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Here we critically evaluate the role of elevated hematocrit as the principal determinant of thrombotic risk in polycythemia and erythrocytosis, defined by an expansion of red cell mass. Since red cell volume determination is no longer readily available, in clinical practice, polycythemia and erythrocytosis are defined by elevated hemoglobin and hematocrit. Thrombosis is common in Chuvash erythrocytosis and polycythemia vera. Although the increased thrombotic risk is assumed to be due to the elevated hematocrit and an associated increase in blood viscosity, thrombosis does not accompany most types of erythrocytosis. We review studies indicating that the occurrence of thrombosis in Chuvash erythrocytosis is independent of hematocrit, that the thrombotic risk is paradoxically increased by phlebotomy in Chuvash erythrocytosis, and that, when compared to chemotherapy, phlebotomy is associated with increased thrombotic risk in polycythemia vera. Inherited and environmental causes that lead to polycythemia and erythrocytosis are accompanied by diverse cellular changes that could directly affect thrombotic risk, irrespective of the elevated hematocrit. The pressing issue in these disorders is to define factors other than elevated hematocrit that determine thrombotic risk. Defining these predisposing factors in polycythemia and erythrocytosis should then lead to rational therapies and facilitate development of targeted interventions.

Hereditary pyropoikilocytosis is a severe hemolytic anemia caused by spectrin deficiency and defective spectrin dimer self-association, typically found in African populations. We describe two Utah families of northern European ancestry including 2 propositi with atypical non-microcytic hereditary pyropoikilocytosis, 7 hereditary elliptocytosis members and one asymptomatic carrier. The underlying molecular defect is a novel mutation in the alpha(α) spectrin gene, SPTAR34P that impairs spectrin tetramer formation. It is inherited in trans to the hypomorphic SPTAαLELY in the 2 propositi and 5 of 7 hereditary elliptocytosis individuals indicating that SPTAαLELY is not the sole determinant of the variable clinical expression. α Spectrin mRNA was mildly decreased in all hereditary elliptocytosis subjects, whereas both hereditary pyropoikilocytosis propositi had a severe decrease to ~10% of normal. Genotyping identified a unique SPTA intragenic crossover and uniparental disomy in one hereditary elliptocytosis individual. Two additional crossover events demonstrated the susceptibility of SPTA gene to rearrangement and revealed a novel segregation of the two SPTAαLELY mutations. We conclude that the profound phenotypic heterogeneity in these families can be attributed to the SPTAR34P mutation in combination with: 1) inheritance in trans of either SPTAαLELY; or 2) the wild-type SPTA; 3) a decrease of α spectrin mRNA; and 4) SPTA intragenic crossover.

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.

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).

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.

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2V617F in polycythemia vera stem cells and that TET2 mutations precede JAK2V617F. We quantified the proportion of TET2, JAK2V617F mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34+ cells. We found that TET2 mutations may also follow rather than precede JAK2V617F as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2V617F and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.

Background While in adults with sickle cell disease an elevation of tricuspid regurgitation velocity is associated with increased mortality, the importance of this finding in children has not been established. The role of intravascular hemolysis in the development of this complication is controversial.Design and Methods We conducted a prospective, longitudinal, multi-center study of 160 individuals aged 3–20 years with hemoglobin SS, performing baseline and follow-up determinations of clinical markers, six-minute walk distance less than tricuspid regurgitation velocity and E/Etdi ratio by echocardiography.Results At baseline, 14.1% had tricuspid regurgitation velocity of 2.60 m/sec or over, which suggests elevated systolic pulmonary artery pressure, and 7.7% had increased E/Etdi, which suggests elevated left ventricular filling pressure. Over a median of 22 months, baseline elevation in tricuspid regurgitation velocity was associated with an estimated 4.4-fold increase in the odds of a 10% or more decline in age-standardized six-minute-walk distance (P=0.015). During this interval, baseline values above the median for a hemolytic component derived from four markers of hemolysis were associated with a 9.0-fold increase in the odds of the new onset of elevated tricuspid regurgitation velocity (P=0.008) and baseline E/Etdi elevation was associated with an estimated 6.1-fold increase in the odds (P=0.039). In pathway analysis, higher baseline hemolytic component and E/Etdi predicted elevated tricuspid regurgitation velocity at both baseline and follow up, and these elevations in turn predicted decline in six-minute-walk distance.Conclusions Further studies should define the long-term risks of elevated tricuspid regurgitation velocity in childhood and identify potential interventions to prevent increased pulmonary artery pressure and preserve function.

Background Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results.Design and Methods JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing.Results A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques – one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean±2SD – with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2V617F.Conclusions Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.

Background Polycythemia vera is a clonal hematopoietic stem cell disorder in which the JAK2 V617F mutation is observed in >95% of patients, but an as yet unidentified process appears to initiate the clonal expansion of hematopoiesis. Because microRNA regulate hematopoietic differentiation, we hypothesized that dysregulated expression of microRNA may contribute to the pathophysiology of polycythemia vera.Design and Methods We performed gene expression profiling in five patients with polycythemia vera and in five controls using CombiMatrix MicroRNA CustomArray. ANOVA identified deregulated microRNA in polycythemia vera, and their expression was studied in a larger set of samples by quantitative reverse transcriptase polymerase chain reaction. The expression of these microRNA was also analyzed in other myeloproliferative disorders.Results We observed down-regulation of let-7a and up-regulation of miR-182 in polycythemia vera granulocytes, up-regulation of miR-143, miR-145 and miR-223 in polycythemia vera mononuclear cells, up-regulation of miR-26b in polycythemia vera platelets, and down-regulation of miR-30b, miR-30c and miR-150 in polycythemia vera reticulocytes. JAK2 V617F frequency was positively correlated with miR-143 expression and inversely correlated with let-7a, miR-30c, miR-342 and miR-150. Transcript level of predicted target genes was determined, and overexpression of IRAK2 was detected in all granulocytes from patients with myeloproliferative disorders and in polycythemia vera reticulocytes. Abnormally high HMGA2 microRNA was found in myelofibrosis granulocytes.Conclusions Our study demonstrates that peripheral blood cells from patients with polycythemia vera have microRNA signatures distinct from those of controls. Our findings of aberrant microRNA expression underline the complexity of the molecular basis of polycythemia vera.

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.

Hemoglobin Monroe (β globin G->C, codon 30) is a missense mutation. We could not detect either the mutant peptide or transcript in reticulocyte-enriched preparation and in expanded erythroid progenitor cells. By quantitative gene expression assay β globin mRNA was found to be reduced by more than 70% in all heterozygous subjects with different haplotypes. We conclude that this mutation also interferes with expression of wild type allele.