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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) but not in CE (nominal 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.

Background. Whether glucose metabolism differs in sickle cell anemia (SCA) patients versus controls is not clear. Transfusional iron overload and Inflammation might enhance glucose intolerance, while lower BMI and increased hypoxic responses might be protective. Methods: We prospectively determined indicators of glucose metabolism in 46 African-American hemoglobin SS or S/β0-thalassemia subjects >35 years of age who did not have the diagnosis of DM, and examined the relationship of these parameters to BMI and serum ferritin concentration. We compared the SCA subjects to 46 African-American controls from the National Health and Nutrition Examination Study (NHANES) who did not have the diagnosis of DM and were matched by age, sex, BMI and serum ferritin to the SCA subjects. Results: The BMI was >25 kg/m2 in 23 SCA subjects (48%) and the serum ferritin was >300 ng/ml in 30 (65%) consistent with increased body iron stores. The fasting blood glucose was >125 mg/dL in 3 of 46 SCA subjects (6.5%) suggesting unsuspected DM. The fasting glucose concentration independently correlated with serum ferritin concentration (r=0.336, P=0.024) and the fasting insulin level correlated independently with both BMI (r=0.583, P=0.00002) and serum ferritin (r=0.300, P=0.016). Measures of glucose metabolism did not differ significantly between the SCA subjects and NHANES controls, even though the SCA subjects had lower blood pressures, WBC counts and blood lipid levels. Conclusions: Measures of glucose metabolism in older SCA patients are similar to controls matched for BMI and iron status despite marked differences in blood pressure, hemoglobin, WBCs and blood lipid levels. Disclosures Gordeuk: Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Imara: Research Funding; Emmaus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

Introduction Sickle cell anemia (SCA) is characterized by hemolysis, a tissue hypoxia-driven increase in erythropoietin production, and a marked reticulocytosis reflecting stress erythropoiesis. Reticulocytes in SCA constitute 4-15% or more of peripheral red blood cells. Sickle reticulocytes express adhesion molecules that are normally cleared by exosome shedding, including α4β1 integrin, which is thought to contribute to vaso-occlusive crises by binding to vascular endothelial cell vascular cell adhesion molecule 1. Our recent study of the reticulocyte transcriptome in SCA versus healthy African Americans, using RNA-Seq, revealed differential genes that were implicated in a broad range of biological processes including terminal erythroid differentiation, the unfolded protein response, and altered membrane systems. In this study, we assessed the genetic association of these differential genes with disease complications relevant to sickle reticulocytes. Methods We analyzed vaso-occlusive complications including episodic severe pain and chronic avascular necrosis. We also analyzed hemolysis and anemia measured by a hemolysis index and hemoglobin concentration, respectively. The patient cohort combined 525 adolescent and adult patients from Walk PHaSST and 377 pediatric patients from PUSH. Genomic DNA isolated from peripheral blood mononuclear cells of Walk-PHaSST and PUSH patients were genotyped using Illumina Human 610-Quad SNP array and the genotypes were phased and imputed to 1000 genomes phase 3 data. Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) &gt;0.01 and imputation r2&gt;0.7 were tested for gene-wise genetic association using SKAT. For severe pain and avascular necrosis, the covariates included age, gender, β globin genotype severity (HHbSS and HbSβ0-thalassemia versus HbSC and HbSβ+-thalassemia), and population stratification. For hemolysis and hemoglobin concentration, the covariates included age, gender, β globin genotype severity, alpha thalassemia, hydroxyurea treatment, recent blood transfusion, and population stratification. Results A total of 111 genes were analyzed. These genes were classified into 13 functional categories based on published research (transcription, post-transcription, translation, histone modification, cell cycle arrest, mitophagy, metabolism, iron homeostasis, antioxidative system, unfolded protein response, membrane skeleton and structural proteins, membrane adhesion molecules and blood group antigens, and membrane transporters). For each functional class, the number of significant gene-wise associations the functional class contains was compared to the number of significant associations from the other classes combined. Defining significance of gene-wise association at nominal P=0.05, we found that the "Unfolded protein response" contained more significant gene-wise associations with severe pain than the other functional classes (one-sided Fisher's exact test P=0.046), driven by genes FUCA1 (alpha-L- fucosidase) and CLN8 (associated with endoplasmic reticulum function). "Membrane skeleton and structural proteins" showed a trend of enrichment for hemolysis (P=0.057), driven by genes MYO5B and CD2AP, and for avascular necrosis (P=0.075), driven by genes MYLK, CAVIN2 and PALLD. Defining the significance of gene-wise association at a less stringent nominal P=0.1, "membrane adhesion molecules and blood group antigens" showed a trend of enrichment in association with severe pain (P=0.097), driven by genes GYPA, ITGA4 and ITGB1, the latter two encoding α4β1 integrin subunits which are abnormally present on the reticulocyte membrane and have been implicated in sickle vaso-occlusion. Regional scans revealed the SNPs with the most significant associations within these genes. Discussion Our results reveal enrichment of genetic association with vaso-occlusive and hemolytic complications in gene functional categories that showed altered expression in sickle reticulocytes. Future study is needed to validate these associations. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding; Ironwood: Research Funding; Novartis: Consultancy.

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.