Josef T. Prchal

Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia

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 α (HIF1α). Our data indicate that the Arg200Trp substitution impairs the interaction of VHL with HIF1α, reducing the rate of degradation of HIF1α 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).

Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera.

OBJECTIVEnClonal stem cell proliferation and increased erythrocyte mass are hallmarks of the myeloproliferative disorder polycythemia vera (PV). The molecular basis of PV is unknown.nnnMETHODSnWe carried out a genome-wide screening for loss of heterozygosity (LOH) and analyzed candidate genes within the LOH loci.nnnRESULTSnThree genomic regions were identified on chromosomes 9p, 10q, and 11q. The presence of these LOHs in both myeloid and lymphoid cells indicated their stem cell origin. The 9pLOH prevalence is approximately 33% and is the most frequent chromosomal lesion described in PV so far. We report that the 9pLOH is due to mitotic recombination and therefore remains undetectable by cytogenetic analysis. Nineteen candidate genes were selected within the 9pLOH region for sequencing and expression analysis. No mutations were found in these genes; however, unexpectedly, increased expression of the transcription factor NFI-B was detected in granulocytes and CD34(+) cells in PV with 9pLOH. Since a member of the NFI gene family (NFI-X) was reported to result in TGF-beta resistance when overexpressed in vitro (TGF-beta is a known inhibitor of hematopoiesis), we transfected the NFI-B gene to the mouse 32D cell line. We found that overexpression of the NFI-B gene confers TGF-beta resistance in vitro.nnnCONCLUSIONSnWe characterized a new region on chromosome 9p frequently involved in LOH in PV. Analysis of genes within this 9pLOH region revealed increased expression of the NFI-B gene. Our in vitro studies suggest that TGF-beta resistance may be the physiologic mechanism of clonal stem cell expansion in PV.

Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation.

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

Divalent metal transporter 1.

Abstract In the last few years, the field of iron metabolism has exploded with the discovery of many new proteins including ferroportin, hephaestin, hepcidin, duodenal cytochrome b and the topic of this review, divalent metal ion transporter 1 (DMT1). DMT1 functions in transport of ferrous iron, and some, but not all divalent metal ions across the plasma membrane and/or out of the endosomal compartment. DMT1 mRNA has been found in every cell type in which it has been sought and its structure is highly conserved in evolution with similar proteins expressed in plants, insects, microorganisms and vertebrate animals. Rodents with defects in iron absorption and utilization were identified long before it was determined that the defect was due to a single nucleotide mutation in DMT1. Study of these animals reveals that transport of iron and other divalent metal ions by DMT1 is pH dependent, but the exact manner in which pH exerts its effect is unknown. The structure of the DMT1 gene is complex. Alternative usage of 3 exons, results in forms with and without iron responsive elements (IREs), while alternative usage of 5 exons and less well defined products of alternative splicing results in an array of isoforms with incompletely defined function. Expression of some isoforms is tissue specific and appears to affect subcellular targeting of the protein. At least one signal for DMT1 expression appears to be intracellular iron status, however, other, as yet undefined signals may also contribute to DMT1 expression. Interestingly, DMT1 function may differ subtly between humans and other animals; the spontaneous DMT1 mutation found in mice and rats appears to limit iron uptake in the intestine and iron utilization in red cell precursors, whereas the only known human mutation has its primary effect on iron utilization by erythroid cells. The importance of DMT1 function at the level of the whole organism and the individual cell and mechanisms of its regulation on a molecular scale are only beginning to be understood; an appreciation of these process will lead to an understanding of the role of iron in various cellular processes and improved treatments for both anemia and iron-overload.

Disruption of ferroportin 1 regulation causes dynamic alterations in iron homeostasis and erythropoiesis in polycythaemia mice

Coding region mutations in the principal basolateral iron transporter of the duodenal enterocyte, ferroportin 1 (FPN1), lead to autosomal dominant reticuloendothelial iron overload in humans. We report the positional cloning of a hypermorphic, regulatory mutation in Fpn1 from radiation-induced polycythaemia (Pcm) mice. A 58 bp microdeletion in the Fpn1 promoter region alters transcription start sites and eliminates the iron responsive element (IRE) in the 5′ untranslated region, resulting in increased duodenal and hepatic Fpn1 protein levels during early postnatal development. Pcm mutants, which are iron deficient at birth, exhibited increased Fpn1-mediated iron uptake and reticuloendothelial iron overload as young adult mice. Additionally, Pcm mutants displayed an erythropoietin (Epo)-dependent polycythemia in heterozygotes and a hypochromic, microcytic anemia in homozygotes. Interestingly, both defects in erythropoiesis were transient, correcting by young adulthood. Delayed upregulation of the negative hormonal regulator of iron homeostasis, hepcidin (Hamp), during postnatal development correlates strongly with profound increases in Fpn1 protein levels and polycythemia in Pcm heterozygotes. Thus, our data suggest that a Hamp-mediated regulatory interference alleviates the defects in iron homeostasis and transient alterations in erythropoiesis caused by a regulatory mutation in Fpn1.

Excessive erythrocytosis, chronic mountain sickness, and serum cobalt levels.

In a subset of high-altitude dwellers, the appropriate erythrocytotic response becomes excessive and can result in chronic mountain sickness. We studied men with (study group) and without excessive erythrocytosis (packed-cell volume >65%) living in Cerro de Pasco, Peru (altitude 4300 m), and compared them with controls living in Lima, Peru (at sea-level). Toxic serum cobalt concentrations were detected in 11 of 21 (52%) study participants with excessive erythrocytosis, but were undetectable in high altitude or sea-level controls. In the mining community of Cerro de Pasco, cobalt toxicity might be an important contributor to excessive erythrocytosis.

Polycythemia vera and other primary polycythemias.

Purpose of reviewDiagnosis 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. Recent findingsErythropoietin 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. SummaryThe 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.

Pathogenetic mechanisms of polycythemia vera and congenital polycythemic disorders

The absolute polycythemias--those with increased red blood cell mass--can be divided into two groups: primary, caused by acquired or inherited mutations leading to a gain-of-function abnormalities expressed within the erythroid progenitors; and secondary, due to circulating serum factors, typically erythropoietin, stimulating erythropoiesis. This overview concentrates on the molecular biology of polycythemia vera (PV) discussed in the context of other polycythemic disorders. Recent advances in the regulation of erythropoiesis, as they may relate to polycythemic states, are discussed as a background for those well-defined polycythemic states wherein the molecular defect has not yet been elucidated. A number of cellular abnormalities associated with PV, including the hyperresponsiveness of PV progenitors to many cytokines as well as decreased expression of the thrombopoietin receptor on platelets and increased expression of Bcl-xL, suggest that the PV defect alters a number of cellular functions and is not restricted to cytokine receptor signal transduction. The increasing number of recognized instances of familial incidence of PV suggests that in these families the predisposition for PV is inherited as a dominant trait, and that PV is acquired as a new mutation that leads to a clonal hematopoiesis and may be due to loss of heterozygosity. The existence of these families provides a unique opportunity for isolation of the mutations in the gene leading to PV. Semin Hemaol 38(suppl 2):10-20.

Search for genetic determinants of individual variability of the erythropoietin response to high altitude.

There is marked variability in the erythropoietin (Epo) and erythrocytic response to extreme high altitude among mountain dwellers, as well as to hypoxic training among athletes, at least in part because of the variation in the erythropoietic response to hypoxia. We hypothesized that this may be genetically determined. Forty-eight athletes were exposed to 24 h of simulated altitude to 2,800 m in a hypobaric chamber. Serum Epo concentrations were determined at baseline and after 24 h. The Epo responses ranged from -41 to 433% of baseline values after 24 h at simulated altitude. The association of the Epo response to hypoxia with the EPO gene and eight genes involved in Epo regulation utilizing 16 polymorphic dinucleotide repeats was examined. Initial analysis showed a possible association between the EPO gene (marker D7S477) and the increase of the Epo level (P = 0.018). We then tested the possibility that sequence abnormalities in the 3 and 5 hypoxia response elements (3 HRE) and (5 HRE) of the EPO gene could explain the differences in Epo response. We found a 3434 C --> T polymorphism in the 3 HRE sequence. However, this polymorphism showed no correlation with the differences in Epo levels. Further, when we analyzed two additional markers flanking the EPO gene by less than 0.3 cM, we found no association of the allelic variants at these loci with the Epo hypoxic response. In conclusion, we could find not convincing association between markers tightly linked to EPO or eight genes involved in Epo regulation and Epo differential responses to hypoxia.

Mitochondrial iron metabolism and sideroblastic anemia.

Sideroblastic anemias are a heterogeneous group of disorders, characterized by mitochondrial iron overload in developing red blood cells. The unifying characteristic of all sideroblastic anemias is the ring sideroblast, which is a pathological erythroid precursor containing excessive deposits of non-heme iron in mitochondria with perinuclear distribution creating a ring appearance. Sideroblastic anemias may be hereditary or acquired. Hereditary sideroblastic anemias are caused by defects in genes present on the X chromosome (mutations in the ALAS2, ABCB7, or GRLX5 gene), genes on autosomal chromosomes, or mitochondrial genes. Acquired sideroblastic anemias are either primary (refractory anemia with ring sideroblasts, RARS, representing one subtype of the myelodysplastic syndrome) or secondary due to some drugs, toxins, copper deficiency, or chronic neoplastic disease. The pathogenesis of mitochondrial iron loading in developing erythroblasts is diverse. Ring sideroblasts can develop as a result of a heme synthesis defect in erythroblasts (ALAS2 mutations), a defect in iron-sulfur cluster assembly, iron-sulfur protein precursor release from mitochondria (ABCB7 mutations), or by a defect in intracellular iron metabolism in erythroid cells (e.g. RARS).