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Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation. We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity. Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034). Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. Disclosures Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

Thrombosis is the major cause of morbidity and mortality in Chuvash erythrocytosis (CE), caused by a hypomorphic R200W mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs). This mutation augments HIF activity even in normoxia. High hematocrit associated with hyperviscosity has been considered a risk factor for thrombosis suggesting that maintaining normal hematocrit may prevent thrombosis. However, our prospective controlled study reported that phlebotomies, but not the high hematocrit, are associated with increased risk of thrombosis (PMID 289208) by a mechanism that remains to be elucidated. Phlebotomies lead to iron deficiency. We explored the potential role of iron deficiency as a cause of thrombosis. Iron deficiency induces HIF activity by inhibiting prolyl hydroxylase domain 2 (PHD2), an inhibitor of HIFs that requires iron as a co-factor (PMID 18066546). The expression of certain prothrombotic genes regulated by HIFs is modestly increased in CE mononuclear cells (PMID 23993337). We hypothesized that further augmentation of already high HIF activity by iron deficiency might further increase expression of prothrombotic genes leading to increased risk of thrombosis in phlebotomized subjects. We reported that, in polycythemia vera (PV) and essential thrombocythemia (ET), the pattern of increase in prothrombotic and inflammatory gene expression differs between granulocytes and platelets (PMID: 32203583), suggesting cell-specific contributions to thrombosis in these disorders. We analyzed the whole transcriptome of the platelets of 10 CE patients, 6 with iron deficiency (ferritin 1). Dysregulated genes as analyzed by Ingenuity Pathway Analysis (Qiagen) were associated with platelet binding, hemostasis and thrombus signaling and decreased bleeding time. To explore our hypothesis further, we quantitated the mRNA of these HIF-regulated prothrombotic genes: THBS1 (thrombospondin 1), SERPINE1 (plasminogen activator inhibitor-1 [PAI-1]), ITGA2B (integrin alpha-IIb), PTGS2 (prostaglandin-endoperoxide synthase 2), SELP (P-selectin), PDGFA (platelet derived growth factor subunit A), and ITGB3 (integrin beta-3). We analyzed granulocytes from 16 CE subjects (8 iron deficient) and platelets from 12 CE subjects (7 iron deficient). In platelets, THBS1, SELP, SERPINE1, and PDGFA mRNA levels were higher in iron deficient CE subjects than those with normal ferritin (p=0.015-0.088). In all CE subjects, the mRNA levels of these four genes correlated inversely with ferritin (figure 1). PTGS2 (known to be down regulated in thrombosis) was down regulated in iron deficient CE patients and correlated positively with ferritin. ITGB3 and ITGA2B mRNA levels were not different between the two groups. In granulocytes, SELP mRNA was augmented in CE patients with iron deficiency and both SELP and ITGB3 mRNA levels correlated inversely with ferritin. We did not find a difference in expression of KLF2, a regulator of thrombotic genes, in iron deficient versus iron sufficient CE patients; see this meeting Song J et al. We then tested our hypothesis of augmentation of thrombosis risk by iron deficiency in granulocytes from 50 PV and ET patients (9 with iron deficiency) and platelets from 41 patients (5 with iron deficiency). In granulocytes, THBS1, SELP, and IRAK1 (Interleukin 1 Receptor Associated Kinase 1) mRNA levels were higher in patients with iron deficiency, and IRAK1, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. In platelets, THBS1, and SERPINE1 mRNA were higher in patients with iron deficiency and SELP, THBS1, and SERPINE1 mRNA levels correlated inversely with ferritin. JAK2V617F allele burden also correlated inversely with ferritin. Our study demonstrates that iron deficiency is associated with increased expression of HIF-regulated prothrombotic genes in CE platelets and granulocytes in a pattern that differs between these two cell types. We also report increased expression of prothrombotic genes in PV and ET patients with iron deficiency. These results underline the potential danger of phlebotomies in attempts to control high hematocrit. We caution against indiscriminate use of therapeutic phlebotomy for treatment of patients with PV and other erythrocytoses. VG &JTP contributed equally Figure Disclosures Gordeuk: Ironwood: Research Funding; Novartis: Consultancy; CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding.

Background:Chuvash erythrocytosis (CE), an inherited autosomal recessive disease endemic to Russia's mid-Volga River region, is caused by a germlineVHLC598T mutation (encoding VHLR200W) that alters oxygen sensing (PMID: 12415268). VHLR200W protein displays impaired degradation of hypoxia inducible transcription factor (HIF)-α subunits leading to increased HIF-1 and HIF-2 in normoxia and increased transcription of many HIF-regulated genes including erythropoietin (PMID: 14726398). CE patients have increased risk of venous and arterial thromboses, which are the major cause of morbidity and mortality (PMID:28104701). Thrombosis occurs despite lower blood pressure, body mass index and white blood cells compared to controls and is not related to the elevation in hematocrit but is increased in patients treated with phlebotomy therapy (PMID: 31289208). We have shown by microarray analysis of CE peripheral blood mononuclear cells modestly increased expression of several HIF-regulated pro-thrombotic genes at false discovery rate Methods:EightVHLC598T homozygotes and 6VHLwild type controls from Cheboksary, Chuvashia, Russia were studied as outpatients under basal circumstances. Blood was collected from these participants by venipuncture into vacutainer tubes containing 0.129 M sodium citrate. Immediately after venipuncture, platelet free plasma (PFP) was prepared by two rounds of centrifugation at 2,500 × g for 15 minutes at room temperature and EVs were isolated from PFP by centrifugation at 20,000 g for 15 minutes at 4 ºC. EV-TF was measured in duplicate by a two-stage Factor Xa generation assay with and without anti-TF antibody using Innovin (Siemens Healthcare Diagnostics) as a standard (PMID: 30656275). We also isolated granulocyte mRNA from 3 of the patients and 2 of the controls at a different time point and measured F3 transcripts by RT-qPCR. Results:We detected EV-TF in 4 of 8 CE patients but in no wild type controls (one-sided P = 0.043). The range of 0.45 to 1.25 pg/ml is similar to the range recently reported in US patients with cancer (PMID: 32548563). MCHC tended to be lower among 4 CE patients with detectable EV-TF than 4 without (one-sided P = 0.12), but this was not the case for serum ferritin. We also detectedF3transcripts in granulocytes, and these levels were higher in the 3 patients than the 2 controls that we analyzed (one-sided P = 0.12). Furthermore,F3mRNA correlated with plasma EV-TF in these five subjects (Spearman rho = 0.71, one-sided P < 0.05). As previously reported, CE patients had higher hemoglobin, hematocrit and red blood cells and lower platelets compared to controls (PMID: 14726398). They also had lower values for serum ferritin and mean corpuscular hemoglobin concentration (MCHC) suggesting iron deficiency, likely induced by phlebotomy therapy or by the presence of gastritis, which is increased in CE. Discussion:The presence of EV-TF in the plasma of 4 out of 8 CE patients and none of six controls, and increasedF3transcripts in CE granulocytes, may point to a potential thrombogenic role, although none of the CE patients in this study had a history of thrombosis. Further studies on larger numbers of patients are warranted to confirm these findings and to clarify the potential role of EV-TF in thrombosis in CE. *NM, JTP & VRG contributed equally Figure Disclosures Gordeuk: CSL Behring:Consultancy, Research Funding;Ironwood:Research Funding;Imara:Research Funding;Global Blood Therapeutics:Consultancy, Research Funding;Novartis:Consultancy.

Sickle cell anemia (SCA) is associated with an increased hypoxic response from anemia and vaso-occlusion-impaired tissue perfusion. The effects of hypoxia are mediated by hypoxia transcription factors (HIFs). Chuvash erythrocytosis (CE) is an inherited condition due to homozygosity for the missense mutation in VHL gene (VHLR200W) that impairs interactions of VHL with HIF-α subunits, thereby augmenting transcription of HIF-regulated genes. CE and SCA share increased expression of erythropoietin (EPO) and other HIF target genes. As HIF-regulation of transcription is tissue and differentiation-stage specific, in this study we used reticulocytes, which are easily accessible and purified peripheral blood erythroid cells. We compared the transcriptomes of SCA and CE reticulocytes to differentiate HIF-mediated dysregulation from non-hypoxic dysregulation of SCA transcripts. Our study revealed reticulocyte gene expression changes that are common to both diseases as well as SCA-specific changes. The reticulocytes were purified from 5 HbSS and 5 age- and gender-matched HbAA African-American individuals, and from 17 VHLR200W homozygotes from Chuvashia and 13 wild type Chuvash individuals. Total RNA was depleted of ribosomal RNA and globin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 100 or 125 bp paired-end sequencing to 30-45 million reads using Illumina HiSeq 2500 or 4000 platform. The sequencing data were mapped to human reference genome version GRCh37 using the splice-aware aligner STAR and analyzed using DESeq2. In separate analyses of the two diseases, we identified 1435 genes differentially expressed in SCA among 6965 analyzed genes, 848 increased and 587 decreased in SCA relative to control individuals. We also identified 1498 genes differentially expressed in CE among 8989 analyzed genes, 862 increased and 636 decreased in CE relative to control individuals. Across all analyzed genes, there was a moderate correlation (r=0.30) of expression changes between the two diseases. Among genes differentially expressed, 258 up-regulated and 155 down-regulated genes overlapped between the two diseases, representing a 1.4-fold enrichment. In a combined analysis of the two diseases, we identified 1228 genes among 6924 analyzed genes that shared altered regulation in both diseases. The 693 genes increased in both diseases were enriched (adjusted P < 0.05) in multiple metabolic, inflammatory, and oxidative pathways. The 535 genes decreased in both diseases were enriched in a cell cycle pathway. Among the commonly increased genes, the expression level of ERFE encoding erythroferrone was increased by 9.4-fold in CE and by 4.3-fold in SCA, suggesting markedly altered iron regulation in CE and SCA. This would be expected as both SCA and CE share upregulated erythropoiesis that is associated with augmented erythroferrone. Among the commonly decreased genes, RPL3L encoding Ribosomal Protein L3 Like was decreased by 68% in CE and by 93% in SCA. These common expression changes reflect hypoxic regulation related to chronic anemia in SCA. To assess disease-specific gene expression change, we tested the disease (SCA versus CE) by genotype (mutation versus wildtype) interaction effect. We identified 822 genes that showed disease-specific expression changes among 6924 analyzed genes. Of these disease-specific genes, 304 were increased and 153 decreased in SCA (adjusted P 0.05). For example, SLC16A1, encoding Solute Carrier Family 16 Member 1 and associated with an erythrocyte lactate transporter defect, was increased in SCA by 11-fold whereas PPBP encoding Pro-Platelet Basic Protein was decreased in SCA by 92%; both genes showing no change in CE. The 304 genes with SCA-specific increased expression were enriched in "Thyroid hormone signaling" (5.1 fold) and "Glioma" (7.0-fold) pathways. The 153 genes with SCA-specific decreased expression were enriched in "Ribosome" (12-fold) pathway. Among the disease-specific genes, only 34 increased and 28 decreased in CE but not in SCA. Our study demonstrates high HIF transcriptional activity in both CE and SCA reticulocytes but also reveals hypoxia-independent gene expression changes in SCA reticulocytes. These results suggest that HIF might be a therapeutic target of SCA. These data also shed light on the different molecular mechanisms underlying SCA complications. Disclosures Gordeuk: Pfizer: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

In Chuvash polycythemia, a homozygous 598CT 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 VHL ( R200W ) 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 VHL ( R200W ) homozygotes. We expanded these observations in VHL ( R200W ) 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.

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 (VHL(R200W)) resulting in elevated hypoxia inducible factor (HIF)-1alpha and HIF-2alpha 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-1alpha and HIF-2alpha. HIF-1alpha inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21(Cip1), thereby inducing its expression. In contrast, HIF-2alpha 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-1alpha, hepatic HIF-2alpha 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 p21(Cip1) 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-2alpha and decreased p21(Cip1) levels leading to increased hepatocyte proliferation.

Homozygosity for the VHLR200W mutation in Chuvash polycythemia (CP) leads to decreased degradation of the α subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. The constitutively activated HIFs directly regulate the transcription of a suite of hypoxic responsible genes, including the principal regulators of erythropoiesis, vessel development, and glycolytic metabolism, which further trigger a downstream cascade of gene expression. Besides these transcriptional factors, cis acting elements play an important role in the hypoxic gene regulatory network. To assess the extent of cis regulatory variation in hypoxic gene expression, we compared allele-specific expression (ASE) in purified reticulocytes between VHLR200W homozygote individuals and age- and gender-matched wild type control individuals living at the same altitude of ~200 meters from the Chuvash population. Cell fractions of reticulocytes were purified from 17 VHLR200W homozygotes and 13 wild type individuals. Total RNA was extracted, depleted of ribosomal RNA and hemoglobin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 125 bp paired-end sequencing to 30-45 million read pairs per sample using Illumina HiSeq 2500 platform. The samples were collected and processed in three batches across two years, with VHL genotype randomized in each batch. The sequencing data were mapped to human reference genome and analyzed for differential expression and differential ASE between VHLR200W homozygotes and wild type individuals. At 5% false discovery rate (FDR, i.e., 5, FDR ASE was analyzed between CP and wild type individuals to assess hypoxic response-dependent genetic effects on gene expression. For the 1,267 genes differentially expressed in the CP, we selected genes containing exonic SNPs with heterozygous alleles for ASE analysis. With a null hypothesis of no cis acting regulation on the gene expression, both alleles are expected to be expressed at the same level, whereas allelic imbalance indicates linked cis regulation. At a given bi-allelic SNP, individuals with ≥2 read counts covering each of the reference and alternative alleles and with ≥20 total counts were included in the analysis. Exonic SNPs with at least one individual in each of the CP and wild type group were further selected to test for differential ASE between the CP and wild type groups, using a generalized linear model. A total of 147 genes passed the filtering and were analyzed, among which 32 were detected to have significant CP-dependent ASE at 5% FDR. Some of these genes may have important roles in hypoxic responses in CP reticulocytes, for example NEIL3, encoding a DNA glycosylase that initiates the first step in base excision repair by cleaving bases damaged by reactive oxygen species, and STOM, encoding an integral membrane protein that localizes to the cell membrane of red blood cells, loss of which is associated with hereditary stomatocytosis. Our study reveals plethora of gene expression changes in CP reticulocytes compared to wild type controls, among which 22% could be regulated by hypoxic response-specific cis genetic variations. These observations indicate the prominence of cis elements in hypoxic response, for which substantial inter-individual differences exist even among a relatively isolated population. Disclosures Gordeuk: Emmaus Life Sciences: Consultancy.

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)-1alpha and HIF-2alpha 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-gamma, granulocyte-monocyte colony-stimulating factor, tumor necrosis factor-alpha) 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.

Environmental variations have strong influences in the etiology of type 2 diabetes mellitus. In this study, we investigated the genetic basis of diabetes in patients with sickle cell disease (SCD), a Mendelian disorder accompanied by distinct physiological conditions of hypoxia and hyperactive erythropoiesis. Compared to the general African American population, the prevalence of diabetes as assessed in two SCD cohorts of 856 adults was low, but it markedly increased with older age and overweight. Meta-analyses of over 5 million single-nucleotide polymorphisms (SNPs) in the two SCD cohorts identified a SNP, rs59014890, the C allele of which associated with diabetes risk at P = 3.2 × 10(-8) and, surprisingly, associated with decreased APOB expression in peripheral blood mononuclear cells (PBMCs). The risk allele of the APOB polymorphism was associated with overweight in 181 SCD adolescents, with diabetes risk in 592 overweight, non-SCD African Americans ≥ 45 years of age, and with elevated plasma lipid concentrations in general populations. In addition, lower expression level of APOB in PBMCs was associated with higher values for percent hemoglobin A1C and serum total cholesterol and triglyceride concentrations in patients with Chuvash polycythemia, a congenital disease with elevated hypoxic responses and increased erythropoiesis at normoxia. Our study reveals a novel, environment-specific genetic polymorphism that may affect key metabolic pathways contributing to diabetes in SCD.

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-1α (HIF-1α), 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-1α 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.

Chuvash polycythemia (CP) is a monogenic disorder characterized by an upregulated hypoxic response at normoxia. Homozygosity for the VHLR200W mutation leads to decreased degradation of the a subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. An array of HIF-regulated genes, including the principal regulators of erythropoiesis and iron metabolism, have altered expression. Previous studies in CP using peripheral blood mononuclear cells (PBMCs), a heterogeneous mixture of cells, identified significant gene expression differences from wild type controls, but the cell linage specificity of these hypoxia-regulated genes remains unknown. In this study, we systematically analyzed gene expression by unbiased deep RNA sequencing in purified reticulocytes, granulocytes and platelets of CP and control individuals living at the same altitude of ~200 meters. Thirty-one samples passed quality control: reticulocytes from 10 individuals (5 VHLR200W homozygotes and 5 wild type controls), platelets from 7 individuals (3 VHL homozygotes and 4 controls) and granulocytes from 14 individuals (5 VHL homozygotes, 1 heterozygote and 8 controls). The samples were analyzed for expression differences (VHL homozygote/heterozygote versus wild type) in each cell type. We found abundant gene expression differences in these three cell types. The differential genes detected in the three cell types showed no more overlap than expected by random (Binomial test P=1 for all pairings of the three cell types), suggesting cell lineage specificity of hypoxic gene expression in CP. At 5% false discovery rate (FDR, i.e., At 5% FDR, 3646 of 12,334 analyzed genes (30%) were differentially expressed in the platelets of VHLR200W homozygotes, 1830 up-regulated and 1816 down-regulated. The up-regulated genes were enriched in pathways of "Lysosome" and "Signaling in immune system". The down-regulated genes were enriched in pathways of "Hemostasis" and "Opioid signaling". At 5% FDR, 3423 of 11,274 analyzed genes (30%) were differentially expressed in the granulocytes of VHLR200W homozygotes, 1490 up-regulated and 1933 down-regulated. The up-regulated genes were enriched in pathways of "Gene Expression", "Metabolism of nucleotides", "Metabolism of proteins", and "Aminoacyl-tRNA biosynthesis". The down-regulated genes were highly enriched in immune pathways including "Chemokine signaling pathway", "Fc gamma R-mediated phagocytosis", "Endocytosis", "Neurotrophin signaling pathway", "B cell receptor signaling pathway", "Fc epsilon RI signaling pathway", as well as several cancer-related pathways. The relative abundance of alternative transcript isoforms differed in VHLR200W homozygotes relative to wild type controls for many genes in these three blood lineages indicating a role for HIFs in regulation of mRNA processing. At 1% FDR, 3121 of 12,514 analyzed genes (25%) in platelets, 233 of 7342 analyzed genes (3%) in reticulocytes, and 224 of 11,306 analyzed genes (2%) in granulocytes contained alternative exon(s) in VHLR200W homozygotes compared to wild type controls. In conclusion, we report marked gene expression variation in three blood cell lineages from individuals with CP, the first described disorder of congenital augmentation of hypoxia sensing. Dysregulated expression of genes not known to be transcriptionally regulated by HIFs may be due to the well-known but poorly defined effects of HIFs on epigenetic regulation of transcription. Our results demonstrate extensive cell lineage specificity in blood gene expression variations induced by augmented signaling of HIFs caused by the VHLR200W mutation. This provides novel insights to our understanding of clinical complications in CP and more broadly of hypoxic gene regulation. Disclosures No relevant conflicts of interest to declare.

To the editor: Hypoxia inducible factor (HIF)-1 and HIF-2 are transcription factors that play a major role in cellular responses to hypoxia. Levels of the α subunits of HIFs are constantly degraded under normoxia by the proteasome through an interaction with their negative regulator, von Hippel

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.

Blood erythropoietin (EPO) increases primarily to hypoxia. In sickle cell anaemia (homozygous HBBE6V; HbSS), plasma EPO is elevated due to hemolytic anaemia-related hypoxia. Hydroxyurea treatment reduces haemolysis and anaemia by increasing foetal haemoglobin, which leads to lower hypoxic transcriptional responses in blood mononuclear cells but paradoxically further increases EPO. To investigate this apparent hypoxia-independent EPO regulation, we assessed two sickle cell disease (SCD) cohorts for genetic associations with plasma EPO, by prioritizing 237,079 quantitative trait loci for expression level and/or transcript isoform variations of 12,727 genes derived from SCD blood mononuclear cells. We found an association between the T allele of SNP rs60684937 and increased plasma EPO (n = 567, combined P = 5.5 × 10 − 8 adjusted for haemoglobin and hydroxyurea) and validated it in independent SCD patients (n = 183, P = 0.018). The T allele of rs60684937 was associated with a relatively increased expression of a non-coding transcript of PRKAR1A (cAMP-dependent protein kinase type I-alpha regulatory subunit) in 58 SCD patients (P = 7.9 × 10 − 7) and 58 HapMap Yoruba samples (P = 0.0011). In conclusion, we demonstrate that plasma EPO elevation with hydroxyurea in SCD is independent of hypoxic responses and that genetic variation at SNP rs60684937 may contribute to EPO regulation through a cAMP-dependent protein kinase A pathway.

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 VHL R200W 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 VHL R200W 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 VHL R200W homozygotes, consistent with increased blood volume, but the elevation in tricuspid regurgitation velocity persisted after adjustment for these variables. Among VHL R200W 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][1] identifier [NCT00495638][2]) . [1]: http://www.clinicaltrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00495638&atom=%2Fhaematol%2F97%2F2%2F193.atom

Background The level of distorted erythrocytes due to polymerization of hemoglobin S in sickle cell disease (SCD) (Science 1949;110:543) is a major determinant of the severity of hemolysis and microvascular occlusion (Lancet 2010;376:2018). Erythropoietin (EPO) is elevated in SCD due to hemolytic anemia and a related increase in hypoxia-inducible factors (HIFs) (Eur J Haematol 2007;78:183). Hydroxyurea (HU) is widely used in the treatment of SCD. HU inhibits ribonucleotide reductase (Semin Oncol 1992;19(3 Suppl 9):1-10) and promotes γ globin synthesis thereby increasing HbF-containing erythrocytes (F cells) while suppressing sickle β hemoglobin production (J Clin Invest 1984;74:652 and 2003;111:231). Increased level of F cells reduces hemolysis and ameliorates clinical complications in SCD. We and others have observed an increase in serum EPO level with HU treatment in SCD despite an increase in the hemoglobin concentration, and we hypothesized that this may be due to the known increased affinity of hemoglobin F for oxygen and related tissue hypoxia (Blood 2009;114:4639). Methods Messenger RNA from peripheral blood mononuclear cells (PBMCs) was profiled using Affymetrix Human Exon 1.0 ST Array. Hypoxic transcriptional alteration was defined in 15 Chuvash polycythemia (CP) patients vs. 17 control individuals. CP leads to constitutive up-regulation of HIFs in the absence of anemia or hypoxia. Transcriptional alteration in SCD was determined in 13 HbSS subjects without HU treatment vs. 16 control individuals, and that induced by HU treatment was determined in 19 HbSS subjects with vs. 13 without HU treatment. For meta-analysis on serum EPO concentration, genomic DNA isolated from PBMCs was hybridized to the Illumina Human 610-Quad SNP array. Genotypes were imputed to 1000 genomes project phase 1 data. A linear regression model was applied adjusting for age, gender, hemoglobin concentration, and HU treatment. Results Gene expression changes by HbSS highly correlated with those associated with homozygous VHLR200W (Pearson's r=0.79, Figure 1A). At 5% false discovery rate (FDR), expression levels of 377 genes were altered in both VHLR200W homozygotes and HbSS by >1.2 fold. For these hypoxic genes, the correlation of expression changes between HbSS and homozygous VHLR200W reached r=0.97 (Figure 1B). In contrast to our hypothesis, HU treatment in general suppressed expression changes induced by HbSS (r=-0.85, Figure 1C), especially for the hypoxic genes (r=-0.95, Figure 1D). In VHLR200W homozygotes, 62 of the hypoxic genes correlated with plasma EPO levels (adjusted P Discussion Our study demonstrates a prominent release from hypoxic transcriptional responses by HU treatment in SCD despite an increase in serum EPO, a defining characteristic of an up-regulated hypoxic response. Our study hypothesizes that hypoxia-independent signals trigger EPO production in the setting of HU therapy and it identifies a potential genetic determinant in this alternative pathway. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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 VHLR200W mutation is common in Chuvashia, Russia, and also occurs elsewhere. VHLR200W homozygotes have elevated hypoxia-inducible factor 1α (HIF-1α) and HIF-2α levels, increased red cell mass, propensity to thrombosis, and early mortality. Ninety VHLR200W 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 VHLR200W. 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 VHLR200W 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.

The germ-line loss-of-function VHL(R200W) mutation is common in Chuvashia, Russia and occurs in other parts of the world. VHL(R200W) 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 VHL(R200W) 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 VHL(R200W) 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 VHL(R200W) heterozygotes compared to controls (95% confidence interval 1.4-22.7; P=0.017). In conclusion, heterozygosity for VHL(R200W) may provide protection from anemia; such protection could explain the persistence of this mutation.

Endothelin-1 has been associated with development of hypoxia-related pulmonary hypertension and vascular endothelial growth factor (VEGF) with protection from this complication. In Chuvash polycythemia, homozygous germline von Hippel-Lindau (VHL) 598C-T leads to up-regulation during normoxia of hypoxia inducible factor-1a and several hypoxia-controlled genes including erythropoietin and VEGF. We postulated that endothelin-1 and pulmonary artery pressure may be elevated in Chuvash polycythemia.Systolic pulmonary artery blood pressure was estimated by Doppler echocardiography and plasma concentrations of endothelin-1, VEGF and erythropoietin were determined in 14 patients with Chuvash polycythemia and 14 controls. Results. Plasma endothelin-1 (p=0.010), VEGF (p=0.022) and erythropoietin (p0.0005) concentrations and Doppler-estimated systolic pulmonary artery pressures (p0.0005) were higher in the patients while systolic systemic blood pressures were lower (p=0.001). Five (36%) patients and no controls had mild pulmonary hypertension defined as systolic pulmonary artery pressure (c) 35 mmHg. Among the patients with Chuvash polycythemia, the trends of association of estimated pulmonary artery pressure with plasma concentrations of endothelin-1 (R = +0.236), VEGF (R = -0.389) and erythropoietin (R = +0.220) were not statistically significant.Estimated systolic pulmonary artery pressure and plasma concentrations of endothelin-1 and VEGF are increased in patients with Chuvash polycythemia patients. The lack of significant associations of estimated systolic pulmonary artery pressure with plasma endothelin-1 and VEGF levels could conceivably be due to the small sample size. Further studies are indicated, especially in view of the reported efficacy of endothelin-1 receptor blockers in treating hypoxia-associated pulmonary hypertension.

Chuvash polycythemia (CP) is an autosomal recessive condition that is endemic in the Russian mid-Volga River region of Chuvashia. We previously found that CP patients may have increased serum erythropoietin (EPO) levels, ruled out linkage to both the EPO and EPO receptor (EPOR) gene loci, and hypothesized that the defect may lie in the oxygen homeostasis pathway. We now report a study of five multiplex Chuvash families which confirms that CP is associated with significant elevations of serum EPO levels and rules out a location for the CP gene on chromosome 11 as had been reported by other investigators or a mutation of the HIF-1 alpha gene. Using a genome-wide screen, we localized a region on chromosome 3 with a LOD score >2. After sequencing three candidate genes, we identified a C to T transition at nucleotide 598 (an R200W mutation) in the von Hippel-Lindau (VHL) gene. The VHL protein (pVHL) downregulates the alpha subunit of hypoxia-inducible factor 1 (HIF-1 alpha), the main regulator of hypoxia adaptation, by targeting the protein for degradation. In the simplest scenario, disruption of pVHL function causes a failure to degrade HIF-1 alpha resulting in accumulation of HIF-1 alpha, upregulation of downstream target genes such as EPO, and the clinical manifestation of polycythemia. These findings strongly suggest that CP is a congenital disorder of oxygen homeostasis.

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).

Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality. 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 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHLR200W-induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman’s ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a >1.15 fold change in both sickle cell anemia and VHLR200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8, encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis. To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures: No relevant conflicts of interest to declare.

Abstract 5039 Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen sensing, is characterized by homozygous germ-line loss-of-function mutation of VHL (598C>T) resulting in elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, increased red cell mass and propensity to thrombosis. Organ size is determined by the size and number of cells, and its molecular mechanisms are not fully elucidated. Previous work from several groups demonstrated that cell proliferation is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing C-MYC from the promoter of the cyclin-dependent kinase inhibitor, p21Cip, and inducing its expression, while HIF-2α promotes MYC activity and cell proliferation. We determined the volumes of the liver, spleen, and kidneys and the diameter of the head of the pancreas by computerized tomography in 30 individuals with Chuvash polycythemia and 30 age- and sex-matched Chuvash controls and calculated the ratio of organ volume (or diameter) to body mass. The subjects with Chuvash polycythemia had larger ratios of the liver (P Disclosures No relevant conflicts of interest to declare.

Abstract 1897 Poster Board I-920 Background: Chuvash polycythemia is caused by homozygosity for the VHL598C>T mutation, which leads to up-regulation of HIF-1a and HIF-2a in normoxia. As the result, circulating concentrations of erythropoietin are elevated. Chuvash polycythemia patients suffer from cardiovascular abnormalities that include pulmonary arterial hypertension, thrombosis and stroke. Phlebotomy is a common therapy for patients to decrease symptoms such as plethora and headache. However, the outcomes of phlebotomy have not been assessed for these patients. The objective of this analysis is to evaluate the effect of phlebotomy on hemoglobin concentration, serum concentrations of ferritin and erythropoietin, and echocardiographically-determined tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure. Methods: One hundred twenty patients homozygous for VHL598C>T and 38 controls of comparable age and gender from Chuvash Republic of the Russian Federation were studied. Clinical and demographic characteristics were determined and echocardiography was performed. Serum ferritin and erythropoietin concentrations were measured by ELISA. Results: The median (interquartile) age for Chuvash polycythemia cases was 36 (22–48) years. They included 68 females (56%). Chuvash polycythemia patients had higher serum erythropoietin concentration (medians of 46 versus 8 mIU/ml, P = 0.0001) and lower serum ferritin concentration (medians of 12 versus 48 ng/ml, P = 0.0001) compared to controls. Tricuspid regurgitation velocity was higher in cases than controls (medians of 2.5 vs. 2.3 m/sec, P = 0.007). Among the cases, 87 (71%) had a history of phlebotomy and 54 of these had phlebotomy within the last year. Phlebotomy was associated with higher erythropoietin concentration (P = 0.033) and lower ferritin concentration (P = 0.024) but no significant difference in hemoglobin concentration (P = 0.9) (Table 1). After adjusting for the effect of age, phlebotomy was associated with significantly higher odds of tricuspid regurgitation velocity ≥2.5 (m/sec) (odds ratio: 3.3; 95% CI: 1.1–9.9). Conclusion: Patients with Chuvash polycythemia tend to mobilize iron stores and increase erythropoietin production to maintain a constant, elevated hemoglobin concentration despite phlebotomy therapy. In this process, estimated pulmonary systolic blood pressure appears to increase. Therefore, phlebotomy therapy might be a risk factor for pulmonary hypertension in the context of Chuvash polycythemia. Disclosures: No relevant conflicts of interest to declare.

Chuvash polycythemia results from a homozygous 598 C>T mutation in exon 3 of the von Hippel-Lindau (VHL) gene. This disrupts the normoxia pathway for degrading hypoxia inducible factor (HIF)-1a and HIF-2a causing altered expression of HIF-1 and HIF-2 regulated 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. Concentrations of Th1 (interleukins-2 and 12, interferon-g, GM-CSF, tumor necrosis factor-a) and Th2 cytokines (interleukins-4, 5, 10 and 13) were measured in plasma using the Bio-Plex multiplex suspension array system. Concentrations of all of these cytokines were elevated in patients with Chuvash polycythemia 598C>T homozygous compared to the control wild type participants. In parallel, peripheral blood concentrations of CD3 positive T-helper cells and CD-4 positive T-helper cells were lower in patients with Chuvash polycythemia compared to controls. The ratios of interferon-g and tumor necrosis factor-a to interleukin-10 did not differ by genotype. Thus, although the upregulated hypoxic response in Chuvash polycythemia is associated with increased plasma products of the Th1 and Th2 pathways, the peripheral blood concentrations of T-helper cells are reduced, perhaps as part of a feed-back mechanism, and the balance between the two pathways may be preserved. Such countervailing responses to unregulated hypoxia sensing may explain why patients with Chuvash polycythemia do not present clinically with immunologic disorders.

Background: Chuvash Polycythemia (CP) is an autosomal recessive disorder that is endemic to the Chuvash region of Russia; however, it does occur worldwide. It is characterized by a mutation (598C>T) of the von Hippel Lindau gene, a negative regulator of hypoxia-sensing, and increased levels of hypoxia inducible factor-alpha during normoxia. CP is manifested by vertebral hemangiomas, varicose veins, hypotension, elevated concentrations of products of hypoxia-induced genes (serum vascular endothelial growth factor and endothelin-1), and premature mortality related to cerebral vascular events and peripheral thrombosis. Echocardiography (echo) studies in adults with CP suggest an increased prevalence of pulmonary hypertension (PH), but these findings have not been validated in children with CP. Methods: This work is supported by an NIH grant evaluating the prevalence, outcomes, pathophysiology, vascular response, and gene polymorphisms of PH in sickle cell disease and CP. We traveled to Cheboksary, Russia to prospectively enroll children with CP and age and gender matched controls to undergo echo for assessment of left ventricular (LV) systolic and diastolic function, LV size and mass, estimated right ventricular (RV) systolic pressure by tricuspid regurgitation velocity (TRV), and the RV to systemic systolic blood pressure (SBP) ratio. Data was compared between CP and control children. Results: 24 CP and 11 control patients were enrolled in the study. 20 CP and 9 control patients had measurable TRV. 4 of 20 CP and none of the control patients had TRV > 2.5 m/sec (max TRV 2.89 m/sec). There was a trend towards higher TRV and estimated RV pressure in CP patients. Systolic function, LV size, and mass were the same in both groups. Diastolic function was normal in both groups but significantly better in CP patients. SBP was the same in both groups. Systemic mean and diastolic BP, adjusted for age, was significantly lower in CP children. Conclusions: Preliminary prospective data show a trend towards higher echo derived estimates of RV pressure in children with CP that can not be explained by hypertension or diastolic dysfunction. Rather, congenital upregulation of the hypoxic response may be responsible. Continued patient recruitment and correlation of TRV with other variables will allow for a better understanding of PH in children with CP. ECHO PARAMETERS CP Controls p TRV (m/sec) 2.24 ± 0.32 2.08 ± 0.14 0.08 RV systolic pressure (mm Hg) 25.4 ± 5.7 22.4 ± 2.4 0.06 RV/systemic pressure ratio 0.26 ± 0.06 0.24 ± 0.03 0.17 LV diastolic dimension z score 0.11 ± 0.91 −0.13 ± 0.79 0.42 LV Mass Index: g/m^2.7 25.8 ± 8.2 24.7 ± 6.5 0.68 Ejection fraction (%) 64.9 ± 3.7 64.7 ± 3.2 0.91 Mitral E/Tissue Doppler E 5.35 ± 0.81 6.48 ± 1.31 0.02

Chuvash polycythemia is characterized by a homozygous 598C>T mutation in VHL and up regulation of HIF-1α during normoxia. Disorders of chronic hypoxia may be complicated by the development of pulmonary hypertension. Because of the up regulation of the hypoxic response in Chuvash polycythemia, we postulated that there may be a tendency to increased pulmonary artery pressures in this condition as well. To test this hypothesis, we analyzed results for Doppler echocardiography in 15 patients with Chuvash polycythemia and 15 Chuvash individuals without polycythemia. The tricuspid regurgitation velocity (TRV) allows estimation of pulmonary artery systolic pressure. A TRV of 2.5 m/sec or higher corresponds to a pulmonary artery systolic pressure of at least 35 mm Hg (normal up to 32 mm Hg), while a TRV of 3.0 m/sec or higher to a pressure of at least 46 mm Hg. The results are summarized in the Table. Pulmonary artery pressures as estimated by tricuspid regurgitation velocity (TRV) in Chuvash subjects with and without polycythemia Chuvash polycythemia (n = 15) Controls (n = 15) P Age in years; mean (SD) 35 (17) 35 (17) 1.0 Female sex in no. (%) 8 (53%) 8 (53%) 1.0 Hemoglobin in g/dL; mean (SD) 16.7 (2.3) 13.3 (1.2) 2.4 m/sec in no. (%) 4 (27%) 0 (0%) 0.1 Most of the patients with Chuvash polycythemia were receiving phlebotomy therapy and therefore many had hemoglobin concentrations in the upper normal range. Four of the patients with Chuvash polycythemia and none of the others had TRV ≥ 2.5 m/sec (range of 2.5 to 3.0), and mean TRVs were significantly higher in the patients with Chuvash polycythemia. Interestingly, the mean ± SD TRV in these 15 patients with Chuvash polycythemia was identical to the mean ± SD TRV that was recently reported in 195 American patients with sickle cell disease (Gladwin et al, NEJM2004;350:886), another hematological condition with a tendency to pulmonary hypertension. While the pulmonary arterial pressures detected so far in Chuvash polycythemia patients are lower than those in patients with primary pulmonary hypertension, our results suggest that pulmonary hypertension may be an unrecognized complication of Chuvash polycythemia.

Background: In Chuvash polycythemia (CP) (Problemi Gematologii I Perelivaniya Krovi 1974, 10:30), impaired degradation of hypoxia inducible factor (HIF)-1α and HIF-2α from a homozygous germline VHLR200W mutation leads to augmented hypoxic responses during normoxia (Nat Genet 2002, 32:614). In addition to elevated hematocrit, CP is marked by leg varices, benign vertebral hemangiomas, decreased systemic blood pressure, increased systolic pulmonary artery pressure, and by the defining phenotypes of thrombosis and early mortality (Blood 2004, 103:3924; Haematologica 2012, 97:193). There is no effective therapy. While phlebotomy has been recommended for idiopathic polycythemia by the British Committee for Standards in Haematology (Br J Haematol 2005, 130:174) and is administered to some CP patients, its benefits are unknown. Phlebotomy-induced iron deficiency inhibits PHD2 enzyme, the principal negative regulator of HIFs, which further augments hypoxic responses. This affects the transcription of many genes (BCMD 2014, 52:35). Hypoxia-regulated IRAK1 is augmented in inflammation and may promote thrombosis (Circ Res. 2013, 112:103). Methods: 165 patients with CP were enrolled in a registry between 2001 and 2009 after providing written informed consent. Survival analysis was used to examine the predictors of new thrombosis and death during the follow-up period. mRNA from peripheral blood mononuclear cells (PBMCs) was profiled by Affymetrix Human Exon 1.0 ST Array in 42 of the subjects. Results: The median age at enrollment was 35 years and 90 participants were females, 25 had a history of one thrombosis, 5 of two thromboses and 3 of three thromboses. In the year prior to study entry, 72 had received phlebotomy therapy (Table 1). In July 2015 the median follow-up was 9.0 years (range 1-14.5). During this follow-up period, 30 (18.2%) participants had one new thrombosis, 6 (3.6%) had two new thromboses and 17 (10.3%) died. The median age of death was 55 years (range 16-76) and deaths were related to thrombotic cerebrovascular accident (n = 4), myocardial infarction (n = 4), mesenteric or portal vein thrombosis (n = 3), other major thromboembolic events (n = 2) and trauma or unknown cause (n = 2). Baseline characteristics of older age, prior thrombosis, pentoxifylline treatment, smoking and splenomegaly were independently associated with greater thrombosis risk during follow-up (P < 0.003). After adjustment for these variables, the estimated probability of new thrombosis at 10 years was 26% in those receiving phlebotomies compared to 12% in those not phlebotomized (log rank P = 0.014) (Figure 1). There was also a trend for increased risk of death with phlebotomy: estimated probability 8.7% versus 3.7% (P = 0.15). Examination of gene transcripts affecting thrombosis by logistic regression identified 12 protective and 16 risk genes at 5% false discovery rate. Upregulation of two mRNAs was of singular significance: 1) IL1RAP, a proximal signaling adaptor of IRAK1 (Immunity 1997, 7: 837) and 2) THBS1, encoding thrombospondin1 (Blood 2015, 125: 399). Both genes have known roles in thrombosis promotion and we previously reported that THBS1 is upregulated in CP (BCMD 2014, 52:35). Further analysis revealed a further upregulation of THBS1 in patients with baseline history of phlebotomy (β=0.41, P=0.046). Conclusion: These findings underscore a high rate of thrombosis and death in patients with CP and reveal a potential role of increased IRAK1/IL1RAP signaling in these complications. They raise the possibility that phlebotomy therapy has a detrimental rather than beneficial effect, possibly contributed to by increased THBS1 expression. Table 1. Baseline characteristics by phlebotomy in the year prior to enrollment. Results in median (interquartile range) or n (%); four without phlebotomy data. No phlebotomy N=89 Received phlebotomy N=72 Age (years) 32 (18-48) 37 (26-49) 0.08 Female gender, n (%) 52 (58%) 34 (47%) 0.16 Smoking, n (%) 18 (20%) 24 (33%) 0.060 History of thrombosis, n (%) 20 (23%) 12 (17%) 0.4 Splenomegaly, n (%) 2 (2.3%) 2 (2.8%) 0.8 ASA treatment, n (%) 27 (30%) 36 (50%) 0.011 Pentoxifylline, n (%) 7 (7.9%) 17 (23.6%) 0.005 BMI (kg/m2) 20.4 (18.3-22.9) 21.6 (19.9-24.6) 0.010 Systolic BP (mm Hg) 109 (100-123) 118 (105-124) 0.6 Diastolic BP (mm Hg) 76 (68-84) 78 (71-83) 0.8 Hemoglobin (g/dL) 18.1 (16.4-21.0) 17.9 (16.0-19.8) 0.5 WBC (per uL) 5.7 (4.6-7.0) 5.5 (4.6-6.7) 0.9 Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.