Alternative titles; symbols
HGNC Approved Gene Symbol: THPO
Cytogenetic location: 3q27.1 Genomic coordinates (GRCh38): 3:184,371,935-184,379,688 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 3q27.1 | Amegakaryocytic thrombocytopenia, congenital, 2 | 620481 | Autosomal recessive | 3 |
| Thrombocythemia 1 | 187950 | Autosomal dominant | 3 | |
| Thrombocytopenia 9 | 620478 | Autosomal dominant | 3 |
The THPO gene encodes thrombopoietin, a lineage-specific cytokine that is an essential regulator of hematopoiesis and the differentiation of progenitors into platelet-forming megakaryocytes (summary by Cornish et al., 2020).
As dramatically indicated by Metcalf (1994), the isolation of a postulated thrombopoietin eluded investigators for more than 30 years. Finally, cDNAs for both human and murine thrombopoietin were cloned and shown to encode a glycoprotein with selective action on megakaryocyte proliferation in vitro. As often happens, the success story had an unlikely beginning in the findings in a different field.
Vigon et al. (1992) cloned the human homolog of the myeloproliferative leukemia virus (MPL; 159530) and found that it showed striking homologies with members of the hematopoietin receptor superfamily. Methia et al. (1993) demonstrated that MPL may have special relevance as a receptor for cells of the megakaryocyte lineage. They did this by using MPL antisense oligodeoxynucleotides to show that these inhibited megakaryocyte colony formation in vitro, but had no inhibitory effects on erythroid or granulocyte-macrophage colonies. Thus, the power of the 'receptor first' approach for detecting growth factors was demonstrated.
Four groups used cell lines engineered to express the MPL receptor as essential tools for monitoring and cloning the MPL ligand. De Sauvage et al. (1994) purified the ligand from the plasma of irradiated pigs by using MPL affinity columns and, with amino acid sequence data, isolated a porcine genomic clone. A human genomic MPL ligand clone was then isolated by PCR, using porcine-based oligonucleotides, and a cDNA clone isolated from a human fetal liver library. De Sauvage et al. (1994) then went on to show that recombinant MPL ligand increases levels of platelets when injected into animals.
Lok et al. (1994) and Kaushansky et al. (1994) adopted a quite different strategy. They based their approach on the observations, made with a number of hemopoietic cell lines, that when autonomous transformation occurs, it is often due to the acquired autocrine production by the cells of the relevant growth factor for a receptor being expressed on the cells. Therefore, they analyzed clones of autonomous mutants of cells expressing the inserted candidate receptor, Mpl, in the hope of detecting one where transformation was based on Mpl ligand production. One such cell line was found and cDNA libraries were prepared from it. Expression screening was undertaken using Mpl-bearing target cells. In this way, they isolated a cDNA for murine MPL ligand. The recombinant protein proved capable of increasing platelet levels in mice to far higher levels than those attainable with known hemopoietic regulators. Analysis in vitro showed that it could also stimulate megakaryocyte formation, particularly in combination with other factors.
Wendling et al. (1994) completed the evidence that MPL ligand is indeed thrombopoietin. They showed that all the megakaryocyte colony-stimulating activity and platelet-elevating activity in thrombocytopenic plasma can be removed through binding to recombinant MPL (the receptor), indicating that both biologic properties can be ascribed to the MPL ligand. Metcalf (1994), a pioneer researcher in the field of hematopoietic factors, commented that 'a busy time now lies ahead for biologists and clinicians....it will also be a busy time for the lawyers arguing who has the patent rights to what should prove to be a lucrative and valuable new therapeutic agent.'
Bartley et al. (1994) reported the identification and cloning of a megakaryocyte growth and development factor (MGDF) from canine, murine, and human sources. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively. Both canine and recombinant human MGDF support the development of megakaryocytes from human CD34(+) progenitor cell populations. MGDF binds to Mpl affinity columns, and its biologic effects are inhibited by the soluble extracellular domain of Mpl, suggesting that MGDF is a cytokine that regulates megakaryocyte development and is a ligand for the MPL receptor.
Foster et al. (1994) isolated a human THPO cDNA by PCR from kidney mRNA. The cDNA was found to encode a protein with 80% identity to murine Thpo.
In addition to the previously described full-length cDNA, 2 cDNA variants were isolated from human fetal liver by Chang et al. (1995). Comparison of these with the genomic sequence indicated that they arise by differential splicing.
The THPO gene is transcribed from 2 different promoters and generates 2 major mRNA isoforms, variants 1 and 5. Variant 1 contains 6 exons with the first ATG in exon 2 and an open reading frame of 353 codons. Variant 5 contains 7 exons with the first ATG in exon 2 and a putative open reading frame of 493 codons; however, this protein has not been isolated, likely because translation is inhibited. Capaci et al. (2023) identified a conserved regulatory region upstream of the transcriptional start site in the THPO gene where the transcription factors ETS1 (164720) and STAT4 (600558) interact with each other in a complex and bind to consensus sequences that regulate the level of gene expression in hepatocellular cells. Further studies suggested that STAT3 (102582) may also play a role in regulating the transcription of THPO. THPO is produced primarily in the liver (Seo et al., 2017).
Sohma et al. (1994) cloned the complete THPO gene by screening a human genomic library using human THPO cDNA as a probe. They found that the gene is 6.2 kb long and contains 6 exons and 5 introns. According to Southern blot analysis, the human genome contains a single copy.
Using a murine cDNA for the mpl ligand as a probe, Foster et al. (1994) isolated a gene encoding human THPO from a human genomic library. They found that the THPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO; 133170). Southern blot analysis of human genomic DNA showed a hybridization pattern consistent with a single gene locus.
Chang et al. (1995) showed that the MGDF gene consists of 7 exons and 6 introns spanning 8 kilobases. The protein is encoded by exons 3 through 7.
Wiestner et al. (1998) identified 2 promoter regions, designated P1 and P2, in the THPO gene. P1 is within exon 1, and P2 is within exon 2. The 5-prime region of THPO contains several short ORFs.
By fluorescence in situ hybridization, Sohma et al. (1994) mapped the THPO gene to 3q27. By fluorescence in situ hybridization, Foster et al. (1994) mapped the THPO gene to 3q26-q27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. By fluorescence in situ hybridization, Chang et al. (1995) mapped the MGDF gene to 3q26.3. Chang et al. (1995) mapped the murine Thpo gene to chromosome 16 by linkage analysis in an interspecific backcross.
Farese et al. (1996) reported that administration of a pegylated form of recombinant MGDF (PEG-MGDF) significantly induced bone marrow regeneration compared with rMGDF untreated with PEG. When combined with recombinant G-CSF, PEG-MGDF significantly enhanced multilineage hematopoietic recovery with no evidence of lineage competition.
Ratajczak et al. (1997) found that in the presence of erythropoietin (133170) and interleukin-3 (147740), THPO was able to stimulate a small increase in erythroid colony formation in culture. Other studies suggested that THPO has little direct stimulatory effect on erythroid progenitor cells but may indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.
Using exon-specific PCR analysis, Dordelmann et al. (2008) found that THPO was expressed from both promoters in HEK293T human embryonic kidney cells and in HepG2 adult liver cells. However, expression from promoter P1, which they called P1a, was cell line-dependent and showed cell line-dependent responses to phorbol ester and cAMP stimulation.
Decker et al. (2018) assessed the physiologic source of thrombopoietin using a TPO(DsRed-CreER) knockin mouse and showed that TPO is expressed by hepatocytes but not by bone marrow cells. Deletion of Tpo from hematopoietic cells, osteoblasts, or bone marrow mesenchymal stromal cells did not affect hematopoietic stem cell number or function. However, when Tpo was deleted from hepatocytes, bone marrow hematopoietic stem cells were depleted. Thus, a cross-organ factor, circulating TPO made in the liver by hepatocytes, is required for bone marrow hematopoietic stem cell maintenance.
Thrombocythemia 1
Thrombocythemia-1 (THCYT1; 187950) is a chronic myeloproliferative syndrome due to sustained proliferation of megakaryocytes, which results in elevated numbers of circulating platelets, thrombotic or hemorrhagic episodes, and occasional leukemic transformation. Because the THPO gene encodes a lineage-restricted growth factor with profound stimulatory effects on megakaryopoiesis and platelet production, Wiestner et al. (1998) tested the hypothesis that mutation in this gene was responsible for thrombocythemia in a Dutch family with 11 affected individuals. THPO protein concentrations in serum were consistently elevated in affected members of this family. Using an intragenic CA marker for the THPO gene, they demonstrated linkage to the disorder; lod score = 3.5 at theta = 0.0. Affected members were found to carry a splice donor mutation in the THPO gene (600044.0001) that led to THPO mRNAs with shortened 5-prime untranslated regions that were more efficiently translated than the normal THPO transcripts.
In affected members of a family with thrombocythemia, Kondo et al. (1998) identified heterozygosity for a 1-bp deletion in the THPO gene (600044.0002).
In affected members of a Japanese family reported by Kikuchi et al. (1995), Ghilardi et al. (1999) identified a heterozygous gain-of-function germline mutation in the THPO gene (600044.0003).
In a Filipino mother and her 2 children with thrombocythemia, Zhang et al. (2011) identified the same intronic mutation identified by Wiestner et al. (1998) in affected members of a Dutch kindred. The same mutation was identified in affected members of a Caucasian family with thrombocytosis by Prouzet-Mauleon et al. (2020).
Thrombocytopenia 9
In 4 individuals from 2 unrelated families with thrombocytopenia-9 (THC9; 620478), Noris et al. (2018) identified a heterozygous nonsense mutation in the THPO gene (R31X; 600044.0004). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the phenotype in the families. The 2 probands were ascertained from a cohort of 86 individuals with idiopathic thrombocytopenia who underwent whole-exome sequencing. The R31X mutation was predicted to result in degradation of the mutant protein and haploinsufficiency; accordingly, serum THPO levels in the individuals with the mutation were at the lower limit of normal. The mutation was found in 1 of 60,000 individuals in the ExAC database, but the authors noted that since the phenotype is mild, that individual may be clinically asymptomatic. Yang et al. (2022) demonstrated that expression of the R31X mutation in thpo-null zebrafish was unable to rescue thrombocytopenia, suggesting that it is a loss-of-function mutation (see ANIMAL MODEL).
In 6 individuals from 3 unrelated families with THC9, Cornish et al. (2020) identified heterozygous mutations in the THPO gene (600044.0005-600044.0007). The mutations, which were found by whole-genome sequencing and confirmed by Sanger sequencing, segregated with the phenotype in the families. There were 2 frameshift mutations resulting in loss of the glycosylated C-terminal domain, and a missense mutation in the N-terminal receptor-binding domain that likely disrupted the protein structure. Studies of HEK293 cells transfected with the mutations showed decreased secretion of THPO compared to controls, and there was evidence of mistrafficking of the mutant proteins with abnormal retention in the cell, resulting in a loss-of-function effect. The probands were ascertained from a cohort of 105 unrelated thrombocytopenia cases who underwent genetic studies.
Congenital Amegakaryocytic Thrombocytopenia 2
In 2 sibs of Micronesian descent with congenital amegakaryocytic thrombocytopenia-2 (CAMP2; 620481), Dasouki et al. (2013) identified a homozygous missense mutation in the THPO gene (R17C; 600044.0008). The mutation, which was found by exome sequencing, segregated with the disorder in the family. Both parents and another sib, who were all heterozygous for the mutation, had asymptomatic mild thrombocytopenia. Molecular modeling predicted that the mutation would disrupt normal disulfide bonding to cause poor receptor binding. In vitro functional studies showed that the mutant protein was expressed, but was defective in activating the MPL (159530) signaling cascade and was unable to maintain proliferation of megakaryocytic cells, consistent with a partial loss of function. The mutant protein did not act in a dominant-negative manner.
In 5 children from 3 unrelated consanguineous families of Middle Eastern descent with CAMT2, Seo et al. (2017) identified homozygous mutations in the THPO gene. Two families (A and B) carried a missense mutation (R99W; 600044.0007) and 1 had a nonsense mutation (R157X; 600044.0010). The mutations, which were found by various sequencing methods, were not present in public databases, including dbSNP (build 141), Exome Variant Server, 1000 Genomes Project, and ExAC. The R99W mutation was present in the heterozygous state in the unaffected mother in family A, but genetic information was not available from the father in family A and the parents in family B. In vitro studies showed that the R99W mutant protein was expressed and functional, but THPO was undetectable in the patients, suggesting that the mutation somehow severely reduces THPO levels. The R157X was predicted to result in a loss of function.
In 3 sibs, born of consanguineous Egyptian parents, with CAMT2, Pecci et al. (2018) identified a homozygous missense mutation in the THPO gene (R119C; 600044.0011). The mutation, which was found by direct sequencing of the THPO gene, segregated with the disorder in the family and was not present in the dbSNP, 1000 Genomes Project, or ExAC databases. In vitro cellular studies showed that the R119C variant interfered with proper THPO secretion and the mutant protein had impaired ability to sustain proliferation of megakaryocyte-derived cells in culture. These abnormalities were associated with defective activation of the THPO receptor MPL and other downstream signaling pathways, consistent with a loss-of-function effect.
In a boy, born of consanguineous Palestinian parents, with CAMT2, Capaci et al. (2023) identified a homozygous c.-323C-T transition in the promoter region of the THPO gene (600044.0012). The mutation, which was found by whole-exome sequencing, segregated with the disorder in the family. The variant was not present in gnomAD, but was found in dbSNP. In vitro studies in transfected HEK293 cells showed that the mutation significantly reduced promoter activity by almost 80% compared to wildtype. The mutation impaired proper binding of the transcription factors ETS1 (164720) and STAT4 (600558), thus reducing expression of the THPO cytokine.
Li et al. (2001) demonstrated that some individuals treated with a recombinant thrombopoietin preparation developed thrombocytopenia (see 620478) due to antibodies to thrombopoietin.
To determine whether Lnk (605093) might act as a physiologic negative regulator of cytokine-induced hematopoietic stem cell (HSC) expansion, Buza-Vidas et al. (2006) analyzed HSC expansion in Lnk -/- mice. They found that Lnk -/- HSCs continued to expand postnatally, up to 24-fold above normal by 6 months of age. Within the stem cell compartment, this expansion was highly selective for self-renewing long-term HSCs, which showed enhanced thrombopoietin responsiveness. Lnk -/- HSC expansion was dependent on thrombopoietin, and 12-week-old Lnk -/- Thpo -/- mice had 65-fold fewer long-term HSCs than Lnk -/- mice. Expansions in multiple myeloid, but not lymphoid, progenitors in Lnk -/- mice also proved to be thrombopoietin-dependent.
Yang et al. (2022) found that knockdown of the thpo gene in zebrafish resulted in early-onset thrombocytopenia, but the animals could survive into adulthood without spontaneous or abnormal bleeding. Treatment with THPO receptor agonists (THPO-RAs) increased the number of thrombocytes.
In affected members of a 4-generation Dutch family with autosomal dominant thrombocythemia-1 (THCYT1; 187950), Wiestner et al. (1998) identified a heterozygous G-to-C transversion at position +1 of intron 3 of the THPO gene.
In a Filipino mother and her 2 children with thrombocythemia, Zhang et al. (2011) identified heterozygosity for the same intronic mutation identified by Wiestner et al. (1998). (Zhang et al. (2011) described the mutation as occurring in intron 2.)
Prouzet-Mauleon et al. (2020) identified the c.433G-A mutation (c.433G-A, NM_001290003) at the donor splice site of intron 3 in 3 affected members of a Caucasian family with thrombocytosis. The variant was predicted to modify RNA splicing, resulting in skipping of exon 3 and more efficient translation of thrombopoietin.
In a family with thrombocythemia-1 (THCYT1; 187950), Kondo et al. (1998) found that serum TPO levels were significantly elevated in affected members as compared with unaffected members. Moreover, they identified a heterozygous 1-bp deletion in the 5-prime untranslated region of the THPO gene in affected but not in unaffected family members. In vitro experiments showed that the identified mutation increased TPO production. Thus, they proposed that this region of the gene plays a crucial role in regulating TPO expression. Affected members were observed in 3 generations of the family. None of the affected members of the family had any thrombotic or hemorrhagic symptoms. In the mutated allele, a guanine at nucleotide 3252 was deleted. This guanine was located 47 bases upstream of the authentic initiation codon.
In a Japanese family with hereditary thrombocythemia-1 (THCYT1; 187950), previously reported by Kikuchi et al. (1995), Ghilardi et al. (1999) found markedly elevated thrombopoietin serum levels and identified a novel heterozygous point mutation in the THPO gene, a G-to-T transversion at position 516 of the THPO mRNA, that cosegregated with the phenotype. This mutation was located in the 5-prime untranslated region of the THPO mRNA. Cell lines transfected with the mutant THPO cDNA secreted up to 8-fold more THPO protein than cells transfected with the normal cDNA. The authors stated that this was the third family in which hereditary thrombocythemia had been found to be caused by the loss of translational inhibition of thrombopoietin mRNA.
Graziano et al. (2009) reported a father and 2 sons with essential thrombocythemia and distal limb defects who were all heterozygous for the 516G-T mutation, which they referred to as 185G-T, in exon 2 of the THPO gene. The father had absence of the right forearm, right hand, and right foot. Both sons had milder lower limb defects of the foot. The paternal grandfather only had thrombocythemia and also carried the mutation. Graziano et al. (2009) postulated that THPO may be involved in disorders of vasculogenesis, which may have led to the transverse limb defects observed in this family.
In 4 individuals from 2 unrelated families with thrombocytopenia-9 (THC9; 620478), Noris et al. (2018) identified a heterozygous c.91C-T transition (c.91C-T, ENST00000204615) in the THPO gene, resulting in an arg31-to-ter (R31X) substitution. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the phenotype in the families. The 2 probands were ascertained from a cohort of 86 individuals with idiopathic thrombocytopenia who underwent whole-exome sequencing. The R31X mutation was predicted to result in degradation of the mutant protein and haploinsufficiency; accordingly, serum THPO levels in the individuals with the mutation were at the lower limit of normal. The mutation was found in 1 of 60,000 individuals in the ExAC database, but the authors noted that since the phenotype is mild, that individual may be clinically asymptomatic.
In 3 individuals spanning 3 generations of a family (family A) with thrombocytopenia-9 (THC9; 620478), Cornish et al. (2020) identified a heterozygous 1-bp duplication (c.610dup, ENST00000204615.7) in the THPO gene, resulting in a frameshift, premature termination (Glu204GlyfsTer123), and loss of the C-terminal domain of glycosylated residues. The mutation, which was found by whole-genome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. Studies of HEK293 cells transfected with the mutation showed decreased secretion of THPO compared to controls. There was abnormal cytoplasmic accumulation of the mutant protein likely due to mistrafficking. Serum THPO concentrations were decreased in the mutation carriers, consistent with a loss-of-function effect.
In 2 individuals spanning 2 generations of a family (family B) with thrombocytopenia-9 (THC9; 620478), Cornish et al. (2020) identified a heterozygous 1-bp duplication (c.805dup, ENST00000204615.7) in the THPO gene, resulting in a frameshift and premature termination (Leu269ProfsTer58) with loss of the C-terminal domain of glycosylated residues. The mutation, which was found by whole-genome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. Studies of HEK293 cells transfected with the mutation showed decreased secretion of THPO compared to controls. There was perinuclear localization of mutant THPO and less punctate cytoplasmic expression, suggesting mistrafficking of the mutant protein, accumulation in the Golgi, and intracellular degradation. Serum THPO concentrations were decreased in the mutation carriers, consistent with a loss-of-function effect.
Thrombocytopenia 9
In a 39-year-old woman (family C) with thrombocytopenia-9 (THC9; 620478), Cornish et al. (2020) identified a heterozygous c.295C-T transition (c.295C-T, ENST00000204615.7) in the THPO gene, resulting in an arg99-to-trp (R99W) substitution at a conserved residue in the N-terminal receptor binding domain (RBD) distinct from residues that interface with the THPO receptor. The mutation was found by whole-genome sequencing and confirmed by Sanger sequencing. The mutation was absent from the unaffected mother; the father was deceased and not studied. Studies of HEK293 cells transfected with the mutation showed decreased secretion of THPO compared to controls. There was perinuclear localization of mutant THPO and less punctate cytoplasmic expression, suggesting alteration of the protein structure resulting in mistrafficking of the mutant protein, accumulation in the Golgi, and intracellular degradation. Serum THPO concentrations were decreased in the mutation carriers, consistent with a loss-of-function effect.
Amegakaryocytic Thrombocytopenia, Congenital, 2
In 4 patients from 2 unrelated consanguineous families of Middle Eastern origin (families A and B) with congenital amegakaryocytic thrombocytopenia-2 (CAMT2; 620481), Seo et al. (2017) identified a homozygous c.295C-T transition (c.295C-T, NM_000460.3) in the THPO gene, resulting in an arg99-to-trp (R99W) substitution at a highly conserved residue in the EPO-like receptor-binding domain. The mutation, which was found by various sequencing methods, was not present in public databases, including dbSNP (build 141), Exome Variant Server, 1000 Genomes Project, and ExAC. The R99W mutation was present in the heterozygous state in the unaffected mother in family A, but genetic information was not available from the father in family A or the parents in family B. In vitro studies showed that the R99W mutant protein was expressed and functional, but THPO was undetectable in the patients, suggesting that the mutation somehow severely reduces THPO levels.
In 2 sibs of Micronesian descent with congenital amegakaryocytic thrombocytopenia-2 (CAMT2; 620481), Dasouki et al. (2013) identified a homozygous c.112C-T transition in the THPO gene, resulting in an arg17-to-cys (R17C) substitution in the receptor-binding domain (RBD) (numbering excludes the 21-residue signal peptide). The mutation, which was found by exome sequencing, segregated with the disorder in the family. The proband presented at age 16 years with menorrhagia, whereas her brother, who was also homozygous for the mutation, was asymptomatic at age 28 years, indicating variable expressivity. He was found to have pancytopenia with macrocytosis. Both parents and another sib, who were all heterozygous for the mutation, had asymptomatic mild thrombocytopenia. Molecular modeling predicted that the mutation would disrupt normal disulfide bonding to cause poor receptor binding. In vitro functional studies showed that the mutant protein was expressed, but was defective in activating the MPL (159530) signaling cascade and was unable to maintain proliferation of megakaryocytic cells, consistent with a partial loss of function. The mutant protein did not act in a dominant-negative manner.
Variant Function
Pecci et al. (2018) found that the R17C (ARG38CYS, R38C) variant identified by Dasouki et al. (2013) interfered with proper THPO secretion. In vitro functional studies showed that the mutant protein had impaired ability to sustain proliferation of megakaryocyte-derived cells in culture. These abnormalities were associated with defective activation of the THPO receptor MPL and downstream signaling pathways.
In a girl, born of consanguineous Saudi Arabian parents (family C), with congenital amegakaryocytic thrombocytopenia-2 (CAMT2; 620481), Seo et al. (2017) identified a homozygous c.469C-T transition (c.469C-T, NM_000460.3) in the THPO gene, resulting in an arg157-to-ter (R157X) substitution in the middle of the protein, predicted to result in absence of the C-terminal glycan domain. The mutation, which was found by whole-exome sequencing, segregated with the disorder in the family and was not present in public databases, including dbSNP (build 141), Exome Variant Server, 1000 Genomes Project, and ExAC. Functional studies of the variant were not performed, but it was predicted to result in a loss of function. Serum THPO levels were undetectable in the patient.
In 3 sibs, born of consanguineous Egyptian parents, with congenital amegakaryocytic thrombocytopenia-2 (CAMT2; 620481), Pecci et al. (2018) identified a homozygous c.355C-T transition (c.355C-T, NM_000460) in the THPO gene, resulting in an arg119-to-cys (R119C) substitution at a highly conserved residue in the N-terminal region that is involved in binding to the MPL (159530) receptor. The mutation, which was found by direct sequencing of the THPO gene, segregated with the disorder in the family and was not present in the dbSNP, 1000 Genomes Project, or ExAC databases. In vitro cellular studies showed that the R119C variant interfered with proper THPO secretion and the mutant protein had impaired ability to sustain proliferation of megakaryocyte-derived cells in culture. These abnormalities were associated with defective activation of the THPO receptor MPL and downstream signaling pathways, consistent with a loss-of-function effect.
In a boy, born of consanguineous Palestinian parents, with congenital amegakaryocytic thrombocytopenia-2 (CAMT2; 620481), Capaci et al. (2023) identified a homozygous c.-323C-T transition (c.-323C-T, NM_000460.4) in the promoter region of the THPO gene upstream of the transcription start site in isoform 1. The mutation, which was found by whole-exome sequencing, segregated with the disorder in the family. The variant was not present in gnomAD, but was found in dbSNP. In vitro studies in transfected HEK293 cells showed that the mutation significantly reduced promoter activity by almost 80% compared to wildtype. The mutation impaired proper binding of the transcription factors ETS1 (164720) and STAT4 (600558), thus reducing expression of the THPO cytokine.
Bartley, T. D., Bogenberger, J., Hunt, P., Li, Y.-S., Lu, H. S., Martin, F., Chang, M.-S., Samal, B., Nichol, J. L., Swift, S., Johnson, M. J., Hsu, R.-Y., and 41 others. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77: 1117-1124, 1994. [PubMed: 8020099] [Full Text: https://doi.org/10.1016/0092-8674(94)90450-2]
Buza-Vidas, N., Antonchuk, J., Qian, H., Mansson, R., Luc, S., Zandi, S., Anderson, K., Takaki, S., Nygren, J. M., Jensen, C. T., Jacobsen, S. E. W. Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK. Genes Dev. 20: 2018-2023, 2006. [PubMed: 16882979] [Full Text: https://doi.org/10.1101/gad.385606]
Capaci, V., Adam, E., Bar-Joseph, I., Faleschini, M., Pecci, A., Savoia, A. Defective binding of ETS1 and STAT4 due to a mutation in the promoter region of THPO as a novel mechanism of congenital amegakaryocytic thrombocytopenia. Haematologica 108: 1385-1393, 2023. [PubMed: 36226497] [Full Text: https://doi.org/10.3324/haematol.2022.281392]
Chang, M., McNinch, J., Basu, R., Shutter, J., Hsu, R., Perkins, C., Mar, V., Suggs, S., Welcher, A., Li, L., Lu, H., Bartley, T., Hunt, P., Martin, F., Samal, B., Bogenberger, J. Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene. J. Biol. Chem. 270: 511-514, 1995. [PubMed: 7822271] [Full Text: https://doi.org/10.1074/jbc.270.2.511]
Chang, M.-S., Hsu, R.-Y., McNinch, J., Copeland, N. G., Jenkins, N. A. The gene for murine megakaryocyte growth and development factor (thrombopoietin, Thpo) is located on mouse chromosome 16. Genomics 26: 636-637, 1995. [PubMed: 7607698] [Full Text: https://doi.org/10.1016/0888-7543(95)80193-p]
Cornish, N., Aungraheeta, M. R., FitzGibbon, L., Burley, K., Alibhai, D., Collins, J., Greene, D., Downes, K., NIHR BioResource, Westbury, S. K., Turro, E., Mumford, A. D. Monoallelic loss-of-function THPO variants cause heritable thrombocytopenia. Blood Adv. 4: 920-924, 2020. [PubMed: 32150607] [Full Text: https://doi.org/10.1182/bloodadvances.2019001293]
Dasouki, M. J., Rafi, S. K., Olm-Shipman, A. J., Wilson, N. R., Abhyankar, S., Ganter, B., Furness, L. M., Fang, J., Calado, R. T., Saadi, I. Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with autosomal recessive aplastic anemia. Blood 122: 3440-3449, 2013. [PubMed: 24085763] [Full Text: https://doi.org/10.1182/blood-2012-12-473538]
de Sauvage, F. J., Hass, P. E., Spencer, S. D., Malloy, B. E., Gurney, A. L., Spencer, S. A., Darbonne, W. C., Henzel, W. J., Wong, S. C., Kuang, W.-J., Oles, K. J., Hultgren, B., Solberg, L. A., Jr., Goeddel, D. V., Eaton, D. L. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369: 533-538, 1994. [PubMed: 8202154] [Full Text: https://doi.org/10.1038/369533a0]
Decker, M., Leslie, J., Liu, Q., Ding, L. Hepatic thrombopoietin is required for bone marrow hematopoietic stem cell maintenance. Science 360: 106-110, 2018. [PubMed: 29622652] [Full Text: https://doi.org/10.1126/science.aap8861]
Dordelmann, C., Telgmann, R., Brand, E., Hagedorn, C., Schroer, B., Hasenkamp, S., Baumgart, P., Kleine-Katthofer, P., Paul, M., Brand-Herrmann, S.-M. Functional and structural profiling of the human thrombopoietin gene promoter. J. Biol. Chem. 283: 24382-24391, 2008. [PubMed: 18617523] [Full Text: https://doi.org/10.1074/jbc.M802198200]
Farese, A. M., Hunt, P., Grab, L. B., MacVittie, T. J. Combined administration of recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor enhances multilineage hematopoietic reconstitution in nonhuman primates after radiation-induced marrow aplasia. J. Clin. Invest. 97: 2145-2151, 1996. [PubMed: 8621805] [Full Text: https://doi.org/10.1172/JCI118652]
Foster, D. C., Sprecher, C. A., Grant, F. J., Kramer, J. M., Kuijper, J. L., Holly, R. D., Whitmore, T. E., Heipel, M. D., Bell, L. A., Ching, A. F. T., McGrane, V., Hart, C., O'Hara, P. J., Lok, S. Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization. Proc. Nat. Acad. Sci. 91: 13023-13027, 1994. [PubMed: 7809166] [Full Text: https://doi.org/10.1073/pnas.91.26.13023]
Ghilardi, N., Wiestner, A., Kikuchi, M., Ohsaka, A., Skoda, R. C. Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene. Brit. J. Haemat. 107: 310-316, 1999. [PubMed: 10583217] [Full Text: https://doi.org/10.1046/j.1365-2141.1999.01710.x]
Graziano, C., Carone, S., Panza, E., Marino, F., Magini, P., Romeo, G., Pession, A., Seri, M. Association of hereditary thrombocythemia and distal limb defects with a thrombopoietin gene mutation. Blood 114: 1655-1657, 2009. [PubMed: 19553636] [Full Text: https://doi.org/10.1182/blood-2009-04-217851]
Kaushansky, K., Lok, S., Holly, R. D., Broudy, V. C., Lin, N., Bailey, M. C., Forstrom, J. W., Buddle, M. M., Oort, P. J., Hagen, F. S., Roth, G. J., Papayannopoulou, T., Foster, D. C. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 369: 568-571, 1994. [PubMed: 8202159] [Full Text: https://doi.org/10.1038/369568a0]
Kikuchi, M., Tayama, T., Hayakawa, H., Takahashi, I., Hoshino, H., Ohsaka, A. Familial thrombocytosis. Brit. J. Haemat. 89: 900-902, 1995. [PubMed: 7772529] [Full Text: https://doi.org/10.1111/j.1365-2141.1995.tb08432.x]
Kondo, T., Okabe, M., Sanada, M., Kurosawa, M., Suzuki, S., Kobayashi, M., Hosokawa, M., Asaka, M. Familial essential thrombocythemia associated with one-base deletion in the 5-prime-untranslated region of the thrombopoietin gene. Blood 92: 1091-1096, 1998. [PubMed: 9694695]
Li, J., Yang, C., Xia, Y., Bertino, A., Glaspy, J., Roberts, M., Kuter, D. J. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98: 3241-3248, 2001. [PubMed: 11719360] [Full Text: https://doi.org/10.1182/blood.v98.12.3241]
Lok, S., Kaushansky, K., Holly, R. D., Kuijper, J. L., Lofton-Day, C. E., Oort, P. J., Grant, F. J., Heipel, M. D., Burkhead, S. K., Kramer, J. M., Bell, L. A., Sprecher, C. A., Blumberg, H., Johnson, R., Prunkard, D., Ching, A. F. T., Mathewes, S. L., Bailey, M. C., Forstrom, J. W., Buddle, M. M., Osborn, S. G., Evans, S. J., Sheppard, P. O., Presnell, S. R., O'Hara, P. J., Hagen, F. S., Roth, G. J., Foster, D. C. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369: 565-568, 1994. [PubMed: 8202158] [Full Text: https://doi.org/10.1038/369565a0]
Metcalf, D. Thrombopoietin: at last. Nature 369: 519-520, 1994. [PubMed: 8202150] [Full Text: https://doi.org/10.1038/369519a0]
Methia, N., Louache, F., Vainchenker, W., Wendling, F. Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 82: 1395-1401, 1993. [PubMed: 7689867]
Noris, P., Marconi, C., De Rocco, D., Melazzini, F., Pippucci, T., Loffredo, G., Giangregorio, T., Pecci, A., Seri, M., Savoia, A. A new form of inherited thrombocytopenia due to monoallelic loss of function mutation in the thrombopoietin gene. Brit. J. Haemat. 181: 698-701, 2018. [PubMed: 28466964] [Full Text: https://doi.org/10.1111/bjh.14694]
Pecci, A., Ragab, I., Bozzi, V., De Rocco, D., Barozzi, S., Giangregorio, T., Ali, H., Melazzini, F., Sallam, M., Alfano, C., Pastore, A., Balduini, C. L., Savoia, A. Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim. EMBO Molec. Med. 10: 63-75, 2018. [PubMed: 29191945] [Full Text: https://doi.org/10.15252/emmm.201708168]
Prouzet-Mauleon, V., Montibus, B., Chauveau, A., Hautin, M., Migeon, M., Ka, C., Laharanne, E., Bidet, A., Corcos, L., Lippert, E. A novel thrombopoietin (THPO) mutation altering mRNA splicing in a case of familial thrombocytosis. Brit. J. Haemat. 190: e104-e106, 2020. [PubMed: 32430933] [Full Text: https://doi.org/10.1111/bjh.16742]
Ratajczak, M. Z., Ratajczak, J., Marlicz, W., Pletcher, C. H., Jr., Machalinski, B., Moore, J., Hung, H., Gewirtz, A. M. Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis. Brit. J. Haemat. 98: 8-17, 1997. [PubMed: 9233556] [Full Text: https://doi.org/10.1046/j.1365-2141.1997.1802997.x]
Seo, A., Ben-Harosh, M., Sirin, M., Stein, J., Dgany, O., Kaplelushnik, J., Hoenig, M., Pannicke, U., Lorenz, M., Schwarz, K., Stockklausner, C., Walsh, T., and 11 others. Bone marrow failure unresponsive to bone marrow transplant is caused by mutations in thrombopoietin. Blood 130: 875-880, 2017. [PubMed: 28559357] [Full Text: https://doi.org/10.1182/blood-2017-02-768036]
Sohma, Y., Akahori, H., Seki, N., Hori, T., Ogami, K., Kato, T., Shimada, Y., Kawamura, K., Miyazaki, H. Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Lett. 353: 57-61, 1994. [PubMed: 7926023] [Full Text: https://doi.org/10.1016/0014-5793(94)01008-0]
Vigon, I., Mornon, J.-P., Cocault, L., Mitjavila, M.-T., Tambourin, P., Gisselbrecht, S., Souyri, M. Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily. Proc. Nat. Acad. Sci. 89: 5640-5644, 1992. [PubMed: 1608974] [Full Text: https://doi.org/10.1073/pnas.89.12.5640]
Wendling, F., Maraskovsky, E., Debili, N., Florindo, C., Teepe, M., Titeux, M., Methia, N., Breton-Gorius, J., Cosman, D., Vainchenker, W. c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 369: 571-574, 1994. [PubMed: 8202160] [Full Text: https://doi.org/10.1038/369571a0]
Wiestner, A., Schlemper, R. J., van der Maas, A. P. C., Skoda, R. C. An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nature Genet. 18: 49-52, 1998. [PubMed: 9425899] [Full Text: https://doi.org/10.1038/ng0198-49]
Yang, L., Wu, L., Meng, P., Zhang, X., Zhao, D., Lin, Q., Zhang, Y. Generation of a thrombopoietin-deficient thrombocytopenia model in zebrafish. J. Thromb. Haemost. 20: 1900-1909, 2022. [PubMed: 35622056] [Full Text: https://doi.org/10.1111/jth.15772]
Zhang, B., Ng, D., Jones, C., Oh, S. T., Nolan, G. P., Salehi, S., Wong, W., Zehnder, J. L., Gotlib, J. A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia. (Letter) Blood 118: 6988-6990, 2011. [PubMed: 22194398] [Full Text: https://doi.org/10.1182/blood-2011-10-386177]