Elevated Erythropoietin Levels Research Articles

Vhl deletion in Dmp1-expressing cells alters MEP metabolism and promotes stress erythropoiesis

IntroductionErythropoiesis occurs in a specialized niche in the bone marrow and is regulated in part by oxygen-sensing signaling pathways. VHL is an E3 ubiquitin ligase that regulates the degradation of hypoxia-inducible factor (HIF) proteins under normoxic conditions. In hypoxia, VHL is inactivated resulting in enhancement of HIF signaling. Methods and resultsTo investigate the effects of HIF-activation in bone cells on erythropoiesis, we performed longitudinal analyses of conditional Vhl knockout mice (Dmp1-Cre;Vhlfl/fl; VhlcKO) mice, in which Vhl is deleted primarily in osteocytes but is retained in hematopoietic cells. VhlcKO mice display elevated red blood cell counts and erythropoietin levels starting at 10 weeks of age, resembling polycythemia. Using flow cytometry, we observed increased frequency of CD71loTER119hiFSClo orthochromatophilic erythroblasts and reticulocytes in 10- and 24-week-old VhlcKO bone marrow. Features of extramedullary hematopoiesis were observed in the spleen, including red pulp hyperplasia, the presence of megakaryocytes, and increased frequency of basophilic and polychromatophilic erythroblasts and of mature stress erythroid progenitors. To investigate the mechanisms that drive the accelerated erythropoiesis in the bone marrow, we enumerated myeloid progenitors and observed higher frequencies of megakaryocyte-erythroid progenitors (MEPs) at 6 and 10 weeks of age, but MEP frequency was similar to controls at 24 weeks. Despite this normalization in MEP frequencies, bulk RNA-Seq of MEPs at this age showed upregulation of Epas1 (Hif2a), consistent with a response to hypoxia, as well as genes involved in erythrocyte development. Surprisingly, genes involved in the response to glucose were also upregulated in VhlcKO MEPs. DiscussionOur studies implicate that HIF-driven alterations in skeletal homeostasis drive changes to the bone marrow microenvironment that influence erythroid cell development and MEP metabolism.

Suspected renal interstitial cell tumor causing polycythemia in two dogs.

Here we report a case series of two dogs diagnosed as renal interstitial cell tumor (RICT) accompanied by elevated serum erythropoietin level and marked polycythemia. RICT is a rare tumor in dogs, originating from renal interstitial cells. While several renal tumors such as renal lymphoma, adenocarcinoma, carcinoma, sarcoma, fibrosarcoma and nephroblastoma may cause polycythemia, polycythemia caused by RICT has never been reported in dogs. The tumors in both dogs were solitary and lied within cortex or cortico-medullary junction. Histopathology revealed spindle-shaped cells suggesting mesenchymal origin, with no mitotic figures suggesting that the tumors in both dogs were benign. Following surgical removal of the affected kidney, serum erythropoietin level and polycythemia normalized in both dogs.

Effect of long-term treatment with trivalent chromium on erythropoietin production in HepG2 cells

Trivalent chromium (Cr(III)) is sometimes taken as a long-term supplement, but its effectiveness is unclear. Recently, Cr(III) reportedly modulates peroxisome proliferator-activated receptor gamma (PPARγ) expression. Our previous study reported that increased PPARγ after 24 h Cr(III) treatment promoted erythropoietin (EPO) production in HepG2 cells. In the current study, we analyzed 4-week Cr(III) treatment effects on PPARγ and EPO production in HepG2 cells. Long-term Cr(III) treatment resulted in significantly elevated mRNA expression levels of PPARγ and EPO compared to controls. Additionally, treatment with a PPARγ inhibitor suppressed EPO mRNA expression. Increased EPO mRNA expression due to stimulation with hypoxia or cobalt was unaffected by long-term Cr(III) treatment. Administration of lipopolysaccharide and pyocyanin which causes oxidative stress, promoted EPO production, but this effect was attenuated in cells treated with Cr(III). Long-term Cr(III) treatment increased hypoxia inducible factor (HIF)-1α and 2α mRNA expression and protein levels. Increased PPARγ, induced by long-term Cr(III) treatment, suppressed sirtuin1 (SIRT1) mRNA expression and increased EPO mRNA expression, suggesting that increased PPARγ attenuated the suppressive effect of SIRT1 on HIF. These results suggest that the sustained increase in PPARγ during long-term Cr(III) treatment maintains increased EPO production through a mechanism different from that observed under hypoxia.

Polycythemia Vera with Elevated Erythropoietin Level: A Review

According to the 2016 World Health Organization (WHO) recommendations, polycythemia vera (PV) should be suspected at hemoglobin levels of 16.5 gm/dl in males and 16 gm/dl in females. Depending on the underlying driving factor, polycythemia is classified to primary and secondary. Secondary polycythemia is driven by erythropoietin (EPO), which can be released due to hypoxemia, as seen in people living at high altitudes, those with chronic lung diseases, obstructive sleep apnea (OSA) and smoking. It can also be directly released by an underlying EPO secreting tumor, such as renal cell carcinoma. On the other hand, primary polycythemia or polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) in which erythrocytosis is driven by an acquired activating mutation that increases sensitivity to EPO or causes EPO independent erythroid colony formation. Manifestations of PV include fatigue, excessive itching, abdominal pain, weight loss, and fever. However, the most critical complications of PV, which impact survival, are thrombotic complications such as stroke and venous thromboembolism and leukemic transformation. The risk for leukemic transformation in patients with PV is about 2-5% over 15 years and is more common in individuals older than 60. History and physical examination should focus on smoking history and signs and symptoms of cardiopulmonary diseases causing chronic or intermittent hypoxemia, as seen in OSA. On the other hand, findings of abdominal pain, early satiety, weight loss, erythromelalgia, and hepatosplenomegaly would be suggestive of PV. Laboratory workup classically starts with measuring EPO level which further directs workup toward PV or secondary polycythemia. If workup is suggestive of PV, the following steps would be testing for activating Janus Kinase 2 (JAK2) mutation (V617F), which has a sensitivity of 97% for PV, and bone marrow biopsy which shows hypercellularity with trilineage growth (panmyelosis) in cases of PV. EPO measurement constitutes an essential step in polycythemia workup. It is widely accepted that elevated EPO levels are associated with secondary polycythemia; and suppressed EPO level was considered a criterion to diagnose PV, according to the WHO 2016 diagnostic criteria for PV. We conducted a systematic review searching the entity of PV with elevated EPO to study the clinical significance and outcomes. Our search yielded 4 cases summarized in Table 1. The cases were of three males and one female, with a mean age of 50. Two of them were healthy, with two with comorbid diseases, and as seen in the table; only one was a smoker. The most common symptom was headache, followed by erythromelalgia. Thrombotic phenomena happened in a single case in the form of Budd Chiari syndrome. Hepatosplenomegaly was found in a single case, and splenomegaly was found in two cases. The mean Hb level was 20.2 gm/dl, and the mean EPO level was 213. EPO is a glycoprotein hormone that is mainly produced by the kidneys. However, it is also produced in small amounts in the liver, spleen, bone marrow, lungs, and brain. The primary regulator of EPO production is the partial pressure of oxygen (PaO2). Multiple case reports and case series suggested an association between PV with elevated EPO and Budd Chiari Syndrome. Thumures et al. described four patients that were diagnosed with Budd Chiari Syndrome with elevated red cell mass and were found to have elevated serum erythropoietin levels. PV was confirmed with JAK2 mutation assay and bone marrow biopsy examination. It is speculated that liver hypoxia with subsequent hepatocyte injury and necrosis caused by Budd Chiari Syndrome leads to EPO level elevation. Accordingly, we assume that patients with PV and elevated EPO have some degree of organ injury that results in the release of EPO into circulation. This highlights the importance of a systematic approach in medicine, where signs and symptoms remain the cornerstone in diagnosis and where lab tests should be taken in their appropriate clinical context. Thus, we recommend testing for JAK2 mutation in the proper clinical scenario regardless of the EPO levels. It still remains to be clarified whether there are any prognostic importance of the elevated EPO level in these patients and whether it would alter the management.

Red Blood Cell Transfusion Dependence Is Associated with Greater Healthcare Resource Utilization, Higher Medical Cost, and Poorer Prognosis in Patients with Lower-Risk Myelodysplastic Syndromes: A 28-Year Retrospective Observation Study Result

Myelodysplastic syndromes (MDS) encompass a heterogeneous group of myeloid neoplasms characterized by cytopenia due to inefficient hematopoiesis, with a considerable risk of progression to acute myeloid leukemia (AML) or early mortality. In lower-risk MDS (LR-MDS) patients, anemia and its associated complications have been the focus of clinical attention. A significant portion of these patients eventually develop red blood cell transfusion dependence (RBC TD), which has been linked to a poorer prognosis. Given the limited efficacy of available treatments in reducing RBC TD, most patients face increased risks of chronic anemia and various complications. Moreover, RBC TD is known to be associated with reduced overall survival (OS) and leukemia-free survival (LFS). This study aimed to assess the impact of RBC TD on the socioeconomic burden and clinical prognosis of LR-MDS patients, utilizing an electronic medical records (EMR) database spanning nearly 30 years of MDS patient data. This retrospective observational study examined adult MDS patients (≥ 18 years of age) treated at Samsung Medical Center in the Republic of Korea. Eligible patients were diagnosed with MDS between Sep 1, 1994, to Apr 1, 2022 (index period), with the observation period spanning from Sep 1, 1994, to Sep 1, 2022. Baseline assessments included estimation of revised international prognostic scoring system (IPSS-R) scores, categorizing patients with very low, low or intermediate risks as lower-risk MDS (LR-MDS). TD was defined as the occurrence of any 16-week period with RBC transfusion of ≥ 2 units per 8 weeks, without consecutive 56-day period without transfusion. Healthcare resource utilization (HCRU), medical costs, and prognosis indicators including overall survival (OS) and AML-free survival (AFS) were analyzed and compared between patients who experiencing TD and those not (non-TD). Out of 831 MDS patients, 349 (42.0%) were classified as lower-risk at baseline. Among LR-MDS patients, 29.5% (103/349) experienced TD while 23.3% (194/831) experienced TD in the entire study population. The median age at diagnosis of MDS was 63 (ranging from 18 to 89), and 65.3% (543/831) were male. In LR-MDS patients, there were no significant differences in either baseline age (61 vs 63, P=0.16) or gender (71.8% vs 61.0% males, P=0.053) between TD and non-TD groups. However, TD group exhibited significantly higher median erythropoietin (EPO) level than non-TD group (290.5U/L vs. 112U/L, P<0.001) among LR-MDS patients with available baseline serum EPO levels (76 TD and 178 non-TD). Among the 76 TD LR-MDS patients with baseline serum EPO levels, 51 (67.1%) exhibited > 200U/L, which is considered a clinical threshold for erythropoiesis-stimulating agent (ESA) treatment. As shown in Table 1, RBC TD was consistently associated with greater HCRU and higher medical costs, especially in LR-MDS patients. TD LR-MDS patients had higher rates of outpatient department and emergency room visits (15676 vs. 8303 visits and 587 vs. 328 visits per 1000 person-years [PY], respectively), and increased hospitalizations (709 vs. 456 per 1000 PY), with longer hospital stays (13343 vs. 8272 days per 1000 PY). Furthermore, TD LR-MDS patients required over four-times of packed RBC units than non-TD patients (31107 vs. 7073 units per 1000 PY). Consequently, TD LR-MDS patients incurred higher total medical cost than their non-TD counterpart (13.5 million vs. 6.1 million USD per 1000 PY). Median OS (58.4 months vs. 103.1 months) and AFS (52.7 months vs. 102.7 months) were both significantly shorter in the TD group compared to the non-TD group among LR-MDS patients, while the differences in OS and AFS did not reach the level of significance in the entire study population (Table 1 and Figure 1). Notably, median OS after the first documented RBC TD event was 33.8 months (95% confidence interval: 24.7-58.5 months) in LR-MDS patients. The findings of this study indicate that RBC TD can be a strong predictor of increased HCRU and medical costs, as well as decreased OS and AFS over a 20-year period following the initial diagnosis of LR-MDS. Given that a majority of LR-MDS patients experiencing TD may not be eligible for ESA treatment due to elevated EPO levels exceeding 200U/L, and the use of cytotoxic agents is not usually recommended for these patients, these results highlight a substantial unmet need for alternative treatment options to address RBC TD in LR-MDS patients.

First use of 18F-FDG PET in TEMPI syndrome: can it be used for treatment assessment? A case report

TEMPI syndrome (TEMPI) compounds telangiectasias and polycythemia with elevated erythropoietin levels, monoclonal gammopathy, perirenal fluid collections, and intrapulmonary shunt. Although the pathophysiology of this syndrome remains unclarified, prior research has been established that it is a plasma cell neoplasm, often containing less than 10% bone marrow plasma cells. 18F-FDG PET serves as a valuable instrument for initial staging and treatment monitoring in multiple myeloma management. Thus, 18F-FDG PET can be legitimately applied for TEMPI assessment. Here, we present the first 18F-FDG PET images for the initial evaluation and treatment monitoring of TEMPI in a 51-year-old woman, who exhibited polycythemia (EPO:5,448 mIU/ml) without JAK2 mutation, telangiectasias, monoclonal IgG lambda gammopathy (13.9) g/L and 7% dysmorphic plasma cells (CD38 + CD138+), occasionally clustered, in favor of tumoral plasmacytomas. The first PET scan exhibited hypermetabolic diffuse bone marrow, potentially related to polycythemia, accompanied by non-lytic bone hypermetabolic lesions in the femoral and humeral diaphysis, and ametabolic peri-renal fluid collections, brown fat, and pleural talcoma. Post-treatment 18F-FDG PET (Daratumumab Bortezomib Thalidomide Dexamethasone) revealed a completely reduced signal of bone lesions, suggesting a complete response, which was substantiated both clinically and biologically, with the concurrent disappearance of telangiectasia and the monoclonal component, and the normalization of the EPO level. In future, additional data will be required to confirm the added value of 18F-FDG PET with TEMPI. Nevertheless, 18F-FDG PET can be a preferred tool for the extension workup and therapeutic evaluation of TEMPI syndrome.

The utility of testing erythropoietin level in polycythemia diagnosis

ABSTRACTObjectives:Polycythemia vera (PV) is classically thought to be associated with low erythropoietin (EPO) levels. Here, we present a review of the utility of using EPO levels in diagnosing polycythemia.Methods:We conducted a systematic literature review of the Medline data through Pubmed and Google Scholar. We included the articles which described confirmed PV associated with elevated EPO level. Our search strategy included the following terms in Pubmed (((polycythemia vera[MeSH Terms]) OR (jak2 protein tyrosine kinase[MeSH Terms])) OR (Myeloproliferative Disorders[MeSH Terms])) AND (Erythropoietin[MeSH Terms]), and ‘polycythemia vera with erythropoietin’ in Google Scholar.Results:Our research yielded four cases of PV with elevated EPO levels. The most common symptom was a headache. Thrombotic phenomena happened in a single case in the form of Budd-Chiari syndrome. The mean Hb level was 20.2 gm/dl, and the EPO level was 213 mlU/mL.Discussion:Although PV is usually associated with low EPO levels, high levels do not exclude this diagnosis. Workup should include testing for JAK2 mutation and bone marrow biopsy in the presence of suggestive signs and symptoms. Novel biomarkers are also being proposed to aid in the diagnosis.Conclusion:Although elevated EPO levels suggest secondary causes of polycythemia, cases where elevated EPO levels were associated with an underlying PV are reported in the literature, and we have summarized a review of them. Workup for polycythemia should include JAK2 mutation testing if signs and symptoms suggest PV even if EPO is elevated.

Prognostic impact of elevated erythropoietin levels in patients with severe aortic stenosis receiving trans-catheter aortic valve implantation

BackgroundElevated endogenous erythropoietin (EPO) levels are associated with worse clinical outcomes in patients with heart failure (HF). The clinical implication of endogenous EPO levels in patients undergoing trans-catheter aortic valve implantation (TAVI) beyond other conventional risk factors remains unknown. MethodsConsecutive patients with EPO measurements who underwent TAVI for the treatment of their severe aortic stenosis at our institute between May 2015 and December 2020 were included. The association between the endogenous EPO levels and the primary outcome consisting of all-cause mortality and HF hospitalization was evaluated. ResultsA total of 263 patients (85.1 ± 5.1 years old, 74 men) were included and tertiled according to the baseline EPO levels. The high EPO group had more advanced anemia, renal impairment, and hypoalbuminemia than the other two tertiled groups (p < 0.05 for both). Patients with high EPO had a significantly higher cumulative incidence of the primary outcomes compared to the other two groups (p = 0.002) with an adjusted hazard ratio of 6.0 (95 % confidence interval 1.9–18.1) in its logarithmic value (p < 0.001). ConclusionsElevated baseline EPO levels were independently associated with mortality and morbidity following TAVI. The clinical implication of aggressive intervention on the elevated EPO levels in this cohort remains the next concern.

Polycythemia with elevated erythropoietin production in a patient with a urinary stone and unilateral hydronephrosis: a case report

BackgroundAbsolute polycythemia can be primary or secondary. Erythropoietin-producing diseases (for example, hypoxia) are the major cause of secondary polycythemia. There are reports of polycythemia secondary to hydronephrosis. However, to our knowledge, there is no report on polycythemia secondary to hydronephrosis due to a urinary stone. Herein, we present a case of polycythemia with an elevated erythropoietin level in a patient with a urinary stone and unilateral hydronephrosis.Case presentationA 57-year-old Japanese man presented with polycythemia and an elevated erythropoietin level. Erythropoietin accumulation was not due to erythropoietin secretion by a tumor as no obvious lesions were detected on contrast-enhanced computed tomography. Abdominal ultrasonography revealed a stone in the left urinary tract and renal hydronephrosis, and 2 weeks later, the patient underwent transurethral ureterolithotripsy without complications. Blood tests 2 weeks after transurethral ureterolithotripsy showed that the erythropoietin level had declined. Hemoglobin concentration decreased from 20.8 mg/dL before and immediately after transurethral ureterolithotripsy to 15.8 mg/dL 3 months after transurethral ureterolithotripsy. This case was diagnosed as erythropoietin elevation due to unilateral hydronephrosis with a urinary stone, resulting in polycythemia.ConclusionsHydronephrosis is a common disease but is not often associated with polycythemia. Further studies are required to elucidate the mechanism and implications of elevated erythropoietin production in hydronephrosis.

A case of AORPA in an adult presenting as secondary polycythemia

BackgroundPolycythemia refers to a condition of an abnormal increase in the red blood cell (RBC) mass. In men with a hematocrit > 60% or women with a hematocrit > 55%, there is 99% likelihood that the RBC mass is elevated. WHO uses a hemoglobin concentration > 16.5 g/dl in men or 16.0 g/dl in women to define an elevated RBC mass, in its criteria for the diagnosis of polycythemia vera. Congenital cardiac anomalies presenting as polycythemia are very rare in adults.Case presentationIn this case report, the patient presented with non-specific complaint and had an incidental finding of secondary polycythemia, ultimately diagnosed as secondary to AORPA. Our patient, a 43-year-old male, presented to our emergency with focal onset seizures without impairment of consciousness involving left upper and lower limbs. On routine investigations, the patient was found to have grade III clubbing and hemoglobin of 19.8 g/dl with elevated Erythropoietin levels. Further workup of secondary polycythemia revealed anomalous origin of the right pulmonary artery (AORPA) from the ascending aorta with tortuous patent ductus arteriosus (PDA) and pulmonary hypertension. Secondary polycythemia, in our patient, was due to right to left shunt causing Eisenmenger phenomenon, following pulmonary hypertension, making it one of the rarest presentation in adults.ConclusionIn the evaluation of secondary polycythemia, possibility of underlying asymptomatic cardiopulmonary shunt must be considered, though rare.

Exploring the Functional Role of an Alternative mRNA Transcript of the EPO Gene

Erythropoietin (EPO) is the key regulator of erythropoiesis and stimulates the proliferation and differentiation of erythroid progenitor cells into mature erythrocytes. In a previous study, we identified a frame-shift mutation in the EPO gene that causes familial erythrocytosis with elevated erythropoietin (EPO) levels by initiating excess production of EPO protein from an alternative EPO mRNA [1]. This alternative non-coding mRNA is transcribed from a promoter (P2) located in intron 1, whereas the full length mRNA that encodes the EPO protein is transcribed from the P1 promoter located upstream of exon 1. Large parts of intron 1 sequences, including the P2 transcript are conserved among different species (Figure 1A), suggesting that the P2 transcript could have an as yet unknown physiological function. To study the putative function of the P2 transcript, we performed experiments in a human cell line, Hep3B, which can produce EPO. We used the CRISPR/Cas9 system to generate mutant Hep3B clones with targeted deletions in intron 1 of the EPO gene. Close to the P2 transcription start site in intron 1, we identified three GATA-consensus motifs that might act as promoter elements for the P2 transcript. In the first round of CRISPR/Cas9 mutagenesis, we targeted the first two GATA elements and expected to knock down the expression of the P2 transcript. Targeted Hep3B cells were sorted and deposited as single cells to derive clones that were genotyped by next generation sequencing. We obtained 2 clones with homozygous deletions in EPO intron 1 and one clone that was compound heterozygous (Figure 1A). In these Hep3B clones we assessed EPO mRNA expression by qPCR and EPO protein levels in the supernatant by ELISA (Figure 1B). Unexpectedly, the P2 transcript was still detectable in all three clones carrying deletions in intron 1 that eliminated GATA boxes 1 and 2 (data not shown). EPO P1 mRNA expression and EPO protein levels in the supernatant were slightly reduced in most of the clones. These data indicate that the GATA box 1 and 2 motifs are not required for generating the P2 transcript. We have now performed a second round of CRISPR/Cas9 mutagenesis. In one series we targeted all three GATA-motifs through a 186 bp deletion, and in a second series we targeted a highly conserved region upstream of the GATA boxes by a 93 bp deletion (Figure 1A). We obtained 4 clones homozygous for the 93 bp deletion and the clones for the 186 bp deletion are currently being expanded. The analysis of EPO mRNA and protein in these additions CRISPR/Cas9 clones will be presented. Our study shows that the GATA motifs 1 and 2 are not required for function of the P2 promoter in intron 1 of the EPO gene. However, deleting these two motifs in two different cell clones decreased the P1 full-length EPO mRNA expression and EPO protein production in Hep3B cells by 70.2% and 37.2%, respectively. Additional clones with deletion of all three GATA motifs and a highly conserved region upstream of the GATA boxes will likely provide more insights into the physiological role of the P2 transcript.

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phlebotomy in Secondary Polycythemia: Does It Improve Clinical Symptoms? a Systematic Review

Introduction: Secondary polycythemia (SP) is the increased number of red blood cells in the body that occurs due to elevated erythropoietin levels usually secondary to chronic hypoxia. Treating the underlying cause is the mainstay of treatment of SP. Phlebotomy is rarely used in patients with SP, especially those with symptoms related to hyperviscosity. We present a systematic review assessing the effectiveness of phlebotomy in SP. Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, a comprehensive literature search was conducted on PubMed, Cochrane, and Clinical trials.gov using MeSH terms and keywords for "Polycythemia, Secondary " AND " Phlebotomy " in July 2022. We did not place any time constraints. Our search produced a total of 232 records, and duplicates were removed. After screening and removing irrelevant and review articles, we included a total of 17 original articles and case reports reporting phlebotomy as a treatment for SP in adult patients. The data were collected for baseline characteristics of the participants and efficacy and safety of the intervention. Results: A total of 86 patients from 17 articles were evaluated. (Table 1) The mean age of participants was 51 years and 89% (n=65/73) were males. Most common cause of SP was chronic obstructive pulmonary disease (COPD) 94.4%, followed by congenital cyanotic heart disease (3.4%), high altitude erythrocytosis (1.1%), and obesity hypoventilation syndrome (1.1%). Most common reported symptoms were dyspnea, confusion, headaches, exercise intolerance, and suppression of hypothalamic-pituitary-testicular axis. Two case reports also reported chorea secondary to SP while there was one case report each for hemi-retinal artery occlusion, angina attacks and portal hypertensive gastropathy. Majority of the studies had a goal hematocrit of <55%. There was subjective improvement in clinical symptoms in 85% (n=73) of the patients while 2.3% (n=2) felt worse after phlebotomy. None of the patients (9/9) with suppression of hypothalamic-pituitary-testicular axis had improvement in hormonal profile after phlebotomy. One patient developed hemiparesis, stiffness, loss of dexterity, chorea after phlebotomy with the symptoms resolving when hematocrit went back to baseline >62.5%. One patient died a week after phlebotomy due to respiratory issues. Conclusion: Phlebotomy results in clinical improvement in symptoms in patients with secondary polycythemia. Majority of the patients report either complete resolution of symptoms or at least subjective improvement. Goal hematocrit is usually <55%. Phlebotomy, although rarely used, may be considered a safe and effective treatment option for symptom management in SP, although its utility in objective improvement needs further prospective studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

POLYCYTHEMIAWITH HYDRONEPHROSIS:A RARE CASE

Secondary polycythaemia results from increased Erythropoietin production which acts on normal erythropoietic progenitors leading to erythrocytosis. This report is about a 64 years old hypertensive and anaemic male who presented with complaints of painful per rectal bleeding with dribbling micturition. On undergoing relevant investigations, he was found to be anaemic with low haemoglobin and haematocrit. Lateron, subsequent follow up visits he was incidentally detected to be polycythemic. On further investigations for the same, he got diagnosed with urinary bladder outlet obstruction, atonic bladder with ureteric narrowing and left hydronephrosis with elevated erythropoietin levels. On treatment, his Haemoglobin count, haematocrit and Erythropoietin levels came back to normal.Unexplained polycythaemia should be thoroughly investigated to rule out renal pathology.

Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice.

Dnm2fl/fl Pf4-Cre (Dnm2Plt–/– ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt–/– platelets. Additionally, Dnm2Plt–/– platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt–/– mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt–/– platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt–/– mice in the Mpl–/– background to generate Mpl–/–Dnm2Plt–/– mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl–/– Dnm2Plt–/– platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl–/– Dnm2Plt–/– mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl–/– Dnm2Plt–/– mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl–/– Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

PB2050: EVALUATION OF NON-CLONAL ERYTHROCYTOSIS: SINGLE CENTRE EXPERIENCE

Background: Secondary erythrocytosis has a broad differential diagnosis that includes hypoxic lung disease, cyanotic congenital heart disease, kidney disease, medications and erythropoietin-producing malignant disorders. Aetiology remains unknown in some patients, they have the so-called idiopathic erythrocytosis (IE). Among the latter, patients with inherited/congenital erythrocytosis can be found, especially if they have a positive family history. Aims: The aim of our study was to analyse the characteristics of non-clonal erythrocytosis in patients referred to our Haematology department in a 5-year period (2016 – 2021) and to compare the IE group to the group with known causes. Methods: We included all individuals who were Janus kinase 2 (JAK2) mutation negative and had erythrocytosis. Data was retrospectively collected with the help of the hospital database and statistically analysed with SPSS. Results: The study included 250 patients, 176 (70%) men and 74 (30%) women, with mean age 58.8 years (21 – 94 years). Their average haemoglobin values within a 1-year time period were 182.5 g/L for men and 169 g/L for women. Average haematocrit levels were 0.53 for men and 0.50 for women. Elevated erythropoietin (EPO) levels were found in 10.5% patients, 83.4% had normal and 6.1% decreased values. Symptoms were reported by 48% of the patients, mostly itchiness (16.4%), sweating (12%) and other (30%) such as tinnitus, myalgia, joint pain and snoring. The most probable reasons for secondary erythrocytosis were smoking/chronic obstructive pulmonary disease (COPD) (32%), IE (29%), dehydration (7%), nephrological aetiology (6%), obesity (4%), sleep apnoea (4%), asthma (4%), infection (4%), heart or pulmonary disease hypoxia (4%), possible hemochromatosis (3%), medications (2%) and high altitude/training (1%). Patients with IE had an average age of 57.2 years (24 – 83 years), 57 (78%) were men and 8 (11%) had a positive family history. EPO levels in this group were elevated in 7.8%, normal in 88.2% and decreased in 3.9%. Past thrombotic events were reported by 23.3% of individuals with IE and 20.3% of patients with known secondary cause. There was no statistical difference in age, gender, EPO, haemoglobin levels and past thrombotic events between the IE group and group with known causes. Patients with IE significantly differed from the group with known causes in haematocrit levels (p=0.0219) with lower levels in IE patients. Summary/Conclusion: Among 250 patients with non-clonal erythrocytosis the majority had acquired aetiology. The most common cause was hypoxia caused by smoking or COPD. Patients with IE significantly differed from the group with known causes in haematocrit levels, while there were no significant differences between groups in age, gender, EPO levels, haemoglobin levels and past thrombotic events.

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

TEMPI Syndrome: Update on Clinical Features, Management, and Pathogenesis.

TEMPI (telangiectasias, elevated erythropoietin level and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting) syndrome is a rare and newly defined multisystemic disease, which belongs to “monoclonal gammopathy of clinical significances”. Due to its rarity, the etiology, pathogenesis, and clinical features of this disease remain largely unknown. Owing to its hidden and diverse clinical manifestations, missed diagnosis and misdiagnosis are common. In recent years, as more patients (including three fatal cases) were identified, some special clinical manifestations other than the typical pentad of TEMPI syndrome have been reported. Meanwhile, several studies attempting to identify the pathogenesis of TEMPI syndrome were conducted. In this review, we summarize the reported clinical characteristics of TEMPI syndrome and discuss the current and potential treatment options for patients with TEMPI syndrome, including those with relapsed/refractory disease. Furthermore, we provide an overview of current knowledge on the pathophysiology of TEMPI syndrome.

Activation of Sympathetic Nervous System Contributes to Erthroprotein Gene Upregulation by Hypobaric Hypoxia

Hypobaric hypoxia (HH) experienced at high altitude initiates a series of physiological adaptations to maintain homeostasis. One such adaptation is the activation of the Epo gene which encodes the glycoprotein hormone erythropoietin (EPO). Increased circulating EPO levels defend against HH by facilitating blood O2 transport through increasing red blood cell production. HH also increases blood pressure (BP) through activation of the sympathetic nervous system and the ensuing elevation of circulating catecholamine levels. The present study examined the role of sympathetic nerve activation in HH‐evoked Epo gene activation and elevated circulating levels of EPO. Studies were performed on adult, male wild‐type (WT) mice treated with 18 hours of HH (0.4 atmospheres), simulating low O2 condition experienced at high altitude. Our results showed increased renal Epo gene abundance and elevated plasma EPO levels. Remarkably, these responses were either markedly attenuated or absent in mice with selective ablation of adrenal medulla as well as after treatment with propranolol, a non‐selective beta‐adrenergic receptor blocker. Moreover, infusion of epinephrine alone activated renal Epo gene and elevated plasma EPO levels. These results suggest that sympathetic nerve activation and the ensuing increase in plasma epinephrine contribute to Epo gene activation by HH.

Ineffective Erythropoietin Response to Anemia in Sepsis.

Background: To determine whether degree of anemia at sepsis onset is predictive of inflammatory cytokine trajectory, erythropoietin response, and recovery. Patients and Methods: Critically ill patients with sepsis were stratified into three groups based on initial hemoglobin (Hgb): Hgb <8 g/dL (severe); 8-10 g/dL (moderate); and >10 g/dL (mild). Granulocyte colony stimulating factor (G-CSF), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, C-reactive protein (CRP), erythropoietin (EPO), and Zubrod scores were measured serially. Results: Thirty-four percent had severe anemia (Hgb, 7.2 ± 0.7g/dL), 35% had moderate anemia (Hgb, 9.1 ± 0.6g/dL), and 31% had mild anemia (Hgb, 11.3 ± 1.1g/dL). All groups experienced persistently high EPO levels without resolution of anemia. IFN-γ and CRP was persistently elevated in all groups. At three, six, and 12 months, the severe anemia group had higher Zubrod scores. Conclusions: Degree of anemia at sepsis onset was not associated with a difference in proinflammatory cytokine trajectory but was associated with a worse functional outcome. Despite initial elevated EPO levels, it did not correlate with resolution of anemia.