Sickle Beta-thalassemia Research Articles

Manual RBC exchange transfusion in a patient of sickle-beta thalassemia syndrome presenting in crisis: a case report

Sickle cell disease and beta thalassemia are caused by abnormal haemoglobin (Hb) derived from mutation of the HBB gene encoding beta-globin. Compound heterozygous status for both mutations results in HbS/beta thalassemia (Sickle- beta thalassemia). Vaso-occlusive phenomena and haemolysis are the clinical hallmarks and major causes of mortality. Here we report a case of successful reduction of HbS level by manual RBC exchange transfusion. Capillary zone electrophoresis showed the case to be Sickle-beta thalassemia. A total of 3 units of 450ml whole blood units were used for manual exchange transfusion done in 2 sittings on consecutive days. Preexchange HbS level was 80.9% of total Hb. HbS level after 24 hours of the second procedure was 44%. In the absence of facility to conduct automated RBC exchange by a cell separator, to reduce HbS in patients presenting with acute complications of SCD and in patients with Vaso-occlusive Crisis, previous stroke, manual RBC exchange can provide a better relief.

Vitamin D supplementation for sickle cell disease.

Vitamin D supplementation for sickle cell disease.

Moyamoya Syndrome with Recurrent Intraparenchymal Hemorrhage from Hemoglobin C Disease

Moyamoya syndrome is an acquired idiopathic non-atherosclerotic vasculopathy that involves the terminal internal carotid artery and its branches. It is associated with numerous medical conditions such as hematologic abnormalities, autoimmune diseases, brain radiation, and tumors. These medical diseases cause chronic endothelial damage to cerebral vasculature resulting in both friability and stenosis of vessels, presenting as cerebral hemorrhage or infarction. There is an established link between moyamoya syndrome and hematologic diseases such as sickle cell and beta thalassemia. A few case reports of rare hemoglobin opathies have caused moyamoya syndrome; however, there are no reports of moyamoya due to hemoglobin C disease. This case report presents a patient with known hemoglobin C disease who presented with her second intracranial hemorrhage, found to have moyamoya syndrome.

New Concepts in Genome Regulation

Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation

INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT.METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student’s t-test for 2 samples at each time point assuming unequal variances.RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD.CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Growth Differentiation Factor-15 Levels Are Increased in Patients with Compound Heterozygous Sickle Cell and Beta-Thalassemia, Correlate with Hepcidin-25/Ferritin Molar Ratio and with Markers of Hemolysis, Endothelial Dysfunction and Angiogenesis

Background: The clinical manifestations of Sickle Cell Disease (SCD) include episodes of vascular occlusion, chronic hemolytic anemia and frequent infections. SCD is also characterized by the presence of chronic inflammation manifested by leukocytosis and monocytosis and increased circulating levels of pro-inflammatory cytokines and chemokines. Growth Differentiation Factor-15 (GDF-15), also known as macrophage inhibitory cytokine 1 (MIC-1) or non-steroidal anti-inflammatory drug-activated gene (NAG-1) is a member of the transforming growth factor- superfamily. Expression of the GDF-15 gene in cardiomyocytes, vascular smooth muscle cells, and endothelial cells is strongly upregulated in response to oxidative stress, inflammation and tissue injury. Recently, GDF-15 and Twisted Gastrulation Protein (TWSG1) have been proposed to be erythroblast-derived factors, although not erythroblast specific, that mediate Hepcidin-25 suppression under conditions of increased erythropoietic activity. High levels of GDF15 associate with ineffective erythropoiesis and may reflect a certain type of bone marrow stress or erythroblast apoptosis. However, the role of GDF15 in Hepcidin-25 regulation under physiologic conditions and in other disorders is unclear and has been debated.Aims: The aim of this study was to evaluate the GDF-15 levels in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) and to explore possible associations with disease features, such as Hepcidin-25 production, hemolysis, inflammation, endothelial dysfunction and angiogenesis.Methods: Seventy-five adult Caucasian patients with HbS/βthal were included in the study, while 20 healthy individuals served as controls. Patients with HbS/βthal divided in two groups: group A included 36 patients under hydroxycarbamide (HC+) treatment and group B included 39 patients without hydroxycarbamide (HC-) treatment. Along with hematology and blood chemistry parameters determination, measurements of circulating levels of GDF-15, hepcidin-25, hs-CRP, vWF-antigen, hs-TnT and Placental Growth Factor (PlGF) were performed in both patients with HbS/βthaland controls using immunoenzymatic techniques.Results: The main results of the study included: GDF-15 levels were elevated in patients with HbS/βthal compared to controls ((1,980.7±159.8 vs 665.4±50.9 pg/mL, p<0.001). Regarding hydroxycarbamide treatment, GDF-15 levels were elevated in (HC+) patients compared to (HC-) patients (p=0.002), or 30/36 vs 21/39 patients had elevated GDF-15 levels (p=0.002). In contrast, Hepcidin-25 levels were significantly lower in patients with HbS/βthal compared to controls (57.2±12.3 vs 67.3±9.1 ng/mL, p<0.01). The reduction of Hepcidin-25 levels was more pronounced in (HC-) patients compared to (HC+) patients (p=0.005). In addition, a markedly low Hepcidin-25/Ferritin molar ratio was obsereved in patients with HbS/βthal compared to controls (p<0.001). Whilst, no direct correlation was found between GDF-15 and hepcidin-25 levels, a significant negative correlation between GDF-15 levels and Hepcidin-25/Ferritin molar ratio was detected in patients with HbS/βthal(p=0.002). GDF-15 levels also correlated significantly with markers of erythropoiesis, such as Hb, HbF, ferritin and reticulocytes (p<0.05), with markers of hemolysis, such as LDH and uric acid (p<0.01), and with markers of endothelial dysfunction and angiogenesis such as vWF-antigen and PlGF (p<0.01). Surprisingly, no correlation was found between GDF-15 and hs-CRP levels.Conclusions: These findings demonstrate a multifactorial role of GDF-15 in patients with HbS/βthal as it correlates with erythropoiesis, hemolysis, endothelial dysfunction and angiogenesis. However, we found no direct specific function, despite the negative correlation with Hepcidin-25/Ferritin molar ratio. Interestingly, the higher GDF-15 levels measured in patients treated with hydroxycarbamide may reflect possible drug induced sub-clinical cardiotoxicity, although this has not been described to-date. To this end, our knowledge is restricted only to doxorubicin-induced cardiotoxicity, where GDF-15 up-regulation seems to be more sensitive than that of hs-TnT, LDH and NT-proBNP. Further studies will reveal the role of GDF-15 in the biology of HbS/βthal. DisclosuresOstland:Intrinsic LifeScienc s: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Terpos:Genesis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria.

Cholelithiasis and its complications in sickle cell disease in a university hospital.

IntroductionThe clinical manifestations of sickle cell disease are related to the polymerization of hemoglobin S. The chronic hemolysis caused by this condition often causes the formation of gallstones that can migrate and block the common bile duct leading to acute abdomen.ObjectiveThis study aimed to evaluate the profile of patients with sickle cell disease and cholelithiasis.MethodsPatients with sickle cell disease were separated into groups according to the presence or absence of cholelithiasis. Socioepidemiological and clinical characteristics, such as gender, age, use of hydroxyurea and the presence of other hemoglobinopathies were researched in the medical records of patients.ResultsA hundred and seven patients with sickle cell anemia were treated at the institution. Of these, 27 (25.2%) had cholelithiasis. The presence of cholelithiasis was higher in the 11–29 age group than in younger than 11 years and over 29 years. No association was found for the presence of cholelithiasis with gender, use of hydroxyurea or type of hemoglobinopathy (hemoglobin SS, hemoglobin SC or sickle beta-thalassemia). Sixteen of the patients had to be submitted to cholecystectomy with 14 of the surgeries being performed by laparoscopy. Complications were observed in three patients and one patient died for reasons unrelated to the surgery.ConclusionA quarter of patients with sickle cell disease had gallstones, more commonly in the 11- to 29-year age range. Patients should be monitored from childhood to prevent cholelithiasis with preoperative, intra-operative and postoperative care being crucial to reduce the risk of complications in these patients.

Clinicohematological profile of hemolytic anaemia in tertiary care hospital in rural Andhra Pradesh

Introduction: Hemolytic anaemias are a group of disorders that cause significant morbidity in children. Method: A cross sectional study was conducted at NRI Medical College for a period of 1 year. All cases of newly diagnosed and old cases of hemolytic anaemia on follow up were included. Results: The study showed beta thalassemia as the most common hemolytic anaemia; followed by malaria, sickle beta thalassemia, thalassemia intermedia, beta thalassemia minor, Sickle cell disease, sickle cell trait, auto immune hemolytic anaemia, and hereditary spherocytosis. The mean hemoglobin at presentation was 5.39 gm/dl. Anthropometric measurements in 32 cases of congenital hemolytic anaemias revealed height < 3rd centile in 9 cases. Weight less than 3rd centile was seen in 11 cases. 13 children had hemolytic facies. Massive splenomegaly causing discomfort, gall stones, heart failure were seen in 2 cases each. In thalassemia major, 10 cases required frequent transfusions, [10-12 per year]. 9 came for less frequent transfusions [6 per year]. Sickle thalassemia, thalassemia intermedia, required one transfusion every 1-2 years. Occasional transfusions were given in sickle cell anaemia, hereditary spherocytosis. Serum ferritin levels varied between 220-1427. Conclusion: Hemoglobin electrophoresis remains the main investigation of choice in diagnosis of hemolytic anaemia. Thalassemia major is the most severe among other hemolytic anaemias encountered in this series. The study emphasizes the need to improve awareness regarding hemoglobinopathies among population, prenatal screening, blood transfusion policies, chelation policies to prevent complications in transfusion dependent patients.

Sickle cell disease in Madhya Pradesh, Central India: A comparison of clinical profile of sickle cell homozygote vs. sickle-beta thalassaemia individuals

Background and objectives: The clinical manifestation in sickle cell disease (SCD) patients varies from one individual to another due to factors like the presence of alpha-thalassaemia mutation, foetal haemoglobin, and β-globin gene haplotype. The present study enumerates the clinical profile of sickle cell anaemia patients from Central India.Methods: Seven hundred seventy-six SCD patients from Jabalpur and surrounding districts (Madhya Pradesh) in central India were registered with the sickle cell clinic of NIRTH, Jabalpur. The present study reveals recorded signs and symptoms of genetically confirmed sickle cell anaemia (404) and sickle beta thalassaemia (92) patients.Results: Majority of the patients were from scheduled caste communities (47.9%) and Gond tribal community (13.8%). Splenomegaly was the most common clinical manifestation observed (71.4%). Overall, 63.5% patients had a history of blood transfusion. The most frequent signs and symptoms observed were Pallor, Icterus, Joint pain, Fever, and Fatigue. Majority of the patients revealed onset of disease prior to attaining the age of 3 years (sickle cell anaemia 44.3% and sickle beta thalassaemia 35.9%). Mean haemoglobin levels among SCA individuals were marginally higher than SBT patients. On the other hand, mean foetal haemoglobin levels among SBT individuals showed the reverse trend. Notably, the present study reports the first incidence of priapism recorded in Central India.Conclusions: The study revealed a high prevalence of SCD among scheduled caste, backward caste, and tribal communities. Dissemination of study findings, screening, pre-marriage counselling, and pre-natal diagnosis are fundamental to preventing or lowering of birth of sickle cell anaemia children in the affected populations.

Prevalence of Hemoglobinopathies and Thalassemia Carriers in Women of Reproductive Age Group Especially the Prospective Mothers: A Single Center Study at West Bengal

Background: Hemoglobinopathies are group of diseases characterized by abnormalities both quantitative and qualitative in the production of hemoglobin. In India, major concerned hemoglobinopathic disorders are sickle cell anemia and beta-thalassemia. The aim of the study was to prevent the birth of first thalassemic child by screening pregnant women before 17 weeks of gestation and offering prenatal diagnosis if needed and to increase the awareness among general population about prevention of birth of genetically abnormal child. Methods: Blood samples were tested for complete blood count and hemoglobin electrophoresis. Results: During the study period, a total of 1,083 women were screened; among them 50 women who carried abnormal pattern (beta-thalassemia) were detected. The prevalence of carriers was 4.61%. Apart from I²-thalassemia and hemoglobin E carrier (1.10%), two additional variants were encountered. Conclusion: The data regarding prevalence and distribution can be useful in prevention and management of various hemoglobinopathies. J Hematol. 2016;5(3):99-102 doi: http://dx.doi.org/10.14740/jh297w Â

Study on the Efficacy of a JAK Inhibitor Pharmacological Agent As Inducer of Fetal Hemoglobin Production in Cultured Erythroid Precursors from Sickle Cell and Beta-Thalassemia Patients

Phenotypic improvement of hemoglobinopathies such as sickle cell disease and beta-thalassemia (beta-thal) has been shown in patients with high levels of fetal hemoglobin (HbF). In sickle cell disease (SCD) the beneficial effects of HbF are due to the inhibition of HbS polymerization and to the dilution of HbS determining the reduction of sickling and vascular occlusion. Moreover, in beta-thal, high levels of gamma-chains combined with the redundant alpha-chains, lead to a reduction of dyserythropoiesis and of the requirement for blood transfusions. The only drug approved for the treatment of adult patients with SCD and that has been entered in clinical practice of patients affected by beta-thal is hydroxyurea (HU); however there is a great variability in the responses of patients to HU, in fact some patients are good responder, while others exhibit little or no change in HbF levels after HU treatment; moreover a decrease in the efficacy during long term treatment was observed. Other pharmacological compounds, including 5-azacytidine and thalidomide have been shown to increase HbF production. Due to concerns about the safety of this agents, their use was limited to severe cases for whom conventional therapy was unfeasible. For this reason the search of new inducers of HbF production is important. Ruxolitinib is a JAK inhibitor and decreases the phosphorilation of STAT (Signal transducers and activators of transcription) family proteins, in particular STAT5 and STAT3. Phosphorylation of STAT5 is essential for basal erythropoiesis and for its acceleration during stress erythropoiesis. STAT3 plays an essential role in regulating gene expression of several genes involved in cell growth and apoptosis, in particular it was demonstrated to inhibit gamma-globin gene expression. The decrease of STAT3 phoshorilation could decrease the inhibition of gamma-globin gene expression; for this reason we considered ruxolitinib a candidate as inducer of HbF production.In our laboratory an ex vivo system was developed predictive of the in vivo response to hydroxyurea treatment by using liquid erythroid cultures, an in vitro culture system that recapitulates the process of human erythropoiesis.To evaluate the efficacy of ruxolitinib in increasing gamma-globin gene expression we carried out a study in vitro using liquid erythroid cultures. In this study we developed and exposed to ruxolitinib liquid erythroid precursors from 4 SCD and 17 beta-thal intermedia (beta-TI) patients. The use of quantitative Real-Time-polymerase chain reaction allowed us to determine the increase in gamma-globin mRNA expression in human erythroid cells treated with ruxolitinib compared to untreated cells. The results are summarized in Table 1 and showed that ruxolitinib at 200nM is able to determine a significant increase of gamma-globin gene expression (3.4±0.1)compared to HU (2.0± 0.2).In conclusion our study suggests that ruxolitinib could be considered an inducer of HbF and could be used in vivo for the treatment of hemoglobinopathies, particularly in patients who do not respond to HU therapy or who show a decreased response after long-term treatment.Table 1Fold increase of Gamma-globin gene expression in presence of Ruxolitinib in erythroid cultured cells.PatientSexGenotypegamma-globin mRNA fold increasein the presence of ruxolitinib#1Mb039/aaa+1#2Fb039/aaa+1.65#3Fb039/b039+1.9#4Fb039/IVS1,110+1.5#5MIVS1,1/aaa+2.5#6MIVS1,110/IVS1,1+9.2#7Mb039/bs+6#8Fbs/b039+1.6#9Fb039/IVS1,6+1.7#10MIVS1,6/frcd6+3#11MIVS1,6/bs+2.5#12MIVS1,6/frcd6+8#13FIVS1,6/b039+9#14MIVS1,1/b039+2.2#15Mdb/IVS1,110+8#16Fdb/IVS1,110+1.8#17FIVS2,1/aaa+3.9#18Mb039/-101+1.4#19MIVS1,6/b039+1#20Mbs/IVS1,110+1.4#21MIVS1,6/IVS1,6+1.9 DisclosuresNo relevant conflicts of interest to declare.

ASH1L: A Novel Beta-Globin Gene Regulator in Humans?

BACKGROUNDWe have previously described a unique English family with beta-thalassemia trait which was not linked to the β-globin gene locus (Thein, Wood, Wickramasinghe, & Galvin, 1993). This suggested involvement of a trans-acting factor required for full activation of the β-globin gene locus. Such a factor is likely to be a modulator of disease severity in sickle cell disease and beta-thalassemia which could provide insights for novel therapeutic targets in the beta-globinopathies. RESULTSWe applied whole genome scan (WGS) to 2 affected and 2 unaffected subjects of the English family. The familial segregation suggested a dominant transmission mode; WGS identified 15 genes as potentially causative to the phenotype, with four genes located on chromosome 1, four on chromosome 3, three on chromosome 20, and one on chromosome 6, chromosome 8, chromosome 10 and chromosome 19.Sanger sequence analysis on 23 family members spanning three generations, including the 4 individuals that were subjected to WGS, revealed that the 15 variants were not artefacts of the WGS and that all variants were present in the 2 affected but not in the 2 unaffected individuals. Furthermore we found that 4 of the 15 variants were consistently and uniquely present in all 9 affected but absent in the unaffected family members. We performed association linkage analysis using the 15 markers in the whole family, and confirmed that the phenotype was closely linked to the 4 genes that were inherited as a block spanning the centromere on chromosome 1. We concluded that the region containing these 4 genes most likely harbours the mutation causing the phenotype. Among the 4 candidate genes, 2 were not expressed in erythroid cells, but the other 2 - one encoding an integral membrane protein (LRIG2) and the other one encoding a methyl transferase (ASH1L)- were expressed in erythroid cells.Functional studies for these two genes were performed on primary human erythroid progenitor cells (hEPCs) in culture. In following the kinetics of the 2 candidates during differentiation of hEPCs, we observed that the expression of ASH1L increased at later stages of differentiation, where LRIG2 displayed a less dramatic change of expression. Moreover, ASH1L has previously been found to occupy transcribed chromatin domains and methylate histone tails in vitro (Gregory et al., 2007; Miyazaki et al., 2013; Tanaka et al., 2011). In undifferentiated mouse embryonic stem cells there is no ASH1L recruitment to the β-globin gene locus but upon erythroid differentiation the protein is recruited to the transcribed portion of the gene (Gregory et al., 2007). This suggests an involvement of ASH1L in beta-globin activation in erythroid lineages. We used shRNA lentiviruses to generate knock-down (KD) of ASH1L and obtained over 65-75% KD of the gene. In hEPCs treated with the shRNA lentivirus, we observed a slight decrease in beta-globin expression compared to the control hEPCs. The α/β-globin and α/(β+γ) globin ratios were also affected by the gene knock-down. ChIP-qPCR was performed to assess the enrichment of the ASH1L protein at β-globin promoter region. The results show that enrichment of ASH1L at the β-globin promoter correlates with the β-globin expression in cells. CONCLUSIONSThese results suggest that ASH1L is responsible for the phenotype observed in the English family and act in differentiating hEPCs as a trans-acting factor for full beta-globin gene activation. Further ChIP analysis to assess the binding of the protein to the beta-globin locus during hEPCs differentiation and under KD condition will provide us with a better understanding of the influence of the methyl transferase on β-globin activation. The replication of the patient mutation in vitro using CRISPR technology will provide the model to study fully the impact of the mutation on the phenotype described in the original paper. These findings could provide new insights for therapeutic targets for beta-globinopathies. DisclosuresNo relevant conflicts of interest to declare.

Gene Editing with Crispr-Cas9 for Treating Beta-Hemoglobinopathies

CRISPR/Cas9 systems provide a powerful tool for precision genome editing due to their highly efficient targeting of specific DNA sequences in a genome. We have designed CRISPR single-guide RNAs (sgRNAs) containing the guide and tracr RNAs to selectively edit the human globin locus for potential therapeutic benefit in treating sickle cell anemia and beta-thalassemia. Multiple sgRNAs have been identified and tested across key sites in the human globin locus, and the gene modification frequencies quantified in 293T, K562 and CD34+ hematopoietic progenitor cells. The nuclease cutting efficiency was determined by the rate of insertions, deletions or mutations (indels) from NHEJ-directed repair. The indel rates for these sgRNAs ranged from <1% to ~70%. We found that there was only a weak correlation between indel rates and deletion frequencies, and the gene modification efficiency varied between cell lines/cell types. Although bioinformatics tools are available for sgRNA design, determining the optimal sgRNAs is still largely empiric and system-dependent, thus better bioinformatics tools are needed. Our results demonstrate that, with careful design and optimization of sgRNAs, efficient genome editing can be achieved with high specificity, which promises effective treatment of beta-hemoglobinopathies. DisclosuresPorteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Cornu:CRISPR Therapeutics: Research Funding. Miccio:CRISPR Therapeutics: Research Funding. Cradick:CRISPR Therapeutics: Employment. Cathomen:CRISPR Therapeutics: Consultancy, Research Funding. Lundberg:CRISPR Therapeutics: Employment. Mavilio:CRISPR Therapeutics: Consultancy, Research Funding.

Modified IPS Cells for Hemoglobinopathies

We have recently used CRISPR-Cas to correct the HBB gene in induced Pluripotent Stem Cells (iPSC) derived from patients with Sickle Cell Disease (SCD) and to correct the JAK3 gene in iPSC derived from patients with Severe Combine Immune Deficiency (SCID). Off-target mutations were minimized, if not eliminated, by use of paired guide RNAs and the Cas9 nickase. When erythroid progenitors (EP) produced from corrected SCD iPSC are transplanted into NSG mice, a complete switch from gamma- to beta-globin gene expression occurs within 24 hours and high levels of beta mRNA are synthesized; the betaA:betaS ratio is 60:40. Hematopoietic progenitors produced from corrected SCID iPSC can be differentiated into T cell populations that express a full repertoire of T Cell Receptors (TCRs). These results suggest that CRISPR-Cas enhanced gene replacement may provide safe and effective therapies for many erythroid and lymphoid disorders. In an alternative approach, we electroporated a preformed, biochemical complex composed of a guide RNA (gRNA), a modified recombinant Cas9 (mrCas9) and a single stranded oligodeoxnucleotide (ssODN) into sickle iPSC or primary sickle CD34+ hematopoietic progenitors. Sixty-five percent of sickle iPSC colonies contained at least one corrected allele. When homozygous corrected colonies were differentiated into erythroid progenitors and transplanted into NSG mice, human erythroid cells expressed 100% HbA. When primary sickle CD34+ hematopoietic progenitors were electroporated with the gRNA/mrCas9/ssODN complex and differentiated into erythroid cells in vitro, HbA was expressed at 30-40%. These results suggest that primary bone marrow CD34+ cells that are isolated from patients with sickle cell disease, beta-thalassemia, SCID, DBA and other hematopoietic disorders may be electroporated with specific gRNA/mrCas9/ssODN complexes and transplanted into patients to rapidly, safely and effectively treat these debilitating disorders. DisclosuresNo relevant conflicts of interest to declare.

Increased Von Willebrand Factor and High Circulating Placental Growth Factor Correlate with Inflammation and Iron Overload in Patients with Compound Heterozygous Sickle Cell and Beta-Thalassemia

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by pathological polymerization of hemoglobin, increased red cell rigidity, poor microvascular blood flow, with consequent tissue ischemia and infarction. Thus, hemolytic anemia, vaso-occlusion and vasculopathy are the hallmarks of its clinical presentation. Several other factors contribute to the clinical variability, which is present in SCD, including leukocyte dysfunction, platelet interactions with endothelial cells, pro-inflammatory cytokines, oxidative stress, reduced nitric oxide (NO) availability and hemostatic activation. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor superfamily, which plays an important role in both inflammation and neoangiogenesis. Recently it has been reported that markers of iron overload are associated with high plasma level of PlGF and early mortality [Wang et al, Blood 2014]. Furthermore, in SCD patients, hemolysis can lead to a prothrombotic state by increasing the activity of von Willebrand factor (vWF), a multimeric plasma glycoprotein secreted by the endothelium [Chen et al, Blood 2011]. ADAMTS-13 is a member of the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats) family that cleaves vWF. Recent observations suggest that SCD patients suffer from an acquired ADAMTS-13 deficiency primarily because Hb competitively binds and blocks the proteolysis of vWF, leading to the accumulation of ultra-large VWF multimers in circulation and on endothelium.The aim of this study was to evaluate inflammation, endothelial dysfunction and angiogenesis in patients with compound heterozygous SCD and beta-thalassemia (HbS/βthal) and explore possible association with iron overload and other disease features.Eighty-nine adult caucasian patients with HbS/bthal were included in the study, while 20 apparently healthy individuals served as controls. Patients with HbS/βthal divided in two groups: group A included 49 patients under hydroxycarbamide (HC+) treatment and group B included 40 patients without hydroxycarbamide (HC-) treatment. Along with hematology and blood chemistry parameters determination, measurements of circulating high-sensitivity C-reactive protein (hs-CRP), vWF, D-Dimers, ADAMTS-13, hs-Troponin-T (hs-TnT) and PlGF were measured in patients with HbS/βthal and controls using immunoenzymatic techniques.Levels of hs-CRP, vWF and PlGF were elevated in patients with HbS/βthal compared to controls (6.9±5.3 vs. 0.4±0.4 mg/L, 170.1±78.3 vs. 85.3±22.1 IU/dL and 20.2±7.6 vs, 15.3±2.4 pg/mL, respectively, p<0.001), while ADAMTS-13 levels were decreased in patients with HbS/βthal compared to controls (965.2±244.0 vs. 1144.2±187.0 pg/mL, p<0.001). No significant differences were found for the above parameters between patients of groups A and B (p=0.211, p=0.710, p=0.589 and p=0.384, respectively. Hs-CRP correlated positively with vWF, PlGF and ADAMTS-13 (r=0.360, p<0.001; r=0.300, p=0.005 and r=0.382, p=0.002, respectively). PlGF levels in patients with HbS/βthal correlated positively with markers of hemolysis such as reticulocyte counts, LDH and with uric acid levels (r=0.674, p<0.001; r=0.425, p<0.001 and r=0.357, p<0.001, respectively). Ferritin levels correlated positively with hs-CRP, vWF, PlGF and D-Dimers (r=0.360, p<0.001; r=0.317, p=0.004; r=0.354, p<0.001; and r=0.344, p=0.002, respectively) as well as with markers of hemolysis (p<0.001). Furthermore, only 6 patients had slightly increased hs-TnT levels and almost all patients had pathologic D-Dimers levels.Our findings demonstrate that patients with HbS/βthal have a significant degree of endothelial dysfunction as assessed by increased vWF. The increased levels of the D-Dimers in almost all patients indicate the activation of coagulation and fibrinolytic systems even in the steady state of the disease. Iron overload and inflammation along with reduced circulating ADAMTS-13 contribute, at least partially, to the increased levels of vWF. Similarly, inflammation and iron overload enhance the production of angiogenesis markers, such as PlGF suggesting a possible pathogenetic role for iron load in SCD biology. Further studies will reveal if iron chelation is able to restore some of the above phenomena and improve endothelial dysfunction and inflammation in this entity. DisclosuresNo relevant conflicts of interest to declare.

Glycoprotein B genotyping of human cytomegalovirus strains isolated from Brazilian patients with sickle cell disease and beta-thalassemia major.

The role of the human cytomegalovirus (HCMV) infection in individuals with hemoglobinopathies is unclear. Our objective was to examine the molecular and genotypic characteristics of HCMV in patients with sickle cell disease, beta-thalassemia major, and volunteer blood donors by viral load quantitation, glycoprotein B (gB) genotyping, and phylogenetic analysis. The patients with sickle cell disease demonstrated the highest HCMV DNA prevalence (13.8%), followed by the patients with beta-thalassemia major (7.6%), and the blood donors (3%). The infection was characterized by a low mean viral load (3.8×10(3) copies/mL), but infections with higher copy numbers were also observed. Genotype gB2 was detected in the majority of cases (90.9%), followed by genotype gB1 (9.1%). No gB3/gB4 genotype was detected. No statistical significance was observed between HCMV DNAemia/gB genotype and hematological alterations or severity of the disease. The high number of sickle cell disease patients with HCMV DNAemia could be due to their partial immune dysfunction (multiple transfusions, spleen dysfunction, hydroxyurea treatment). The extensive HCMV gB2 prevalence in patients with hemoglobinopathies is probably due to HCMV epidemiologic characteristics in the examined region, and can be important during the clinical management of these patients.

Doppler assessment of renal hemodynamic alterations in homozygous sickle cell disease and sickle Beta-thalassemia.

We evaluated the renal vascular indices in children and adolescents with sickle cell disease (SCD) using Doppler ultrasonography. We also assessed the renal hemodynamics alterations in patients with homozygous SCD and sickle beta-thalassemia (sickle β-thalassemia). We studied 75 patients (age range = 3-20 years; M = 9.95 ± 4.15) with SCD: 42 patients suffering from homozygous SCD and 33 patients diagnosed with sickle β-thalassemia. Thirty, age- and sex-matched, normal subjects were also included as a control group. Both patients and control groups had Doppler assessment of pulsatility (PI) and resistivity (RI) indices of main renal, segmental, interlobar, and arcuate arteries. Both PIs and RIs were significantly higher in SCD patients, compared with the control group. Among patients, PIs and RIs in the main renal, segmental, interlobar, and arcuate arteries were significantly higher in patients with homozygous SCD as compared with those with sickle β-thalassemia (p values <0.01, <0.001, <0.001, and <0.001 for PIs and <0.001, <0.001, <0.001, and <0.01 for RIs, respectively). We concluded that renal vascular resistance is raised in children and adolescents with SCD. This is more pronounced in patients with homozygous SCD as compared with those with sickle β-thalassemia.

Steroid Responsive T-Cell Mediated Pure Red Cell Aplasia Following Allogeneic Hematopoietic Stem Cell Transplantation

Pure red cell aplasia (PRCA) is an unusual complication following allogeneic hematopoietic stem cell transplantation (HSCT), the true incidence of which is not known. Almost all cases reported in the literature describe major ABO-incompatibility between the recipient and the donor as the major risk factor for this complication. Delayed recovery of reticulocyte counts and hypoplasia of erythroblasts in the marrow is attributed to incompatible hemagglutinins in recipients. There are reports of successful treatment of PRCA using different strategies include rapid tapering of calcineurin inhibitors, corticosteroids, donor lymphocyte infusion, rituximab, and/or plasma exchange. Additional causes of PRCA following allogeneic HSCT include parvovirus B19 infection and graft-versus-host disease (GVHD). The pathogenesis of PRCA in parvovirus infection is thought to be viral-mediated suppression of erythroid precursors in the bone marrow and this is due to the tropism of this virus for erythroid progenitor cells. Nonmyeloablative and reduced intensity conditioning regimens have been reported to have an increased risk of PRCA due to persistence of recipient lymphocytes. Here we report two cases of PRCA following allogeneic HSCT from major ABO mismatched donors. Both cases were correlated with the presence of a T-cell clone in the peripheral blood and bone marrow, and there was complete resolution of anemia with a short course of steroid treatment, as well as disappearance of the T-cell clone. Patient # 1 – A 24 year old female of Mediterranean descent underwent an HLA- 8/10 matched and major ABO-mismatched sibling donor bone marrow transplant for Sickle-Beta Thalassemia. She received a myeloablative but reduced toxicity conditioning regimen consisting of busulfan 16mg/kg, fludarabine 140mg/m2, cyclophosphamide 105mg/kg, and alemtuzumab 52mg/m2. Her immediate post-transplant course was relatively benign and significant for a mild CMV colitis which responded with resolution after treatment with anti-viral therapy. Her red cell transfusion needs had significantly decreased to every four weeks by day 130. However, at day 146 from HSCT, she started requiring red cell transfusions every two weeks, with a drop in the reticulocyte count to 0.6%. No antibodies were detected, and the patient was negative for parvovirus B19 and did not have any evidence of GVHD. However, her bone marrow showed an marked erythroid hypoplasia, and was positive for a T-cell clone with gamma chain gene rearrangement in the T-cell receptor. Due to a new onset of transfusion need, she was treated with a short course of steroids with an excellent response and disappearance of the T-cell clone from peripheral blood. She remained on immunosuppression during this entire period with a calcineurin inhibitor. Patient# 2 – A 10 year old Hispanic girl underwent a 10/10 HLA-matched and major ABO-mismatced sibling donor bone marrow transplant for idiopathic acquired severe aplastic anemia. Her preparative regimen consisted of 200 mg/kg of cyclophosphamide and 16mg/kg of rabbit-anti-thymocyte globulin. Her immediate post-transplant course was complicated by E.coli bacteremia. She had prompt engraftment and became transfusion independent on day 86. A cyclosporine taper was initiated at day 100 but held at day 119 when anemia was first noted. Peripheral blood showed a T-cell clone with gamma and beta chain gene rearrangement. She was started on a moderate dose of steroids with a good response, and has been tapered down to physiologic replacement dose of steroids with normal Hb and transfusion independence. With the resolution of anemia, the gamma chain gene rearrangement is not seen in the T-cell clonality assay, however, the beta chain gene rearrangement persists. To our knowledge, this is the first report of a T-cell mediated PRCA following allogeneic HSCT. Moreover, the PRCA was steroid responsive and not associated with GVHD in both the patients. Disclosures:No relevant conflicts of interest to declare.

To respond or not to respond to hydroxyurea in thalassemia: a matter of stress adaptation?

In hereditary hemoglobinopathies, such as sickle cell anemia (SCA) and beta-thalassemia (BT), the absolute level of fetal hemoglobin (HbF) production is a critical predictor of severity. High HbF is protective against sickling and vaso-occlusive complications in SCA, and high levels of gamma-globin

Hypoxaemia affects male reproduction: a case study of how to differentiate between primary and secondary hypoxic testicular toxicity due to chemical exposure

Classification for fertility is based on two conditions, namely on evidence of an adverse effect on sexual function and fertility and that the effect is not secondary to other toxic effects. To decide on an adverse effect is a relatively simple day-to-day decision in toxicology but whether this effect is secondary often leads to serious controversy. As the seminiferous epithelium operates on the verge of hypoxia, oxygen deficit can lead to secondary impairment of testicular function. This is well known from healthy mountaineers exposing themselves to high altitude. They have reduced blood oxygen content that goes in parallel with impairment of testicular function and this effect remains for some time in spite of a compensatory polycythaemia. Similar findings are described for experimental animals exposed to hypobaric oxygen/high altitude. In addition, testicular function is affected in severe diseases in humans associated with systemic oxygen deficit like chronic obstructive pulmonary disease, sickle cell disease or beta-thalassaemia as well as in transgenic animals simulating haemolytic anaemia or sickle cell disease. The problem of insufficient oxygen supply as the underlying cause for testicular impairment has received relatively little attention in toxicology, mainly because blood oxygen content is generally not measured in these animal experiments. The difficulties associated with the decision whether testicular toxicity is primary or secondary to hypoxia are exemplified by the results of inhalation studies with nickel subsulphide and gallium arsenide (GaAs). Both of these particulate substances lead to severe lung toxicity that might impair oxygen uptake, but testicular toxicity is only observed with GaAs. This may first be explained by different effects on the blood: nickel subsulphide inhalation leads to a compensatory erythropoiesis that may mitigate pulmonary lack of oxygen uptake. In contrast, GaAs exposure is associated with microcytic haemolytic anaemia thereby aggravating any possible oxygen undersupply. Furthermore, the predominant pulmonary effect caused by GaAs (but not by nickel subsulphide) is alveolar proteinosis. Pulmonary alveolar proteinosis is also known as a severe disease in humans associated with hypoxaemia. Therefore, we conclude that the testicular effects observed after GaAs are secondary to hypoxaemia caused by the combination of pulmonary proteinosis and haemolytic anaemia. This publication tries to raise awareness to the severe consequences of hypoxaemia on testicular function that may already be caused by reduced oxygen pressure at high altitude without any chemical exposure.