Adelina I. Sergueeva

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).

Chuvash polycythemia VHLR200W mutation is associated with down-regulation of hepcidin expression.

Hypoxia is known to reduce the expression of hepcidin, the master regulator of iron metabolism. However, it is not clear whether this response is primarily related to increased erythropoiesis driven by hypoxically stimulated erythropoietin or to a more direct effect of hypoxia on hepcidin expression. The germline loss-of-function VHL(R200W) mutation is common in Chuvashia, Russia, and also occurs elsewhere. VHL(R200W) homozygotes have elevated hypoxia-inducible factor 1α (HIF-1α) and HIF-2α levels, increased red cell mass, propensity to thrombosis, and early mortality. Ninety VHL(R200W) homozygotes and 52 controls with normal VHL alleles from Chuvashia, Russia, were studied under basal circumstances. In univariate analyses, serum hepcidin concentration was correlated positively with serum ferritin concentration and negatively with homozygosity for VHL(R200W). After adjustment for serum erythropoietin and ferritin concentrations by multiple linear regression, the geometric mean (95% confidence interval of mean) hepcidin concentration was 8.1 (6.3-10.5) ng/mL in VHL(R200W) homozygotes versus 26.9 (18.6-38.0) ng/mL in controls (P < .001). In contrast, a significant independent relationship of serum erythropoietin, hemoglobin, or RBC count with hepcidin was not observed. In conclusion, up-regulation of the hypoxic response leads to decreased expression of hepcidin that may be independent of increased erythropoietin levels and increased RBC counts.

Decreased serum glucose and glycosylated hemoglobin levels in patients with Chuvash polycythemia: a role for HIF in glucose metabolism

In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel–Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia-inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzyme genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wild-type VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc, but not Pdk2, was decreased, and skeletal muscle expression of Glut1, Pdk1, and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.

Hypoxic response contributes to altered gene expression and precapillary pulmonary hypertension in patients with sickle cell disease.

Background— We postulated that the hypoxic response in sickle cell disease (SCD) contributes to altered gene expression and pulmonary hypertension, a complication associated with early mortality. Methods and Results— To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 subjects with SCD with hemoglobin SS genotype and 15 subjects with Chuvash polycythemia (VHLR200W homozygotes with constitutive upregulation of hypoxia-inducible factors in the absence of anemia or hypoxia). At a 5% false discovery rate, 1040 genes exhibited >1.15-fold change in both conditions; 297 were upregulated and 743 downregulated including MAPK8 encoding a mitogen-activated protein kinase important for apoptosis, T-cell differentiation, and inflammatory responses. Association mapping with a focus on local regulatory polymorphisms in 61 patients with SCD identified expression quantitative trait loci for 103 of these hypoxia response genes. In a University of Illinois SCD cohort, the A allele of a MAPK8 expression quantitative trait locus, rs10857560, was associated with precapillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 mm Hg and pulmonary capillary wedge pressure ⩽15 mm Hg at right heart catheterization (allele frequency, 0.66; odds ratio, 13.8; n=238). This association was confirmed in an independent Walk–Treatment of Pulmonary Hypertension and Sickle Cell Disease With Sildenafil Therapy cohort (allele frequency, 0.65; odds ratio, 11.3; n=519). The homozygous AA genotype of rs10857560 was associated with decreased MAPK8 expression and present in all 14 of the identified precapillary pulmonary hypertension cases among the combined 757 patients. Conclusions— Our study demonstrates a prominent hypoxic transcription component in SCD and a MAPK8 expression quantitative trait locus associated with precapillary pulmonary hypertension.

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W)

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations.

Altered cytokine profiles in patients with Chuvash polycythemia

Chuvash polycythemia results from a homozygous 598C>T mutation in exon 3 of the von Hippel‐Lindau (VHL) gene. This disrupts the normoxia pathway for degrading hypoxia inducible factor (HIF)‐1α and HIF‐2α causing altered expression of HIF‐1 and HIF‐2 inducible genes. As hypoxia induces expression of pro‐inflammatory cytokines, we hypothesized that there might be an elevation of Th1 cytokines in the setting of Chuvash polycythemia. We analyzed plasma concentrations of Th1 (interleukins‐2 and 12, interferon‐γ, granulocyte‐monocyte colony‐stimulating factor, tumor necrosis factor‐α) and Th2 cytokines (interleukins‐4, 5, 10, and 13) using the Bio‐Plex multiplex suspension array system in 34 VHL598C>T homozygotes and 32 VHL wild‐type participants from Chuvashia. Concentrations of all the Th1 and Th2 cytokines measured were elevated in the VHL598C>T homozygotes compared with the control wild‐type participants, but the ratios of Th1 to Th2 cytokines did not differ by genotype. In parallel, peripheral blood concentrations of CD4 positive T‐helper cells and CD4/CD8 ratio were lower in the VHL598C>T homozygotes. In conclusion, the up‐regulated hypoxic response in Chuvash polycythemia is associated with increased plasma products of both the Th1 and Th2 pathways, but the balance between the two pathways seems to be preserved. Am. J. Hematol., 2009.

Pulmonary artery pressure and iron deficiency in patients with upregulation of hypoxia sensing due to homozygous VHLR200W mutation (Chuvash polycythemia)

Background Patients with Chuvash polycythemia, (homozygosity for the R200W mutation in the von Hippel Lindau gene (VHL)), have elevated levels of hypoxia inducible factors HIF-1 and HIF-2, often become iron-deficient secondary to phlebotomy, and have elevated estimated pulmonary artery pressure by echocardiography. The objectives of this study were to provide a comprehensive echocardiographic assessment of cardiovascular physiology and to identify clinical, hematologic and cardiovascular risk factors for elevation of tricuspid regurgitation velocity in children and adults with Chuvash polycythemia. Design and Methods This cross-sectional observational study of 120 adult and pediatric VHLR200W homozygotes and 31 controls at outpatient facilities in Chuvashia, Russian Federation included echocardiography assessment of pulmonary artery pressure (tricuspid regurgitation velocity), cardiac volume, and systolic and diastolic function, as well as hematologic and clinical parameters. We determined the prevalence and risk factors for elevation of tricuspid regurgitation velocity in this population and its relationship to phlebotomy. Results The age-adjusted mean ± SE tricuspid regurgitation velocity was higher in VHLR200W homozygotes than controls with normal VHL alleles (2.5±0.03 vs. 2.3±0.05 m/sec, P=0.005). The age-adjusted left ventricular diastolic diameter (4.8±0.05 vs. 4.5±0.09 cm, P=0.005) and left atrial diameter (3.4±0.04 vs. 3.2±0.08 cm, P=0.011) were also greater in the VHLR200W homozygotes, consistent with increased blood volume, but the elevation in tricuspid regurgitation velocity persisted after adjustment for these variables. Among VHLR200W homozygotes, phlebotomy therapy was associated with lower serum ferritin concentration, and low ferritin independently predicted higher tricuspid regurgitation velocity (standardized beta=0.29; P=0.009). Conclusions Children and adults with Chuvash polycythemia have higher estimated right ventricular systolic pressure, even after adjustment for echocardiography estimates of blood volume. Lower ferritin concentration, which is associated with phlebotomy, independently predicts higher tricuspid regurgitation velocity (www.clinicaltrials.gov identifier NCT00495638).

Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1α and HIF-2α

Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHLR200W) resulting in elevated hypoxia inducible factor (HIF)-1α and HIF-2α levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21Cip1, thereby inducing its expression. In contrast, HIF-2α promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a+/− mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1α, hepatic HIF-2α mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21Cip1 mRNA levels were 9.5-fold lower in Hif1a+/− mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2α and decreased p21Cip1 levels leading to increased hepatocyte proliferation.

Elevated homocysteine, glutathione and cysteinylglycine concentrations in patients homozygous for the Chuvash polycythemia VHL mutation.

Up-regulated hypoxia sensing may influence multiple steps in thiol metabolism and result in hyperhomocysteinemia. In Chuvash polycythemia, homozygous von Hippel-Lindau (VHL) 598C>T leads to increased hypoxia inducible factor-1α and 2α, thromboses and lower systemic blood pressures. Circulating homocysteine, glutathione, γ-glutamyltransferase and cysteinylglycine concentrations were higher in 34 VHL598C>T homozygotes than in 37 normal controls and cysteine was lower. Multivariate analysis showed elevated homocysteine independently associated with higher mean systemic blood pressures and elevated glutathione was associated with lower pressures to a similar degree. Among VHL598C>T homozygotes, homocysteine was elevated with low and normal folate concentrations, consistent with a possible defect in the remethylation pathway. The elevated glutathione and γ-glutamyltranserase levels correlated positively with cysteinylglycine, consistent with possible upregulation of a glutathione synthetic enzyme and γ-glutamyltransferase. Cysteinylglycine correlated inversely with cysteine, consistent with possible reduced cysteinyldipeptidase activity. We conclude that up-regulated hypoxia-sensing may influence multiple steps in thiol metabolism. The effects of the resultant elevated levels of homocysteine and glutathione on systemic blood pressure may largely balance each other out.

The heterozygote advantage of the Chuvash polycythemia VHLR200W mutation may be protection against anemia

The germ-line loss-of-function VHLR200W mutation is common in Chuvashia, Russia and occurs in other parts of the world. VHLR200W homozygotes have elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, increased hemoglobin concentration, propensity to thrombosis and early mortality. Because the mutation persists from an ancient origin, we hypothesized that there is a heterozygote advantage. Thirty-four VHLR200W heterozygotes and 44 controls over 35 years of age from Chuvashia, Russia were studied. Anemia was defined as hemoglobin less than 130 g/L in men and less than 120 g/L in women. Mild anemia was present in 15% of VHLR200W heterozygotes and 34% of controls without a mutated VHL allele. By multivariate logistic regression, the odds of anemia were reduced an estimated 5.6-fold in the VHLR200W heterozygotes compared to controls (95% confidence interval 1.4–22.7; P=0.017). In conclusion, heterozygosity for VHLR200W may provide protection from anemia; such protection could explain the persistence of this mutation.