plasma of irradiated rats by using a quantitative in vitro assay and then determined its partial amino acid sequences. Based on the sequence information, we isolated a rat TPO cDNA, a human TPO cDNA and the human TPO gene. The human TPO protein comprises 353 amino acids, including a 21 amino acid residue signal peptide. The protein displays two domains, with an amino-terminal region essential for biological activity and a carboxyl-terminal region containing six potential sites for N-glycosylation. The expression of TPO mRNA is the highest in the liver among various tissues tested, indicating that the liver is the primary organ of TPO production. It is now evident that TPO is a lineage-dominant hematopoietic factor that primarily regulates megakaryocytopoiesis and thrombopoiesis. Data from in vitro studies have shown that TPO exhibits both megakaryocyte colony-stimulating and megakaryocyte maturation activities. In vivo, TPO or PEG-rHuMGDF (a truncated form of TPO, chemically modified with polythyelene glycol) markedly increases platelet production in normal animals. Administration of PEG-rHu-MGDF or TPO is highly effective in improving thrombocytopenia in myelosuppressed animal models, suggesting the therapeutic potential of this molecule.

Characterization of the Human Thrombopoietin Gene Promoter

Characterization of the Human Thrombopoietin Gene Promoter

Translation initiation region plays an important role in the expression of human thrombopoietin in Escherichia coli.

A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site-specific mutagenesis based on the PCR technique. This mutant TPO gene encoded the same amino acid sequence as wild-type TPO gene. The wild-type TPO gene was expressed in E. coli with very low efficiency. The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E. coli, which was much higher than the wild-type gene. The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding.

Low levels of erythroid and myeloid progenitors in thrombopoietin-and c- mpl-deficient mice

Thrombopoietin (TPO), the ligand for the c-mpl receptor, has been shown to be the major regulator of platelet production. Mice deficient in either c-mpl or TPO generated by homologous recombination show a dramatic decrease in platelet counts, but other blood cell counts are normal. Because TPO treatment of myelosuppressed mice not only enhances the recovery of platelets but also accelerates erythroid recovery, we investigated the levels of myeloid and erythroid progenitor cells in TPO-or c-mpl-deficient mice. Our results show that the number of megakaryocyte, granulocyte-macrophage, erythroid, and multilineage progenitors are significantly reduced in the bone marrow, spleen, and peripheral blood of either TPO-or c-mpl-deficient mice. Administration of recombinant murine TPO to TPO-deficient mice and control littermate mice significantly increased the absolute number of myeloid, erythroid, and mixed progenitors in bone marrow and spleen. This increase was especially apparent in TPO-deficient mice where numbers were increased to a level greater than in diluent-treated control mice and approached or equaled that in the TPO-treated control mice. Moreover, TPO- administration greatly increased the number of circulating progenitors as well as platelets in both TPO-deficient and control mice. Furthermore, the megakaryocytopoietic activity of other cytokines in the absence of a functional TPO or c-mpl gene was shown both in vitro and in vivo.