HbF Concentration Research Articles

Interference of hemoglobin variants with HbA1c measurements by six commonly used HbA1c methods.

Glycated hemoglobin, or hemoglobin A1c (HbA1c), serves as a crucial marker for diagnosing diabetes and monitoring its progression. We aimed to assess the interference posed by common Hb variants on popular HbA1c measurement systems. A total of 63 variant and nonvariant samples with target values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-calibrated methods were included. We assessed 6 methods for measuring HbA1c in the presence of HbS, HbC, HbD, HbE, and fetal hemoglobin (HbF): 2 cation-exchange high-performance liquid chromatography (HPLC) methods (Bio-Rad D-100 and HLC-723 G8), a capillary electrophoresis (CE) method (Sebia Capillarys 3 TERA), an immunoassay (Roche c501), an enzyme assay system (Mindray BS-600M), and a boronate affinity method (Primus Premier Hb9210). The HbA1c results for nonvariant samples from the 6 methods were in good agreement with the IFCC-calibrated method results. The Bio-Rad D-100, Capillarys 3, Mindray BS-600M, Premier Hb9210, and Roche c501 showed no interference from HbS, HbC, HbD, and HbE. Clinically significant interference was observed for the HLC-723 G8 standard mode. Elevated HbF levels caused significant negative biases for all 6 methods, which increased with increasing HbF concentration. Elevated levels of HbF can severely affect HbA1c measurements by borate affinity, immunoassays, and enzyme assays.

Detection of Asymptomatic Sickle Cell Hemoglobin Carriers and Fetal Hemoglobin Regulating Genetic Variants in African Descendants from Oaxaca, Mexico.

Sickle cell anemia has been classified as a noninfectious neglected tropical disease and, although not exclusively, affects African descendants more frequently. This study aimed to detect asymptomatic sickle cell hemoglobin carriers (HbAS) in marginalized and vulnerable populations during a public health screening in African descendants from Oaxaca, Mexico, and to validate an amplification refractory mutation system (ARMS)-PCR methodology to detect fetal-hemoglobin (HbF)-regulating genetic variants in BCL11A toward affordable routine association of single nucleotide variants (SNVs) with HbF concentrations. To this aim, hemoglobin variants were detected by acidic citrate agar and alkaline cellulose acetate electrophoreses. SNVs in the hemoglobin subunit beta gene (HBB) were identified by the β-globin mutation detection assay (β-GMDA) and ARMS-PCR, respectively, and validated by Sanger sequencing. The association between genotypes and HbF concentrations was evaluated using Spearman's correlation coefficient. The results obtained during a directed screening in 140 self-identified African descendants revealed 42 HbS-carriers (30%), of which 39 showed normal total hemoglobin concentrations (92.8%), only 3 presented anemia (7.2%), and 9 showed quantifiable HbF concentration (21.4%). As validated by Sanger sequencing, the designed ARMS-PCR efficiently detected homozygous and heterozygous variants in BCL11A. In a cohort of 42 heterozygous (HbAS) and 27 healthy (HbAA) individuals from the same population, only one SNV (rs766432) showed statistically significant association with increasing HbF concentration, and two new unrelated homozygous silent variants were identified. This study reveals the need to raise coverage of HbS screening in vulnerable populations and shows a feasible low-cost ARMS-PCR methodology to determine the presence of SNVs in quantitative trait loci affecting HbF.

Impact of Foetal Haemoglobin on Disease Severity among Sickle Cell Subjects

Objective: Haemolysis is a common complication of Sickle cell disease, exhibiting considerable variability among affected individuals. Established haemolysis markers including lactate dehydrogenase activity (LDH) and bilirubin have been reported as reliable indicators of disease severity. Foetal haemoglobin plays a pivotal role in Sickle Cell Disease by neutralizing Haemoglobin-S (HbS) polymerization, consequently extending the lifespan of red blood cells and influencing overall clinical outcomes. This study aims to investigate the impact of foetal haemoglobin on disease severity among sickle cell subjects. Method: This study involved forty (40) sickle cell subjects and thirty (30) normal subjects. Blood samples from the participants were analysed for lactate dehydrogenase activity, bilirubin concentration, and foetal haemoglobin concentration. The alkali denaturation technique was employed for HbF analysis, while diagnostic kits were used for LDH and bilirubin assays. Results: Foetal haemoglobin concentrations below 10% have been defined as low. The HbF concentration of the sickle cell subjects was relatively low (5.23%) but significantly higher than the normal subjects (1.84%). Similarly, there was a significant elevation in markers of haemolysis in the sickle cell subjects compared to the normal subjects. Subjects with HbF>10% demonstrated a noteworthy increase in haemolysis markers compared to those with HbF<5%. Additionally, a significant inverse relationship was observed between HbF concentration and haemolysis markers. Conclusion: The study highlights a significant inverse correlation between foetal haemoglobin concentration and disease severity among sickle cell subjects and also contributes valuable insights to our understanding of sickle cell disease pathology.

AsCas12a Gene Editing of HBG1/2 Promoters with EDIT-301 Results in Rapid and Sustained Normalization of Hemoglobin and Increased Fetal Hemoglobin in Patients with Severe Sickle Cell Disease and Transfusion-Dependent Beta-Thalassemia

Background: Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are hereditary blood disorders caused by mutations in the β-globin gene. Clinical evidence has demonstrated that increased fetal hemoglobin (HbF, α2γ2) can reduce or eliminate SCD and TDT complications. EDIT-301, an investigational gene-edited autologous hematopoietic stem cell medicine, has a unique genomic modification that mimics naturally occurring mutations of hereditary persistence of fetal hemoglobin in the γ-globin gene ( HBG1/2) promoters. These mutations reactivate γ-globin expression and increase HbF production. EDIT-301 is manufactured with a highly efficient and specific, proprietary gene editing nuclease, AsCas12a. In preclinical studies, edited CD34 + cells from patients with SCD or TDT increased HbF production in red blood cells (RBCs) sufficient for a therapeutic effect, including reduced sickling (SCD) and improved erythropoiesis (TDT). Aim: RUBY (NCT04853576) and EdiThal (NCT05444894) are Phase I/II, multicenter, open-label, single-arm studies evaluating the safety, efficacy, and tolerability of EDIT-301 in patients with severe SCD and TDT, respectively. As of June 28, 2023, 7 SCD patients and 2 TDT patients have received EDIT-301 treatment. Preliminary clinical efficacy and safety data are reported. Methods: Patients aged 18-50 years with severe SCD (defined as ≥2 severe vaso-occlusive events [VOEs] per year in the 2 years prior to informed consent [IC]) and patients aged 18-35 years with TDT (defined as at least 100 mL/kg/year or 10 U/year of packed RBC transfusions in the 2 years prior to IC) were eligible to enroll in RUBY and EdiThal, respectively. Autologous CD34 + hematopoietic stem and progenitor cells collected by apheresis after plerixafor (RUBY) or plerixafor + filgrastim (EdiThal) mobilization were edited at the HBG1/2 promoters with AsCas12a. After myeloablative conditioning with busulfan, patients received a single infusion of EDIT-301 (a minimum of 3 × 10 6 CD34 + cells/kg), and were monitored for engraftment, total hemoglobin (Hb), HbF, mean HbF concentration/F-cell (MCH-F/F-cell), percentage of F-cells, markers of hemolysis, transfusion requirement, VOEs (SCD only), and adverse events (AEs) for 24 months. Results: Based on a data cut of June 28, 2023, Patients 1-4 with SCD were 12-, 9-, 4-, and 4-months post-EDIT-301 infusion, respectively. Patients 5-7 with SCD were <1-month post-EDIT-301 infusion. Patients 1-2 with TDT were 3- and <1-months post-EDIT-301 infusion, respectively. Neutrophil and platelet engraftment were achieved after a mean (range) of 25 (23-29) and 27 (19-37) days in Patients 1-4 with SCD and on Day 23 and 26 in Patient 1 with TDT, respectively. There were no VOEs in SCD patients post-EDIT-301 infusion, compared with a mean (range) of 4.2 (3.0-5.5) VOEs/year in the 2 years before enrollment (n=6). Following EDIT-301 infusion, Hb levels rapidly increased to 14.2 (12.4-15.7) g/dL by Month 4 (n=4), reaching the normal physiological range, from a mean (range) of 10.5 (8.5-11.9) g/dL at baseline (n=5). By Month 4, the mean (range) HbF concentration was 6.8 (5.7-7.6) g/dL and HbF was >40% (n=4); Patients 3 and 4 had >50% HbF. Percentage of F-cells and MCH-F/F-cell also increased. Key markers of hemolysis improved or normalized in all patients with SCD. Patient 1 with TDT had a HbF concentration of 7.2 g/dL by Month 3, stopped receiving RBC transfusions 20 days after EDIT-301 infusion, and remained transfusion free through the 3-month period. Patient 2 with TDT also showed early improvements. The safety profile of EDIT-301 in both SCD and TDT was consistent with myeloablative conditioning with busulfan. No serious AEs were reported after EDIT-301 infusion. Conclusion: These data demonstrated successful engraftment, a rapid and sustained normalization of Hb as early as 4 months after infusion, an increase in HbF and percentage of F-cells, resolution of VOEs (SCD) and transfusion independence (TDT). In addition, there were improvements in key markers of hemolysis (SCD) and a favorable safety profile in EDIT-301-treated patients. EDIT-301 treatment showed promising results for the first clinical use of AsCas12a in both SCD and TDT patients after gene editing of the γ-globin gene ( HBG1/2) promoters. These findings support further investigation of EDIT-301 in the RUBY and EdiThal trials. Updated data with additional outcomes will be presented.

Association of β-Globin Gene Haplotypes with Haematological Parameters and Foetal Haemoglobin among Patients with Sickle Cell Disorder in Raipur, Chhattisgarh, India

Introduction: Sickle cell disease is caused by a single nucleotide substitution in the β-globin gene. The variations in Foetal Haemoglobin (HbF) levels, β-globin gene cluster haplotype have been used as predictors of disease severity in sickle cell disease patients. Aim: To determine the frequency of β-globin gene haplotypes in sickle cell disease patients and also to establish their association with haematological parameters and HbF. Materials and Methods: The present cross-sectional study was conducted in Department of Biochemistry, Government Medical College, Jagdalpur, Chhattisgarh, India, in collaboration with Department of Biotechnology, Government Nagarjuna PG College of Science, Raipur, Chhattisgarh, India, from April 2021 to May 2022. A total of 100 patients with Sickle Cell Disease (SCD) and 50 with sickle cell traits were included in the study. Haplotypes were identified by Restriction Fragment Length Analysis (RFLP) method for seven polymorphic sites in β-globin gene cluster. Haematological parameters such as Hb, Haematocrit (HCT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH) and HbF levels were estimated. Data was analysed using various statistical tests such as ShapiroWilk test, Levene’s test, student t-test, Mann-Whitney test and Kruskal-Wallis tests as per analysis requirement. Results: In the present study, 51 (51%) males and 49 (49%) females were in sickle cell disease group (SS), while 25 (50%) males and 25 (50%) females were in sickle cell trait group (AS). The mean age of the sickle cell disease patients was 23.84±8.38 years and for sickle cell trait group was 26.3±7.37 years. There was a significant difference (p-value <0.0001) in HbF levels among haplotypes. Additionally, higher HbF concentration was found in Arab-Indian haplotypes in SCD patients. No significant association was observed between the haplotypes and haematological parameters. Conclusion: The findings suggested that haematological parameters were not significantly associated with β-globin gene haplotypes. The β-globin gene haplotypes influence the HbF levels in sickle cell patients.

Individual Red Blood Cell Fetal Hemoglobin Concentrations Determined By Imaging Flow Cytometry and the Number of F Cells Are Very Good Predictors of Clinical Response to Increased Level of HbF and Are Associated with Hemolysis, Tricuspid Regurgitant Jet Velocity, and Sickle Retinopathy in Adult Sickle Cell Disease Patients

Sickle cell disease (SCD) patients with similar total fetal hemoglobin (HbF) levels may present with different severity, which is attributed to the heterogeneous distribution of HbF among red blood cells (RBCs) with varying percentages of circulating HbF-containing RBCs (% F cells). Steinberg et al. (2014) proposed that an individual HbF concentration per cell (iHbF) above 10pg inhibits the polymerization of HbS, so the development of precise methods to quantify iHbF and the percentage of F cells may allow clinicians to use those parameters as targets to monitor patient treatment. We devised a novel procedure to very accurately measure and quantify iHbF using imaging flow cytometry (Amnis Imagestream) and investigated its correlations with clinical manifestations. Samples from 54 adult SCD patients (22 [41%] female, 39 [72%] on hydroxyurea) were used to acquire images from RBCs labeled with fluorescent anti-HbF antibody and were analyzed using IDEAS software v 6.2. Along with the hematimetric data and HPLC quantification of total HbF of each sample, we modified the formulas by Maier-Redelsperger et al. (1994) and Horiuchi et al. (1995), with minimization of interference from autofluorescence of non-F cells, correction for area and fluorescence intensity, and analyzed RBC subpopulations with varying levels of HbF expression. Statistical analysis was performed using R considering a p value <0.05 to be statistically significant. We did not find associations of iHbF with alpha thalassemia carrier state or beta globin cluster haplotypes Benin or Bantu, the most common in our population. Perpendicular cutoff correlation studies defined two patient populations: one group with lower mean iHbF (9.9pg) and lower % F cells (28.2%) and another group with a higher mean iHbF (15.8pg) and higher mean % F cells (72.5%). The latter group had a lower frequency of vaso-occlusive crises (p=0.011). That was further validated with our finding that the cutoffs of % F cells>30% and iHbF>10pg were strongly associated with lower absolute reticulocyte counts (p<0.001), LDH (p=0.002), and indirect bilirubin (p<0.001). The association between hemolysis and iHbF was further explored regarding pulmonary hypertension, showing that a value of iHbF>10pg was associated with a lower median tricuspid regurgitant jet velocity than in patients with iHbF<10pg (2.72m/s vs. 2.29m/s, p=0.046). We also found an association of a mean iHbF<8pg with the presence of sickle retinopathy (p=0.002). Patients with retinopathy had a lower mean iHbF and % F cells than those without it (7.08pg vs. 8.75pg, p=0.032; 48.5% vs. 71.0%, p=0.043, respectively), suggesting increments in iHbF and % F cells may be protective against sickle retinopathy, a complication not usually associated with high hemolytic rates. Our results suggest that a percentage of F cells above 30% along with individual RBC HbF concentration of 10pg may be protective against complications in SCD patients and, therefore, should be explored as goals for hydroxyurea therapy in this population. In addition, the data presented here suggest further studies on the efficacy of HbF induction therapy against the development of sickle retinopathy may be performed in a large population of sickle cell disease patients.

Fetal hemoglobin-boosting haplotypes of BCL11A gene and HBS1L-MYB intergenic region in the prediction of clinical and hematological outcomes in a cohort of children with sickle cell anemia.

Single nucleotide polymorphisms (SNPs) of BCL11A gene and HBS1L-MYB intergenic region (named HMIP-2) affect both fetal hemoglobin (HbF) concentration and clinical outcomes in patients with sickle cell anemia (SCA). However, no previous study has examined the interaction among these SNPs in the regulation of HbF. We examined whether HbF-boosting haplotypes combining alleles of functional SNPs of BCL11A and HMIP-2 were associated with clinical outcomes and hematological parameters, and whether they interact to regulate HbF in a cohort of Brazilian children with SCA. The minor haplotype of BCL11A ("TCA", an allele combination of rs1427407, rs766432, and rs4671393) was associated with higher HbF, hemoglobin and lower reticulocytes count compared to reference haplotype "GAG". The minor haplotype of HMIP-2 ("CGC", an allele combination of rs9399137, rs4895441, and rs9494145) was associated with higher HbF and hemoglobin compared to reference haplotype "TAT". Subjects carrying minor haplotypes showed reduced rate of clinical complications compared to reference haplotypes. Non-carriers of both minor haplotypes for BCL11A and HMIP-2 showed the lowest HbF concentration. Subjects carrying only the minor haplotype of BCL11A showed significantly higher HbF concentration than non-carriers of any minor haplotype, which showed no significant difference compared to subjects carrying only the minor haplotype of HMIP-2. Interestingly, subjects carrying both minor haplotypes of BCL11A ("TCA") and HMIP-2 ("CGC") showed significantly higher HbF levels than subjects carrying only the minor haplotype of BCL11A. Our novel findings suggest that HbF-boosting haplotypes of BCL11A and HMIP-2 can predict clinical outcomes and may interact to regulate HbF in patients with SCA.

BCL11A POLYMORPHISMS ASSOCIATED WITH INCREASED FETAL HEMOGLOBIN IN RESPONSE TO HYDROXYUREA TREATMENT IN A COHORT OF PEDIATRIC PATIENTS WITH SICKLE CELL ANEMIA

Fetal Hemoglobin (HbF) is a major modifier of clinical events in patients with sickle cell anemia (SCA). Hydroxyurea (HU) improves their clinical course by raising HbF level. There is variability in the response to HU treatment among SCA patients. Genome-wide association studies (GWAS) have identified the BCL11A gene and HBS1L-MYB intergenic region (HMIP-2) as the main regulators of baseline HbF production, and polymorphisms in both loci have been associated with HbF levels. However, it is unknown whether genetic polymorphisms involved in regulation of baseline HbF can modulate HbF in response to HU treatment. Therefore, we examined whether polymorphisms in BCL11A (rs11886868, rs766432, rs4671393, rs1427407, rs7557939 and rs7599488) and HMIP-2 (rs9399137, rs9402686, rs4895441 and rs9494145) were associated with HbF changes in a retrospective cohort of 110 pediatric patients diagnosed with SCA (HbSS) by the Newborn Screening Program of Minas Gerais, and followed up at Center of Hematology and Hemotherapy of Minas Gerais (HEMOMINAS), Belo Horizonte. Participants have been on HU treatment for at least one year. We assessed “delta HbF”for each patient as the “final HbF relative concentration”(last available test during HU treatment and at least three months after blood transfusion) “minus baseline HbF”(last available test before HU treatment and after the age of 5 years, and at least 3 months after blood transfusion). Genotypes were assessed by Taqman assays and real-time PCR or PCR-RFLP. Univariate associations were performed using Mann-Whitney U Test. For the 110 enrolled SCA patients (59/53.6% were females), we calculated the average age at start of HU (10.0±2.8 years), average HU dose (24.40±4.41 mg/kg/day) and the average HU treatment time (4.23±2.01 years). Mean of baseline HbF was 12.01±6.05% and final HbF concentration 18.97±8.75%. While we found no significant differences at final HbF or delta HbF among genotypes of HMIP-2 polymorphisms, carriers of the minor alleles of rs9402686, rs4895441 and rs9494145 showed higher baseline HbF when compared to wild-type homozygous patients (all p 0.05). Moreover, carriers of the minor allele T (GT or TT genotypes) of rs1427407 showed higher baseline HbF (p = 0.002) and delta HbF (p = 0.049) when compared to GG homozygous patients. Similarly, carriers of the minor allele C (AC or CC genotypes) of rs766432 showed higher delta HbF when compared to AA homozygous patients (p = 0.009). Finally, carriers of the minor allele T (CT or TT genotypes) of rs7599488 showed higher final HbF (p = 0.003) and delta HbF (p = 0.002) when compared to CC homozygous patients. BCL11A is a negative regulator of HbF expression. BCL11A polymorphisms were associated with increased HbF after HU treatment in pediatric patients with SCA, suggesting they modulate HbF levels in response to HU. Understanding genetic factors underlying HU response may clarify the interpatient variability on HbF induction upon HU treatment and help to guide therapy.

Efficacy of Thalidomide Treatment in Children With Transfusion Dependent β-Thalassemia: A Retrospective Clinical Study.

Background: Thalidomide has been reported as a promising treatment for reducing transfusion volume in adults with β-thalassemia. However, the evidence about the safety and efficacy of thalidomide on children with transfusion dependent β-thalassemia (TDT) is scarce.Methods: Seventy-seven children with TDT treated with thalidomide at least for 6 months were included and retrospectively analyzed. Oral dose was started at 2.5 mg·kg-1·d-1. Blood volume for maintenance of hemoglobin above 90 g·L-1 compared with pre-treatment volume is the evaluation index for response.Results: After the sixth month treatment, 51/77 (66.2%) maintained Hb over 90 g·L-1 without transfusion. Adverse events were reported in 48 (63.2%) patients. Age, sex, genotype category, dosage, and transfusion interval before thalidomide treatment were not correlated to treatment response. The AUC was 0.806 for the HbF at the third month of treatment in predicting probability of major responders at the sixth month treatment. Based on Youden’s index algorithm in the ROC curve, 47.298 g·L-1 was the optimal cut-off value of the HbF at the third month of treatment in predicting major responders at the sixth month treatment, with sensitivity of 67.5% and specificity of 93.3%.Conclusions: The dose of thalidomide between 2.5 mg·kg-1·d-1 and 3.6 mg·kg-1·d-1 is effective in TDT children. Severe side effects are uncommon. HbF concentration of 47.298 g·L-1 at the third month is recommended as the predictor for further major responders.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.

Functional polymorphisms of BCL11A and HBS1L-MYB genes affect both fetal hemoglobin level and clinical outcomes in a cohort of children with sickle cell anemia.

Fetal hemoglobin (HbF) ameliorates clinical severity of sickle cell anemia (SCA). The major loci regulating HbF levels are HBB cluster, BCL11A, and HMIP-2 (HBS1L-MYB). However, the impact of noncoding single-nucleotide polymorphisms (SNPs) in these loci on clinical outcomes and their functional role on regulating HbF levels should be better elucidated. Therefore, we performed comprehensive association analyses of 14 noncoding SNPs in five loci with HbF levels and with clinical outcomes in a cohort of 250 children with SCA from Southeastern Brazil, and further performed functional annotation of these SNPs. We found SNPs independently associated with HbF levels: rs4671393 in BCL11A (β-coefficient = 0.28), rs9399137 in HMIP-2A (β-coefficient = 0.16), and rs4895441 in HMIP-2B (β-coefficient = 0.15). Patients carrying minor (HbF-boosting) alleles for rs1427407, rs93979137, rs4895441, rs9402686, and rs9494145 showed reduced count of reticulocytes (p < 0.01), while those carrying the T allele of rs9494145 showed lower white blood cell count (p = 0.002). Carriers of the minor allele for rs9402686 showed higher peripheral saturation of oxygen (p = 0.002). Patients carrying minor alleles in BCL11A showed lower risk of transfusion incidence rate ratio (IRR ≥ 1.3; p < 0.0001). This effect was independent of HbF effect (p = 0.005). Carriers of minor alleles for rs9399137 and rs9402686 showed lower risk of acute chest syndrome (IRR > 1.3; p ≤ 0.01). Carriers of the reference allele for rs4671393 showed lower risk of infections (IRR = 1.16; p = 0.01). In conclusion, patients carrying HbF-boosting alleles of BCL11A and HMIP-2 were associated with milder clinical phenotypes. Higher HbF concentration may underlie this effect.

Factors affecting health-related quality of life and its association with the Xmn1-Gγ polymorphism among adolescents with transfusion-dependent beta thalassemia and HbE/β-thalassemia in East Coast Malaysia

IntroductionXmn1-Gγ polymorphism has been significantly associated with disease severity in HbE/β-thalassemia patients. The aim of this study is to determine the factors affecting health related quality of life (HRQoL) and its association with Xmn1-Gγ polymorphism among transfusion-dependent β-thalassemia and HbE/β-thalassemia adolescents. Materials and MethodsA cross-sectional study was conducted from day care thalassemia centers and thalassemia clinics of tertiary centers in East Coast Malaysia. The Malay version of the Pediatric Quality of Life Inventory™ 4.0 Generic Core Scale was used to evaluate quality of life. Associated factors were analyzed using multivariate regression analysis. ResultsThe lowest QoL mean score was school functioning at 59.69 (16.23). Xmn1-Gγ polymorphism was significantly associated with physical functioning (p = 0.018) and emotional functioning (p = 0.013). Frequency of blood transfusion per year was found to be significantly associated with social functioning (p = 0.005) while gender and age onset of anemia were significantly associated with school functioning (p = 0.009 and p = 0.009). Xmn1 heterozygous CT was significantly associated with a higher physical functioning score by 0.192 and a higher emotional functioning score by 0.202 compared to Xmn1 homozygous wild-type CC. It is reportedly significantly higher concentration of HbF in patients with a “T” nucleotide change at the -158 locus at the Gγ-globin promoter compared to those without the polymorphism. ConclusionThe precise factors that influence HRQoL empowers a better understanding to the effectiveness of thalassemia disease management in the country.

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 &amp;gt;80% F-cells, and 17/36 (47%) had &amp;gt;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 &amp;gt;16 pg was associated with a mean hemoglobin &amp;gt;10 g/dL and mean absolute reticulocyte count &amp;lt;100x106/mm3. Bland-Altman analysis of total F-cells and F-retics showed a mean bias of 33.5 percentage points (total F-cells &amp;gt; F-retics), indicating the difference between F-cell production (F-retics) and peripheral survival advantage of F-cells. For F-cells &amp;gt;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.

Targeting Transcriptional Co-Activator PGC-1a for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is a FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). Transcriptional co-activator PGC-1α has recently been showed to play a crucial role in globin gene regulation and erythropoiesis by our group. Loss of function in the PGC-1α knock out mice significantly reduced the expression of murine embryonic εy- and βh1-globin genes; both of them are homologues of the human ε- and γ-globin genes. Forced overexpression of PGC-1α in vitroby adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. These results directly validate a role for PGC-1α in human γ-globin induction and provide significant support for the hypothesis that modulating PGC-1α activity, or the signaling pathways that it regulates, in SCD patients may be an effective approach that could therapeutically benefit individuals with SCD. Here we report the effects of PGC-1α in inducing HbF in human primary erythoid progenitor CD34+cells. We infected CD34+cells with lentivirus carrying PGC-1α and found that over-expression of PGC-1α resulted in significantly increased F-cells in the culture, from 1.6% up to 41.1%. We further discovered several PGC-1α agonists that are highly selective for PGC-1α. We found that one of the PGC-1α agonists, Compound SR-18292, induces PGC-1α mRNA expression in a dose dependent manner (2.6-fold at 1 μM, 5.7-fold at 10 μM) in cultured CD34+cells without effecting cell viability. Consistent with the increase of PGC-1α levels, the percentage of F-cells increased from 2.76% (vehicle control) to 32.1% when cells exposed to 1 µM SR-18292, which is comparable to that of 25 µM HU (32.2%). The mean fluorescence intensity of F-cells in SR-18292 treated cells was higher than in controls, suggesting that the treatment not only increased the number of F-cells but also increased the concentration of HbF in F-cells. The increased levels of PGC-1α reactivates the expression of fetal γ-globin genes resulting in significant HbF accumulation in adult erythrocytes, suggesting that the transcriptional co-activator PGC-1α comprises a new molecular target for possible therapeutic intervention in treating SCD. Disclosures No relevant conflicts of interest to declare.

BCL2L1 is associated with γ-globin gene expression

AbstractFetal hemoglobin (HbF) expression is partially governed by the trans-acting quantitative trait loci BCL11A and MYB and a cis-acting locus linked to the HBB gene cluster. Our previous analysis of the Genotype-Tissue Expression database suggested that BCL2L1 was associated with HbF gene expression. In erythroid progenitors from patients with sickle cell disease, BCL2L1 messenger RNA (mRNA) levels were positively correlated with HBG mRNA and total HbF concentration (r2 = 0.72, P = .047 and r2 = 0.68, P = .01, respectively). Inhibition of BCL2L1 protein activity in HbF-expressing HUDEP-1 cells decreased HBG expression in a dose-dependent manner. Overexpression of BCL2L1 in these cells increased HBG expression fourfold (P < .05) and increased F cells by 13% (P < .05). Overexpression of BCL2L1 in erythroid progenitors derived from primary adult CD34+ cells upregulated HBG expression 11-fold (P < .05), increased F cells by 18% (P < .01), did not significantly affect cell differentiation or proliferation, and had a minor effect on survival. Although the mechanism remains unknown, our results suggest that BCL2L1 is associated with HbF gene activation.

Clinical and Laboratory Benefits of Early Initiation of Hydroxyurea with Pharmacokinetic Guided Dosing for Young Children with Sickle Cell Anemia

Background: Hydroxyurea is now the standard of care for children with sickle cell anemia (SCA). Results from the BABY HUG study and recommendations from the 2014 NHLBI Guidelines have led to early initiation (increasingly before 1 year of age) of hydroxyurea for many patients. Given the known variability in hydroxyurea pharmacokinetics (PK), treatment response (HbF%), and maximum tolerated dose (MTD), we hypothesized that individualized dosing would provide the optimal treatment approach.Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154) is a prospective study of a personalized, PK-guided dosing model of hydroxyurea for children with SCA. Using population PK model-based Bayesian estimation, each participant's PK data are used to generate an individualized starting hydroxyurea dose that targets an area under the curve associated with actual MTD. Clinical follow-up and subsequent dose adjustments target MTD, usually defined by ANC<3.0x109/L. We analyzed clinical and laboratory data for TREAT participants who started hydroxyurea before 2 years of age, to allow for comparison to published results from BABY HUG, which included a similar young cohort but with conservative weight-based dosing of 20 mg/kg/day. TREAT participants had ongoing clinical and research evaluations of organ function, including transcranial doppler (TCD) studies, RBC pit counts, and cystatin C measurements.Results:The analysis of children starting hydroxyurea before 2 years of age included 33 participants (of 47 total TREAT enrollments), who contributed a total of 59.5 patient-years of hydroxyurea therapy. The mean age (±SD) at hydroxyurea initiation was 1.0±0.4 years of age. The average PK-guided, individualized starting dose was 27.8±5.3 mg/kg/day, higher than conventional and BABY HUG initial dosing (20 mg/kg/day). For children who have completed 12 months of therapy (n=24), effects in hematologic laboratory data are remarkable with average 35.9±8.9% HbF and hemoglobin concentration of 10.2±1.1 g/dL after 12 months of therapy (compared to 29.3±8.8% and 9.2±1.3 g/dL at baseline). The majority (70%) of these participants have HbF>30% and almost half achieved HbF>40% after 12 months of hydroxyurea. This hematological response is more robust than what was observed in BABY HUG (HbF=22.4%, Hb=9.1 g/dL after two years of therapy, Wang WC et al. Lancet 2011). In the TREAT cohort, there were no episodes of dactylitis, acute splenic sequestration, or stroke. There were 111 emergency room or sick outpatient clinic visits for this young cohort; 107 ED/clinic visits (without subsequent hospitalization) were for fever, URI symptoms, GI illness, or other non-specific complaints unrelated to SCA, while only 4 (3.6%) visits were for pain. There were 38 hospitalizations in 17 participants, mostly for routine evaluation of fever (66%), but no positive blood cultures and no admissions for febrile neutropenia. The average length of hospitalization was 2.8±2.4 days with 81% of participants discharged within 72 hours of admission. There were 3 episodes of acute chest syndrome in 2 patients, two of whom required PRBC transfusion. Including all types of visits, there were only 6 pain events, equivalent to 10.1 pain events per 100 patient-years, which is much lower than the published 94 events per 100 patient-years in the hydroxyurea treatment arm of BABY HUG (Thornburg CD et al. Blood 2012). There were 37 TCD exams performed in 16 participants, all normal except for one patient with conditional velocities that normalized with hydroxyurea. There were no significant differences from baseline to month 12 in either RBC pit counts or cystatin C values.Conclusions: Hydroxyurea initiation at an early age using PK-guided dosing provides significant clinical benefits for young children with sickle cell anemia. These TREAT study data suggest that initiating hydroxyurea around one year of life using a personalized dosing strategy can provide better clinical and laboratory benefits than starting at the conventional 20 mg/kg/day weight-based dose. Very high HbF levels are observed at modest and well-tolerated doses of hydroxyurea, perhaps because treatment was initiated before the process of HbF inactivation is complete. Continued long-term follow-up of these patients will determine whether these will be sustained and able to prevent both short- and long-term complications of SCA. DisclosuresMalik:CSL Behring: Patents & Royalties. Quinn:Silver Lake Research Corporation: Research Funding; Global Blood Therapeutics: Research Funding; Amgen: Research Funding. Ware:Biomedomics: Research Funding; Nova Laboratories: Consultancy; Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Global Blood Therapeutics: Other: advisory board; Agios: Other: advisory board; Novartis: Membership on an entity's Board of Directors or advisory committees.

XmnI polymorphism in Egyptian patients with β-thalassemia major and its correlation with the HbF level

Clinical severity of β-thalassaemia depends on the types of β-gene mutations involved. It can also be influenced by genetic factors like concomittant α-thalassaemia and increased γ-chain production. Several loci are implicated in higher production of HbF. The XmnI restriction site at −158 position of the Gγ-gene is associated with increased expression of the Gγ-globin gene and higher production of HbF. This study aims to determine the frequency of the Gγ XmnI polymorphism in β-thalassaemia patients in Egypt and its correlation to the HBF level and clinical severity of the disease. We investigated the XmnI polymorphism in 100 children with β-thalassaemia major using polymerase chain reaction (PCR-RFLP)-restriction fragment length polymorphism. We found that ninety-four children had XmnI (−/−) genotype (94%) and six children had XmnI (+/−) genotype. On the other hand, the study found that the presence of this polymorphism influences HbF concentration and ameliorate the clinical severity of the disease.

The ‑α3.7 deletion in α‑globin genes increases the concentration of fetal hemoglobin and hemoglobin A2 in a Saudi Arabian population

The regions of Al‑Qatif and Al‑Ahssa in the Eastern Province of Saudi Arabia are known for their high prevalence of hemoglobinopathies, including β‑thalassemia and sickle cell anemia. Previously, the α‑gene deletion has been demonstrated as highly prevalent among populations residing in these two regions. The present study was conducted in order to investigate the implications of the α‑globin gene deletion on fetal hemoglobin (HbF) and hemoglobin α2 (HbA2) concentrations in patients with transfusion‑dependent β‑thalassemia. A total of 166 Saudi patients with transfusion‑dependent β‑thalassemia and 337 healthy Saudi patients were included in the study. The ‑α3.7, ‑α4.2, -‑FIL, -‑SEA, -‑MED and -‑(20.5) gene deletions were identified using multiplex α‑globin deletion polymerase chain reaction. The present study revealed that the ‑α3.7 gene deletion is the most prevalent (43.5%) in the Saudi populations that were analyzed and is characterized by the deletion of 3,804 base pairs. Numerous genotypes, namely ‑3.7α2/α1α2, ‑3.7α2/α1α12, ‑3.7α2/‑3.7α2, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/α1‑4.2, ‑3.7α2/α1polyA‑1α2, ‑3.7α12/α1α12, ‑‑FIL/‑3.7α2 and ‑3.7α2/‑3.7α2Hb Villiers le Bel were also identified in the investigated population. Furthermore, a gradual increase in the concentration of HbF and HbA2 in patients with β‑thalassemia and the number of α‑gene deletions was demonstrated; whereas in healthy patients the level of HbA2 was demonstrated to decrease as the number of α‑gene deletions increased. Therefore, it can be concluded that the high HbF concentration in the present study is predominantly associated with other mutations associated with β‑thalassemia rather than α‑globin deletions. Furthermore, the results of the present study also revealed novel α‑gene deletion genotypes prevalent in the population studied, namely α1α2/α1α2HphI, α1α2HphI/α1α2HphI, α1α2/α1α2Hb Handsworth, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/‑3.7α2Hb Villiers le Bel and ‑-MED/α1α2HphI.

Influence of Genetic Modifiers on Levels of Invariant Natural Killer T-Cells in Patients with Sickle Cell Anemia

Although patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been associated with five different beta S-globin gene cluster haplotypes (βS-haplotypes) that show different clinical expression. Because genetic modifiers can modulate treatment response, we hypothesized that βS-haplotypes can affect levels of invariant natural killer T (iNKT) cells, which are known to play a key role in the pathogenesis of SCD, and are being considered as a potential target to treat acute crises in SCD patients. Herein we evaluated the impact of βS-haplotypes and HbF concentration on iNKT cell and dendritic cell (DC) subsets in a well-defined group of patients with SCD in a steady state. Sickle cell anemia patients were selected based on the history of patients, clinical examination and hematological findings. The βS-haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism analysis for seven restriction sites. The iNKT cell subsets were characterized by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells using flow cytometry and cell culture. The myeloid DC (mDC) and plasmacytoid DC (pDC) cells were identified by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells by flow cytometry. Comparisons among βS-haplotypes were performed using ANOVA and unpaired t test, while the Spearman’s correlation was used to assess associations. Among the 125 sickle cell anemia patients studied, Benin haplotype (40%) was the most common followed by Bantu (21%), Arab/Indian (18%), Atypical (12%) and Benin/Bantu or Benin/Arab-Indian or Bantu/Arab-Indian (9%). The majority of subjects with Benin/Benin βS-haplotype had a severe to moderate clinical profile similar to Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.23). A trend toward increased levels of CD3iNKT, CD4iNKT and CD8iNKT cell subsets was observed in the subjects with the Benin/Benin βS-haplotype, when compared to other βS-haplotype groups (P=0.06). Interestingly, subjects with the Benin/Benin βS-haplotype exhibited slightly higher levels of Th1-like cells, but not Th2-like and Th17-like cells, when compared to subjects with the Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.047). Comparisons between the levels of mDC and pDC cell subsets, as well as the expression of CD1d on these DC cells, showed no statistically significant differences among the βS-haplotype groups (P=0.42). Similarly, levels of iNKT cell subsets, Th1-like, Th2-like, and Th17-like cells as well as DC subsets were similar among patients who received or not hydroxyurea therapy, independently of βS-haplotype groups (P= 0.09). Likewise, coexistence of high HbF and βS-haplotypes did not show any significant association with iNKT cell subsets. Collectively, our findings suggest that neither the βS-haplotypes or HbF levels nor the hydroxyurea therapy or clinical severity appeared to be associated with iNKT cell or DC cell subsets in patients with SCD. The effects of other genetic modifiers such as alpha/beta-thalassemia and G6PD deficiency on iNKT cell subsets need to be evaluated in future studies. DisclosuresNo relevant conflicts of interest to declare.

Severity of Brazilian sickle cell disease patients: severity scores and feasibility of the Bayesian network model use.

The integration of the several clinical and laboratory dimensions and the influence of each parameter on the sickle cell disease (SCD)-related mortality is useful for predicting the phenotype of an individual. This study evaluated the feasibility of the SCD severity calculator use to measure disease severity in Brazilian patients. The study group was composed of 500 SCD patients (440 HbSS and 60 HbSC) diagnosed by molecular biology. We observed a decrease in severity scores in 72 SCD patients assessed before and after the hydroxyurea (HU) use. Furthermore, the HU influenced the increase of mean corpuscular volume (MCV) and HbF concentration, and the decrease of leukocytes and total bilirubin. We found 180 (36.0%) patients with intermediate phenotype, 170 (34.0%) mild phenotype and 150 (30.0%) with severe phenotype. Patients with ages >40 years had higher mean score (0.778±0.177) than patients between 18 and 40 years (0.562±0.152) and patients between 5 and 17 years (0.322±0.145). We observe that there is a tendency of individuals with leg ulcers, avascular necrosis and cardiac complications with increasing age. Correlation analysis showed relations between severity scores with leukocytes, reticulocytes, bilirubin, lactate dehydrogenase, HbS, hemoglobin and hematocrit (p<0.05). Several comparisons involving age groups, SCD genotype and phenotypic classification had satisfactory results and this classification will be used for future studies involving genetic polymorphisms, response to treatment with HU and oxidative stress markers in SCD.