Burden of central nervous system complications in sickle cell disease: A systematic review and meta-analysis.

Sickle cell disease (SCD) patients are at high risk of central nervous system (CNS) complications and may experience significant morbidity. The study was conducted to describe the comprehensive burden of SCD-related CNS complications and to identify patient-reported outcome (PRO) instruments for future research. The review included 32 studies published from January 2000 to 2020, evaluating humanistic and economic outcomes. Twenty-three studies reported humanistic outcomes, 16 of which measured cognitive function using Wechsler Intelligence Scales. A meta-analysis was conducted, finding full-scale intelligence quotient (IQ) was significantly lower in: overt stroke versus controls: -12.6 (p < .001); silent cerebral infarct (SCI) versus controls: -5.7 (p < .001); overt stroke versus SCI: -9.4 (p = .008); and any event versus controls: -7.6 (p < .001). This review quantified the cognitive deficits associated with CNS complications in pediatric SCD populations and highlights the need for improved prevention/treatment. As PRO evidence was limited, we discussed areas for future research.

Comparisons of oxygen gradient ektacytometry parameters between sickle cell patients with or without α-thalassaemia.

The present study tested the impact of α-thalassaemia on oxygen gradient ektacytometry in sickle cell anaemia (SCA). Three SCA groups were compared: (i) no α-thalassaemia (four α-genes, n = 62), (ii) silent α-thalassaemia (three α-genes, n = 35) and (iii) homozygous α-thalassaemia (two α-genes, n = 12). Red blood cell (RBC) deformability measured in normoxia was not different between the three groups. The lowest RBC deformability reached at low oxygen partial pressure (pO2 ) was greater and the pO2 at which RBC started to sickle was lower in the two α-genes group compared to the other groups. Our present study showed an effect of α-thalassaemia on oxygen gradient ektacytometry in SCA.

Highly efficient editing of the ß-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the ß-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells.

Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2ßS2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified FOXO3 as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34+ hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. FOXO3 gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34+ cell-derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.

The vaso-occlusive pain crisis in sickle cell disease: Definition, pathophysiology, and management.

Early diagnosis, treatment, and prevention of a vaso-occlusive crisis (VOC) are critical to the management of patients with sickle cell disease. It is essential to differentiate between VOC-associated pain and chronic pain, hyperalgesia, neuropathy, and neuropathic pain. The pathophysiology of VOCs includes polymerization of abnormal sickle hemoglobin, inflammation, and adhesion. Hydroxyurea, L-glutamine, crizanlizumab, and voxelotor have been approved by the US Food and Drug Administration for reducing the frequency of VOCs; the European Medicines Agency has approved only hydroxyurea. Other novel treatments are in late-stage clinical development in both the United States and the European Union. The development of agents for prevention and treatment of VOCs should be driven by our understanding of its pathophysiology.

Pediatric to Adult Transition in Sickle Cell Disease: Survey Results from Young Adult Patients.

BACKGROUND/AIMS: We surveyed sickle cell disease (SCD) patients who transitioned from pediatric care at Texas Children's Hematology Center (TCHC) to adult care to determine the characteristics of patients with an adult SCD provider, continuation rates of pre-transition therapies, and patient perceptions of the transition process. METHODS: A cross-sectional study was conducted by telephone survey of 44 young adults with SCD, aged 19-29 years, who transitioned from TCHC to adult care within the last 15 years. RESULTS: Findings of the 23-item questionnaire revealed that transitioned patients with current adult providers (68.2%) were more likely to have seen a provider within 6 months of transition (p = 0.023) and to have been on hydroxyurea and/or monthly blood transfusions pre-transition (p = 0.021) than transitioned patients without a provider; 83% of patients on pre-transition hydroxyurea reported continuing hydroxyurea after transition. Transition challenges included inadequate preparation, difficulty finding knowledgeable adult providers, and lack of healthcare insurance/coverage. CONCLUSION: Transition to adult providers is predicted by establishing care with an adult SCD provider within 6 months of transition and being on pre-transition disease-modifying therapy. Transition may be improved if pediatric hematology centers assist and verify adult provider contact within 6 months of transition and engage patients of all disease severity during transition.

Fetal haemoglobin induction in sickle cell disease.

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.

Whole-exome sequencing of sickle cell disease patients with hyperhemolysis syndrome suggests a role for rare variation in disease predisposition.

BACKGROUND: Hyperhemolysis syndrome (HHS) is an uncommon, but life-threatening, transfusion-related complication of red blood cell transfusion. HHS has predominantly been described in patients with sickle cell disease (SCD) and is difficult to diagnose and treat. The pathogenesis of HHS, including its occurrence in only a subset of apparently susceptible individuals, is poorly understood. We undertook whole-exome sequencing (WES) of 12 SCD-HHS patients to identify shared genetic variants that might be relevant to the development of HHS. STUDY DESIGN AND METHODS: DNA from adults with SCD having at least one previous episode of HHS were subject to WES. High-quality variants were passed through a series of bioinformatics filters to identify variants that were uncommon among African populations represented in public databases. Recurrent, putative loss-of-function variants occurring in biologically plausible genes were prioritized and then genotyped in a larger, ancestry-matched cohort of non-HHS controls. RESULTS: A rare, heterozygous stop-gain variant (p.Glu210Ter) in MBL2 was significantly enriched among HHS cases (p = 0.002). This variant is predicted to result in a premature termination codon that escapes nonsense-mediated mRNA decay, potentially leading to a novel phenotype. We also observed a complex insertion-deletion variant in the final exon of KLRC3 that was enriched among cases (p = 0.0019), although neither variant was found among seven pediatric SCD-HHS patients. CONCLUSION: Our results suggest a potential role for rare genetic defects in the development of HHS among adult SCD patients. Such enriched variants may ultimately be useful for identifying high-risk individuals and informing therapeutic approaches in HHS.

A Retrospective Analysis of Sociodemographic and Hematologic Characteristics Associated With Achieving Optimal Hydroxyurea Therapy in Children With Sickle Cell Disease.

Hydroxyurea (HU) has proven hematologic and clinical benefits, especially when escalated to the maximum tolerated dose (MTD). We reviewed clinical data from patients with sickle cell disease (January 2011 to 2016) to determine baseline sociodemographic and laboratory parameters associated with reaching HU MTD without significant delays. In total, 210 patients (mean HU start age, 6.6 y) were included. Initial Kaplan-Meier event analysis showed 1 year to be an inflection point for reaching MTD. In total, 116 patients (55%) reached MTD in <1 year, with 56 (27%) taking >1 year to reach MTD and 38 (18%) patients not successfully reaching MTD during follow-up. In both crude and adjusted analyses, age at HU start was found to be significantly and inversely associated with reaching MTD within 1 year. The data presented, specifically the inflection point of reaching MTD at 1 year and the association of young HU start age with reaching MTD within a year, suggest that successful achievement of MTD may be facilitated by starting patients on HU at a young age and that older patients should receive additional intervention to attain MTD within 1 year. Patients who do not achieve MTD within a year may need the most extensive intervention.

The role of BCL11A and HMIP-2 polymorphisms on endogenous and hydroxyurea induced levels of fetal hemoglobin in sickle cell anemia patients from southern Brazil.

High levels of fetal hemoglobin (HbF) reduce sickle cell anemia (SCA) morbidity and mortality. HbF levels vary considerably and there is a strong genetic component that influences HbF production. Genetic polymorphisms at three quantitative trait loci (QTL): Xmn1-HBG2, HMIP-2 and BCL11A, have been shown to influence HbF levels and disease severity in SCA. Hydroxyurea (HU) is a drug that increases HbF. We investigated the influence of single nucleotide polymorphisms (SNPs) at the Xmn1-HBG2 (rs7482144); BCL11A (rs1427407, rs4671393 and rs11886868); and HMIP-2 (rs9399137 and rs9402686) loci on baseline and HU-induced HbF levels in 111 HbSS patients. We found that both BCL11A and HMIP-2 were associated with increased endogenous levels of HbF. Interestingly, we also found that BCL11A was associated with higher induction of HbF with HU. This effect was independent of the effect of BCL11A on baseline HbF levels. Additional studies will be needed to validate these findings and explain the ample inter-individual variations in HbF levels at baseline and HU-induced in patients with SCA.

Genetic mapping and exome sequencing identify 2 mutations associated with stroke protection in pediatric patients with sickle cell anemia.

Stroke is a devastating complication of sickle cell anemia (SCA), occurring in 11% of patients before age 20 years. Previous studies of sibling pairs have demonstrated a genetic component to the development of cerebrovascular disease in SCA, but few candidate genetic modifiers have been validated as having a substantial effect on stroke risk. We performed an unbiased whole-genome search for genetic modifiers of stroke risk in SCA. Genome-wide association studies were performed using genotype data from single-nucleotide polymorphism arrays, whereas a pooled DNA approach was used to perform whole-exome sequencing. In combination, 22 nonsynonymous variants were identified and represent key candidates for further in-depth study. To validate the association of these mutations with the risk for stroke, the 22 candidate variants were genotyped in an independent cohort of control patients (n = 231) and patients with stroke (n = 57) with SCA. One mutation in GOLGB1 (Y1212C) and another mutation in ENPP1 (K173Q) were confirmed as having significant associations with a decreased risk for stroke. These mutations were discovered and validated by an unbiased whole-genome approach, and future studies will focus on how these functional mutations may lead to protection from stroke in the context of SCA.

Hepatitis C and neutropenia.

PURPOSE OF REVIEW: This review describes the pathogenesis and therapeutic implications of neutropenia in patients with hepatitis C. RECENT FINDINGS: Mild-to-moderate neutropenia is increasingly recognized as the hepatitis C population has caused increased cirrhosis. Multiple mechanisms for the neutropenia have been postulated, with recent evidence pointing toward a combination of hypersplenism, autoimmunity, and direct viral infection of bone marrow cells. Advances in antiviral therapy are associated with worsened neutropenia and dose modification. Severe neutropenia is underreported and is generally not associated with increased rates of infection. SUMMARY: Although neutropenia is common in hepatitis C patients it generally has a benign course and may not prohibit antiviral therapy.

Can we use biomarkers to identify those at risk of acute pain from sickle cell disease?

INTRODUCTION: Acute pain episodes, also known as vaso-occlusive crises (VOC), are a major symptom of sickle cell disease (SCD) and lead to frequent hospitalizations. The diagnosis of VOC can be challenging, particularly in adults with SCD, 50% of whom have chronic pain. Several potential biomarkers have been proposed for identifying individuals with VOC, including elevation above the baseline of various vascular growth factors, cytokines, and other markers of inflammation. However, none have been validated to date. AREAS COVERED: We summarize prospective biomarkers for the diagnosis of acute pain in SCD, and how they may be involved in the pathophysiology of a VOC. Previous and current strategies for biomarker discovery, including the use of omics techniques, are discussed. EXPERT OPINION: Implementing a multi-omics-based approach will facilitate the discovery of objective and validated biomarkers for acute pain.

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a ß-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.