The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway.

Erythropoietin regulates the proliferation and differentiation of erythroid precursor cells. Its effect is mediated by the erythropoietin receptor (EPOR), a member of a large family of cytokine receptors. The EPOR gene has recently been cloned, sequenced, and characterized. As shown experimentally, its intracellular C-terminal part contains a domain exerting negative control on erythropoiesis. Here we describe a G to A transition in nucleotide 6002 of the EPOR gene that converts a TGG codon for tryptophan into a TAG stop codon, predicting the truncation of the 70 C-terminal amino acids of the EPOR molecule. The mutation occurs in heterozygous form in the germ-line DNA of members of a large kindred in which primary erythrocytosis is segregating as a mild autosomal dominant trait. The mutation cosegregates with the disease phenotype in all 29 affected family members studied; it occurs in no unaffected family members or unrelated controls. This appears to be an example of a human condition caused by an EPOR mutation. Striking similarities exist between the human phenotype described here and phenotypes of cell lines expressing similarly truncated EPOR molecules produced experimentally. By analogy with these in vitro studies, one can hypothesize that the truncated EPOR molecules are activated by suppression of phosphorylation leading to loss of the down-modulation exerted by intact EPOR molecules. Experimental modifications of the EPOR gene may eventually have therapeutic applications.

Chuvash polycythemia is an autosomal recessive disorder that is endemic to the mid-Volga River region. We previously mapped the locus associated with Chuvash polycythemia to chromosome 3p25. The gene associated with von Hippel-Lindau syndrome, VHL, maps to this region, and homozygosity with respect to a C-->T missense mutation in VHL, causing an arginine-to-tryptophan change at amino-acid residue 200 (Arg200Trp), was identified in all individuals affected with Chuvash polycythemia. The protein VHL modulates the ubiquitination and subsequent destruction of hypoxia-inducible factor 1, subunit alpha (HIF1alpha). Our data indicate that the Arg200Trp substitution impairs the interaction of VHL with HIF1alpha, reducing the rate of degradation of HIF1alpha and resulting in increased expression of downstream target genes including EPO (encoding erythropoietin), SLC2A1 (also known as GLUT1, encoding solute carrier family 2 (facilitated glucose transporter), member 1), TF (encoding transferrin), TFRC (encoding transferrin receptor (p90, CD71)) and VEGF (encoding vascular endothelial growth factor).

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5' flanking sequence and 0.7 kilobase of 3' flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.

Adaptation to hypoxia is critical for survival and regulates multiple processes, including erythropoiesis and vasculogenesis. Chuvash polycythemia is a hypoxia-sensing disorder characterized by homozygous mutation (598C>T) of von Hippel-Lindau gene (VHL), a negative regulator of hypoxia sensing. Although endemic to the Chuvash population of Russia, this mutation occurs worldwide and originates from a single ancient event. That VHL 598C>T homozygosity causes elevated normoxic levels of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), serum erythropoietin and hemoglobin is known, but the disease phenotype has not been documented in a controlled manner. In this matched cohort study, VHL 598C>T homozygosity was associated with vertebral hemangiomas, varicose veins, lower blood pressures, and elevated serum vascular endothelial growth factor (VEGF) concentrations (P <.0005), as well as premature mortality related to cerebral vascular events and peripheral thrombosis. Spinocerebellar hemangioblastomas, renal carcinomas, and pheochromocytomas typical of classical VHL syndrome were not found, suggesting that overexpression of HIF-1alpha and VEGF is not sufficient for tumorigenesis. Although hemoglobin-adjusted serum erythropoietin concentrations were approximately 10-fold higher in VHL 598C>T homozygotes than in controls, erythropoietin response to hypoxia was identical. Thus, Chuvash polycythemia is a distinct VHL syndrome manifested by thrombosis, vascular abnormalities, and intact hypoxic regulation despite increased basal expression of hypoxia-regulated genes.

The congenital polycythemic disorders with elevated erythropoietin (Epo) have been until recently an enigma, and abnormality in the hypoxia-sensing pathway has been hypothesized as a possible mechanism. The tumor suppressor von Hippel-Lindau (VHL) participates in the hypoxia-sensing pathway, as it binds to the proline-hydroxylated form of the hypoxia-inducible factor 1alpha (HIF-1alpha) and mediates its ubiquitination and proteosomal degradation. The loss of VHL function may result in the accumulation of HIF-1alpha and overproduction of HIF-1 downstream target genes including Epo. VHL syndrome is an autosomal dominant disorder predisposing to the development of tumors, due to inherited mutations in the VHL gene. Some rare patients with VHL syndrome have polycythemia, which has been attributed to Epo production by a tumor. It was recently found that homozygosity for the VHL Arg200Trp mutation is the cause of Chuvash polycythemia, an autosomal recessive polycythemic disorder characterized by elevated serum Epo and hypersensitivity of erythroid cells to Epo. We evaluated the role of VHL in 8 children with a history of polycythemia and an elevated serum Epo level and found 3 different germline VHL mutations in 4 of them. One child was homozygous for the Arg200Trp VHL mutation, and another compound heterozygous for the Arg200Trp and the Val130Leu mutations. Two children (siblings) were heterozygous for an Asp126Tyr mutation, one of them fulfilling some criteria of VHL syndrome. We propose that mutations of the VHL gene represent an important cause of pediatric sporadic polycythemias with an inappropriately high serum Epo concentration.

Primary familial and congenital polycythemia (PFCP) is characterized by erythrocytosis with normal arterial PO2, blood P50, and serum erythropoietin (EPO) levels. In two PFCP families EPO receptor (EPOR) polymorphisms cosegregated with PFCP. A heterozygous insertion of G at EPOR nucleotide 5975 was identified in genomic DNA from polycythemic members of family no. 2. 5974insG shifts the reading frame at codon 430, predicting amino acid substitutions and truncation of the last 64 amino acids. Wild-type and mutant EPOR transcripts were detected in erythroid progenitors from affected individuals. Burst-forming units-erythroid from patients exhibited increased colony size and sensitivity to EPO. Transfected Ba/F3 cells expressing EPOR 5974insG exhibited increased EPO sensitivity compared with cells expressing wild-type EPOR. The functional effect of this EPOR mutation was directly compared with the other C-terminal mutations reported in unrelated PFCP families by expression in Ba/F3 cells. The transfected cells with another primary polycythemia associated EPOR mutant construct (G6002A) also exhibited increased sensitivity to EPO.

Familial and congenital polycythemia, not due to high oxygen affinity hemoglobin or reduced 2,3-diphosphoglycerate in erythrocytes, is common in the Chuvash population of the Russian Federation. Hundreds of individuals appear to be affected in an autosomal recessive pattern. We studied six polycythemic Chuvash patients <20 years of age from unrelated families and 12 first-degree family members. Hemoglobins were markedly elevated in the index subjects (mean +/- standard deviation [SD] of 22.6 +/- 1.4 g/dL), while platelet and white blood cell counts were normal. Although performed in only three of the index subjects, serum erythropoietin concentrations determined by both radioimmune and functional assays were significantly higher in polycythemic patients compared with first-degree family members with normal hemoglobin concentrations. Southern blot analysis of the Bgl 2 erythropoietin gene polymorphism showed that one polycythemic subject was a heterozygote, suggesting the absence of linkage of polycythemia with the erythropoietin gene, assuming autosomal recessive inheritance. Polymerase chain reaction (PCR) amplification of the GGAA and GA minisatellite polymorphic regions of the erythropoietin receptor gene showed no evidence of linkage of phenotype with this gene. We conclude that Chuvash polycythemia may represent a secondary form of familial and congenital polycythemia of as yet unknown etiology. This condition is the only endemic form of familial and congenital polycythemia described.

Primary polycythemias are caused by an acquired or inborn mutation affecting hematopoietic/erythroid progenitors that results in an abnormal response to hematopoietic cytokines. Primary familial and congenital polycythemia (PFCP; also known as familial erythrocytosis) is characterized by elevated red blood cell mass, low serum erythropoietin (EPO) level, normal oxygen affinity of hemoglobin, and typically autosomal dominant inheritance. In this study we screened for mutations in the cytoplasmic domain of the EPO receptor (EPOR; exons 7 and 8 of the EPOR gene) in 27 unrelated subjects with primary or unidentified polycythemia. Two new EPOR mutations were found, which lead to truncation of the EPOR similarly to previously described mutations in PFCP subjects. The first is a 7-bp deletion (del5985-5991) found in a Caucasian family from Ohio. The second mutation (5967insT) was found in a Caucasian family from the Czech Republic. In both cases the EPO dose responses of the erythroid progenitors of the affected subjects were examined to confirm the diagnosis of PFCP. In one of these families, the in vitro behavior of erythroid progenitors in serum-containing cultures without the addition of EPO mimicked the behavior of polycythemia vera progenitors; however, we show that antibodies against either EPO or the EPOR distinguish the in vitro growth abnormality of polycythemia vera erythroid progenitors from that seen in this particular PFCP family. We conclude that PFCP is a disorder that appears to be associated in some families with EPOR mutations. So far, most of the described EPOR mutations (6 out of 8) associated with PFCP result in an absence of the C-terminal negative regulatory domain of the receptor.