Plasmodium falciparum malaria causes morbidity and mortality in African children with sickle cell anemia (SCA), but comparisons of host responses to P falciparum between children with SCA (homozygous sickle cell disease/hemoglobin SS [HbSS]) and normal hemoglobin genotype/hemoglobin AA (HbAA) are limited. We assessed parasite biomass and plasma markers of inflammation and endothelial activation in children with HbAA (n = 208) or HbSS (n = 22) who presented with severe anemia and P falciparum parasitemia to Mulago Hospital in Kampala, Uganda. Genotyping was performed at study completion. No child had known SCA at enrollment. Children with HbSS did not differ from children with HbAA in peripheral parasite density, but had significantly lower sequestered parasite biomass. Children with HbSS had greater leukocytosis but significantly lower concentrations of several plasma inflammatory cytokines, including tumor necrosis factor α (TNF-α). In contrast, children with HbSS had threefold greater concentrations of angiopoietin-2 (Angpt-2), a marker of endothelial dysregulation associated with mortality in severe malaria. Lower TNF-α concentrations were associated with increased risk of postdischarge mortality or readmission, whereas higher Angpt-2 concentrations were associated with increased risk of recurrent clinical malaria. Children with SCA have decreased parasite sequestration and inflammation but increased endothelial dysregulation during severe anemia with P falciparum parasitemia, which may ameliorate acute infectious complications but predispose to harmful long-term sequelae.

It is critical to characterize the natural history of albuminuria in patients with sickle cell anemia (SCA); however, these data are currently lacking and affecting evidence-based guidelines. We performed a natural history study of the development of pediatric albuminuria. We identified participants with hemoglobin SS/SB0 thalassemia ≥5 years with albumin to creatinine ratio (ACR) measurements performed at a steady-state clinic visit. Participants were characterized as either persistent, intermittent, or never albuminuria. We determined the prevalence of persistent albuminuria, use of ACR ≥100 mg/g as a predictor, and variation in ACR measurements. We mirrored this study to determine the variation in albuminuria measurements in the SCA murine model. Among 355 participants with HbSS/SB0 thalassemia with 1728 ACR measurements, we identified 17% with persistent and 13% with intermittent albuminuria. Thirteen percent of participants with persistent albuminuria developed an abnormal ACR before 10 years of age. A single ACR measurement ≥100 mg/g was associated with 55.5 times (95% confidence interval, 12.3-527) higher odds of having persistent albuminuria. Among participants with ACR ≥100 mg/g, we identified significant variability in the results of repeated measurements. The median ACR at the initial and next measurements were 175.8 mg/g (interquartile range [IQR], 135-242) and 117.3 mg/g (IQR, 64-292). The human variability in ACR was mirrored by ∼20% variability in albuminuria in murine model. This evidence suggests adopting standards for repeating ACR measurements, consider screening for ACR before 10 years of age, and using an ACR >100 mg/g as a risk factor for progression. Pediatric and murine renoprotective clinical trials need to consider the high variability in repeated ACR measurements.

We conducted one of the first prospective studies to test the hypothesis that the clinical history of priapism underestimates priapism incidence compared with a priapism pain diary. Eligibility criteria were men with sickle cell anemia (SCA) between 18 and 40 years of age who have had at least 3 episodes of priapism in the past 12 months. Seventy-one men with SCA completed the diary for at least 3 months. The first 3 months of the priapism diary were included in the analysis. A total of 298 priapism episodes were recorded, and 80% (57 of 71) of the participants had at least 1 priapism event. Priapism severity was reported in the range of moderate to the worst imaginable pain in 81.5% (263 of 298), and a total 57 participants (80%) had a median pain rating of 6 (interquartile range: 5-8) on a scale from 1 to 10. The monthly incidence rate of priapism per participant based on history versus self-report pain diary was 2.0 (95% confidence interval, 1.9-2.1) and 1.4 (95% confidence interval, 1.2-1.6), respectively (P < .001). For participants that had a prior priapism episode, 80% had another episode during the 3-month interval follow-up. The median time to that second episode was 27.5 days. Major priapism occurred in 9.9% of episodes and was associated with the sum of all prospective priapism events. Men with SCA and at least 3 priapism episodes in the past 12 months are at significant risk for recurrent priapism in the following 3 months.

In sickle cell disease, the relative importance of reduced hemoglobin (Hb) and peripheral oxygen saturation on brain structure remains uncertain. We applied graph-theoretical analysis to diffusion magnetic resonance imaging data to investigate the effect of structural brain connectivity on cognitive function, alongside the presence or absence, number, and volume of silent cerebral infarction. In patients, we investigated the relationships between network properties, blood oxygenation, and cognition (working memory and processing speed indices). Based on streamline counts and fractional anisotropy, we identified a subnetwork with weakened connectivity in 92 patients with sickle cell disease (91 homozygous for HbS [HbSS], 1 heterozygote with HbSβ0 thalassemia; 49 males; aged 8.0 to 38.8 y), compared with 54 control subjects (22 males; aged 6.7 to 30.6 y). Multiple regression analyses showed a significant effect of Hb on full-network edge density (P < .05) and of peripheral oxygen saturation on streamline-weighted subnetwork efficiency (P < .01). There were effects of fractional anisotropy-weighted full-network and subnetwork efficiency on working memory index (both P < .05), and of streamline-weighted subnetwork efficiency on processing speed index (P = .05). However, there were no effects of presence, number or volume of silent cerebral infarcts. Streamline-weighted efficiency was progressively lower with lower oxygen saturation, with a downstream effect on the processing speed index. In path analysis, indirect relationships between blood oxygenation and cognition, mediated by network properties, were better supported than direct alternatives, with an indirect relationship between low oxygen saturation and processing speed index in patients, mediated by structural connectivity efficiency in a subnetwork of the brain differing from control subjects. Our findings are consistent with the notion that cognitive impairment is primarily mediated by hypoxic-ischemic effects on normal-appearing white matter and highlight the utility of network-based methods in providing biomarkers of cognitive dysfunction in patients with sickle cell disease.

Data from small clinical trials in the United States and India suggest zinc supplementation reduces infection in adolescents and adults with sickle cell anemia (SCA), but no studies of zinc supplementation for infection prevention have been conducted in children with SCA living in Africa. We conducted a randomized double-blind placebo-controlled trial to assess zinc supplementation for prevention of severe or invasive infections in Ugandan children 1.00-4.99 years with SCA. Of 252 enrolled participants, 124 were assigned zinc (10 mg) and 126 assigned placebo once daily for 12 months. The primary outcome was incidence of protocol-defined severe or invasive infections. Infection incidence did not differ between treatment arms (282 vs. 270 severe or invasive infections per 100 person-years, respectively, incidence rate ratio of 1.04 [95% confidence interval (CI), 0.81, 1.32, p=0.78]), adjusting for hydroxyurea treatment. There was also no difference between treatment arms in incidence of serious adverse events or SCA-related events. Children receiving zinc had increased serum levels after 12-months, but at study exit, 41% remained zinc deficient (<65 μg/dL). In post-hoc analysis, occurrence of stroke or death was lower in the zinc treatment arm (adjusted hazard ratio (95% CI), 0.22 (0.05, 1.00); p=0.05). Daily 10 mg zinc supplementation for 12 months did not prevent severe or invasive infections in Ugandan children with SCA, but many supplemented children remained zinc deficient. Optimal zinc dosing and the role of zinc in preventing stroke or death in SCA warrant further investigation. This trial was registered at clinicaltrials.gov as #NCT03528434.

Undernutrition is a risk factor for under-5 mortality and is also postulated to be a risk factor for mortality in older children and adults with sickle cell anemia (SCA). We tested the hypothesis that underweight is associated with mortality in children aged 5 to 12 years with SCA. We performed a secondary analysis of participants in the Primary Prevention of Stroke in Children with Sickle Cell Disease in Nigeria trial, a double-blind, parallel-group randomized controlled trial for low-dose or moderate-dose hydroxyurea in children with abnormal transcranial Doppler velocities and a comparison group of participants with nonelevated transcranial Doppler velocities in northern Nigeria. Nutritional status was classified as underweight (weight-for-age z score), stunting (height-for-age z score), and wasting (body mass index z score) using the World Health Organization growth reference. The mean weight-for-age z score was lower in children who died during the study than in those who survived. Otherwise, the baseline characteristics of children who died during the study were not significantly different from those of the children who survived. A pooled analysis of participants demonstrated that a lower weight-for-age z score was associated with an increased hazard of death. Underweight participants (weight-for-age z score <-1) had a greater probability of death during follow-up than those who were not underweight. Underweight status in school-aged children with SCA is a previously unrecognized risk factor for early mortality in Nigeria and can be easily applied to screen children at risk for death. This trial was registered at www.clinicaltrials.gov as #NCT02560935.

Sickle cell hemoglobin SC (HbSC) disease is the second most frequent sickle cell disease (SCD) genotype after sickle cell anemia (HbSS). Globally, ∼55 000 newborns with HbSC are delivered annually, with the highest HbC gene frequency in West Africa. In Ghana, 40% of adults visiting the Ghana Institute of Clinical Genetics SCD clinic have HbSC. Unlike HbSS, hydroxyurea use is not routinely recommended for individuals with HbSC because of the perceived high-risk to benefit ratio. To test the hypothesis that at least 5% of adults with HbSC will meet the American Society of Hematology criteria for severe disease, we conducted a retrospective descriptive cohort study of all individuals with HbSC (≥18 years) who visited the clinic in 2019. Adults with HbSC aged from 18 to 45 years were selected. We identified a comparison group of 639 individuals with HbSS and matched the frequency based on the age and sex of individuals with HbSC. Severe disease was defined as a history of ≥3 SCD-associated moderate or severe pain episodes per year, history of acute chest syndrome, and severe symptomatic chronic anemia that interferes with daily activities or quality of life. The study end points were the proportion of individuals with SCD who met the definition of severe disease and were eligible for hydroxyurea. In total, 64 of 639 (10.0%) individuals with HbSC met the eligibility criteria for hydroxyurea therapy compared with 154 of 639 (24.1%) individuals with HbSS. Less than 1% and 3% of individuals with severe HbSC and HbSS, respectively, were routinely prescribed with hydroxyurea in this tertiary care medical center.

The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional regulator of β-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds γ-globin messenger RNA (mRNA), and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal hemoglobin (∼22% HbF) without affecting β-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and β-thalassemia. In support of this idea, we report elevated levels of HbF in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain (p.(His1090Profs∗16); c.3267_3270delTCAC), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching.

In 2020, the American Society of Hematology published evidence-based guidelines for cerebrovascular disease in individuals with sickle cell anemia (SCA). Although the guidelines were based on National Institutes of Health-sponsored randomized controlled trials, no cost-effectiveness analysis was completed for children with SCA and silent cerebral infarcts. We conducted a cost-effectiveness analysis comparing regular blood transfusion vs standard care using SIT (Silent Cerebral Infarct Transfusion) Trial participants. This analysis included a modified societal perspective with direct costs (hospitalization, emergency department visit, transfusion, outpatient care, and iron chelation) and indirect costs (special education). Direct medical costs were estimated from hospitalizations from SIT hospitals and unlinked aggregated hospital and outpatient costs from SIT sites by using the Pediatric Health Information System. Indirect costs were estimated from published literature. Effectiveness was prevention of infarct recurrence. An incremental cost-effectiveness ratio using a 3-year time horizon (mean SIT Trial participant follow-up) compared transfusion vs standard care. A total of 196 participants received transfusions (n = 90) or standard care (n = 106), with a mean age of 10.0 years. Annual hospitalization costs were reduced by 54% for transfusions vs standard care ($4929 vs $10 802), but transfusion group outpatient costs added $22 454 to $137 022 per year. Special education cost savings were $2634 over 3 years for every infarct prevented. Transfusion therapy had an incremental cost-effectiveness ratio of $22 025 per infarct prevented. Children with preexisting silent cerebral infarcts receiving blood transfusions had lower hospitalization costs but higher outpatient costs, primarily associated with the oral iron chelator deferasirox. Regular blood transfusion therapy is cost-effective for infarct recurrence in children with SCA. This trial is registered at www.clinicaltrials.gov as #NCT00072761.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.