Alternative titles; symbols
HGNC Approved Gene Symbol: EPO
Cytogenetic location: 7q22.1 Genomic coordinates (GRCh38): 7:100,720,468-100,723,700 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 7q22.1 | ?Diamond-Blackfan anemia-like | 617911 | Autosomal recessive | 3 |
| {Microvascular complications of diabetes 2} | 612623 | 3 | ||
| Erythrocytosis, familial, 5 | 617907 | Autosomal dominant | 3 |
Human erythropoietin is an acidic glycoprotein hormone with a molecular mass of 34 kD. As the prime regulator of red cell production, its major functions are to promote erythroid differentiation and to initiate hemoglobin synthesis.
Lee-Huang (1984) cloned human erythropoietin cDNA in E. coli. McDonald et al. (1986) and Shoemaker and Mitsock (1986) cloned the mouse gene and the latter workers showed that coding DNA and amino acid sequence are about 80% conserved between man and mouse. This is a much higher order of conservation than for various interferons, interleukin-2, and GM-CSF. Sherwood and Shouval (1986) described a human renal carcinoma cell line that continuously produces erythropoietin.
Romanowski and Sytkowski (1994) reviewed the molecular structure of human erythropoietin in historical perspective. The EPO gene encodes a deduced 193-amino acid propolypeptide. A 27-amino acid leader sequence is cleaved off the amino terminus of the propeptide, yielding the functional 166-amino acid protein. However, recombinant human EPO (rhEPO) expressed in Chinese hamster ovary cells contains only 165 amino acids, having lost arg166. The mechanism for this was undefined, and whether EPO circulating in the plasma also lacked arg166 was not known. Both the nucleotide and amino acid sequences of EPO are highly conserved among mammals.
Romanowski and Sytkowski (1994) stated that the EPO gene has 5 exons.
Law et al. (1986) assigned EPO to chromosome 7 by Southern blot analysis of DNA from human/Chinese hamster cell hybrids with a cDNA clone for the entire coding region of the gene. Further localization to 7q11-q22 was achieved by in situ hybridization. They found a RFLP with a frequency of about 20% in a Chinese population. By hybridization analysis (dot-blot) of DNA from human chromosomes isolated by high resolution dual laser sorting, Powell et al. (1986) also located EPO on chromosome 7. By somatic cell hybrid analysis, Watkins et al. (1986) placed EPO on the proximal half of 7q, closely linked to COL1A2 (120160) and to DNA markers linked to CF (219700). Because of the close linkage of EPO to COL1A2 and markers linked to CF, it is probably justified to narrow the assignment of EPO to 7q21-q22.
By in situ hybridization and by genetic analysis using RFLPs in interspecific mouse backcross DNAs, Lacombe et al. (1988) demonstrated that EPO is located on chromosome 5 in the mouse.
Eschbach et al. (1987) demonstrated the effectiveness of recombinant human erythropoietin in treating the anemia of end-stage renal disease.
In the central nervous system, neurons express EPO receptor (EPOR; 133171) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from NMDA receptor-mediated glutamate toxicity. Sakanaka et al. (1998) reported in vivo evidence that EPO protects neurons against ischemia-induced cell death. They presented findings suggesting that EPO may exert its neuroprotective effect by reducing the nitric oxide-mediated formation of free radicals or antagonizing their toxicity. Siren et al. (2001) presented data suggesting that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. They suggested that evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is indicated.
Novel erythropoiesis-stimulating protein (NESP) stimulates erythropoiesis in the same manner as human recombinant EPO. NESP is distinct from EPO in that it has additional sialic acid which has been shown to confer an increased terminal half-life in animal models, patients with chronic renal failure, and cancer patients receiving multiple cycles of chemotherapy (Macdougall et al., 1999). In studies of 89 patients with nonmyeloid malignancies, Smith et al. (2001) found that NESP was well tolerated, with response rates ranging from 61 to 83%, depending on dosage.
In addition to its role as a kidney cytokine regulating hematopoiesis, EPO is also produced in the brain after oxidative or nitrosative stress. The transcription factor HIF1 (603348) upregulates EPO following hypoxic stimuli. Digicaylioglu and Lipton (2001) demonstrated that preconditioning with EPO protects neurons in models of ischemic and degenerative damage due to excitotoxins and consequent generation of free radicals, including nitric oxide. Activation of neuronal EPO receptors (133171) prevents apoptosis induced by NMDA or nitric oxide by triggering crosstalk between the signaling pathways JAK2 (147796) and NFKB (see 164011). Digicaylioglu and Lipton (2001) demonstrated that EPO receptor-mediated activation of JAK2 leads to phosphorylation of the inhibitor of NFKB (see I-kappa-B-alpha, 164008), subsequent nuclear translocation of the transcription factor NFKB, and NFKB-dependent transcription of neuroprotective genes. Transfection of cerebrocortical neurons with a dominant interfering form of JAK2 or an I-kappa-B-alpha superrepressor blocks EPO-mediated prevention of neuronal apoptosis. Thus, neuronal EPO receptors activate a neuroprotective pathway that is distinct from previously well characterized JAK and NFKB functions. Moreover, this EPO effect may underlie neuroprotection mediated by hypoxic-ischemic preconditioning.
Celik et al. (2002) undertook studies in a rabbit model to determine whether exogenous EPO might have a protective effect in injuries to the spinal cord. Immunocytochemistry performed using human spinal cord sections showed abundant EPO receptor immunoreactivity of capillaries, especially in white matter, and motor neurons within the ventral horn. Spinal cord ischemia was produced in rabbits by occlusion of the abdominal aorta. Recombinant human EPO was administered intravenously immediately after the onset of reperfusion. The authors found both an acute and a delayed beneficial action of recombinant human EPO in ischemic spinal cord injury.
Erythropoietin is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and also apoptosis of brain neurons (Siren et al., 2001). Grimm et al. (2002) showed in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor-2 (134920), and vascular endothelial growth factor (192240) via HIF1 stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 (147678) activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1 (see 165160), an early event in the light-stressed retina, is not affected by hypoxia. The erythropoietin receptor (133171), required for EPO signaling, localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied erythropoietin that crosses the blood-retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of EPO may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.
In rats, Junk et al. (2002) conducted parallel studies of recombinant EPO in a model of transient global retinal ischemia induced by raising intraocular pressure, which is a clinically relevant model for retinal diseases. They observed abundant expression of EPOR throughout the ischemic retina. Neutralization of endogenous EPO with soluble EPOR exacerbated ischemic injury, which supports a crucial role for an endogenous EPO/EPOR system in the survival and recovery of neurons after an ischemic insult. Systemic administration of recombinant EPO before or immediately after retinal ischemia not only reduced histopathologic damage but also promoted functional recovery as assessed by electroretinography. Exogenous EPO also significantly diminished terminal deoxynucleotidyltransferase-mediated dUTP end labeling of neurons in the ischemic retina, implying an antiapoptotic mechanism of action. These results further established EPO as a neuroprotective agent in acute neuronal ischemic injury.
Becerra and Amaral (2002) reviewed the role of erythropoietin as an endogenous retinal survival factor. It is a multifunctional protein that has erythropoietic, neuroprotective, and angiogenic activities. Becerra and Amaral (2002) hypothesized that identification and separation of the structural determinants of those 3 activities within the erythropoietin molecule could elucidate additional ways to minimize side effects associated with local administration of erythropoietin to the eye, an approach that offers advantages over systemic administration.
Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Erbayraktar et al. (2003) stated that enthusiasm for rhEPO as a potential neuroprotective therapeutic needed to be tempered by the knowledge that it also enlarges circulating red cell mass and increases platelet aggregability. They examined whether erythropoietic and tissue-protective activities of rhEPO are dissociated by a variation of the molecule. They demonstrated that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. At doses and frequencies at which rhEPO exhibited erythropoiesis, asialoEPO, in marked contrast with rhEPO, did not increase the hematocrit of mice or rats. AsialoEPO promptly appeared within the cerebrospinal fluid after intravenous administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibited a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush.
Because of the generalized neuroprotective and neurotrophic actions of EPO, Bianchi et al. (2004) tested the efficacy of rhEPO in preventing and reversing nerve dysfunction in streptozotocin (STZ)-induced diabetes in rats. They found that EPO both protected from and reversed experimental diabetic neuropathy (MVCD2, 612623). Although in acute brain injury such as cerebral ischemia or brain trauma, a single injection of rhEPO was sufficient to obtain a protective effect (Brines et al., 2000), Bianchi et al. (2004) administered the agent over a long period. With this schedule, a marked increase in the hematocrit was observed. This could represent a potentially serious side effect and increase the risk of cerebrovascular accidents. Therefore nonerythropoietic analogs of EPO would be important for use in chronic diseases.
Leist et al. (2004) found that carbamylated EPO did not bind to EPOR and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, carbamylated EPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.
Although vascular endothelial growth factor (VEGF; 192240) is a primary mediator of retinal angiogenesis, VEGF inhibition alone is insufficient to prevent retinal neovascularization. Hence, it was postulated that there are other potent ischemia-induced angiogenic factors. Erythropoietin possesses angiogenic activity, but its potential role in ocular angiogenesis had not been established. Watanabe et al. (2005) measured both erythropoietin and VEGF levels in the vitreous fluid of 144 patients. Vitreous proliferative potential was measured according to the growth of retinal endothelial cells in vitro and with soluble erythropoietin receptor. In addition, a murine model of ischemia-induced retinal neovascularization was used to evaluate erythropoietin expression and regulation in vivo. They found that the median vitreous erythropoietin level in 73 patients with proliferative diabetic retinopathy (MVCD2, 612623) was significantly higher than that in 71 patients without diabetes. The median VEGF level in patients with retinopathy was also significantly higher than that in patients without diabetes. Multivariate logistic-regression analyses indicated that erythropoietin and VEGF were independently associated with proliferative diabetic retinopathy and that erythropoietin was more strongly associated with the presence of proliferative diabetic retinopathy than was VEGF. They concluded that their data suggested that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy. Watanabe et al. (2005) suggested that erythropoietin blockade might be beneficial in the treatment of proliferative diabetic retinopathy, but cautioned that erythropoietin blockade may be hazardous for retinal diseases that involve apoptosis of retinal photoreceptors since erythropoietin is a survival factor for retinal photoreceptors and acts as a neurologic protection factor in diabetic neuropathy.
Casals-Pascual et al. (2008) compared the levels of EPO, VEGF, and TNF (191160) in paired samples of plasma and cerebrospinal fluid of Kenyan children with cerebral malaria (see 611162) who died or who survived with or without developing neurologic sequelae. They found that plasma EPO of more than 200 units per liter was associated with a more than 80% reduction in the risk of developing neurologic sequelae. Admission with profound coma and convulsions after admission were independently associated with neurologic sequelae. Casals-Pascual et al. (2008) concluded that age-dependent EPO responses to anemia and age-dependent protective effects may influence the clinical epidemiology of cerebral malaria, suggesting that EPO may be useful in adjuvant therapy.
Becker et al. (2010) showed by mathematical modeling of quantitative data and experimental validation that rapid ligand depletion and replenishment of the cell surface receptor are characteristic features of the EPO receptor (EPOR; 133171). The amount of EPO-EPOR complexes and EPOR activation integrated over time corresponds linearly to ligand input; this process is carried out over a broad range of ligand concentrations. This relation depends solely on EPOR turnover independent of ligand binding, which suggests an essential role of large intracellular receptor pools. Becker et al. (2010) concluded that these receptor properties enable the system to cope with basal and acute demand in the hematopoietic system.
Minamishima and Kaelin (2010) showed that loss of all 3 PHDs (PHD1, 606424; PHD2, 606425; and PHD3, 606426) in the liver dramatically increased EPO and hematocrit values to concentrations vastly in excess of those achieved after renal PHD2 inactivation. Minamishima and Kaelin (2010) found that PHD2 inactivation is sufficient to induce near maximal renal EPO production, whereas inactivation of all 3 PHDs is needed to reactivate hepatic EPO production.
Microvascular Complications Of Diabetes, Susceptibility To, 2
Tong et al. (2008) found an association between the T allele of a SNP in the promoter of the EPO gene (rs1617640; 133170.0001) and microvascular complications of diabetes, including proliferative diabetic retinopathy and diabetic end-stage renal disease (MVCD2; 612623). They also observed a 7.5-fold higher EPO concentration in vitreous samples from normal subjects with the TT risk genotype than in those with the GG genotype, and studies in cultured HEK293 cells showed that the T allele enhanced luciferase reporter expression by 25-fold compared with that of the G allele (p = 4.7 x 10(-29)).
Familial Erythrocytosis 5
In 10 affected members of a 4-generation Norwegian family with autosomal dominant familial erythrocytosis-5 (ECYT5; 617907), Zmajkovic et al. (2018) identified a heterozygous 1-bp deletion (c.32delG; 133170.0002) in exon 2 of the EPO gene, within the signal peptide. The mutation, which was found by linkage analysis and candidate gene sequencing, segregated with the disorder in the family. The authors were unable to study patient tissue, so they used CRISPR to introduce the mutation into Hep3B human cells. The supernatant of mutant cells contained an 8- to 10-fold increase in biologically active EPO, indicating that the mutation paradoxically results in a gain of function, not a loss of function. Analysis of mRNA in wildtype Hep3B cells identified the wildtype EPO transcript produced from the physiologic promoter (P1) as well as 2 additional noncoding transcripts, a long and a short transcript, from use of an alternative promoter (P2) in intron 1. These 2 alternative transcripts were detected at higher levels in cells with the c.32delG mutation, and further studies indicated that the P2 transcripts in mutant cells had increased stability compared to wildtype and produced functional transcripts using another start codon in exon 2, resulting in excess production of EPO.
In a 3-year-old girl with ECYT5, Camps et al. (2016) identified a heterozygous 1-bp deletion (c.19delC; 133170.0003) in the EPO gene. The mutation, which was found by targeted next-generation sequencing of 125 patients with erythrocytosis, was confirmed by Sanger sequencing. Her affected father also carried the mutation. Functional studies of the variant were not performed. However, Zmajkovic et al. (2018) demonstrated that the mutation resulted in the use of an alternative promoter (P2) in intron 1 causing the production of functional transcripts and increased amounts of biologically active EPO compared to controls. The mechanism was similar to that observed with another mutation in the same region (c.32delG; 133170.0002).
Diamond-Blackfan Anemia-Like
In 2 sibs, born of consanguineous Turkish parents, with Diamond-Blackfan anemia-like (DBAL; 617911), Kim et al. (2017) identified a homozygous missense mutation in the EPO gene (R150Q; 133170.0004). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. In vitro functional expression studies showed that the R150Q mutation significantly altered the kinetics of binding to the EPO receptor, and that increased concentration of the R150Q mutated protein was unable to compensate for the defect in erythropoiesis. Intracellular flow cytometry and Western blot analysis examining downstream EPO receptor pathways showed a selective decrease in JAK2 (147796)-mediated phosphorylation of STAT1 (600555) and STAT3 (102582) resulting from reduced ability of the mutant EPO to promote dimerization of the EPO receptor. Kim et al. (2017) discussed the role of functional selectivity and biased agonists in receptor signaling.
Synthesis of erythropoietin in the kidney and liver in response to hypoxia depends on both protein synthesis and heme synthesis. Goldberg et al. (1988) proposed a model in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulates the production of erythropoietin. Semenza et al. (1989) generated transgenic mice containing the human erythropoietin gene and found increased erythropoietin mRNA expression not only in liver and kidney but in all other transgenic tissues analyzed. The mice were polycythemic, with increased erythroid precursors in hematopoietic tissues and increased erythrocytic indices in peripheral blood. From further studies in these transgenic mice, Semenza et al. (1989) concluded that different DNA sequences flanking the EPO gene control liver versus kidney expression of the gene and that some of these sequences are located 3-prime to the gene.
Erythropoiesis occurs in 2 distinct waves during embryogenesis: the primitive wave in the extraembryonic yolk sac followed by the definitive wave in the fetal liver and spleen. Even though progenitors for both cells types are present in the yolk sac blood islands, only primitive cells are formed in the yolk sac during early embryogenesis. Lee et al. (2001) presented results that led them to propose that erythropoietin expression and the resultant erythropoietin receptor activation regulate the timing of the definitive wave. They demonstrated that Epo and EpoR gene expression are temporally and spatially segregated: though EPOR is expressed early (embryonic days 8.0-9.5) in the yolk sac blood islands, no EPO expression could be detected in this extraembryonic tissue. Only at a later stage can EPO expression be detected intraembryonically, and the onset of EPO expression correlates with the initiation of definitive erythropoiesis. By 'knocking in' a constitutively active form of EpoR, R129C, they demonstrated further that the activation of the EPOR signaling pathway can lead to earlier onset of definitive erythropoiesis in the yolk sac. The observations provided insight into the in vivo mechanism by which 2 erythroid progenitor populations can coexist in the yolk sac yet always differentiate successively during embryogenesis.
Naffakh et al. (1995) examined whether the secretion of erythropoietin from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to EPO. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine cDNA was expressed under the control of the murine phosphoglycerate kinase promoter. 'Neo-organs' containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit and elevated serum EPO concentration were observed in recipient animals over a 10-month observation period. The approach was considered applicable to the treatment of human anemias.
Osborne et al. (1995) investigated in rats the expression and biologic effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat erythropoietin cDNA. Vector-derived Epo secretion caused increases in reticulocytes followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely downregulated in kidney and absent from liver. These results indicated to the authors that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.
Similar experiments were performed by Kessler et al. (1996), who demonstrated that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase gene into adult mice, protein expression was detected in myofibers for at least 32 weeks. Furthermore, a single intramuscular administration of an AAV vector containing a gene for human erythropoietin into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myocytes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo.
To evaluate whether recombinant EPO improves functional outcome if administered after spinal cord injury, Gorio et al. (2002) studied 2 rodent models. In the first model, a moderate compression was produced by applying an aneurysm clip at level T3 and administering recombinant EPO immediately after release of compression; partial recovery of motor function began within 12 hours after injury and was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model, recombinant EPO administration given 1 hour after injury also produced a superior recovery of function compared with saline-treated controls after a contusion at level T9. In the latter model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by recombinant EPO administration and associated with reduced cavitation within the spinal cord. Gorio et al. (2002) suggested that EPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory, and antiapoptotic functions.
High (2005) diagrammed the details of a successful strategy, devised by Takacs et al. (2004), to treat anemia in mice by stimulating production of erythropoietin and its secretion into the bloodstream. Takacs et al. (2004) used a genetically modified, antigen-specific B cell to supply erythropoietin to a mouse deficient in erythropoietin. Transgenic mice were engineered to express human erythropoietin under the control of a B cell-specific element. The mouse was then immunized with a single antigen, phycoerythrin, resulting in a pool of antigen-sensitive B cells. Lymphocytes from the donor mouse were injected into an erythropoietin-deficient mouse. Subsequent injection of antigen into the erythropoietin-deficient mice stimulated the proliferation of B cells expressing erythropoietin, with a resultant increase in serum erythropoietin levels and hematocrit values.
Zhong et al. (2007) studied the efficacy of erythropoietin in providing neuroprotection to retinal ganglion cells (RGCs) in the DBA/2J glaucoma mouse model. EPO promoted RGC survival in these mice without affecting intraocular pressure, suggesting that EPO is be a potential therapeutic neuroprotectant in glaucoma.
With the use of formal backcross analysis, van Wijngaarden et al. (2007) investigated the inheritance of susceptibility to oxygen-induced retinopathy in the rat. Their results indicated that segregation of the susceptibility trait to oxygen-induced retinopathy in the Dark Agouti and Fisher 344 rat strains was associated with pigmentation and erythropoietin expression and could be modeled using an autosomal dominant pattern of inheritance.
In a cohort of 374 patients with type 2 diabetes (125853) and microvascular complications of diabetes, including proliferative diabetic retinopathy (PDR) and end-stage renal disease (ESRD) (MVCD2, 612623), and 239 age- and ethnicity-matched diabetic controls, Tong et al. (2008) found significant association between the T allele of rs1617640, a SNP in the promoter of the EPO gene, and PDR and ESDR (corrected p = 0.036). The association with diabetic microvascular complications was confirmed in 365 patients with type 1 diabetes (222100) with both PDR and ESRD, 500 with nephropathy and retinopathy without progression to PDR and ESRD, and 574 type 1 diabetic control patients without nephropathy or retinopathy (p = 2.66 x 10(-8)), as well as in a third cohort involving 379 type 1 diabetics with both PDR and nephropathy and 141 diabetic controls (p = 0.021). The EPO concentration in vitreous samples was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype, and studies in cultured HEK293 cells showed that the T allele enhanced luciferase reporter expression by 25-fold compared with that of the G allele (p = 4.7 x 10(-29)).
In 10 affected members of a 4-generation Norwegian family with autosomal dominant familial erythrocytosis-5 (ECYT5; 617907), Zmajkovic et al. (2018) identified a heterozygous 1-bp deletion, c.32delG (chr7.100,319,199GG-G, GRCh37) in exon 2 of the EPO gene, within the signal peptide. The mutation was predicted to cause a frameshift with disruption of the signal peptide, resulting in premature termination after 51 amino acids. The mutation, which was found by linkage analysis and candidate gene sequencing, segregated with the disorder in the family and was not found in the gnomAD database. The authors were unable to study patient tissue, so they used CRISPR to introduce the mutation into Hep3B human cells. The supernatant of mutant cells contained an 8 to 10-fold increase in biologically active EPO, indicating that the mutation paradoxically results in a gain of function, not a loss of function. Analysis of mRNA in wildtype Hep3B cells identified the wildtype EPO transcript produced from the physiologic promoter (P1) as well as 2 additional noncoding transcripts, a long and a short transcript, from use of an alternative promoter (P2) in intron 1. These 2 alternative transcripts were detected at higher levels in cells with the c.32delG mutation, and further studies indicated that the P2 transcripts in mutant cells had increased stability compared to wildtype and produced functional transcripts using another start codon in exon 2, resulting in excess production of EPO.
In a 3-year-old girl with familial erythrocytosis-5 (ECYT5; 617907), Camps et al. (2016) identified a heterozygous 1-bp deletion, c.19delC (chr7.100,319,185TC-T, GRCh37) in the EPO gene. The mutation was predicted to result in a frameshift (P7fs). The mutation, which was found by targeted next-generation sequencing of 125 patients with erythrocytosis, was confirmed by Sanger sequencing. Her affected father also carried the mutation. Functional studies of the variant were not performed, but the variant was not found in the dbSNP (build 142) or ExAC databases.
Zmajkovic et al. (2018) demonstrated that the c.19delC mutation resulted in the use of an alternative promoter (P2) in intron 1, causing the production of functional transcripts and increased amounts of biologically active EPO compared to controls. The mechanism was similar to that observed with another mutation in the same region (c.32delG; 133170.0002).
In 2 sibs, born of consanguineous Turkish parents, with Diamond-Blackfan anemia-like (DBAL; 617911), Kim et al. (2017) identified a homozygous c.530G-to-A transition in exon 5 of the EPO gene (chr7:100,320,704G-A), resulting in an arg150-to-gln (R150Q) substitution. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family and was not found in the ExAC database. Mutations in genes known to be involved in DBA were excluded in the proband. In vitro functional expression studies using recombinant wildtype and mutant EPO showed that the R150Q mutation only mildly affected overall affinity to the EPO receptor (EPOR; 133171), but significantly altered the kinetics of binding to the receptor, with significantly faster dissociation compared to wildtype. Studies on cultured erythroid cells and CD34+ hematopoietic stem cells showed that increased concentration of the R150Q mutated protein was unable to compensate for the defect in erythropoiesis. Intracellular flow cytometry and Western blot analysis examining downstream EPO receptor pathways showed a selective decrease in JAK2 (147796)-mediated phosphorylation of STAT1 (600555) and STAT3 (102582). Further studies indicated that the R150Q mutant had reduced ability to promote dimerization of the EPO receptor, resulting in decreased JAK2 activation, even at maximally potent concentrations, and impaired downstream signal transduction. At age 6 years, the proband underwent bone marrow transplant and achieved full donor chimerism, but still required transfusions. He died of transplant complications. A younger sib was subsequently diagnosed clinically and molecularly with the same disorder. She was successfully treated with recombinant EPO. Kim et al. (2017) discussed the role of functional selectivity and biased agonists in receptor signaling.
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