Low HbF Levels Research Articles

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

Molecular understanding of unusual HbE-β+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels

Background Low HbF expression in HbE-β+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the β- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-β+-thalassemia from HbE heterozygotes have been investigated. Participants and Methods A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize β- and α-globin genes. DNA polymorphism at position −158 nt 5′ to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-β+-thalassemia from HbE heterozygotes were determined. Results Five β+-thalassemia mutations trans to βE-gene (β−87(C>A), β−31(A>G), β−28(A>G), β19(A>G), and β126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000–1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden’s index for differentiating HbE-β+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-β+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at −158 nt 5′ to Gγ-globin was found to be negatively associated with HbF expression. Conclusions HbE-β+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-β+-thalassemia diagnosis.

High Performance Liquid Chromatographic Pattern of Children with Sickle Cell Anaemia in Enugu Nigeria

Aim: To identify the quantity of haemoglobin (Hb) ‘S’(HbS), ‘A2’(HbA2), ‘F’(HbF) and other Hb phenotypes of sickle cell anaemia (SCA) children in Enugu Nigeria using High Performance Liquid Chromatography(HPLC).
 Introduction: The inheritance of two abnormal Hb genes one of which must be the Hb ‘S’ gene result in Sickle cell disease (SCD). Co-inheritance of two Hb S genes results in homozygous SCD/SCA), coinheritance of Hb ‘S’ with Hb ‘C’ gene gives HbSC disease and with β-thalassemia allele gives HbSβ-thalassemia (Hb S/β Th). SCA is the most common and most severe of the SCD. The phenotypic expressions of Hb vary unpredictably in the same individual and contribute to the varied clinical severity of SCA among other variables. It is therefore imperative to characterize the Hb variants in children with SCA for proper risk stratification necessary for best outcome management. HPLC is the most validated method for screening, detection and quantification of various Hb subtypes.
 Methodology: A cross-sectional, descriptive study involving 75 SCA children aged 6 months to 17years, on follow-up at the clinic. Patients on hydroxyurea or who received blood transfusion within the previous four months were excluded. Following due ethical protocols, the D-10 HPLC machine (BIO-RAD D-10) was used to identfy the Hb phenotype in venous blood samples based on their ionic gradients and quantify them by the principle of variable absorbance. The participants’ sociodemographic data were recorded. Participants were grouped into 3 socioeconomic classes (SECs) as proposed by Oyedeji.
 Results: There were 48 females (62.7%) and 27 males (37.3%) in age range 6 months – 17 years in lower (16.0%), middle (57.3%) and upper (26.7%) SECs. Majority had HbF below 10% (46.7%), HbS above 80% (43%) and HbA2 of 4% and below (84%). No other Hb variant was identified. The proportion with HbS/HbA2/HbF levels suggestive of beta thalassemia was 16%, 25% males compared to 10.6% females. Females had higher HbF levels while males had higher HbS and HbA2 levels. However the gender differences in HbF, HbS, HbA2 and SECs did not attain statistical significance. A significant negative relationship was found between age and HbF (r = -.424, p < .001) while a significant positive relationship between age and HbS (r = .287, p = .013) and between age and HbA2 (r = .265, p = .022).
 Conclusion: Irrespective of gender, high HbS and low HbF levels at direct variance (HbS) and indirect variance (HbF) with age may be found in children with SCA. Observed Hb phenotypes suggest co-existent β- thalassaemia in this subset of southeast Nigerian SCA children.

BORN study: a multicenter randomized trial investigating cord blood red blood cell transfusions to reduce the severity of retinopathy of prematurity in extremely low gestational age neonates

BackgroundExtremely low gestational age neonates (ELGANs, i.e., neonates born before 28 weeks of gestation) are at high risk of developing retinopathy of prematurity (ROP), with potential long-life visual impairment. Due to concomitant anemia, ELGANs need repeated red blood cell (RBC) transfusions. These produce a progressive replacement of fetal hemoglobin (HbF) by adult hemoglobin (HbA). Furthermore, a close association exists between low levels of HbF and severe ROP, suggesting that a perturbation of the HbF-mediated oxygen release may derange retinal angiogenesis and promote ROP.Methods/designBORN (umBilical blOod to tRansfuse preterm Neonates) is a multicenter double-blinded randomized controlled trial in ELGANs, to assess the effect of allogeneic cord blood RBC transfusions (CB-RBCs) on severe ROP development. Recruitment, consent, and randomization take place at 10 neonatology intensive care units (NICUs) of 8 Italian tertiary hospitals. ELGANs with gestational age at birth comprised between 24+0 and 27+6 weeks are randomly allocated into two groups: (1) standard RBC transfusions (adult-RBCs) (control arm) and (2) CB-RBCs (intervention arm). In case of transfusion need, enrolled patients receive transfusions according to the allocation arm, unless an ABO/RhD CB-RBC is unavailable. Nine Italian public CB banks cooperate to make available a suitable amount of CB-RBC units for all participating NICUs. The primary outcome is the incidence of severe ROP (stage 3 or higher) at discharge or 40 weeks of postmenstrual age, which occurs first.DiscussionBORN is a groundbreaking trial, pioneering a new transfusion approach dedicated to ELGANs at high risk for severe ROP. In previous non-randomized trials, this transfusion approach was proven feasible and able to prevent the HbF decrease in patients requiring multiple transfusions. Should the BORN trial confirm the efficacy of CB-RBCs in reducing ROP severity, this transfusion strategy would become the preferential blood product to be used in severely preterm neonates.Trial registrationClinicalTrials.gov NCT05100212. Registered on October 29, 2021

Early Effects of a Repurposed Therapeutic PB-04 in Beta Thalassemia Intermedia and in Transgenic Sickle Mice

Background: Biochemical, epidemiologic, genetic, and clinical data over 7 decades have shown that renewed or persistent expression of fetal globin reduces clinical severity in beta-thalassemias (BTI) and hemoglobinopathies (sickle cell disease, SCD). Hydroxyurea (HU) and epigenetic therapies which act through HDAC and DNMT-1 inhibition have demonstrated beneficial clinical activity; butyrate, a Class I HDAC inhibitor which suppresses BCL11A, increased HbF by 3-fold and total hemoglobin (Hb) by 1 gm/dl in SCD and eliminated transfusions in beta thalassemia patients, and decitabine strongly induces HbF, increases total Hb, and reduces biomarkers of sickling. Active erythropoiesis, basal EPO levels, available iron, and select mutations or modifiers are associated with responses. Additional small molecule therapies, especially those with additive activity with HU, are considered desirable for many patients world-wide. Methods: Seeking oral noncytotoxic therapeutic candidates with low development risks, we utilized a high-throughput screen with a gamma globin promoter-reporter and cytotoxicity readout for unrecognized fetal globin-inducing activity in libraries of all FDA- and EMA-approved or clinical stage therapeutics (for any medical indication). PB-04, a candidate with a benign long-term safety profile (used to inhibit L-dopa metabolism in Parkinson's disease), does not inhibit proliferation of erythroid progenitors and suppresses or displaces 4 repressors of the fetal globin gene promoter, BCL11A, HDAC3, KLF1, LSD-1, indicating targeted molecular activity. PB-04 has additive effects with HU in inducing F-cells in sickle cell erythroid progenitors. PB-04 was evaluated in 3 predictive animal models and in 3 escalating dose cohorts in adult subjects with BTI. Results: In 3 animal models, PB-04 increased fetal globin mRNA by 12-to 20-fold and F-cells by 20% in anemic baboons, F-cells and mean Hb F/cell by 2 to 7-fold in beta-YAC mice, and fetal globin mRNA by 1.8-fold and % F-cells by 2.4-fold without toxicity in Townes SCD mice, similar to the changes induced by HU (both given 5 days/wk for 4 wks). In six beta thalassemia (BTI) subjects who completed ten 12-week courses at 1, 3, or 5 mg/kg doses given once/day, 3 times/wk, increases from baseline in F-reticulocytes (mean 5.2-fold, range 1.5-15.7), F-cells, (mean 4.4-fold, range 2.3 to 7.3-fold), and HbF/cell (MFI), (mean 6.6-fold, range 3 to 11.3-fold), were observed (p<0.03 for all parameters). Increases were observed even in subjects with low baseline HbF levels. HbF increases by HPLC were 2.7 to 14%. Treatment emergent adverse effects (TEAEs) have been Gr 1-2 and largely transient in these subjects receiving 12 weeks' therapy. Other dosing regimens are being investigated. Conclusions: Initial studies in beta globin disorders indicate PB-04 induces fetal globin protein expression assayed by F-reticulocytes, F-cells, and mean HbF/cell in BTI patients and in transgenic SCD mice without toxicity. These findings indicate PB-04, which has complementary activity to all the approved SCD therapeutics, merits clinical evaluation in patients with sickle cell disease.

Effects of fetal haemoglobin on systemic oxygenation in preterm infants and the development of retinopathy of prematurity PacIFiHER Report No. 2

Background/aimsFetal haemoglobin (HbF) has an oxyhaemoglobin dissociation curve that may affect systemic oxygenation and the development of retinopathy of prematurity (ROP). The study aim is to characterise the effects of...

Unique Polymorphisms at BCL11A, HBS1L-MYB and HBB Loci Associated with HbF in Kuwaiti Patients with Sickle Cell Disease.

Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10–44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (β = −1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.

Priapism in sickle cell disease: Associations between NOS3 and EDN1 genetic polymorphisms and laboratory biomarkers.

Priapism is a urologic emergency characterized by an uncontrolled, persistent and painful erection in the absence of sexual stimulation, which can lead to penile fibrosis and impotence. It is highly frequent in sickle cell disease (SCD) associated with hemolytic episodes. Our aim was to investigate molecules that may participate in the regulation of vascular tone. Eighty eight individuals with SCD were included, of whom thirty-seven reported a history of priapism. Priapism was found to be associated with alterations in laboratory biomarkers, as well as lower levels of HbF. Patients with sickle cell anemia using hydroxyurea and those who received blood products seemed to be less affected by priapism. Multivariate analysis suggested that low HbF and NOm were independently associated with priapism. The frequency of polymorphisms in genes NOS3 and EDN1 was not statistically significant between the studied groups, and the presence of the variant allele was not associated with alterations in NOm and ET-1 levels in patients with SCD. The presence of the variant allele in the polymorphisms investigated did not reveal any influence on the occurrence priapism. Future studies involving larger samples, as well as investigations including patients in priapism crisis, could contribute to an enhanced understanding of the development of priapism in SCD.

Novel Genetic Loci That Influence Fetal Hemoglobin Expression in Children with Sickle Cell Anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p&amp;lt;0.05) in both datasets were then selected for two independent replication steps; these were designated either African-American (N=157) or African (N=269). Third, the SNVs that were significant in both dual discovery and at least one of the replication cohorts were then verified using an additional Caribbean cohort (N=149) with TaqMan techniques for genotyping specific variants. Through this multistep process, we searched for genomic loci with consistent HbF associations across multiple cohorts. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p&amp;lt;10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p&amp;lt;0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p&amp;lt;0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Expected HbF associations with BCL11A (rs1427407) and HBS1L-MYB (rs4895441) were identified. Among other 18 novel candidate variants, the rs77737207 variant (allele frequency ~0.10) near the RUNX1T1 locus was strongly associated with lower HbF levels, while coding variant rs2279587 (allele frequency ~0.03) in the ITGA1 gene approached statistical significance (p&amp;lt;0.08) in the final verification cohort and was associated with higher levels of HbF. Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

Investigation of miR-144-3p expression levels in HbSS cases with high and normal HbF

Abstract Background Sickle cell anemia (HbSS) is a hereditary blood disease that affects millions of people worldwide. Increased the HbF levels affects the clinical course of HbSS positively. The aim of this study was to investigate the association between miR-144-3p and HbSS. Materials and methods In this study 84 cases (44 HbSS/HbS combination and 40 HbAA) were performed. The expression of miR-144-3p was determined by RT-PCR. Statistical analysis was performed by the Mann-Whitney U test (SPSS 20.00 for Windows and p&lt;0.005). Results The miR-144-3p expression levels were higher in the HbSS cases (p≤0.001). Additionally, it was determined that the expression of miR-144-3p was higher in the cases with HbF&lt;3 (p=0.043). Discussion In our study, the increase in the miR-144-3p levels in low HbF levels may be associated with the severity of the disease. Conclusion Considering these results, suppressing miR-144-3p may be considered as a new treatment option in HbSS.

Optical coherence tomography (OCT) and OCT angiography allow early identification of sickle cell maculopathy in children and correlate it with systemic risk factors.

To determine the presence of sickle cell retinopathy and maculopathy and to identify associations between markers of hemolysis and systemic and ocular manifestations in children affected by sickle cell disease. Eighteen children with sickle cell disease, aged 5-16 years, underwent complete eye examination including best-corrected visual acuity, slit-lamp biomicroscopy, ophthalmoscopy after pharmacological mydriasis, spectral-domain optical coherence tomography (SD-OCT), and optical coherence tomography angiography (OCTA). Blood test results and clinical history information were collected for each child, including fetal hemoglobin (HbF), hemoglobin (Hb), hematocrit (Htc), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), reticulocytes percentage (%ret), lactic dehydrogenase (LDH), total and direct bilirubin, glomerular filtration rate, number of painful crises, acute chest syndromes, and splenic sequestration. Therapeutic regimen and transfusion therapy were also evaluated. Sixteen of 36 eyes (44.4%) had non-proliferative sickle cell retinopathy on ophthalmoscopic evaluation. No patients had proliferative sickle cell retinopathy. In 13 of 36 eyes (36.1%), SD-OCT and OCTA detected signs of sickle cell maculopathy. Nine eyes (25%) presented sickle cell retinopathy and maculopathy, 7 eyes (19.4%) sickle cell retinopathy alone, and 4 eyes (11.1%) sickle cell maculopathy alone. A statistically significant association was found between sickle cell retinopathy; lower levels of HbF, Hb, and Htc; and higher MCV and percentage of reticulocytes. Sickle cell maculopathy was associated with lower values of H and Htc and higher levels of reticulocytes and total bilirubin. We identified early signs of sickle cell retinopathy and maculopathy in a pediatric population with SD-OCT and OCTA. These two retinal complications were more frequent in children with higher hemolytic rates.

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania

BackgroundSickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation.MethodsWe investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1.ResultsResults revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis.ConclusionsThis study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.

Lower foetal haemoglobin levels at 31- and 34-weeks post menstrual age is associated with the development of retinopathy of prematurity : PacIFiHER Report No. 1 PacIFiHER Study Group (Preterm Infants and Fetal Haemoglobin in ROP).

Previous studies have suggested that lower mean foetal haemoglobin (HbF) levels is associated with an increased risk for developing retinopathy of prematurity (ROP). Lower HbF levels may lead to high oxygen exposure to the developing retina thereby increasing the risk of acute ROP. In this study, we characterize the temporal relationship of HbF levels and the development of ROP. This is a single institution prospective observational cohort study. Preterm infants (born <31 weeks gestational age or <1500 g) with HbF measured at birth (cord blood), 31-, 34-, and 37-weeks post menstrual age (PMA); and at least one ROP exam, were enrolled. A total of 60 preterm infants (28 females, 47%) were enrolled. At 31-, 34-, 37-weeks PMA, infants with ROP (mild = Type 2 or less severe and severe = Type 1 ROP) had statistically lower percentages of HbF than infants with no ROP (28.2 ± 15 and 9.7 ± 2.9 vs 67.1 ± 29.6; p < 0.0001; 23.3 ± 14.7 and 32.5 vs 60.1 ± 25; p < 0.005; 31.9 ± 15.8 and 41.6 vs 60.2 ± 20.0; p < 0.0019). Infants with HbF levels in the lowest tercile at 31-weeks PMA were 7.6 times more likely to develop mild and severe ROP (95% CI 2.1-24.0, p value = 0.0006) and this risk increased to 12.3 times (95% CI: 2.6-59.0, p value = 0.0017) at 34-weeks PMA. Low HbF levels at 31- and 34-weeks PMA are associated with significantly increased risk of developing ROP. The decrease in HbF precedes the development of ROP and may be important in its pathogenesis.

Microfluidic assessment of red blood cell mediated microvascular occlusion.

Abnormal red blood cell (RBC) deformability contributes to hemolysis, thrombophilia, inflammation, and microvascular occlusion in various circulatory diseases. A quantitative and objective assessment of microvascular occlusion mediated by RBCs with abnormal deformability would provide valuable insights into disease pathogenesis and therapeutic strategies. To that end, we present a new functional microfluidic assay, OcclusionChip, which mimics two key architectural features of the capillary bed in the circulatory system. First, the embedded micropillar arrays within the microchannel form gradient microcapillaries, from 20 μm down to 4 μm, which mimic microcapillary networks. These precisely engineered microcapillaries retain RBCs with impaired deformability, such that stiffer RBCs occlude the wider upstream microcapillaries, while less stiff RBCs occlude the finer downstream microcapillaries. Second, the micropillar arrays are coupled with two side passageways, which mimic the arteriovenous anastomoses that act as shunts in the capillary bed. These side microfluidic anastomoses prevent microchannel blockage, and enable versatility and testing of clinical blood samples at near-physiologic hematocrit levels. Further, we define a new generalizable parameter, Occlusion Index (OI), which is an indicative index of RBC deformability and the associated microcapillary occlusion. We demonstrate the promise of OcclusionChip in diverse pathophysiological scenarios that result in impaired RBC deformability, including mercury toxin, storage lesion, end-stage renal disease, malaria, and sickle cell disease (SCD). Hydroxyurea therapy improves RBC deformability and increases fetal hemoglobin (HbF%) in some, but not all, treated patients with SCD. HbF% greater than 8.6% has been shown to improve clinical outcomes in SCD. We show that OI associates with HbF% in 16 subjects with SCD. Subjects with higher HbF levels (HbF > 8.6%) displayed significantly lower OI (0.88% ± 0.10%, N = 6) compared with those with lower HbF levels (HbF ≤ 8.6%) who displayed greater OI (3.18% ± 0.34%, N = 10, p < 0.001). Moreover, hypoxic OcclusionChip assay revealed a significant correlation between hypoxic OI and subject-specific sickle hemoglobin (HbS) level in SCD. OcclusionChip enables versatile in vitro assessment of microvascular occlusion mediated by RBCs in a wide range of clinical conditions. OI may serve as a new parameter to evaluate the efficacy of treatments improving RBC deformability, including hemoglobin modifying drugs, anti-sickling agents, and genetic therapies.

PB2402 BETA‐GLOBIN GENE CLUSTER HAPLOTYPES ASSOCIATED WITH HBF VARIATION IN β‐THALASSEMIA CARRIERS OF PORTUGUESE ORIGIN

Background:Genetic association studies showed that HbF levels variation in haemoglobinopathies, such as sickle cell disease and β‐thalassemia (β‐thal), are under the influence of single nucleotide polymorphisms (SNPs) in several genomic loci. The first described polymorphism influencing gamma globin gene expression was XmnI in HBG2 promoter −158C &gt; T (rs7482144). Several reports showed that β‐thal carriers with HbF raised levels have a significantly higher rs7482144 minor allele frequency than patients with low HbF levels. The functional significance of HBG2‐XmnI mutation has never been clearly defined and it is possible that it reflects a marker in linkage disequilibrium (LD) with other functional polymorphism elsewhere in the β‐globin gene cluster. It has been suggested that the role assigned to XmnI could be played by the rs16912979 A &gt; G polymorphism in the palindromic region of 5′HS4 β‐globin locus control region (Neishabury et al., Blood Cells Mol Dis. 2012;48:1‐5).Aims:The main goal of this work was to define haplotype backgrounds in β‐globin gene cluster that could explain HbF variation in a population sample of β‐thal carriers.Methods:We studied 79 Portuguese β‐thal carriers (39 males, 40 females), aged between 2 to 70 years old, with HbF levels ranging from 0.2% to 5.1%. HbA2 levels were 3.4 to 5.8% (mean 4.71). Six different HBB mutations were identified at heterozygous state, the two most commons were c.92+6T &gt; C [IVSI‐6 (T &gt; C)] (n = 30) and c.118C &gt; T [CD39 CAG &gt; TAG)] (n = 23). The remaining mutations were c.48G &gt; A [CD15 TGG &gt; TGA] (n = 11), c.92 + 1G &gt; A [IVS‐I‐1 (G &gt; A)] (n = 10), c.93‐21G &gt; A [IVSI‐110 G &gt; A] (n = 3) and c.20delA [CD 6 ‐A] (n = 2). HbA2 and HbF levels were determined by HPLC. The two SNPs rs16912979 and rs7482144 were genotyped by PCR‐RFLP and the HBB gene mutations were identified by Sanger sequencing. Associations of SNPs with HbF levels were assessed by linear regression models using PLINK. Informed consent was provided by all the participants.Results:The two SNPs HBG2‐XmnI rs7482144 and 5′HS4 rs16912979 were in moderate LD (r2 = 0.65): from a total of 41 rs16912979 G alleles, 73.2% were linked to an rs7482144 T‐allele. Linear regression, in the additive model, showed significant association between the XmnI rs7482144 minor T‐allele and increased levels of HbF (β = 0.648; P = 0.0004). A marginal association was found between the 5’HS4 rs16912979 minor G‐allele and HbF (β = 0.648; P = 0.039), however, no significant association was found after conditioning on XmnI rs7482144 (P = 0.192). The common mutation HBB: c.118C &gt; T [CD39] was found strongly associated with increased levels of HbF (β = 0.948; P = 6.4e‐5), remaining significant after conditional analysis on XmnI rs7482144 (P = 0.0002). Haplotypes combining the HbF‐associated alleles on SNPs rs16912979 and rs7482144 (haplotype GT) showed a significant association with raised levels of HbF (P = 0.0008), explaining 13.7% of HbF levels variation. Combining rs16912979, rs7482144 and mutation HBB: c.118T, three haplotypes were found significantly associated with HbF: GTT (β = 1.346; P = 0.00089), ACT (β = 0.784; P = 0.017) and GTC (β = 0.492; P = 0.032), explaining 13.4%, 7.2% and 5.9% of HbF levels variance, respectively.Summary/Conclusion:Our data suggest that in Portuguese β‐thal carriers the HBG2‐XmnI polymorphism and HBB: c.118C &gt; T [CD39] mutation are associated with HbF levels, showing independent effects. The marginally significant effect on HbF levels variation of SNP 5’HS4 rs16912979 appear to be mediated by the effect of XmnI polymorphism.

Hemoglobinopathies in the Fetal Position

(N Engl J Med. 2018;379:1675–1677) Hemoglobinopathies are disorders caused by mutations in genes that encode globins and include sickle cell disease and β-thalassemia. It has been reported that adults with these hemoglobinopathies who have comparatively high levels of fetal hemoglobin (HbF) have better clinical outcomes than those with low levels of HbF. While drugs like hydroxyurea increase HbF levels, thereby reducing the severity of symptoms of hemoglobinopathies, a drug that more effectively increases HbF levels is the holy grail of hemoglobinopathy research.

Functional Analysis of the FOXO3 Gene on the Induction of Fetal Hemoglobin in K562 Cells

The β-hemoglobinopathies are known as genetic disorders of high impact and wide distribution worldwide, with chronic and variable clinical prognosis. These pathologies have their symptoms and sequelae alleviated by increased levels of fetal hemoglobin (HbF; α2γ2). The Hydroxyurea, a drug used for the treatment of β-hemoglobinopathies, is effective for the maintenance and control of the development of the main clinical manifestations, however, it needs some prerequisites for its use, which indicates the need for new therapeutic approaches for this group of individuals. The family of Forkhead Box O transcription factors (FOXO), composed of FOXO1, FOXO3, FOXO4 and FOXO6, plays a key role in the regulation of several biological processes such as: regulation of the cell cycle, modulation oxidative stress, regulation mechanisms of response to DNA damage, controlling apoptosis and inflammatory response. Among members of the FOXO family, FOXO3 has been described as a physiological regulator of the erythroid maturation process and indicated as a positive regulator of HbF levels. In the present work, we evaluated the expression levels of the FOXO3 gene in patients with hemoglobinopathies and, in an attempt to better understand the pathophysiology of the disease, we performed a subcloning of the FOXO3 gene into a lentiviral expression system for its constitutive expression in vitro and subsequent evaluation of its activity as a possible transcription factor for γ globin genes (HBG1 and HBG2). In our clinical study, FOXO3 expression was significantly higher in patients with sickle cell anemia when compared to healthy donors (HbAA) (P <0.001) . In addition, a differential expression of FOXO3 was associated with the development of lower limb ulcer (P <0.005). Although we observed a tendency to increase FOXO3 expression in patients with 2 or more clinical manifestations, this association was not statistically significant (P = 0.06). The same was observed when we compared the number of clinical complications presented by patients last year with HbF levels: patients with 2 or more complications had lower levels of HbF, although this difference did not reach statistical significance (P = 0.182). There was no correlation between FOXO3 expression levels and HbF dosage (r = -0.12). Because of the activity of the FOXO3 gene in the control of oxidative stress, experiments were performed to evaluate the ROS and NO rates in the cell culture and a significant role was found, the FOXO3 increasing NO production (P = 0.0001) as well as in control of ROS produced in mitochondria. DisclosuresNo relevant conflicts of interest to declare.

Proteomic analysis of plasma from children with sickle cell anemia and silent cerebral infarction.

Silent cerebral infarction is the most common neurological abnormality in children with sickle cell anemia, affecting 30-40% of 14 year olds. There are no known biomarkers to identify children with silent cerebral infarcts, and the pathological basis is also unknown. We used an unbiased proteomic discovery approach to identify plasma proteins differing in concentration between children with and without silent cerebral infarcts. Clinical parameters and plasma samples were analysed from 51 children (mean age 11.8 years, range 6-18) with sickle cell anemia (HbSS). A total of 19 children had silent cerebral infarcts and 32 normal MRI; the children with silent infarcts had lower HbF levels (8.6 vs. 16.1%, P=0.049) and higher systolic blood pressures (115 vs. 108.6, P=0.027). Plasma proteomic analysis showed 13 proteins increased more than 1.3 fold in the SCI patients, including proteins involved in hypercoagulability (α2-antiplasmin, fibrinogen−γ chain, thrombospondin-4), inflammation (α2-macroglobulin, complement C1s and C3), and atherosclerosis (apolipoprotein B-100). Higher levels of gelsolin and retinol-binding protein 4 were also found in the population with silent infarcts, both of which have been linked to stroke. We investigated the genetic basis of these differences by studying 359 adults with sickle cell disease (199 with silent cerebral infarcts, 160 normal MRIs), who had previously undergone a genome-wide genotyping array. None of the genes coding for the differentially expressed proteins were significantly associated with silent infarction. Our study suggests that silent cerebral infarcts in sickle cell anemia may be associated with higher systolic blood pressure, lower HbF levels, hypercoagulability, inflammation and atherosclerotic lipoproteins.

Genotyping of BCL11A and HBS1L-MYB Single Nucleotide Polymorphisms in β-thalassemia/HbE and Homozygous HbE Subjects with Low and High Levels of HbF

Whether multiple single nucleotide polymorphisms (SNPs) of BCL11A and HBS1L-MYB genes affect hemoglobin (Hb)F production and hematological parameter variation in β-thalassemia/HbE and homozygous HbE in Thai subjects with low and high HbF levels are still unclear. Three SNPs of BCL11A gene (rs1427407, rs10189857 and rs11886868) and 3 SNPs of HBS1L-MYB gene (rs4895441, rs9399137 and rs28384513) were analyzed in 45 β-thalassemia/HbE patients who had HbF levels lower and higher than 15 %, and in 50 homozygous HbE who had HbF levels lower and higher than 5 %. Their hematological parameters were measured using automated blood counter. The HbF level was analyzed using high performance liquid chromatography (HPLC). There were no statistical significant differences of allele and genotype frequencies of 3 SNPs in the BCL11A gene between the groups of β-thalassemia/HbE patients and homozygous HbE subjects with low and high HbF levels. Significant differences in the allele frequencies in HBS1L-MYB SNP rs4895441 (p = 0.041) and rs9399137 (p = 0.048) were observed in homozygous HbE subjects with HbF £ 5 % and &gt; 5 %. Moreover, significant differences in MCV (p = 0.005) and trends towards significant differences in MCH (p = 0.057) and HbF levels (p = 0.051) were found in HBS1L-MYB SNP rs9399137 of homozygous HbE subjects. Therefore, the HBS11L-MYB SNPs especially rs9399137 had an effect on HbF production and the variation of hematological parameters in homozygous HbE subjects, but not in β-thalassemia/HbE patients.

MiR‐16 Mediated MYB Gene Silencing Induces Fetal Hemoglobin Expression

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the β‐globin gene affecting about 100,000 people in the United States and millions worldwide. Individuals living with this disease suffer from hemolytic anemia, progressive organ damage, and acute painful episodes. Fetal hemoglobin (HbF), a major genetic modifier of clinical presentation and progression of SCD, is of great interest due to its ability to inhibit sickle hemoglobin (HbS) polymerization and reducing erythrocyte damage. Variation in HbF levels among sickle cell patients is highly heritable and has been linked to the presence of single nucleotide polymorphisms (SNPs) in several genetic regulatory regions. In this study, DNA isolated from two cohorts of African American SCD patients with high and low HbF levels were analyzed by PCR‐based SNP analysis in the HBB, BCL11A, and HBS1L‐MYB (HMIP) gene regions. The presence of SNP alleles was then correlated with their clinical phenotype data to identify those associated with increased levels of HbF. Three SNPs in the BCL11A and HBB gene regions were identified in the high HbF patient cohort that may contribute to milder phenotypes in African Americans. To complement the SNP studies, genome wide microRNA (miRNA) data was generated which showed increased miR‐16 levels associated with high HbF. Furthermore, MYB, the negative regulator of γ‐globin expression, is silenced by miR‐16 through binding the 3′‐untranslated region. RT‐qPCR and western blot were used to measure γ‐globin mRNA and HbF protein levels respectively after transfection of miR‐16 mimic into KU812 cells. Preliminary data revealed dose dependent miR‐16 mediated MYB suppression and γ‐globin activation. The discovery of regulatory regions that modulate HbF expression and miRNA mediated suppression of negative regulators of γ‐globin, provides expanded understanding of gene regulation, which can be used to define novel treatment options for individuals with SCD.Support or Funding InformationSponsored by the Medical College of Georgia Medical Scholars Program