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The clinical severity of sickle cell disease (SCD) is strongly influenced by the level of fetal haemoglobin (HbF) persistent in each patient. Three major HbF loci (BCL11A, HBS1L-MYB, and Xmn1-HBG2) have been reported, but a considerable hidden heritability remains. We conducted a genome-wide association study for HbF levels in 1006 Nigerian patients with SCD (HbSS/HbSß0), followed by a replication and meta-analysis exercise in four independent SCD cohorts (3,582 patients). To dissect association signals at the major loci, we performed stepwise conditional and haplotype association analyses and included public functional annotation datasets. Association signals were detected for BCL11A (lead SNP rs6706648, ß = -0.39, P = 4.96 × 10-34) and HBS1L-MYB (lead SNP rs61028892, ß = 0.73, P = 1.18 × 10-9), whereas the variant allele for Xmn1-HBG2 was found to be very rare. In addition, we detected three putative new trait-associated regions. Genetically, dissecting the two major loci BCL11A and HBS1L-MYB, we defined trait-increasing haplotypes (P < 0.0001) containing so far unidentified causal variants. At BCL11A, in addition to a haplotype harbouring the putative functional variant rs1427407-'T', we identified a second haplotype, tagged by the rs7565301-'A' allele, where a yet-to-be-discovered causal DNA variant may reside. Similarly, at HBS1L-MYB, one HbF-increasing haplotype contains the likely functional small indel rs66650371, and a second tagged by rs61028892-'C' is likely to harbour a presently unknown functional allele. Together, variants at BCL11A and HBS1L-MYB SNPs explained 24.1% of the trait variance. Our findings provide a path for further investigation of the causes of variable fetal haemoglobin persistence in sickle cell disease.

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Background: Fetal haemoglobin (HbF) remains a major sickle cell disease modifier. The mechanism of HbF synthesis has been studied for several decades with the intention of increasing interventions for sickle cell disease (SCD), including drugs. However, the complex mechanism of HbF synthesis is influenced by multiple genetic factors interacting with environmental factors. In order to capture useful genetic information, especially with limited resources, one has to carefully design the study. This includes choosing the relevant participants, the correct phenotyping, the choice of samples, and the right genomic assays. This paper describes the approach undertaken as part of preparations for a reticulocyte transcriptome study intended to discover genes associated with HbF decline in newborns in Tanzania. Results: Of the 152 newborns enrolled in the larger study, 40 babies were selected for the reticulocyte transcriptome study based on their HbF levels at birth and later stage of life. Of these, 30 individuals were included under the category of high HbF levels ranging from 72.6-90% and the remaining 10 under the category of low HbF levels ranging from 5.9 - 10.3%. The reticulocyte enrichment recovery purity ranged from 85% - 97%. The total RNA concentrations obtained were >250 ng total RNA, with the average purity of 1.9 (A 260/280) respectively. The total concentration obtained was sufficient for the transcriptome and other downstream assays. Conclusion: We have documented important steps and factors to consider in identifying the relevant participants and required laboratory sample processes prior to the final stage, which involves total reticulocyte RNA sequencing.

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Objective: Fetomaternal hemorrhage (FMH) is an alloimmunization resulting caused by the incompatibility between fetal and maternal blood. For the prevention of newborn haemolytic disease (HDN), it is crucial to quantify the amount of fetomaternal hemorrhage. However, the classical Kleihauer-Betke test (K-B test) for detecting fetomaternal hemorrhage is limited by experimental tools and conditions and is not suitable for routine clinical use. Consequently, the method of prenatal diagnosis of fetomaternal hemorrhage applicable to the clinic is a topic worthy of further study. Therefore, it is worthwhile to further investigation on the clinically applicable prenatal diagnosis method for fetomaternal hemorrhage. Methods: This experiment demonstrates hydrogel's ability to separate sensitized red blood cells from soluble antibodies. Using flow cytometry the fluorescence values of sensitized red blood cells and fluorophore-labeled antibodies were measured, and the testing steps for the detection products of a novel technology were determined. The properties of a hydrogel fluoroimmunoassay were evaluated by distinguishing between the amounts of fetal and adult haemoglobin. The precision of this technology is evaluated using the Kleihauer-Betke test as a comparison. Results: This experiment compared the detection of haemoglobin fluorescence in adults (n = 2) and fetuses (n = 6). At the same time, the fluorescence intensity of different fetal haemoglobin (HbF) in adult haemoglobin (HbA) was calculated. The fluorescence value is 1.6% when the fetal hemoglobin concentration is 0.1%. Conclusion: The novel hydrogel fluoroimmunoassay can accurately determine the fluorescence intensity by flow cytometry to differentiate fetal haemoglobin from adult haemoglobin, quantitatively prenatally diagnose fetal haemoglobin, address the incompatibility between fetal and maternal blood types, and prevent alloimmunization.

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Neurocognitive impairment is common in sickle cell disease (SCD) and is associated with significant functional limitations. In a cross-sectional analysis, we examined the association between hydroxyurea (HU) treatment and neurocognitive functioning from school-age to young adulthood in individuals with SCD. A total of 215 patients with HbSS/HbSß0 -thalassaemia (71% HU treated) and 149 patients with HbSC/HbSß+ -thalassaemia (20% HU treated) completed neurocognitive measures at one of four developmental stages: school-age (age 8-9 years), early adolescence (age 12-13 years), late adolescence (age 16-17 years) and young adulthood (ages 19-24 years). For participants with multiple assessments, only the most recent evaluation was included. In multivariable analysis adjusted for social vulnerability, HU treatment and sex, older age was associated with a reduction in overall intelligence quotient (IQ) of 0·55 points per year of life [standard error (SE) = 0·18, false discovery rate adjusted P value (PFDR) = 0.01] for patients with HbSS/HbSß0 -thalassaemia. Earlier initiation of HU (n = 152) in HbSS/HbSß0 -thalassaemia was associated with higher scores on neurocognitive measures across most domains, including IQ [estimate (SE) 0·77 (0·25)/year, PFDR = 0·01], after adjusting for social vulnerability, sex and treatment duration. These results support the early use of HU to limit the detrimental neurocognitive effects of SCD, while highlighting the need for additional measures to further mitigate neurocognitive deterioration.

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Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

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Dr. John Herrick described the first clinical case of sickle cell anaemia (SCA) in the United States in 1910. Subsequently, four decades later, Ingram and colleagues characterized the A to T substitution in DNA producing the GAG to GTG codon and replacement of glutamic acid with valine in the sixth position of the ßS -globin chain. The establishment of Comprehensive Sickle Cell Centers in the United States in the 1970s was an important milestone in the development of treatment strategies and describing the natural history of sickle cell disease (SCD) comprised of genotypes including homozygous haemoglobin SS (HbSS), HbSß0 thalassaemia, HbSC and HbSß+ thalassaemia, among others. Early drug studies demonstrating effective treatments of HbSS and HbSß0 thalassaemia, stimulated clinical trials to develop disease-specific therapies to induce fetal haemoglobin due to its ability to block HbS polymerization. Subsequently, hydroxycarbamide proved efficacious in adults with SCA and was Food and Drug Administration (FDA)-approved in 1998. After two decades of hydroxycarbamide use for SCD, there continues to be limited clinical acceptance of this chemotherapy drug, providing the impetus for investigators and pharmaceutical companies to develop non-chemotherapy agents. Investigative efforts to determine the role of events downstream of deoxy-HbS polymerization, such as endothelial cell activation, cellular adhesion, chronic inflammation, intravascular haemolysis and nitric oxide scavenging, have expanded drug targets which reverse the pathophysiology of SCD. After two decades of slow progress in the field, since 2018 three new drugs were FDA-approved for SCA, but research efforts to develop treatments continue. Currently over 30 treatment intervention trials are in progress to investigate a wide range of agents acting by complementary mechanisms, providing the rationale for ushering in the age of effective and safe combination drug therapy for SCD. Parallel efforts to develop curative therapies using haematopoietic stem cell transplant and gene therapy provide individuals with SCD multiple treatment options. We will discuss progress made towards drug development and potential combination drug therapy for SCD with the standard of care hydroxycarbamide.

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Repeated red blood cell (RBC) transfusions in preterm neonates are associated with poor outcome and increased risk for prematurity-associated diseases. RBC transfusions cause the progressive replacement of fetal haemoglobin (HbF) by adult haemoglobin (HbA). We monitored HbF levels in 25 preterm neonates until 36 weeks of post-menstrual age (PMA); patients received RBC units from allogeneic cord blood (cord-RBCs) or from adult donors (adult-RBCs), depending on whether cord-RBCs were available. Primary outcome was HbF level at PMA of 32 weeks. Twenty-three neonates survived until this age: 14 received no transfusions, two only cord-RBCs, three only adult-RBCs and four both RBC types. HbF levels in neonates transfused with cord-RBCs were significantly higher than in neonates receiving adult-RBCs (P < 0·0001) or both RBC types (P < 0·0001). Superimposable results were obtained at PMA of 36 weeks. Every adult-RBCs transfusion increased the risk for an HbF in the lowest quartile by about 10-fold, whereas this effect was not evident if combined adult- and cord-RBCs were evaluated. Overall, these data show that transfusing cord-RBCs can limit the HbF depletion caused by conventional RBC transfusions. Transfusing cord blood warrants investigation in randomised trials as a strategy to mitigate the severity of retinopathy of prematurity (NCT03764813).

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Pulse oximetry for detecting critical CHD produces more false positive tests at high altitudes than at sea level, because at altitude the average resting saturation is lower and the variability is higher. This increases diagnostic difficulties, especially in small isolated communities without paediatric echocardio-graphy, and requires expensive transport to a regional medical centre. One way of reducing diagnostic errors is to measure arterial oxygen saturation while the infant is breathing 100% oxygen. In the absence of right-to-left shunting through the heart, the ductus, or the lungs, arterial oxygen tension will exceed 150 mmHg and arterial oxygen saturation will be 100%. With right-to-left shunting, arterial oxygen tension will be <100 mmHg, and thus <96% (usually much lower).

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Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.

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A laboratory haematology analyser is the gold standard for measuring haemoglobin concentration but has disadvantages, especially in neonates. This study compared alternative blood-sparing and non-invasive methods of haemoglobin concentration measurement with the gold standard. Haemoglobin concentrations were measured using a laboratory haematology analyser (reference method), blood gas analyser, HemoCue® using venous and capillary blood samples and a newly developed non-invasive sensor for neonates < 3 kg. A total of 63 measurements were performed. Body weight (2190 (1820-2520 [967-4450]) g) and haemoglobin concentration (12.3 (10.6-15.2 [8.2-20.5]) g.dl-1 ) varied widely. Bias/limits of agreement between the alternative methods and reference method were -0.1/-1.2 to 1.0 g.dl-1 (blood gas analyser), -0.4/-1.8 to 1.1 g.dl-1 (HemoCue, venous blood), 0.7/-1.9 to 3.2 g.dl-1 (HemoCue, capillary blood) and -1.2/-4.3 to 2 g.dl-1 (non-invasive haemoglobin measurement). Perfusion index, body weight and fetal haemoglobin concentration did not affect the accuracy of the alternative measurement methods, and these were successfully applied in term and preterm infants. However, the accuracies of non-invasive haemoglobin measurement and HemoCue of capillary blood especially lacked sufficient agreement with that of the reference method to recommend these methods for clinical decision making.

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Among the many vascular complications of sickle cell disease (SCD), retinopathy is the most prevalent and represents a leading cause of blindness. Hydroxycarbamide therapy ameliorates many symptoms of SCD, and high fetal haemoglobin (HbF) levels have been shown to protect against the development of retinopathy in children with HbSS. Its effect on adults with SCD, who are at a much higher risk of developing retinopathy, has not been studied. We aimed to investigate the effect of hydroxycarbamide use and HbF level on sickle cell retinopathy development in adults. We performed a retrospective cross-sectional study and collected demographics, comorbidities, and ocular and haematological data from 300 adult sickle cell subjects examined at the Henkind Eye Institute at Montefiore Medical Center during a 5-year period, from October 2012 to November 2017. The cohort was comprised mainly of Black and Hispanic subjects with all SCD genotypes, aged 18-71 years. Results show that in HbSS patients treated with hydroxycarbamide, those with retinopathy had significantly lower HbF levels compared to patients without retinopathy (P = 0·018). Our study identified the optimal HbF cut-off point for retinopathy protection to be 14·87%. Thus, a HbF level of 15% appears to be the threshold above which the odds for developing retinopathy in SS patients are reduced by 50%.

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The major ß-haemoglobinopathies, sickle cell disease and ß-thalassaemia, represent the most common monogenic disorders worldwide and a steadily increasing global disease burden. Allogeneic haematopoietic stem cell transplantation, the only curative therapy, is only applied to a small minority of patients. Common clinical management strategies act mainly downstream of the root causes of disease. The observation that elevated fetal haemoglobin expression ameliorates these disorders has motivated longstanding investigations into the mechanisms of haemoglobin switching. Landmark studies over the last decade have led to the identification of two potent transcriptional repressors of γ-globin, BCL11A and ZBTB7A. These regulators act with additional trans-acting epigenetic repressive complexes, lineage-defining factors and developmental programs to silence fetal haemoglobin by working on cis-acting sequences at the globin gene loci. Rapidly advancing genetic technology is enabling researchers to probe deeply the interplay between the molecular players required for γ-globin (HBG1/HBG2) silencing. Gene therapies may enable permanent cures with autologous modified haematopoietic stem cells that generate persistent fetal haemoglobin expression. Ultimately rational small molecule pharmacotherapies to reactivate HbF could extend benefits widely to patients.

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Fetal haemoglobin (HbF, α2γ2) induction has long been an area of investigation, as it is known to ameliorate the clinical complications of sickle cell disease (SCD). Progress in identifying novel HbF-inducing strategies has been stymied by limited understanding of gamma (γ)-globin regulation. Genome-wide association studies (GWAS) have identified variants in BCL11A and HBS1L-MYB that are associated with HbF levels. Functional studies have established the roles of BCL11A, MYB, and KLF1 in γ-globin regulation, but this information has not yielded new pharmacological agents. Several drugs are under investigation in clinical trials as HbF-inducing agents, but hydroxycarbamide remains the only widely used pharmacologic therapy for SCD. Autologous transplant of edited haematopoietic stem cells holds promise as a cure for SCD, either through HbF induction or correction of the causative mutation, but several technical and safety hurdles must be overcome before this therapy can be offered widely, and pharmacological therapies are still needed.

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Theories about fetal respiration began in antiquity. Aristotle characterized pneuma as warm air, but also as the enabler of vital functions and instrument of the soul. In Galen's system of physiology, the vital spirit was carried by the umbilical arteries, the nutrients by the umbilical vein from the placenta to the fetus. In 1569 Aranzio postulated that the maternal and fetal vasculatures are distinct. From 1670 to 1690, a century before the discovery of oxygen, researchers understood that during respiration some form of exchange with the air must occur, naming the substance biolychnium, phlogiston, sal-nitro, or nitro-aerial particles. An analogy of placental and pulmonary gas exchange was described in 1674 by Mayow. In 1779, Lavoisier understood the discovery of oxygen, discarded the phlogiston theory, and based respiration physiology on gas exchange. With the invention of the spectroscope, it became possible to measure hemoglobin oxygenation, and in 1876 Zweifel proved fetal oxygen uptake. Major progress in understanding fetal gas exchange was achieved in the 20th century by the physiologists Barcroft in Cambridge and Dawes in Oxford.

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The single base molecular substitution characterizing sickle cell haemoglobin, ß6glu→val, might be expected to result in predictable haematological and clinical features. However, the disease manifests remarkable diversity believed to reflect the interaction with other genetic and environmental factors. Some of the genetic modifiers include the beta globin haplotypes, alpha thalassaemia, factors influencing the persistence of fetal haemoglobin and the effects of the environment are addressed in this review. It is concluded that much of the genetic data present conflicting results. Environmental factors such as climate and infections, and psychological, educational and social support mechanisms also influence expression of the disease. These interactions illustrate how the expression of a 'single gene' disorder may be influenced by a variety of other genetic and environmental factors.

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