Males with sickle cell disease have higher risks of cerebrovascular disease, increased inflammation, and a reduced response to hydroxyurea.

Biological sex is important. Male sex is associated with worse outcomes in most measures, including cerebrovascular disease, hospital admissions, and blood transfusions, but not survival. Females also appear to have a better response to hydroxyurea therapy, reduced markers of inflammation, and better liver function.

Oxygen gradient ektacytometry does not predict pain in children with sickle cell anaemia.

The loss of red blood cell (RBC) deformability in sickle cell anaemia (SCA) is considered the primary factor responsible for episodes of acute pain and downstream progressive organ dysfunction. Oxygen gradient ektacytometry (Oxygenscan) is a recently commercialised functional assay that aims to describe the deformability of RBCs in SCA at differing oxygen tensions. So far, the Oxygenscan has been evaluated only by a small number of research groups and the validity and clinical value of Oxygenscan-derived biomarkers have not yet been fully established. In this study we examined RBC deformability measured with the Oxygenscan in 91 children with SCA at King's College Hospital in London. We found a significant correlation between Oxygenscan-derived biomarkers and well-recognised modifiers of disease severity in SCA: haemoglobin F and co-inherited α-thalassaemia. We failed, however, to find any independent predictive value of the Oxygenscan in the clinical outcome measure of pain, as well as other important parameters such as hydroxycarbamide treatment. Although the Oxygenscan remains an intriguing tool for basic research, our results question whether it provides any additional information in predicting the clinical course in children with SCA, beyond measuring known markers of disease severity.

The significance of spleen size in children with sickle cell anemia.

It is well established that splenic dysfunction occurs in early childhood in sickle cell anemia (SCA), although the determinants and consequences of splenic injury are not fully understood. In this study, we examined spleen size and splenic function in 100 children with SCA aged 0-16 years at King's College Hospital in London. Spleen size was assessed by abdominal ultrasound (US) and splenic function by pitted red blood cells (PIT counts). In our cohort, 5.6% of children aged 6-10 years and 19.4% of children aged 11-16 years had no visible spleen on US (autosplenectomy). Splenomegaly was common in all age groups, with 28% of children overall having larger spleens than the average for their age. Only one child had a PIT count suggesting preserved splenic function. We found no correlation between hemoglobin F levels and spleen size, nor was there any difference in spleen size between children treated with or without hydroxyurea. Although there was a trend toward increased spleen length in children with co-inherited α-thalassemia, this did not reach statistical significance. Finally, we found a strong association between erythrocyte deformability measured with oxygen gradient ektacytometry, spleen size, and PIT counts. In conclusion, our results do not agree with the general perception that most children with SCA undergo autosplenectomy within the first decade of life and indicate that loss of erythrocyte deformability contributes to loss of splenic filtration capacity in SCA, as well as phenotypical variations in spleen size.

The pleiotropic effects of α-thalassemia on HbSS and HbSC sickle cell disease: Reduced erythrocyte cation co-transport activity, serum erythropoietin, and transfusion burden, do not translate into increased survival.

α-Thalassemia is one of the most important genetic modulators of sickle cell disease (SCD). Both beneficial and detrimental effects have been described previously. We use a 12-year data set on a large cohort of patients with HbSS (n = 411) and HbSC (n = 146) to examine a wide range of these clinical and laboratory associations. Our novel findings are that α-thalassemia strongly reduces erythrocyte potassium chloride co-transporter (KCC) activity in both HbSS and HbSC (p = .035 and p = .00045 respectively), suggesting a novel mechanism through which α-thalassemia induces a milder phenotype by reducing red cell cation loss. This may be particularly important in HbSC where reduction in mean cell hemoglobin concentration is not seen and where KCC activity has previously been found to correlate with disease severity. Additionally, we show that α-thalassemia not only increases hemoglobin in patients with HbSS (p = .0009) but also reduces erythropoietin values (p = .0005), demonstrating a measurable response to improved tissue oxygenation. We confirm the reno-protective effect of α-thalassemia in patients with HbSS, with reduced proteinuria (p = .003) and demonstrate a novel association with increased serum sodium (p = .0004) and reduced serum potassium values (p = 5.74 × 10-10 ). We found patients with α-thalassemia had a reduced annualized transfusion burden in both HbSS and HbSC, but α-thalassemia had no impact on annualized admission rates in either group. Finally, in a larger cohort, we report a median survival of 62 years in patients with HbSS (n = 899) and 80 years in those with HbSC (n = 240). α-thalassemia did not influence survival in HbSS, but a nonsignificant trend was seen in those with HbSC.

Genome wide association study of silent cerebral infarction in sickle cell disease (HbSS and HbSC).

Not available.

The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia.

Abnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.

Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia.

Phosphatidylserine (PS) exposure increases as red cells age, and is an important signal for the removal of senescent cells from the circulation. PS exposure is elevated in red cells from sickle cell anaemia (SCA) patients and is thought to enhance haemolysis and vaso-occlusion. Although precise conditions leading to its externalisation are unclear, high intracellular Ca2+ has been implicated. Red cells from SCA patients are also exposed to an increased oxidative challenge, and we postulated that this stimulates PS exposure, through increased Ca2+ levels. We tested four different ways of generating oxidative stress: hypoxanthine and xanthine oxidase, phenazine methosulphate, nitrite and tert-butyl hydroperoxide, together with thiol modification with N-ethylmaleimide (NEM), dithiothreitol and hypochlorous acid (HOCl), in red cells permeabilised to Ca2+ using bromo-A23187. Unexpectedly, our findings showed that the four oxidants significantly reduced Ca2+ -induced PS exposure (by 40-60%) with no appreciable effect on Ca2+ affinity. By contrast, NEM markedly increased PS exposure (by about 400%) and slightly but significantly increased the affinity for Ca2+ . Dithiothreitol modestly reduced PS exposure (by 25%) and HOCl had no effect. These findings emphasise the importance of thiol modification for PS exposure in sickle cells but suggest that increased oxidant stress alone is not important.

The super sickling haemoglobin HbS-Oman: a study of red cell sickling, K+ permeability and associations with disease severity in patients heterozygous for HbA and HbS-Oman (HbA/S-Oman genotype).

Studying different sickle cell genotypes may throw light on the pathogenesis of sickle cell disease (SCD). Here, the clinical profile, red cell sickling and K+ permeability in 29 SCD patients (15 patients with severe disease and 14 with a milder form) of HbA/S-Oman genotype were analysed. The super sickling nature of this Hb variant was confirmed. The red cell membrane permeability to K+ was markedly abnormal with elevated activities of Psickle , Gardos channel and KCl cotransporter (KCC). Results were consistent with Ca2+ entry and Mg2+ loss via Psickle stimulating Gardos channel and KCC activities. The abnormal red cell behaviour was similar to that in the commonest genotype of SCD, HbSS, in which the level of mutated Hb is considerably higher. Although activities of all three K+ transporters also correlated with the level of HbS-Oman, there was no association between transport phenotype and disease severity. The super sickling behaviour of HbS-Oman may obviate the need for solute loss and red cell dehydration to encourage Hb polymerisation, required in other SCD genotypes. Disease severity was reduced by concurrent α thalassaemia, as observed in other SCD genotypes, and represents an obvious genetic marker for prognostic tests of severity in young SCD patients of the HbA/S-Oman genotype.

Human cellular model systems of ß-thalassemia enable in-depth analysis of disease phenotype.

ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

Determinants of severity in sickle cell disease.

Sickle cell disease is a very variable condition, with outcomes ranging from death in childhood to living relatively symptom free into the 8th decade. Much of this variability is unexplained. The co-inheritance of α thalassaemia and factors determining HbF levels significantly modify the phenotype, but few other significant genetic variants have been identified, despite extensive studies. Environmental factors are undoubtedly important, with socio-economics and access to basic medical care explaining the huge differences in outcomes between many low- and high-income countries. Exposure to cold and windy weather seems to precipitate acute complications in many people, although these effects are unpredictable and vary with geography. Many studies have tried to identify prognostic factors which can be used to predict outcomes, particularly when applied in infancy. Overall, low haemoglobin, low haemoglobin F percentage and high reticulocytes in childhood are associated with worse outcomes, although again these effects are fairly weak and inconsistent.

Automating Pitted Red Blood Cell Counts Using Deep Neural Network Analysis: A New Method for Measuring Splenic Function in Sickle Cell Anaemia.

The spleen plays an important role in the body's defence against bacterial infections. Measuring splenic function is of interest in multiple conditions, including sickle cell anaemia (SCA), where spleen injury occurs early in life. Unfortunately, there is no direct and simple way of measuring splenic function, and it is rarely assessed in clinical or research settings. Manual counts of pitted red blood cells (RBCs) observed with differential interference contrast (DIC) microscopy is a well-validated surrogate biomarker of splenic function. The method, however, is both user-dependent and laborious. In this study, we propose a new automated workflow for counting pitted RBCs using deep neural network analysis. Secondly, we assess the durability of fixed RBCs for pitted RBC counts over time. We included samples from 48 children with SCA and 10 healthy controls. Cells were fixed in paraformaldehyde and examined using an oil-immersion objective, and microscopy images were recorded with a DIC setup. Manual pitted RBC counts were performed by examining a minimum of 500 RBCs for pits, expressing the proportion of pitted RBCs as a percentage (%PIT). Automated pitted RBC counts were generated by first segmenting DIC images using a Zeiss Intellesis deep learning model, recognising and segmenting cells and pits from background. Subsequently, segmented images were analysed using a small ImageJ macro language script. Selected samples were stored for 24 months, and manual pitted RBC counts performed at various time points. When comparing manual and automated pitted RBC counts, we found the two methods to yield comparable results. Although variability between the measurements increased with higher %PIT, this did not change the diagnosis of asplenia. Furthermore, we found no significant changes in %PIT after storing samples for up to 24 months and under varying temperatures and light exposures. We have shown that automated pitted RBC counts, produced using deep neural network analysis, are comparable to manual counts, and that fixed samples can be stored for long periods of time without affecting the %PIT. Automating pitted RBC counts makes the method less time consuming and results comparable across laboratories.

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion.

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

Pathophysiological Relevance of Renal Medullary Conditions on the Behaviour of Red Cells From Patients With Sickle Cell Anaemia.

Red cells from patients with sickle cell anaemia (SCA) contain the abnormal haemoglobin HbS. Under hypoxic conditions, HbS polymerises and causes red cell sickling, a rise in intracellular Ca2+ and exposure of phosphatidylserine (PS). These changes make sickle cells sticky and liable to lodge in the microvasculature, and so reduce their lifespan. The aim of the present work was to investigate how the peculiar conditions found in the renal medulla - hypoxia, acidosis, lactate, hypertonicity and high levels of urea - affect red cell behaviour. Results show that the first four conditions all increased sickling and PS exposure. The presence of urea at levels found in a healthy medulla during antidiuresis, however, markedly reduced sickling and PS exposure and would therefore protect against red cell adherence. Loss of the ability to concentrate urine, which occurs in sickle cell nephropathy would obviate this protective effect and may therefore contribute to pathogenesis.

Genetic Analysis of Patients With Sickle Cell Anemia and Stroke Before 4 Years of Age Suggest an Important Role for Apoliprotein E.

BACKGROUND: Ischemic stroke is a devastating complication affecting children with sickle cell anemia. Genetic factors are likely to be important in determining the risk of stroke but are poorly defined. METHODS: We have studied a cohort of 19 children who had an overt ischemic stroke before 4 years of age. We predicted genetic determinants of stroke would be more prominent in this group. We performed whole exome sequencing on this cohort and applied 2 hypotheses to our variant filtering. First, we looked for strong, potentially mono- or oligogenic variants for ischemic stroke, and second, we considered that more common polygenic variants will be enriched in our cohort. Candidate variants emerging from both strategies were validated in a cohort of 283 patients with sickle cell anemia and known pediatric cerebrovascular outcomes. We used principal component analysis in this cohort to control for relatedness and population substructure. RESULTS: Our primary finding was that the Apoliprotein E genotypes ε2/ε4 and ε4/ ε4, defined by the interplay of rs7412 and rs429358, were associated with increased stroke risk, with an odds ratio of 4.35 ([95% CI, 1.85-10.0] P=0.0011) for ischemic stroke in the validation cohort. We also found that rs2297518 in NOS (NO synthase) 2 (odds ratio, 2.25 [95% CI, 1.21-4.19]; P=0.014) and rs2230123 in signal transducer and activator of transcription (odds ratio, 2.60 [95% CI, 1.30-5.20]; P=0.009) both had increased odds ratios for ischemic stroke, although these two variants were below the threshold for statistical significance after correction for multiple testing. CONCLUSIONS: These data identify new loci for future functional investigations into cerebrovascular disease in sickle cell anemia. Based on African population reference allele frequencies, the Apoliprotein E genotypes would be present in about 10% of children with sickle cell anemia and represent a genetic risk factor that is potentially modifiable by both dietary and pharmaceutical manipulation of its dyslipidemic effects.

Evaluation of oxidative stress-related genetic variants for predicting stroke in patients with sickle cell anemia.

Overt stroke in adults with sickle cell anemia (SCA) continues to be a major cause of morbidity and mortality, while no evidence-based strategy for prevention has been reached so far. Although transcranial Doppler ultrasonography represents the most important tool for identifying young patients with SCA at risk of primary stroke, strategies for stroke prediction in adulthood remain challenging. Emerging data suggest that oxidative stress may exert a pivotal role in the pathogenesis of ischemic brain injury. Combining these pieces of evidences with the well-known genetic contribution to the development of stroke in SCA, we hypothesized that genetic variants related to the biology of oxidative stress could be used to identify adult patients at higher risk of stroke. Overall, 499 unrelated patients with SCA aged >18 years were genotyped for SOD2 Val16Ala (rs4880), GPX3 T-568C (rs8177404), GPX3 T-518C (rs8177406), GPX3 T-65C (rs8177412), and CAT01 C-262 T (rs1001179) polymorphisms, along with α-thalassemia status and ß-globin gene haplotypes. Of these, only the SOD2 Val16Ala polymorphism was associated with stroke. SOD2 Val16Ala polymorphism was independently associated with risk of stroke (odds ratio: 1.98; 95% confidence interval [CI]: 1.18-3.32; P = .009) and with the long-term cumulative incidence of stroke (hazard ratio: 2.24, 95% CI: 1.3-3.9; P = .004). In summary, we provide evidence that oxidative stress-related genetic variants, in particular, the SOD2 Val16Ala polymorphism, may represent a simple and inexpensive alternative for identifying patients at risk of stroke.

The effect of the antisickling compound GBT1118 on the permeability of red blood cells from patients with sickle cell anemia.

Sickle cell anemia (SCA) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O2 ) affinity and reduce sickling. One of these, voxelotor (GBT440), is currently in advanced clinical trials. A structural analogue, GBT1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA, the effect of GBT1118 on RBC cation permeability was also studied. Activities of Psickle , the Gardos channel and the KCl cotransporter (KCC) were all reduced. Gardos channel and KCC activities were also inhibited in RBCs treated with Ca2+ ionophore or the thiol reagent N-ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O2 affinity, GBT1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA.

The effect of xanthine oxidase and hypoxanthine on the permeability of red cells from patients with sickle cell anemia.

Red cells from patients with sickle cell anemia (SCA) are under greater oxidative challenge than those from normal individuals. We postulated that oxidants generated by xanthine oxidase (XO) and hypoxanthine (HO) contribute to the pathogenesis of SCA through altering solute permeability. Sickling, activities of the main red cell dehydration pathways (Psickle , Gardos channel, and KCl cotransporter [KCC]), and cell volume were measured at 100, 30, and 0 mmHg O2 , together with deoxygenation-induced nonelectrolyte hemolysis. Unexpectedly, XO/HO mixtures had mainly inhibitory effects on sickling, Psickle , and Gardos channel activities, while KCC activity and nonelectrolyte hemolysis were increased. Gardos channel activity was significantly elevated in red cells pharmacologically loaded with Ca2+ using the ionophore A23187, consistent with an effect on the transport system per se as well as via Ca2+ entry likely via the Psickle pathway. KCC activity is controlled by several pairs of conjugate protein kinases and phosphatases. Its activity, however, was also stimulated by XO/HO mixtures in red cells pretreated with N-ethylmaleimide (NEM), which is thought to prevent regulation via changes in protein phosphorylation, suggesting that the oxidants formed could also have direct effects on this transporter. In the presence of XO/HO, red cell volume was better maintained in deoxygenated red cells. Overall, the most notable effect of XO/HO mixtures was an increase in red cell fragility. These findings increase our understanding of the effects of oxidative challenge in SCA patients and are relevant to the behavior of red cells in vivo.