F-cell Numbers Research Articles

Protein Phosphatase 6C (PPP6C) Loss Significantly Raises Fetal Hemoglobin Levels and Reduces Cell Sickling

Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor.PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD.To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed.Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. DisclosuresBlobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Pharmacokinetics-Guided Dosing of Hydroxyurea Can Achieve Near-Pancellular Fetal Hemoglobin Expression in Sickle Cell Anemia: F-Cell Analysis As a Benchmark for Disease-Modifying Therapy

Background: Hydroxyurea is the standard of care for children with sickle cell anemia (SCA). We have developed and prospectively validated a population pharmacokinetics (PK)-based dosing model to initiate hydroxyurea at a personalized dose that achieves maximum tolerated dose (MTD) quickly and produces a more robust HbF response than traditional weight-based dosing. HbF is distributed unequally across RBCs, and most mature RBCs have no detectable HbF. Genetic traits and hydroxyurea increase the number of HbF-containing RBCs. Measurement of HbF alone reflects an average of the HbF content across all RBCs in a lysate, so the varying, cell-specific compartmentalization of HbF across RBCs is not evaluated. In contrast, flow cytometry can be used to detect HbF-containing RBCs (F-cells) and non-F-cells, and the amount of HbF per F-cell (F/F-cell) can also be quantified. Prior studies indicate that an F/F-cell of 10 pg should completely prevent sickling. Methods:We studied participants in the Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154), which is a prospective study with the primary objective of developing and evaluating a population PK model to predict the MTD of hydroxyurea for individualized dosing. Individuals with genotype-confirmed HbSS or sickle-β0-thalassemia (both classified here as SCA) were enrolled; none had gene-deletion HPFH. We recently developed a multiparametric flow cytometric assay (F-cell analysis) to measure the amount and distribution of HbF in all RBCs and in immature (CD71+) reticulocytes. The total number of HbF-containing RBCs (F-cells) and HbF-containing immature reticulocytes (F-retics) were enumerated. Mean concentration of HbF per F-cell (F/F-cell) was calculated from F-cell number, Hb F quantitation by capillary zone electrophoresis, and automated blood counts (RBC count and hemoglobin concentration). Results:To date, 36 TREAT patients have had F-cell analysis with a mean age of 5.6 years (median 4.1; range 1.2-21.1). Mean age at start of hydroxyurea was 3.2 years (median 1.0; range 0.5-19.5) with a mean duration of hydroxyurea therapy of 2.4 years (median 2.1; range 0.5-4.1). Mean dose of hydroxyurea at the time of F-cell analysis was 27.0 mg/kg (median 27.6; range 17.8-38.6). HbF induction was robust with a mean HbF level of 30.8% (median 32; range 7.7-52.6). HbF level had a curvilinear association with F-cell fraction (Figure A). Mean total F-cell fraction (percent of all RBCs that are F-cells) was 82.2% (median 89.8; range 33.6-99.0); 24/36 (67%) had >80% F-cells, and 17/36 (47%) had >90% F-cells, indicating the ability to achieve a near pancellular distribution of HbF with PK-guided hydroxyurea therapy in a significant fraction of patients, especially those with high adherence (Figure B). The mean F/F-cell was 11.9 pg (median 11.5; range 5.1-19.4; Figure C), and 26/36 (72%) had an mean F/F-cell ≥10 pg, an amount of HbF in an individual RBC that completely prevents sickling (mean F/F-cell does not reflect the variance of F/F-cell across the RBC population). All patients who had a HbF level ≥30% (22/36, 61%) had mean F/F-cell ≥10 pg (Figure D). Of those with total F-cells ≥80%, 22/24 (91%) had mean F/F-cell ≥10 pg. As expected, higher total F-cells and mean F/F-cell were associated with higher hemoglobin concentration and lower reticulocyte counts. Mean F/F-cell >16 pg was associated with a mean hemoglobin >10 g/dL and mean absolute reticulocyte count <100x106/mm3. Bland-Altman analysis of total F-cells and F-retics showed a mean bias of 33.5 percentage points (total F-cells > F-retics), indicating the difference between F-cell production (F-retics) and peripheral survival advantage of F-cells. For F-cells >80%, however, the difference between F-cell and F-retic fraction decreased rapidly, consistent with little non-F-cell production by the bone marrow. Discussion: PK-guided dosing of hydroxyurea at MTD can achieve near-pancellular HbF expression in a large fraction of patients with sickle cell anemia. Our data suggest that an F-cell fraction of 80% and a mean F/F-cell of 10 pg should be the minimum goals for hydroxyurea therapy to minimize or prevent sickling, hemolysis, and anemia. These same minimum goals should apply to any HbF-inducing therapy, including genetic therapies, and F-cell analysis should be included in the standard laboratory analysis for these treatments. Figure Disclosures Quinn: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Research Support. Ware:Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB.

Evaluation of some haemolymph biochemical properties and F‐cell prevalence in hepatopancreas of white leg shrimp (Litopenaeus vanammei) after fed diets containing apple cider vinegar and propionic acid

The present study aimed to evaluate the effect of dietary ACV (apple cider vinegar) and PA (propionic acid) supplementation on some biochemical properties of haemolymph and fibrillar cell (F-cell) number in the hepatopancreas of shrimp. Five different diets supplemented with 0%, 1%, 2%, 4% ACV and 0.5% PA diets were fed to Litopenaeus vanammei. Some biochemical parameters of haemolymph (cortisol, alkaline phosphatase, albumin, urea, uric acid, creatinine, phosphorus, total hemocyte count) and prevalence of F-cell in hepatopancreas were examined after 60 days feeding trial. The albumin concentration and total hemocyte count in 2% and 4% ACV and PA groups were significantly higher than those of other groups (p < .05). Moreover, the highest phosphorus concentration was detected in shrimp fed 4% ACV and PA experimental diets (p < .05). No significant differences in haemolymph cortisol, urea, uric acid, creatinine, alkaline phosphatase and tubule area were observed among the experimental groups (p > .05). 2% ACV group showed more F-cell number in hepatopancreas than other groups while the reduction in F-cell number was observed in the PA group. Based on these observations, the tested ACV and PA diets remarkably exhibited positive impacts on nutritional value and they may be a potential for adding to shrimp diet.

Environmental stress upon hepatopancreatic cells of freshwater prawns (Decapoda: Caridea) from the floodplain of Paran&amp;#225; River

In order to evaluate the influence of stressed environments on hepatopancreatic cells of fre- shwater prawns, Macrobachium borellii Nobili, 1896 and Palaemonetes argentinus Nobili 1901, (Crustacea, Decapoda, Palaemonidae) were collected at three different aquatic environments with different relationship to urban development in Argentina. Furthermore the effects of several cypermethryn concentration on hepa- topancreatic cell of M. borellii and P. argentinus were evaluated in a laboratory assays. The “N° 1” lake (Santa Fe Argentina) which is more affected by the anthropogenic pressure and “Don Felipe” lake which is still not strongly by human activities were the studied sites from the floodplain of Paraná river. While Alejandra lake was the intermedia effects sites. Different damaged ultra-structures were found in F- and R-cells of prawns in the stressed lake. The predominant features were: disrupted the microvillous border, swelled mitochondria, reduction of endoplasmic reticulum, dyctiosomes, glycoproteins, desna-turalization of vacuole membrane and premature autolysis. Moreover the F-cell number was higher in the environment near to city than in the others sites. Similar effects were observed in the cypermethryn assays. The observations clearly indicate that the ultrastructure of midgut gland in the both palaemonids varies depending on the site from which animals are collected and the biocid presence. So, in this case it can be stated that the hepatopancreas histology of fre- shwater prawns is a good tool to monitor the impact of a stressed environment upon freshwater prawns.

BCL11A represses HBG transcription in K562 cells

BCL11A on chromosome 2p16 was recently shown to be a major quantitative trait locus for Hb F level and F-cell number in several populations with or without β-hemoglobinopathy. We now show that BCL11A isoforms are expressed in K562 cells. Butyrate induction of HBG globin production in K562 is associated with reduced BCL11A. Conversely, augmented expression of BCL11A in K562 cells through transfection of BCL11A expression vector results in more than 50% reduction of HBG promoter transcription activity. This transcription repression can be abrogated by sodium butyrate. BCL11A binds to GGCCGG motif in nucleotide −56 to −51 on the HBG proximal promoter. Together, these data are consistent with BCL11A being able to bind to a core motif in the HBG proximal promoter, recruit and interact with partners to form a repression complex, leading to deacetylation of histones and down-regulation of the HBG transcription.

BCL11A is a major HbF quantitative trait locus in three different populations with β-hemoglobinopathies

Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of β-thalassemia major and sickle cell anemia. Recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with β-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with β-thalassemia trait, and with HbF levels in Thais with either β-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.

Variation and heritability of Hb F and F‐cells among β‐thalassemia heterozygotes in Hong Kong

Enhanced fetal hemoglobin (Hb F) production can partially compensate for the lack of adult hemoglobin (Hb A) in patients with beta-thalassemia major or intermedia, and ameliorate the clinical severity of these diseases. To further elucidate factors governing Hb F levels, we evaluated demographic, clinical, laboratory, and genetic characteristics in 241 unrelated adult beta-thalassemia carriers in Hong Kong. They had wide variations in Hb F and F-cell numbers skewing toward higher levels. Individuals who coinherited the Xmn IT-allele in the (G)gamma-globin gene promoter had higher Hb F and more F-cells compared with those lacking the Xmn I T-allele. However, both groups exhibited a similarly wide spread of Hb F and F-cells. The correlation of Hb F and F-cells corresponded well to both linear and exponential models, suggesting multiple mechanisms for Hb F augmentation. The heritabilities of Hb F and F-cells were calculated in 66 families (111 parents who were beta-thalassemia carriers and 82 asymptomatic offspring) to be 0.7 to 0.9. The Xmn I polymorphism accounted for 9% of the Hb F and 13% of the F-cell heritabilities. These results suggest that these family members are well suited for genome wide association studies that will identify genetic loci regulating Hb F production, and likely novel pharmacological targets for reactivating Hb F production in adults.

Fetal Hemoglobin Protects Against Activation of Hemostasis Endothelial and Circulating Blood Cells in Sickle Cell Anemia.

To determine whether fetal hemoglobin (HbF) protects against activation of hemostasis, endotheliium, and circulating blood elements, infants, children and young adults with homozygous SS disease (n=35) were studied. F-cell numbers, biomarkers of endothelial activation (including sVCAM-1, sE- and sP-selectin), white cell and platelet activation (sL-selectin and sP-selectin respectively) and markers of hemostatic activation, including D-Dimer and prothrombin fragment F1.2 were analyzed. Subjects with HbSS demonstrated significant increases (P<0.001) in all cell activation and hemostatic biomarkers. Analyses revealed a dominant, statistically significant correlation between F-cell and biomarkers of endothelial, white cell and hemostatic activation. F-cells correlated inversely with sVCAM-1 (R = −0.64, P<0.00005), sE-selectin (R = −0.51, P = 0.03) and sP-selectin (R = −0.41, P<0.02). Since HbF protects against HbS polymerization, our data suggests a role for the sickle erythrocyte in endothelial activation and is consistent with reported decreases in sVCAM-1 following hydroxyurea therapy or a chronic transfusion regimen. sP- and sE-selectin levels correlated with neutrophil counts (R = 0.63 and R = 0.42, P<0.00006 and P<0.02 respectively) while no relationship between platelets and sP-selectin was noted. An additional correlation between sP- and sE-selectin (R = 0.50, P<0.003) was also observed, reflecting enhanced neutrophil-endothelial interaction as part of an inflammatory and prothrombotic HbSS phenotype. Surprisingly, a positive correlation between sL-selectin and F-cells occurred (R = 0.4, P<0.02) with the young infant with HbSS demonstrating higher levels of this leucocyte activation marker than the older subject. A dysregulation of L-selectin shedding following neutrophil activation has been proposed to occur in sickle cell anemia. Our results suggest that HbF protects against this dysregulation. D-Dimer levels correlated negatively with F-cells (R = −0.76, P<0.000001), with a striking relationship between D-Dimer and markers of endothelial activation, including sVCAM-1 (R = 0.53, P<0.001), sE-selectin (R = 0.39, P<0.03) and sP-selectin (R = 0.41, P<0.02). Such findings are in keeping with D-Dimer's well-documented role as a biomarker in other inflammatory and ischemia-related disorders including coronary, cerebrovascular and peripheral arterial ischemic states. This report expands our knowledge regarding the protective effects of HbF against endothelial and hemostatic activation, and suggests a role for D-Dimer as a prothrombotic and proinflammatory marker in sickle cell anemia.

Analysis of the X-Linked F-Cell Production Locus.

This laboratory has previously identified a locus on the X chromosome at Xp22.2–22.3 (F-cell production locus or FCP) that is responsible for approximately 40% of the genetic variability in F-cell number in patients with sickle cell disease (SCD). We have re-examined the association of this region with F-cell production by multipoint linkage analysis. We have confirmed linkage to Xp22.2–22.3 and refined the candidate locus to a region of approximately 3 cM, between markers DXS452 and DXS410, with a maximum LOD score of 3.315. Linkage to a more extended region of 11 cM with an average LOD score of 1.5 could not be excluded. In an effort to identify candidate genes within this region that influence F-cell production, we screened known genes within this region for transcription factor binding sites that had relevance to HbF production. We first searched for CREB binding sites given the involvement of cAMP in of HbF induction in CD34 cells recently identified by this laboratory. Of the 15 ESTs containing CREB sequences, several also had binding sites for NFE2 or NFE2-like transcription factors. We examined 4 genes, 3 within the refined candidate locus (KIAA1280, TBL1X, and KAL1) and one in the more extended locus region (EGFL6) for expression levels within pedigrees of known F-cell phenotypes. Results indicate that, within families, a reduced level of KIAA1280 message is associated with high F-cell phenotypes in lymphoblastoid cells from patients with sickle cell anemia. The other genes examined had no such correlation.KIAA1280, also designated BMO42 is a gene of unknown function that is expressed in bone marrow. The predicted protein contains a region homologous to a conserved protein kinase C domain. While not yet definitive, these studies provide the first suggestive evidence for a candidate gene within the FCP locus. Further studies will attempt to define the function of this gene and explore it's involvement in F-cell regulation.

Molecular and Cellular Mechanisms for Fetal Hemoglobin Induction by Hydroxyureain Normal Erythroid Progenitors.

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production. HU-induced HbF production is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia. Various cell culture systems have been used to elucidate the implicated mechanisms. We have previously demonstrated in both K562 human erythroleukemic cells and human erythroid cells (hAEC) that HU increases the transcription of low-expressed globin genes as well as the splicing efficiency of primary globin transcripts; however, other post-transcriptional effects could not be excluded. In this study, we extended our analysis in order to further understand the effect of HU on erythropoiesis and the molecular mechanisms involved in globin gene elevation. Peripheral blood hAEC from normal individuals were cultured in a two-phase liquid culture. Erythropoietin was added only to phase II. Erythroid cells in phase II were exposed to different HU concentrations 25/50/100/300 μM at day 5 (proerythroblast stage) either continuously (1– 3 days) or were pulse treated [initially exposed to HU (1–3 days), then washed and harvested at day 8–14)]. Hb-containing cell number, measured by flow cytometry (FC), was affected by continuous as well as pulse HU treatment in a dose- and-time dependent fashion. HbF-cell numbers were determined from day 0 of phase II throughout phase II culture by both FC and the immunocytochemical StreptABComplex/AP staining procedure. At day 0 of phase II no HbF cells were detected by either method; at day 8 of phase II, F-cell numbers increased 2-fold in cultures exposed to 100 μM/300 μM HU for 3 days. Hbs and globin chains were separated and measured by ion exchange and reverse-phase HPLC, respectively. HU (300 μ M) treatment caused a 1.5-fold increase in γ-globin chain production and preferentially elevated the Gγ-chain. Quantification of γ-mRNA was performed by real-time PCR. While addition of low HU concentrations (25 and 50 μM) for 1–3 days had no effect, treatment with high concentrations (100/300 μM) caused a significant time- and dose-dependent increase in γ-mRNA (100 μM HU, 1– 3 days: 1.2 to 2.1-fold; 300 μM, 1–3 days: 1.5 to 2.2-fold). Pulse treatment with 100 μM HU (cells exposed for 1 day, washed and harvested 2 days later) led to a greater increase in γ-mRNA levels (2.2-fold) than continuous exposure for 3 days (1.6-fold). HU effects on γ-mRNA stability were analyzed in hAEC cultures-treated with HU and actinomycin-D (actD: 5μg/ml) for 1hr to 24hrs. HU-treated (100 μM) cells exhibited a 3.9-fold increase in γ-mRNA 24 hrs post-actD addition. Exposure at higher HU concentrations and/or prolongation of actD treatment had a toxic effect (decrease in both γ-mRNA and cell numbers). In conclusion, the present study provides evidence that HU can stimulate erythropoiesis and increase Hb production in a dose- and-time dependent fashion; HU effects are mediated by transcriptional and post-transcriptional modulation of γ-globin gene expression.

Maintenance of elevated fetal hemoglobin levels by decitabine during dose interval treatment of sickle cell anemia

We have previously demonstrated that 5-aza-2'-deoxycytidine (decitabine) augments fetal hemoglobin (HbF) levels in patients with sickle cell anemia (SS) who did not respond to hydroxyurea (HU). The present study was designed to determine the effect of repeated decitabine dosing on HbF levels and hematologic toxicity over a 9-month treatment period. Seven patients (5 HU nonresponders) were entered. One patient had alpha-thalassemia sickle cell anemia. Decitabine was administered by intravenous infusion at a starting dose of 0.3 mg/kg per day, 5 days a week for 2 weeks, followed by a 4-week observation period. If the absolute neutrophil count dropped below 1000, the dose was reduced by 0.05 mg/kg per day in the next cycle. A drug dose was obtained for each patient, and it resulted in an elevated HbF without neutropenia (absolute neutrophil count nadir greater than 1500) or evidence of cumulative toxicity. Average HbF and average maximal HbF levels attained during the last 20 weeks of treatment for the 6 SS patients increased to 13.93% +/- 2.75% and 18.35% +/- 4.46%, respectively, from a pretreatment mean of 3.12% +/- 2.75%. Mean and mean maximal hemoglobin (Hb) levels increased from 7.23 +/- 2.35 g/dL to 8.81 +/- 0.42 g/dL and 9.73 +/- 0.53 g/dL, respectively. Individual maximal F-cell number observed during the trial was 69% +/- 10.12%. The absence of cumulative toxicity may allow shorter intervals between drug treatments, which may lead to higher hemoglobin and HbF levels after several treatment cycles and, therefore, to greater clinical improvement.

Fetal hemoglobin in sickle cell anemia: relationship to erythrocyte adhesion markers and adhesion

AbstractTo assess whether fetal hemoglobin (HbF) modulates the adhesion of sickle erythrocytes to endothelium, children with homozygous sickle cell anemia (SS disease) were studied, using this physiologically crucial period to evaluate the relationships between HbF and the major erythrocyte adhesion markers. The mean level of CD36+ erythrocytes was 2.59% ± 2.15% (± SD, n = 40) with an inverse relationship between CD36 positivity and F cells (R = −0.76, P < .000 00 002). In univariate analyses, significant correlations with various hematologic parameters and age were noted. Multiple regression analyses, however, revealed a relationship solely with F cells. Minimal levels of very late activation antigen-4+ (VLA4+) erythrocytes (0.31% ± 0.45%, n = 40) with relationships similar to those noted for CD36+ cells were also observed. The subpopulation of strongly adhesive stress reticulocytes was further assessed, using CD71 as their marker. The mean level of CD71+ erythrocytes was 5.81% ± 4.21%, with statistical correlates in univariate and multivariate analyses similar to those discussed above. When adhesion ratios were evaluated, inverse correlations were noted between basal and plasma-induced adhesion and F-cell numbers (R = −0.54, P < .0005;R = −0.53, P < .0006, n = 39). In addition, in analyses where basal or plasma-induced adhesion was the dependent variable and the independent variables included F cells and the various adhesion-related parameters, significant relationships solely with F cells were noted. The results demonstrate that SS patients with higher levels of F cells have concomitant decreases in the numbers of CD36+, VLA4+, and CD71+ erythrocytes and that these findings translate into less adherent erythrocytes. These findings extend knowledge regarding the protective effects of HbF in the pathophysiology of sickle cell disease.

Effects of hydroxurea, stem cell factor, and erythropoietin in combination on fetal hemoglobin in the baboon.

Augmentation of the level of fetal hemoglobin (HbF) is considered therapeutic for patients with sickle cell disease. The objective of this study was to determine the effect of treatment with a combination of erythropoietin (Epo), stem cell factor (SCF), and hydroxyurea (HU) on HbF levels. The effect of treatment with a combination of Epo, SCF, and HU on HbF, F-cell numbers, and globin chain synthesis was evaluated in a baboon model. Treatment with a combination of SCF+Epo resulted in a two-fold increase in HbF, F-cells, and F-reticulocytes compared to Epo alone. The combination of SCF+Epo+HU resulted in an additional two-fold increase in HbF, whereas F-cells and F-reticulocytes increased only 25% compared to the SCF+Epo regimen. Measurement of differential globin chain synthesis indicated that the SCF+Epo+HU treatment also increased the I gamma/V gamma (homologous to human G gamma and A gamma) synthetic ratio toward the fetal ratio. HU can effectively augment growth factor-induced HbF synthesis in vivo. Because I gamma/V gamma ratios are unaffected by erythropoietic stress and similar increases in this ratio have only been observed following administration of 5-azacytidine, we suggest that these two agents may share a common mechanism of action involving the recruitment of a similar target cell population to terminal erythroid differentiation.

Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P <.000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P <.001) and F cells (P <.01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

AbstractIn sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

Dissection of the association status of two polymorphisms in the β-globin gene cluster with variations in F-cell number in non-anemic individuals

Expression of fetal hemoglobin (Hb F) is under polygenic control involving determinants both linked and unlinked to the beta-globin gene cluster on chromosome 11. Variations in the DNase I-hypersensitive site 2 of the locus control region (LCR-HS2) and a C --> T change at position -158 from the Ggamma-gene (detected as an XmnI polymorphism) correlate with the high level of Hb F expression in patients with sickle-cell anemia and beta-thalassemia. Interpretation of data under these conditions of anemic stress is difficult because the preferential survival of Hb F-containing erythrocytes (F-cells) may not reflect the true status of Hb F expression. We investigated the relationship between these markers and Hb F expression in terms of F-cell levels in 48 unrelated non-anemic AS heterozygotes from Sicily. The betaS-chromosome of all these individuals was of the Benin haplotype and they differed only by their betaA chromosomes. We demonstrate that F-cell expression is more strongly associated with LCR-HS2 polymorphism than with XmnI polymorphism. The observed association between XmnI polymorphism and Hb F expression is very likely to be due to linkage disequilibrium with LCR-HS2 sequences.

DNA Sequence Variation in a Negative Control Region 5’ to the β-Globin Gene Correlates With the Phenotypic Expression of the βs Mutation

The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5' to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.

DNA sequence variation in a negative control region 5' to the beta- globin gene correlates with the phenotypic expression of the beta s mutation

The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5′ to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.

Variation of HbF and F-cell number with the G-gamma Xmn I (C-T) polymorphism in normal individuals [letter

Variation of HbF and F-cell number with the G-gamma Xmn I (C-T) polymorphism in normal individuals [letter

Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal gamma-gene expression in association with beta thalassemia and linkage relationship with the beta-globin gene cluster.

We report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to beta thalassemia over two or three generations. The HPFH + beta thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second beta thalassemia trait gives rise to a mild clinical picture characterized by 9-12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or beta-thal trait was observed, and in one case the study of the DNA polymorphisms within the gamma delta beta gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the A gamma chain (the A gamma T chain) allowed us to demonstrate that the increased gamma chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes.