Congenital Polycythemia Research Articles

Clinical study of myocardial damage induced by neonatal jaundice in normal birth weight term infants

To clarify whether neonatal jaundice may cause myocardial damage to term infants with normal birth weight (BW). Totally 178 term neonates admitted during March, 2004 to December, 2010 with normal BW were enrolled. Infants with antenatal or neonatal asphyxia, temperature abnormality, septicemia, antenatal viral infection, congenital dysmorphia, congenital heart disease, 21-trisomy, and polycythemia were excluded. There was no maternal complications during the pregnancy. Serum total bilirubin (TB), creatine kinase (CK), MB isoenzymes of creatine kinase (CK-MB), and cardiac troponin-I (cTnI) were measured. Patients with transcutaneous bilirubin level (TcB) ≥ 342 µmol/L (20 mg/dl) were in Group A (n = 32), and those with TcB below phototherapy level at matched time point were in Group B (n = 25). ECG, for correct Q-T intervals (QTc) and correct QT intervals dispersion (QTcd), and ECHO, for left ventricular ejection fraction (EF), the ratio of the peak velocity of early stage and advanced stage of diastolic phase at the mitral orifice (E/A), were applied to patients in Group A and B. SPSS 13.0 software was used for the data analysis. The coefficients of correlation among age in hours on admission (hr), TB, CK, CK-MB, CK-MB/CK, and cTnI were studied by multiple and partial correlation analysis. Data in Group A and B were compared by independent-samples Mann-Whitney U test (nonparametric method) or Student t-test. When the data were analyzed by multiple correlation, there were significant correlation between TB and cTnI, CK-MB, respectively (r = 0.212, -0.161, respectively, all P < 0.05). But, when the data were analyzed by partial correlation, there was no correlation between TB and cTnI, CK-MB, respectively (r' = 0.112, -0.112, respectively, all P > 0.05), negative correlation between hr and TB, cTnI, respectively (r' = -0.490, P = 0.000; r' = -0.162, P = 0.032). There was no significant difference in CK (Z = -1.384, P = 0.166), CK-MB (Z = -0.821, P = 0.412), cTnI (Z = -1.159, P = 0.246), QTc (t = 1.146, P = 0.257), QTcd (t = 1.342, P = 0.185), EF (t = 1.558, P = 0.125), E/A (t = -0.640, P = 0.525) between group A and B. There was significant difference in CK-MB/CK (Z = -3.187, P = 0.001) between group A and B with a lower value in group A [0.075 (0.032 - 0.102)] comparing to that in group B [0.160 (0.073 - 0.284)]. There is no sufficient evidence to support the hypothesis that neonatal jaundice may induce myocardial damage in normal birth weight term infants.

A novel mutation of the erythropoietin receptor gene associated with primary familial and congenital polycythaemia

Primary familial and congenital polycythaemia (PFCP) is a rare form of inherited erythrocytosis caused by heterozygous mutations in the erythropoietin receptor gene (EPOR). We present a novel mutation in the EPOR in a 15-year-old male who was referred to our clinic for investigation of a persistently elevated haemoglobin level. A significant family history of unexplained erythrocytosis spanning four generations of the patient's family was established. The family history was also significant for an apparent increased rate of cerebrovascular disease in individuals with erythrocytosis. The mutation detected in our patient resides in exon 8 of EPOR, similar to all other EPOR mutations responsible for PFCP. These mutations result in truncation of the cytoplasmic domain of the receptor and impair down-regulation of signalling via the erythropoietin receptor (EPOR). Clinical manifestations in published cases have varied widely and there is a paucity of firm recommendations regarding the management of affected patients. Given the strong family history of complications attributable to erythrocytosis we have recommended venesection with a haematocrit target of ≤0.45 for our patient.

Genetic Investigation by Shared Genomic Segment and Linkage Study of a Unique Family with Primary Familial and Congenital Polycythemia

AbstractAbstract 4783 Introduction:Polycythemia/Erythrocytosis may be congenital and/or familial, or acquired. The first recognized familial polycythemias are associated with mutations in the globins gene resulting in increased affinity of Hb to oxygen, which could also be caused by congenital deficiency of 2, 3 DPG. Subsequently gain-of-function of erythropoietin receptor gene mutations have been shown to cause familial dominantly inherited polycythemia. More recently, mutation in the genes involved in hypoxia sensing pathway have been described as causes of congenital polycythemia; these include recessively inherited VHL (Chuvash polycythemia), and dominantly inherited HIF2a and PHD2 mutations. Yet unexplained familial polycythemias still exist. Here we investigated a family with dominant polycythemia present in five generations wherein all known mutations in polycythemia-causing genes were excluded through linkage analyses and direct sequencing. Familial clustering of the disease provided us an opportunity to identify a novel genetic cause modulating erythropoiesis. We utilized a shared genomic segment (SGS) and linkage analysis approach, to provide evidence of chromosomal candidate regions for further efforts to localize the causative gene. Methods:A five generation family pedigree consisting of 26 phenotyped members (10 affected/16 unaffected) with 16 subjects (5 affected and 11 unaffected) genotyped on an Affymetrix v5.0 SNP platform. A conventional two-point linkage analysis did not identify a potential causative genomic region. Multipoint linkage analysis was subsequently performed because this allows for optimal use of the genetic SNP data to infer inheritance patterns and leads to lower false positive rates than two-point linkage methods. In order to identify potential causative regions for harboring the polycythemia-causing defect, we also performed shared genomic segment (SGS) analysis that recognizes a region identical by descent must also be shared by identical by state. We analyzed 409,426 reference dbSNPs with a 94% call rate for genotyped subjects to determine runs of loci at which affected individuals share a common allele. Parametric multipoint linkage analysis used a pruned set of 8,394 non-monomorphic markers with minimum spacing of 100 kb, minimum heterozygosity of 0.3 and maximum pairwise r2 of 0.16 based on HapMap CEU data. We used a Markov chain Monte Carlo linkage method (MCLINK) which allows for fully informative multilocus linkage analysis on extended pedigrees. Results:SGS identified five runs of shared loci that achieved chromosome-wide significance among all five affected subjects (chromosome, run length): 18, 1980 SNPs; 14, 1292 SNPs and 1095 SNPs; 16, 1060 SNPs and 20, 500 SNPs. The SGS regions on chr. 14 (1292 and 1095 run lengths out of 13,149 total SNPs) begins at (NCBI build 36.1) 55,956,582,765bp (rs17254216) through 73,408,302bp (rs1778222) and contain regions of 80 and 79 consecutive SNPs at which all cases share homozygously from a homozygous shared genomic segment analysis (HGS). Multipoint linkage identified a peak LOD score of 3.41 on chr. 16 at 78,033,240bp (rs4888031) achieving genomewide significance which is contained within an observed SGS region of interest (77,507,467bp to 81,049,285bp). [Display omitted] Conclusion:We submit that genetic epidemiological analyses (SGS and Linkage) augment likelihood of identifying the disease-causing genomic regions that are good candidates for the underlying familial susceptibility variant(s). We observed an 8Mb region of interest on chromosome 14 which may contain a segment of loss-of-heterozygosity, and a 3.9Mb region on chromosome 16 both are now being analyzed by sequencing studies. Disclosures:No relevant conflicts of interest to declare.

A Novel PHD2 Mutation Associated with Tibetan Genetic Adaptation to High Altitude Hypoxia.

AbstractAbstract 2602The ability of the body to maintain oxygen hemostasis is essential for its survival. The body is required to adapt to the physiologic constraints imposed by a hypoxic environment in order to ensure optimal physiological functions. The study of indigenous high-altitude residents provides an opportunity to identify genes that may have play a role in hypoxia adaptation, and recent studies indicate that Tibetans have genetically adapted to the extremely hypoxic environment at high altitude. Interestingly, Tibetan adaption includes protection from polycythemia. We recently reported genome-wide scans of selection (Affymetrix Human SNP chips v6.0) for Tibetans (Simonson, Science July, 2010). At least 10 chromosomal regions were identified as likely targets for high-altitude adaptation, and two of these regions, EGLN1 (PHD2) and PPARA, were associated with protection against high-altitude polycythemia which is unique to this indigenous highland population. PHD2 is a negative regulator of the alpha subunits of HIF-1 and HIF-2 transcription factors regulating many essential functions including erythropoiesis, iron metabolism, development, in addition to other functions. A gain of function mutation of the alpha subunit of HIF-2 is associated with congenital polycythemia. We sequenced the exons of PPARA and PHD2 by direct sequencing and found two missense mutations in exon 1 of the PHD2 gene: the first is located within codon 4, and changes Aspartate to Glutamate (Asp4Glu) and the second is an unvalidated SNP (rs12097901) at codon 127 that changes Cysteine to Serine (Cy127Ser). The samples with high Hb show a heterozygous change, while the subject with normal Hb exhibited a homozygous change in both codons of the PHD2 gene.The association of these two missense PHD2 mutations to hemoglobin level is now being evaluated by studies of a larger population of Tibetan natives and the functional effects on levels and activities of HIF-1 and HIF-2 genes evaluated by transfection of these mutated PHD2 cDNA constructs in reporter cells.Table 1List of samples and controls screened for mutation in PHD2.SampleRaceSexHbPHD2 MutationAsp4GluCys127SerT03TibetanF194HeteroHeteroT43TibetanM194HeteroHeteroT48TibetanM101HeteroHomoT53TibetanM159HomoHomoControlCaucasianM?WTWTControlCaucasianF?WTWT Disclosure:No relevant conflicts of interest to declare.

Abstract 5011: Potential tumor suppressor role of PHD2 : functional study of mutations identified in germline DNA of patients with congenital polycythemia with or without paraganglioma

Abstract The Hypoxia Inducible Factor (HIF) plays a major role in tissue response to hypoxia. HIF induces expression of many genes involved in anaerobic glycolysis, angiogenesis, erythropoiesis and cell proliferation. HIF-alpha subunit is tightly regulated by a family of specific hydroxylases in normoxia. PHD (prolyl-hydroxylase domain) proteins hydroxylate HIF-alpha, thereby allowing its attachment to the VHL protein, a component of an E3 ubiquitin ligase complex involved in protein ubiquitination and degradation. Mutations in HIF regulators have been described and lead to the stabilization of HIF, activation of target genes and development of either tumors or polycythemia. Heterozygous germline mutations in the VHL gene predispose to the von Hippel-Lindau disease characterized by a panel of highly vascularized tumors (central nervous system and retinal hemangioblastomas, pheochromocytomas, renal cell carcinoma, pancreatic endocrine tumors) whereas homozygous or compound heterozygous VHL mutations are responsible for isolated polycythemia (”Chuvash polycythemia”) with high circulating erythropoietin levels. Recently, heterozygous germline mutations in the PHD2 gene have been described in few patients with such congenital polycythemia and a particular mutation (H374R) has been identified in a patient with a polycythemia and a recurrent paraganglioma1. This observation raised the question of a possible role of PHD2 loss of function in the genesis of tumors usually seen in the VHL disease. We report here four new heterozygous PHD2 germline mutations in patients with familial or apparently sporadic polycythemia. We performed a functional study of these mutants compared to the H374R mutant and we showed differences in the capacity of the PHD2 mutants to downregulate HIF-alpha. These differences, tested in various conditions, are highly reproducible and suggest that there may be a genotype/phenotype correlation for the PHD2 mutants like the VHL mutants with their different ability to regulate HIF associated with different phenotypes in the VHL disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5011.

Advances in understanding the pathogenesis of primary familial and congenital polycythaemia

Primary familial and congenital polycythemia (PFCP) is an autosomal-dominant proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to erythropoietin (Epo). Several lines of evidence suggest a causal role of truncated erythropoietin receptor (EpoR) in this disease. In this review, we discuss PFCP in the context of erythrocytosis and EpoR signalling. We focus on recent studies describing mechanisms underlying Epo-dependent EpoR down-regulation. One mechanism depends on internalization mediated through the p85 regulatory subunit of the Phosphoinositide 3-Kinase, and the other utilizes ubiquitin-based proteasomal degradation. Truncated PFCP EpoRs are not properly down-regulated upon stimulation, underscoring the importance of these mechanisms in the pathogenesis of PFCP.

Genetic analysis of von Hippel-Lindau disease

Mutations in the von Hippel-Lindau (VHL) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types as well. Reports of VHL mutations are dispersed throughout original articles and databases that have not been recently updated. We compiled a comprehensive mutation table of 1,548 germline and somatic VHL mutations, derived from this protein of only 213 amino acids. We describe detailed phenotype and gene mutation information for 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). These data represent the most extensive catalog of germline VHL mutations to date. We also review VHL disease, known and theorized pathogenesis of common VHL manifestations, and genotype-phenotype correlations. Analysis of all VHL families, excluding germline mutations resulting in congenital polycythemias, describes the spectrum of mutation types: 52% missense, 13% frameshift, 11% nonsense, 6% in-frame deletions/insertions, 11% large/complete deletions, and 7% splice mutations. This easy-to-use compilation of VHL mutations is intended to facilitate research and function as a necessary adjunct for physicians when providing patient information.

Enrichment of Sca1+ hematopoietic progenitors in polycythemic mice inhibits leukemogenesis

Polycythemia vera (PV) is a myeloproliferative disorder characterized by a pronounced increase in the number of erythroid cells. However, despite this aberrant proliferation, the incidence of erythroleukemia is paradoxically rare in PV patients. In this study, we show that the progression of Friend virus-induced erythroleukemia is delayed in a mouse model of primary familial congenital polycythemia in which the wild-type Epo-receptor (EpoR) gene is replaced with a truncated human EPOR gene. Herein, we show that these mice exhibit enrichment of Sca1(+)/cKit(-) progenitors and several mature immune cells, such as dendritic cells and macrophages. In cotransplantation experiments, Sca1(+)/cKit(-) progenitors inhibit the tumorigenicity of Sca1(-)/cKit(+) erythroleukemic cells. A cell line established from Sca1(+)/cKit(-) progenitors is also capable of inhibiting leukemic proliferation in culture and in mice. This phenomenon of leukemic inhibition, also detected in the serum of PV patients, is partially attributed to increased nitric oxide secretion. In addition, the administration of erythropoietin into leukemic mice induces a polycythemia-like state associated with the expansion of Sca1(+)/cKit(-) progenitors and derivative immune cells, thereby inhibiting leukemia progression. This study indicates that a combination therapy incorporating the enrichment of Sca1(+)/cKit(-) progenitors may serve as a novel approach for the treatment of leukemia.

Ligand-induced EpoR internalization is mediated by JAK2 and p85 and is impaired by mutations responsible for primary familial and congenital polycythemia

AbstractEpo-induced endocytosis of EpoR plays important roles in the down-regulation of EpoR signaling and is the primary means that regulates circulating Epo concentrations. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Both JAK2 kinase activity and EpoR cytoplasmic tyrosines are important for ligand-dependent EpoR internalization. Phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain bind p85 subunit of PI3 kinase on Epo stimulation and individually are sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85α and p85β or expression of their dominant-negative forms, but not inhibition of PI3 kinase activity, dramatically impaired EpoR internalization, indicating that p85α and p85β may recruit proteins in the endocytic machinery on Epo stimulation. Furthermore, mutated EpoRs from primary familial and congenital polycythemia (PFCP) patients lacking the 3 important tyrosines do not bind p85 or internalize on stimulation. Addition of residues encompassing Y429 and Y431 to these truncated receptors restored p85β binding and Epo sensitivity. Our results identify a novel PI3 kinase activity-independent function of p85 in EpoR internalization and support a model that defects of internalization in truncated EpoRs from PFCP patients contribute to Epo hypersensitivity and prolonged signaling.

The complete evaluation of erythrocytosis: congenital and acquired

The approach to a patient with erythrocytosis is greatly simplified by assessing the clonality of the process upfront. In this regard, there has been a dramatic shift toward genetic testing and away from traditional tests, such as measurement of red cell mass. Clonal erythrocytosis is the diagnostic feature of polycythemia vera (PV) and is almost always associated with a JAK2 mutation (JAK2V617F or exon 12). All other scenarios represent non-clonal erythrocytosis, often referred to as secondary erythrocytosis. Serum erythropoietin (Epo) level is usually normal or elevated in secondary erythrocytosis and subnormal in PV. Therefore, in a patient with acquired erythrocytosis, it is reasonable to begin the diagnostic work-up with peripheral blood JAK2 mutation analysis and serum Epo measurement to distinguish PV from secondary erythrocytosis. Conversely, the patient with life-long erythrocytosis is more likely to suffer from congenital polycythemia and should therefore be evaluated for germline mutations that result in enhanced Epo effect (for example, Epo receptor mutations), altered intracellular oxygen sensing (for example, mutations involving the von Hippel-Lindau tumor suppressor gene) or decreased P50 (for example, high-oxygen-affinity hemoglobinopathy). The order of tests in this instance depends on the clinical scenario and serum Epo level.

Congenital and Acquired Polycythemias

Polycythemias are characterized by an increased concentration of red blood cells. Because blood cell counts are a routine investigation, these disorders present to non-hematologic physicians. Polycythemia vera (PV), an acquired stem cell disease, is the most important variant. Selective literature review and the authors' own clinical experiences. Erythropoietin, which is produced in the kidneys, and its receptor system in the bone marrow, are of critical importance in polycythemia. Congenital polycythemias are caused by mutations of the Erythropoietin-receptor gene, hemoglobin variants, 2,3-bisphosphoglycerate mutase deficiency or by disturbances of renal oxygen sensing. Acquired polycythemias can occur secondary to hypoxia at high altitudes, or primarily through acquired mutations in the EPO-receptor signaling system (JAK2 mutations). Alternatively they may be caused by pulmonary or renal disease. An artificial erythrocytosis is induced by athletes through doping. Differential diagnosis comprises erythropoietin determination, JAK2 mutation analysis and if necessary hemoglobin electrophoresis. Only PV requires immediate treatment, because of a high thromboembolic risk. Epidemiological studies on polycythemias in German speaking countries are urgently needed.

Truncated Human Erythropoietin Receptor Causes Fetal Polycythemia and Is Associated with a Prolonged Primitive Erythropoiesis and Hyperactive Definitive Erythropoiesis.

Gain-of-function mutations that cause truncations of the cytoplasmic domain of the human erythropoietin receptor (EpoR) result in the dominantly inherited disorder, primary familial congenital polycythemia. The EpoR truncation causes polycythemia, which is not always seen at birth, and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR (MEpoR) gene with either a wild-type human EPOR (wthEPOR) gene or a mutant human EPOR gene (mthEPOR). This mutation, initially identified in a family with polycythemia, produces a truncated EpoR with a deletion of the cytoplasmic C-terminal domain after the first tyrosine residue. To investigate the effects of wthEPOR and mthEPOR genes on fetal erythropoiesis, we examined mouse fetuses homozygous for either wthEPOR or mthEPOR from E12.5 up to 5 weeks postnatally (PN). The mthEPOR fetuses were polycythemic and the wthEPOR fetuses were anemic during the early embryonic stage (E12.5∼E16.5) and at the adult stage (3 weeks and older). However, during the perinatal period (E18.5∼PN2 weeks), both of the phenotypes temporarily disappeared. To study the pathophysiology of the phenotypes, we analyzed erythropoiesis using differential expression of CD71 and TER119 surface antigens by FACS in the peripheral blood (PB) and fetal liver (FL). PB from wthEPOR and mthEPOR had a significantly higher population of earlier progenitors (proerythroblasts and early basophilic erythroblasts) from E12.5 to E16.5 compared to MEpoR. From E18.5 on, however, all genotypes showed a similar pattern. At E14.5, FL of wthEPOR and mthEPOR homozygotes were similar to MEpoR FLs at E12.5. Since a dynamic switch of primitive to definitive erythropoiesis occurs around E14.5, we evaluated nucleated erythroid cell populations in PB from E12.5∼E15.5 using the DNA dye DRAQ5 followed by FACS analysis. From E13.5 to E14.5, mthEPOR fetuses contained more nucleated cells than MEpoR fetuses (43 to 55% vs 37 to 29%; mthEPOR vs MEpoR), whereas wthEPOR fetuses had comparable numbers to MEpoR fetuses (36 to 25% vs 37 to 29%; wthEPOR vs MEpoR). Interestingly, all genotypes from E15.5 and thereafter contained over 90% enucleated cells with no significant difference in their PB. To understand the molecular mechanism of these features, we have analyzed Stat5 phosphorylation after Epo stimulation in FL from E14.5 and E18.5. At both stages, the mthEPOR FL had a markedly increased and sustained Epo-sensitive Stat5 phosphorylation compared to MEpoR FL. In contrast, in wthEPOR FL Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. The membrane-localized EpoR levels were measured by fluorescence labeled anti-EpoR antibody. All genotypes showed comparable levels of EpoR on the membrane. We demonstrate here that with the exception of the perinatal period, mthEPOR causes polycythemia whereas wthEPOR causes anemia, even in fetal life. We propose that the polycythemia of mthEPOR during early embryogenesis (E12.5∼E14.5) is due to a delayed switch from primitive to fetal erythropoiesis, followed by hyperactive definitive erythropoiesis. These characteristics (the prolonged primitive erythropoiesis and hyperactive definitive erythropoiesis) could be explained, at least in part, by hyperactive and sustained Stat5 activation.

Evaluation of “Increased” Hemoglobin in the JAK2 Mutations Era: A Diagnostic Algorithm Based on Genetic Tests

Recent discoveries in the molecular pathogenesis of both polycythemia vera (PV) and congenital polycythemia (CP) underline the prospect of a genetic diagnosis in these disorders. At the forefront are the mutually exclusive exon 14 (JAK2V617F) and exon 12 JAK2 mutations that are almost always present in PV but not in polycythemias of other causes. Similarly, the molecular basis of CP is being unraveled, and several cases are now associated with germline mutations involving the von Hippel-Lindau (VHL) or erythropoietin receptor (EPOR) genes. Therefore, current diagnostic work-up for acquired polycythemia should start with peripheral blood JAK2 mutation screening, whereas VHL and/or EPOR mutations should be considered when CP is suspected. In all instances, serum erythropoietin measurement provides complementary information; the serum erythropoietin level is expected to be decreased in PV regardless of JAK2 mutation status, increased in VHL mutation-associated CP, and decreased or normal in the presence of an EPOR mutation.

Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.

Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA 1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol 1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband's parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood 1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol 2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.

Is a little hypoxia good for you?

Comment on Perrotta et al, page [514][1] In this issue, Perrotta and colleagues from the Second University of Naples report on their finding of endemic congenital polycythemia due to homozygous VHL 598C > T on the Italian island of Ischia. The condition leads to increased expression of HIF-1, the

Validation of JAK2 and New Clinical Criteria for the Diagnosis of Polycythemia Vera (PV).

Although the pervasive presence of JAK2 in patients (pts) with PV has been noted in at least 7 published studies, its frequency has ranged from 66 to 97%. Although part of the reason for this wide range is false negative results due to the low sensitivity of sequence analysis, varying criteria used for the clinical diagnosis of PV are also likely to be an important factor. In more than 300 studies, the criteria of the Polycythemia Vera Study Group has been used, although many authors, including ourselves, have commented on their inadequacy. Alternative classifications have been the subject of debate mostly centered about using the rbc-Cr51 mass. Our criteria included the use of CR51 labeled red cells to establish first, an increase in red blood cell volume and second, I125 to establish an increase in plasma volume. Exception was made for men with a HCT ≥60%, and women ≥56%. If hypervolemic, the major causes of secondary polycythemia were excluded: congenital polycythemias and those secondary to impaired oxygenation, or benign or malignant tumors. This implied a serum erythropoietin level of ≤5u/ml. The aforementioned and 3 of the following 5 were required: splenomegaly, WBC, ≥12,000/ul, platelet count ≥600,000/ul, abnormal marrow histology. We used them to clinically diagnose 61 patients subsequently undergoing therapeutic trials whose blood was analyzed for the JAK2 V617F mutation by allele specific PCR from 679 patients with myeloproliferative diseases, including normal controls. Of 61 patients, with the phenotypic characteristics of p. vera, 59 (97%) were positive for the JAK2 mutation. The 2 remaining patients, although phenotypically characteristic of PV, were negative on repeat examinations. One pt was newly diagnosed and untreated and the other had a 6 year remission after rIFNα followed by a 1 year remission on imatinib. We conclude that determination of JAK2 is consistent with PV in virtually all cases clinically diagnosed appropriately. Our data, to be presented, indicate that the combination of increased red cell mass, demonstration of JAK2 and increased platelet count establish the diagnosis of PV in virtually all cases.

Truncated Human EpoR Causes Polycythemia in Fetal Erythropoiesis through Stat5 Hyperactivation.

Gain-of-function mutations causing truncations of the cytoplasmic domain of the human erythropoietin receptor (EPOR) result in a dominantly inherited disorder-primary familiar congenital polycythemia. The consequence of EpoR truncation is polycythemia and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR gene with a wild-type human EPOR (whEPOR) gene or a mutant human EPOR gene (mthEPOR) that we initially identified in a family with polycythemia. This mutation produces a truncated EpoR mutant with deleted cytoplasmic C-terminal domain after the first tyrosine residue of the intracellular domain. We reported that the mice containing mthEPOR allele have polycythemia and the adult whEPOR mice have anemia. To study the effects of these genotypes on fetal erythropoiesis, we examined fetuses E14.5 homozygotes wild type mouse EpoR, and human EPOR alleles. Hematocrit results show: MthEPOR fetuses were polycythemic compared to mouse EpoR (mthEPOR vs mouse EpoR; 34.58 ± 1.24 % vs 27.49 ± 0.90 %, P<0.001 by t-test), and whEPOR were anemic (whEPOR vs mouse EpoR; 18.61 ± 0.79 % vs 27.49 ± 0.89 %, P<0.001 by t-test). To study the pathophysiology of these phenotypes, we analyzed the erythroid differentiation using differential expression of CD71 and TER119 surface antigens by FACS in the fetal liver cells (FLs). FLs from mthEPOR homozygote had increased population on early and late basophilic erythroblast stage compared to mouse EpoR, but the populations of differentiated erythroid progenitors from mthEPOR homozygote were significantly lower than mouse EpoR and whEPOR homozygotes. We proposed that the erythroid progenitors from mthEPOR exit fetal liver to fetal blood circulation prematurely. The peripheral blood from mthEPOR and mouse EpoR s was analyzed by morphology and FACS. We show that the majority of population in peripheral blood from mthEPOR homozygote were earlier erythroid progenitors (high CD71/low TER119 population in mthEPOR 47.53 %, vs 13.14 % mouse EpoR; high CD71/high TER119 population from mthEPOR 11.49 % vs 65.00 % mouse EpoR). To evaluate the Epo signal transduction we tested the kinetics and dose responses of Epo at Stat5 phosphorylation level. The mthEPOR expressing FLs showed a markedly increased and sustained Epo sensitivity compared to mouse EpoR expressing FLs. In contrast, in whEPOR expressing FLs, Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. We conclude that mthEPOR causes fetal polycythemia and is associated with premature exit of early erythroid progenitors from fetal liver to fetal circulation. In contrast whEPOR is associated with more severe anemia than we observed in adult erythropoiesis. The degree of Epo induced Stat5 activation plays an important role in generation of anemic and polycythemic phenotypes.

Lessons From Familial Myeloproliferative Disorders

By definition, myeloproliferative disorders (MPDs) are caused by an acquired somatic mutation of a hematopoietic progenitor/stem cell and have sporadic occurrence. However, well-documented families exist with first-degree relatives acquiring one or several MPDs. It is reasonable to assume that the germ-line mutation(s) or genetic background must facilitate or predispose for one or several somatic mutation(s) that lead to the MPD that is indistinguishable from the sporadic form. This is best documented in familial polycythemia vera (PV), which appears to be inherited as an autosomal dominant disorder with incomplete penetrance. However, there are also families wherein members develop any combination of MPDs, including PV, essential thrombocythemia (ET), chronic myelocytic leukemia (CML), and idiopathic myelofibrosis (IMF). A separate group of familial diseases is the familial thrombocythemias, wherein germ-line mutations in the genes for thrombopoietin or its receptor, MPL, cause polyclonal hereditary thrombocythemia, which may be clinically indistinguishable from ET. Patients with the congenital polycythemic condition "primary familial and congenital polycythemia" (PFCP) have characteristically decreased erythropoietin (Epo) levels similar to PV, hypersensitive erythroid progenitors, and low Epo levels; as such, this condition is often confused with PV. Therefore, PFCP will also be discussed here, while other congenital polycythemic states such as the Chuvash polycythemia that have elevated or inappropriately normal Epo levels will be omitted from this review in view of their distinct phenotype and unique laboratory features.

Erythropoietin hypersensitivity in primary familial and congenital polycythemia: Role of tyrosines Y285 and Y344 in erythropoietin receptor cytoplasmic domain

Erythropoietin receptor (EPOR) gene mutations leading to truncations of the cytoplasmic, carboxy-terminal region of EPOR have been described in some patients with primary familial and congenital polycythemia (PFCP), a disorder characterized by isolated erythrocytosis and increased sensitivity of erythroid progenitors to Epo. We studied the role of EPOR in the pathogenesis of PFCP and the requirement for intracytoplasmic tyrosine residues Y285 and Y344 in generation of Epo hypersensitivity phenotype. Interleukin-3-dependent hematopoietic cells were engineered to express variant human EPORs using retrovirus-mediated gene transfer. We introduced tyrosine to phenylalanine substitutions in EPOR-ME, a naturally occurring, mutant human EPOR (G5881T), truncated by 110 carboxy-terminal amino acids and associated with autosomal dominantly inherited PFCP. Cells expressing EPOR-ME exhibited increased Epo sensitivity compared to cells expressing wild type EPOR. Mutation of Y285 alone had a relatively minor effect on Epo hypersensitivity whereas mutation of Y344 resulted in loss of increased Epo sensitivity. Expression of a tyrosine-null truncated EPOR conferred further decrease of Epo-mediated proliferation suggesting that both Y285 and Y344 may contribute to proliferation signals. In the context of EPOR-ME, Y344 was required for Epo-induced Stat5 tyrosine phosphorylation. The positive effect of either Y285 or Y344 on cellular proliferation was associated with Epo-induced tyrosine phosphorylation of Stat1. These findings suggest that both tyrosine residues Y285 and Y344 in the cytoplasmic domain of EPOR-ME may contribute to increased Epo sensitivity that is characteristic of PFCP phenotype.

Polycythemia vera and other primary polycythemias

Diagnosis and therapy of polycythemia vera are controversial since the molecular basis of polycythemia vera remains unknown. Distinguishing between polycythemia vera and other polycythemic disorders can be very challenging. The purpose of this review is to discuss the recent progress in this area and critically review the published data in context of our knowledge of other polycythemic disorders. Erythropoietin is the principal regulator of regulator of erythropoiesis; its production is regulated by the degree of hypoxia. Our knowledge of cellular responses to hypoxia has recently exploded and led to the elucidation of the molecular basis of a polycythemia caused by augmentation of hypoxic sensing, Chuvash polycythemia. Similar progress in understanding the molecular basis of polycythemia vera has been elusive. A simple, readily available laboratory test to establish a diagnosis of polycythemia vera would be highly desirable; however, none exists. The value of quantization of neutrophil PRV-1 mRNA, platelet c-mpl expression, in vitro assays of erythroid progenitor cells, serum erythropoietin levels, establishing clonality in female subjects using assays employing X-chromosome-based polymorphism assays, and the progress in the chromosomal location of the gene is discussed. Integration of this information underlies the complexity of the molecular biology of polycythemia vera and indicates likely interaction of multiple genetic events in the genesis of polycythemia vera. The existence of family clustering of PV may facilitate the search for PV molecular basis. Only collaborative interaction of clinical researchers and laboratory scientists will lead to meaningful progress in determining the molecular basis of PV.