HbSS Genotype Research Articles

Acute Pancreatitis in Individuals with Sickle Cell Disease: A Systematic Review.

Background/Objectives: Sickle cell disease (SCD) impacts about 100,000 people in the US. SCD increases the risk of cholelithiasis and microvascular ischemia, which could increase the risk of acute pancreatitis (AP). Abdominal pain is a common presenting symptom of AP and sickle cell vaso-occlusive crisis. The purpose of our systematic review is to estimate the prevalence and determine the severity of AP in individuals with SCD compared to the general population. Methods: Multiple electronic databases were searched. We included studies that included children and adults (population) and addressed the association of SCD (exposure) with AP (outcome) compared to the same population without SCD (control). Two authors screened titles and abstracts independently, and data were abstracted in duplication from included studies. We registered this protocol in PROSPERO-CRD42023422397. Results: Out of 296 studies screened from multiple electronic databases, we identified 33 studies. These studies included 17 case reports, one case series, and 15 retrospective cohort studies, and 18 studies included children. Eight of the AP case reports were in patients with HbSS genotype, two with sickle beta thalassemia, and one with HbSoArab, and in six case reports, a genotype was not specified. Complications were reported in 11 cases-respiratory complication (in at least four cases), splenic complications (three cases), pancreatic pseudocyst (two cases) and death from AP (one case). Of the four AP cases in the case series, three had HbSS genotype, and two cases had complications and severe pancreatitis. AP prevalence in SCD was estimated to be 2% and 7% in two retrospective studies, but they lacked a comparison group. In retrospective studies that evaluated the etiology of AP in children, biliary disease caused mostly by SCD was present in approximately 12% and 34%, respectively. Conclusions: Data on the prevalence of AP in individuals with SCD are limited. Prospectively designed studies aiming to proactively evaluate AP in individuals with SCD who present with abdominal pain are needed to improve timely diagnosis of AP in SCD and outcomes.

Hospital acquired venous thromboembolism in children with sickle cell disease

Sickle cell disease (SCD) is well recognized as a hypercoagulablestate, however, it remains unclear whether a subgroup of children with SCD at higher risk of venous thromboembolic event (VTE) during hospitalization may benefit from thromboprophylaxis. Our objectives were to describe the clinical characteristics, outcomes and recurrence of hospital acquired VTE in patients with SCD younger than 21 years. This was a single center retrospective study. Data regarding demographics, reason for admission, location of VTE, risk factors like central venous catheter (CVC), intensive care unit (ICU) admission among others were extracted from electronic medical records over a 10-year study period (2011–2021). Recurrence of VTE at 1 and 5 years was assessed. Descriptive statistics were used as indicated. We identified a total of 20 VTE events over the 10-year study period. Six of these events occurred in those younger than 12 years of age. Fourteen (70%) VTE events occurred in the HbSS or HbSβThal0 genotypes compared to 6 (30%) in HbSC. Most common VTE was isolated pulmonary embolism (PE) (n = 10, 50%). VTE were most often associated with acute chest syndrome (ACS) (n = 14, 70%), ICU admissions (n = 10, 50%) and CVC (n = 5/9, 55%). One patient died from the VTE event. One patient with additional underlying risk factors had a recurrent VTE at 13 months. Our study suggests that ICU admission, ACS and presence of CVC increases the risk of VTE in children and young adults with SCD, but larger studies are indicated to validate our findings.

Genotypic Variations in Exercise-Induced Hemoglobin Oxygen Desaturation in Sickle Cell Disease: The Role of Hemoglobin Levels

Background: Sickle cell disease (SCD) presents abnormal hemoglobin S (HbS) and frequent vaso-occlusive crises. Previous studies reveal exercise intolerance in SCD patients. However, the role of exercise-induced hemoglobin oxygen desaturation (EIHOD) and its predictors remains unclear. This study investigates EIHOD in adult SCD patients, focusing on potential predictors, aligning with the hypothesis that EIHOD is prevalent and varies across genotypes, with hemoglobin as a predictor. Methods: A cross-sectional study was conducted on adult patients with HbSS and HbSC genotypes in a steady state at the Korle-Bu Teaching Hospital, Ghana. Clinical, hematological, and haemolytic markers were assessed. Lung function was evaluated through spirometry, and a six-minute walk test (6MWT) was performed to assess EIHOD. Logistic regression was used to identify predictors of EIHOD. We hypothesis that, EIHOD is common in SCD, varying across genotypes, with hemoglobin predicting its occurrence. Results: The prevalence of EIHOD was 41% in HbSS patients and 36.1% in HbSC patients. No significant differences were observed in age, white blood cell count, platelet count, hemolytic markers, hemoglobin levels, or lung function parameters between SCD patients with and without EIHOD. In the logistic regression model, none of the variables predicted EIHOD in HbSS patients. In HbSC patients, reduced hemoglobin was the only predictor of EIHOD (OR=0.551; 95% CI=0.315-0.964; p-value= 0.037). Discussion: The study highlights a high prevalence of EIHOD within the HbSS genotype, emphasizing the importance of hemoglobin levels as a predictor of EIHOD in SCD. Contrarily, hemolytic markers and lung function were not predictors of EIHOD across either genotype. These results suggest the potentially heightened significance of EIHOD-related complications in patients with the HbSS genotype. Conclusion: EIHOD is common in SCD, more so in HbSS. Reduced hemoglobin predicts EIHOD in HbSC. Lung function and hemolytic markers don't. Understanding the impact of EIHOD in SCD may guide interventions to improve exercise tolerance and reduce complications in this population. No Funding. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beyond pulmonary embolism: Alternative diagnosis and incidental findings on CT pulmonary angiography in sickle cell disease.

Sickle cell disease (SCD) is a genetic hematological disorder associated with severe complications, such as vaso-occlusive crises, acute chest syndrome (ACS), and an increased risk of thromboembolic events, including pulmonary embolism (PE). The diagnosis of PE in SCD patients presents challenges due to the overlapping symptoms with other pulmonary conditions. Our previous study revealed that nearly 96% of computed tomography pulmonary angiography (CTPA) scans in SCD patients were negative for PE, highlighting a gap in understanding the significance of CTPA findings when PE is absent. In this retrospective follow-up study conducted at the Salmaniya Medical Complex in Bahrain, we examined SCD patients with HbSS genotypes who underwent CTPA from January 1, 2018, to December 31, 2021, for suspected PE, but the results were negative. The aim of this study was to identify alternative diagnoses and incidental findings from CTPA scans. Experienced radiologists reviewed the CTPA images and reports to assess potential alternative diagnoses and incidental findings, incorporating an additional analysis of chest X-rays to evaluate the diagnostic value of CTPA. Incidental findings were classified based on their location and clinical significance. Among the 230 evaluated SCD patients (average age 39.7 years; 53% male) who were CTPA negative for PE, 142 (61.7%) had identifiable alternative diagnoses, primarily pneumonia (49.1%). Notably, 88.0% of these alternative diagnoses had been previously suggested by chest radiographs. Furthermore, incidental findings were noted in 164 (71.3%) patients, with 11.0% deemed clinically significant, necessitating immediate action, and 87.8% considered potentially significant, requiring further assessment. Notable incidental findings included thoracic abnormalities such as cardiomegaly (12.2%) and an enlarged pulmonary artery (11.3%), as well as upper abdominal pathologies such as hepatomegaly (19.6%), splenomegaly (20.9%), and gallstones (10.4%). This study underscores the limited additional diagnostic yield of CTPA for identifying alternative diagnoses to PE in SCD patients, with the majority of diagnoses, such as pneumonia, already suggested by chest radiographs. The frequent incidental findings, most of which necessitate further evaluation, highlight the need for a cautious and tailored approach to using CTPA in the SCD population.

Evaluation of Anthropometric Parameters and Lipid Profile in Subjects with Sickle Cell Traits at Madonna University, Elele, Nigeria

Sickle cell trait is a genetic condition that affects a substantial portion of the global population, particularly individuals of African descent. This study is aimed at evaluating anthropometric parameters and lipid profile parameters in subjects with sickle cell traits in Madonna University Teaching Hospital (MUTH), Elele. 72 subjects consisting of 30 HbAA (control), 21 HbAS, and 21 HbSS, all aged 18–40 years, were recruited for the study. Ethical clearance and informed consent were duly obtained from those involved. Blood samples were collected via venipuncture. Total cholesterol (TCH), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were analyzed using enzymatic spectrophotometric method. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Data were analyzed using Statistical Package for Social Sciences (SPSS) version 21 for Windows 7. The result from this study suggests that there are statistically significant differences (p < 0.05) among the genotype variants, HbAA, HbAS, and HbSS, for the TCH, TG, HDL, LDL, and hemoglobin (Hb) levels. For the control group (HbAA), the TCH is 4.51 mmol/L, TG is 1.21 mmol/L, HDL is 0.95 mmol/L, LDL is 2.81 mmol/L, and Hb is 14.02 g/dL. For the HbAS, the TCH is 4.16 mmol/L, TG is 0.73 mmol/L, HDL is 0.92 mmol/L, LDL is 2.82 mmol/L, and Hb is 13.02 g/dL. For the HbSS, the TCH is 3.39 mmol/L, TG is 0.59 mmol/L, HDL is 0.63 mmol/L, LDL is 2.59 mmol/L, and Hb is 10.85 g/dL. Also, the BMI (kg/m2) showed no statistically significant differences among the genotype variants, HbAA, HbAS, and HbSS (p > 0.05). This concludes that lipid metabolism may be altered in red cell genetic disorders such as sickle cell anemia.

Real-world experience of patients with sickle cell disease treated with crizanlizumab.

In 2019, crizanlizumab was approved by the Food and Drug Administration (FDA) to reduce the rate of vaso-occlusive crisis in patients with sickle cell disease (SCD). We aimed to study the real-world effectiveness of crizanlizumab in our comprehensive sickle cell center. This was a retrospective cohort analysis of patients with SCD who received at least two consecutive doses of crizanlizumab. Clinically significant improvement was captured using the patient global impression of change scale (PGI-C). As of December 2022, there were 18 patients eligible for analysis with a median age of 30.5 years. Eight patients had the HbSS genotype, 7 HbSC, and 3 HbSB null genotype. Median duration of exposure to crizanlizumab was 53.6 weeks, and 16 (88.9%) patients received crizanlizumab for ≥26 weeks. Crizanlizumab was very well tolerated with no serious adverse events (grade ≥3) related to treatment. There was no significant difference in laboratory parameters. There was a remarkable improvement in patients' subjective response to crizanlizumab infusion. The median PGI-C score of our patients was 5, signifying moderately better with slight but noticeable changes. The morphine equivalent daily doses (MEDD) were lower after crizanlizumab infusion. MEDD prior to crizanlizumab was 90; after ≥2 consecutive crizanlizumab doses, it was 60. There was also a reduction in the hospital admissions, emergency, and urgent care visit for acute pain crisis in 6 (28%) patients. This study shows that crizanlizumab was associated with improvement in patients' response, both directly and indirectly related to the reduction of opioids used, which is consistent with results from the SUSTAIN trial.

Sickle cell disease in Australia: a snapshot from the Australian Haemoglobinopathy Registry.

Sickle cell disease (SCD) is the most common monogenic disorder worldwide. In deoxygenated conditions, the altered beta chain (haemoglobin S [HbS]) polymerises and distorts the erythrocyte, resulting in pain crises, vasculopathy and end-organ damage. Clinical complications of SCD cause substantial morbidity, and therapy demands expertise and resources. Optimising care for patients and planning resource allocation for the future requires an understanding of the disease in the Australian population. The Australian Haemoglobinopathy Registry (HbR) is a collaborative initiative of specialist centres collating and analysing data on patients with haemoglobin disorders. To provide a snapshot of SCD in Australia over a 12-month period based on data from the HbR. Patients with a clinically significant sickling disorder across 12 clinical sites were included for analysis. Data include demographic and diagnostic details, as well as details of the clinical management of the condition over a 12-month period. Data on 359 SCD patients demonstrate a shift in the demographic of patients in Australia, with a growing proportion of sub-Saharan African ethnicities associated with the HbSS genotype. Acute and chronic complications are common, and patients require significant outpatient and inpatient support. Prevalence of disease complications and therapeutic trends are in keeping with other high-income countries. This study provides the first national picture of SCD in Australia, describing the characteristics and needs of SCD patients, elucidating demand for current and novel therapy and facilitating the planning of services for this vulnerable population.

Real-world observational study on the long-term effect of L-glutamine treatment on renal parameters of adult and pediatric patients with sickle cell disease.

Sickle cell disease (SCD) is a rare genetic blood condition affecting millions worldwide. Oxidative stress is a key player in the pathogenesis of SCD and its comorbid consequences. Renal function impairment is a common complication of SCD in both pediatric and adult patients with serious consequences leading to increased risk of mortality. In this observational real-world study, we are reporting the long-term (120 weeks) renal function in 10 patients treated with L-glutamine. Ten patients (4 pediatric and 6 adults), with confirmed diagnoses of SCD (HbSS genotype), were enrolled, these included four patients from Qatar with Arab Indian haplotype and six patients from French Guiana with African haplotype. All patients were treated with L-glutamine oral powder (~0.3 g/kg body weight, Endari®) twice daily for 120 weeks. Clinical events and laboratory parameters (renal function, hemoglobin, reticulocytes, and lactate dehydrogenase [LDH]) were measured at baseline, 48, and 120 weeks. The study showed that with L-glutamine treatment there were improvements in renal and hematological parameters with no vaso-occlusive crisis at both 48-and 120-week follow-up time points in all 10 patients. Improvements were seen in the albumin creatinine ratio (ACR) from baseline to 48 weeks (mean [Standard deviation SD] ACR: -4.19 [9.81] mg/g) and 120 weeks (mean [SD] ACR: -12.31 [21.09] mg/g). Mean (SD) increase in hemoglobin concentrations from baseline to 48 weeks and 120 weeks was 0.72 (1) g/dL and 1.41 (0.79) g/dL, respectively. Mean (SD) reticulocyte counts and LDH levels decreased from baseline to 48 weeks (mean [SD] change from baseline to 48 weeks, reticulocyte counts: -40.30 [101.58] × 109 cells/L; LDH levels: -259 [154.93] U/L) and 120 weeks (mean [SD] change from baseline to 120 weeks, reticulocyte counts: -58.30 [128.38] × 109 cells/L; LDH levels: -344.80 [274.63] U/L). This is one of the first studies that assessed the long-term renal outcomes in SCD using L-glutamine. L-glutamine improved the renal function in patients with SCD along with improvements in clinical outcomes and hemolysis, from 48 weeks and sustained through 120 weeks of treatment.

Religiosity, Anxiety, Depression, and Suicidal Ideation in Brazilian Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy that can evolve with time, in some individuals, as a debilitating chronic pain syndrome with emotional dysfunctions. Objective: To evaluate different types of religiosities: organizational (ORA), non-organizational (NORA), and intrinsic religiosity (IR), and their correlation with mental health in individuals with SCD. The variables analyzed were depression, anxiety, and catastrophic and suicidal thoughts. Method: This is a descriptive cross-sectional study, which is part of a crossover randomized clinical trial. We recruited adults among individuals with SCD from Bahia-Brazil. We used: Duke's religiosity index, Hospital Anxiety and Depression Scale (HADS), and Brazilian Portuguese Pain Catastrophizing Scale (BP-PCS) data. The Spearman correlation and Fisher exact test were used for statistics considering alpha 95%. Results: Of the 131 individuals approached for participation, 75 completed all questionnaires with genotype HbSS and HbSC. Of them, 49 (65,3%) were women, with an average age of 34.13 +10.02, 66 (88.0%) self-declared black, 63 (84.0%) declared to belong to a religious group, 67 (89.3%) with a high form of religious involvement. The mean of intense pain was 3.86 + 2.74; 40 (53.33%) had anxiety, 25 (33.33%) had depression, and 15 (20.0%) declared having suicidal thoughts. There was a negative correlation between depression and IR (r = -0.240, p = 0.038) and a correlation between average pain and NORA (r = 0.301, p = 0.009). An association between NORA and chronic pain was verified (p = 0.023), OR (p < 0.001), NORA (p = 0.042), and IR (p = 0.004) with evangelical SCD subjects. Conclusion: This study highlights the need for mental health care in patients with SCD due to the high rates of anxiety and depression, with the need to include religiosity since it is a frequent and important element in the lives of people with SCD.

Associations between Epigenetic Age Acceleration and Psychoneurological Symptoms in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) affects approximately 100,000 predominantly Black or African American individuals in the United States. The disease has several acute and chronic complications that lead to a reduced median lifespan of 42 years, which is significantly shorter than the general US population. In this shortened lifespan, people experience deleterious psychoneurological symptoms including pain, sleep disturbances, depressive symptoms, and cognitive impairment. The high occurrence of these symptoms has negative effects on quality of life and function, and contributes to health disparities in this population. Evidence suggests a person's epigenetic age may be associated with inter-individual variability in psychoneurological symptoms in SCD. Epigenetic age is a promising biomarker because it reflects the cumulative effects of exposures to stressors at the psychosocial, physical and environmental level, by capturing the molecular processes occurring with declining tissue function through globally assessing DNA methylation patterns that account for the pace of cellular aging. Epigenetic age and age acceleration in relation to disease characteristics and symptom burden has been assessed in several chronic conditions such as cancer, obesity and Alzheimer's. None have assessed this association in SCD. To address this gap, our study aims to determine whether epigenetic age acceleration is associated with psychoneurological symptom burden (pain impact, emotional impact, sleep impact, and cognition) in SCD. Methods: Psychoneurological symptoms and DNA methylation were assessed in a subset of participants aged between 18 - 45 years enrolled in one of the Sickle Cell Disease Implementation Consortium sites. DNA methylation data were generated on the Infinium MethylationEPIC beadchip (Illumina) from the extracted DNA of blood specimens and used to calculate epigenetic age using five epigenetic clocks (Horvath, Hannum, PhenoAge, GrimAge, & DunedinPACE). Psychoneurological symptoms were evaluated using validated instruments: the Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me), and Quality of Life in Neurological Disorders (NeuroQoL). Data were converted to T-scores and were used to determine associations with epigenetic age acceleration for each of the epigenetic clocks. Higher T-scores in ASCQ-Me and NeuroQoL indicate more desirable (better) outcomes. Results: Among participants (n=89) included in the analysis, the mean (SD) chronologic age was 30.6 (8.0) years. A majority identified as female (60.7%), non-Hispanic Black or African American (92%), never married (76.4%), had Hb SS genotype (68.5%), were taking hydroxyurea (60.5%), and had at least 4 pain events in past year (53.5%). Two epigenetic clocks showed mean age acceleration (Horvath: 0.2 ±4.6 years; GrimAge: 12.6 ±4.0 years, whereas two clocks showed mean age deceleration (Hannum: -4.27 ±3.91 years; PhenoAge: -8.43 ±6.0 years). The DunedinPACE clock is on a different scale than other clocks, but also demonstrated evidence for accelerated aging whereby the epigenetic age increased 1.14 years for every chronologic year. Increased age acceleration was associated with worse pain impact scores (GrimAge β=-0.25, p= 0.02), worse emotional impact scores (GrimAge β=-0.24, p=0.03; Dunedin β= -0.30, p=0.01) greater sleep disturbance (Dunedin β=-0.22, p=0.04) and more depressive symptoms (Dunedin β=0.29, p=0.01) (Table 1). Conclusion: This was the first study, to our knowledge, that explored the relationship between epigenetic age and psychoneurological symptoms in SCD. Epigenetic age acceleration was associated with worse symptom scores. Our preliminary findings identified several variations in the associations between symptom outcomes and epigenetic age across the five clocks. These variations may be due to the differences in the models used to develop the clocks, as well as inherent differences in epigenetic loci used to calculate epigenetic age for each clock. Future assessment of those discrepancies is warranted to effectively characterize individuals at higher risk of age acceleration and higher symptom burden in SCD. Our findings also highlight the potential use of epigenetic age acceleration as a biological target for interventions aimed towards preventing age acceleration and symptom development, improving the quality of life of individuals with SCD.

Preliminary Pharmacodynamic Results from a Multicenter Phase 2/3 Study of Next-Generation HbS Polymerization Inhibitor GBT021601 for the Treatment of Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a lifelong inherited disorder resulting in polymerization of sickle hemoglobin (HbS) that leads to red blood cell sickling/damage. SCD is characterized by chronic hemolytic anemia, vaso-occlusive crises, and cumulative organ damage. Voxelotor is a first-in-class HbS polymerization inhibitor approved in the US for the treatment of SCD in patients aged ≥4 years and in Europe for the treatment of hemolytic anemia due to SCD in patients aged ≥12 years as monotherapy or with hydroxyurea. GBT021601 is a next-generation HbS polymerization inhibitor with improved pharmacokinetic properties that increases hemoglobin (Hb)-oxygen affinity and stabilizes Hb in the oxygenated state to inhibit polymerization. GBT021601 has the potential for higher Hb occupancies at lower doses than voxelotor and could potentially reduce treatment burden while improving clinical outcomes. Here we report preliminary pharmacodynamic (PD) data from an ongoing phase 2/3 study of orally administered GBT021601 (NCT05431088) in patients with SCD. Methods:This is a 3-part, multicenter, phase 2/3 study: Part A is a randomized, open-label, 12-week, dose-finding study of GBT021601 in adult participants; Part B is a randomized, double-blind, placebo controlled 48-week study and will evaluate GBT021601 vs placebo in adult and adolescent participants; and Part C will evaluate open-label GBT021601 in pediatric participants. Herein, we present Week 6 and Week 12 data from Part A, which enrolled patients aged 18-65 years with SCD (HbSS or HbSB 0 genotype) and had Hb 5.5-10.5 g/dL at screening. Eligible patients were randomly assigned 1:1 to GBT021601 100 mg or 150 mg and received a loading dose twice daily for 4 days followed by once-daily maintenance doses through Week 12. The primary endpoint was change from baseline (BL) in Hb at Week 12. Clinical laboratory parameters, including RBC count, Hb, hematocrit (Hct), and mean corpuscular volume (MCV), were assessed. PD assessments included red blood cell (RBC) deformability (Oxygenscan, expressed as elongation index [EI]) as a function of partial pressure of oxygen [pO 2]). Results: At data cutoff (June 20, 2023), 28 of the 35 patients who received treatment in Part A of the study completed 12 weeks of treatment. The 100-mg group consisted of 17 patients, with a mean (range) age of 29.1 (18-49) years. The 150-mg group had 18 patients, with a mean (range) age of 30.2 (18-59) years. Most patients (32/35 [91.4%]) were homozygous for SCD (HbSS genotype). At BL, 16/35 patients (45.7%) were taking hydroxyurea. Patients treated with 100- and 150-mg doses of GBT021601 showed robust improvements from BL in several RBC parameters at 6 and 12 weeks ( Table). After 12 weeks of treatment, the mean (SD) increase in Hb from BL was 2.67 (1.52) g/dL for the 100-mg group (n=12) and 3.17 (1.82) g/dL for the 150-mg group (n=11). Additionally, the mean (SD) Hct gradually increased 7.87 (4.25) % in the 100-mg (n=12) and 9.61 (4.60) % in the 150-mg group (n=11), and RBC counts increased by 1.32 (0.79) 10 12/L in the 100-mg group (n=12) and 1.53 (0.60) 10 12/L, in the 150-mg group (n=11), thus approaching the normal range for these RBC parameters. At 12 weeks, mean (SD) MCV decreased from BL by 8.91 (6.49) fL in the 100-mg group (n=12) and 12.70 (9.05) fL in the 150-mg group (n=11). Ektacytometry results with GBT021601 showed marked improvement in RBC deformability with increases from BL in median EI max at Week 6 (100 mg: 0.37 to 0.55 [n=7]; 150 mg: 0.42 to 0.55 [n=7]) and Week 12 (100 mg: 0.37 to 0.53 [n=6]; 150 mg: 0.42 to 0.50 [n=8]) and an increase in median EI min from BL at Week 6 (100 mg: 0.09 to 0.42 [n=7]), which was indicative of improvement in RBC deformability. The median point of sickling during deoxygenation decreased from BL at Weeks 6 and 12 with dosing of GBT021601, indicating HbS polymerization was likely delayed ( Figure). Conclusions: In patients with SCD, GBT021601 maintenance doses of 100 mg and 150 mg led to increased Hb, increased Hct, and improvements in RBC deformability by 6 weeks, which were maintained through to Week 12. Analyses of PD data show promising evidence of efficacy with near normalization of Hb and oxygenscan parameters consistent with the improved mechanism of the drug. PD data from Part A of this phase 2/3 study demonstrated blood parameters approaching normal ranges and support for the ongoing clinical development of GBT021601 as a potential treatment for individuals with SCD.

Myocardial Fibrosis Improves in Young Patients with Sickle Cell Disease after Hematopoietic Cell Transplantation

Introduction: Cardiovascular complications are a leading cause of early mortality in patients with sickle cell disease (SCD). Chronic anemia, iron overload and repeated microvascular ischemic insults with reperfusion injury often result in progressive myocardial injury and maladaptive remodeling in SCD patients. Patients with SCD develop diffuse myocardial fibrosis, which can be detected by quantifying the myocardial extracellular volume (ECV) using cardiovascular magnetic resonance (CMR) imaging. Our group and others have previously shown that diffuse myocardial fibrosis is highly prevalent in both children and young adults with SCD, including those who have normal ventricular size and function by echocardiography. Additionally, we have shown that the disease modifying therapies (DMT) such as hydroxyurea or chronic transfusions do not appear to be protective against development of diffuse myocardial fibrosis. In a retrospective study, we found no difference in the myocardial ECV in patients who began DMT early (less than 6 years of age) versus later in life (at least 6 years or after) (Morin CE and Sharma A et al. Blood 2023.). Hematopoietic cell transplantation (HCT) can cure SCD, but myocardial effects of the therapy are unknown. Our goal was to evaluate cardiovascular parameters of function and injury, especially the progression of diffuse myocardial fibrosis, by comparing serial CMR studies performed before and after HCT in patients with SCD. Methods: We performed prospective CMR evaluation of children and young adults with SCD who received a matched sibling donor (MSD) or haploidentical donor (HAPLO) HCT following a reduced toxicity conditioning on a clinical trial (NCT04362293). CMR was performed pre-HCT, and at 1 month, and 12 months post-HCT. CMR-derived ventricular and atrial volumes were calculated. ECV was measured from T1 maps with a modified Look-Locker inversion recovery (MOLLI) sequence in short-axis. T2 and T2* maps were obtained. Regions of interest in the mid-ventricular septum were identified for analysis. We report descriptive statistics for CMR measures along with paired t-tests comparing patient ECV levels at 1-month and 12-months post-HCT to their respective baseline ECV. Results: Our study cohort consisted of 14 patients with SCD who underwent HCT and had serial CMR evaluations available. Thirteen patients had HbSS genotype and one had HbSC genotype. All patients were receiving DMT, either with hydroxyurea or chronic transfusions prior to HCT. Participants were majority male (57%) with median age of 15.2 years (range 6.8-21.3) at the pre-HCT CMR. Six received an MSD HCT and 8 received a HAPLO HCT. Ten patients each had follow up CMR evaluations performed at 1 month, and 12 months post-HCT. At 1 and 12 months post-HCT ejection fraction was preserved while biventricular volume and cardiac index decreased (Table 1). Compared to pre-HCT, ECV decreased from median 29% [range 27-38] to 28% (range 21-35, P= 0.061) at 1 month and 27% (range 24-31, P= 0.043) at 12 months post-HCT. There was no difference in the cardiac chamber sizes or the ECV measurements of those who received a MSD versus HAPLO HCT. Conclusions: We observed an improvement in cardiac chamber sizes with preservation of cardiac function as early as one month after HCT in patients with SCD. We further noted a reduction in ECV indicating a decrease in diffuse myocardial fibrosis in these young patients with SCD. To our knowledge, this is the first study to show that diffuse myocardial fibrosis can improve in patients with SCD after HCT. In addition to further corroborating the organ function protective effects of HCT in patients with SCD, these data further demonstrate the use of CMR as an essential non-invasive tool to assess the effects of therapeutic interventions on SCD-cardiac injury.

Experiences with Working: A Letter from Older Adults with Sickle Cell Disease

Introduction With medical advancements over the past decades, the life expectancy for people with sickle cell disease (SCD) is longer than ever before. As they age, people with SCD acquire both SCD-complications and age-related comorbidities that lead to premature functional impairment, requiring individuals to make complex decisions that affect their work and career ambitions. There is a gap in the literature on work experiences in older adults with SCD. The purpose of this study is to describe the work experiences of older adults with SCD. Methods We enrolled 19 older adults with SCD (age ≥ 50) from a comprehensive sickle cell center. We conducted semi-structured interviews by phone and in-person. Participants were asked open-ended questions about their experiences living with SCD and their single biggest problem faced with having SCD. Participants described obstacles faced when working and strategies to continue working as older adults living with SCD. Audio-recorded interviews were transcribed verbatim. Conventional content analysis was used to analyze the transcripts. Results The mean age of participants was 58 years (range 50-71) and 47% were women. Nearly half were externally employed (47%), 5% unemployed, 11% retired, 32% disabled, and 5% self-employed. Only 21% had a high school education or less, 37% completed some college, associates, or technical school, 26% had a bachelor's degree, and 16% had an advanced degree. For household income, 37% earned ≤ $30,000, 16% earned $30,001-$50,000, and 42% earned > $50,001. The majority were the head of their household (53%) and 21% reported living alone. Within the last 6 months, 32% were hospitalized and 26% missed at least a day of work due to a pain crisis. Most participants (53%) had HbSS genotype, 37% had HbSC, and 11% had HbSβ+-thalassemia. There were two major work-related themes that emerged from the interviews (Figure 1). Theme 1 was “ Obstacles faced when working ”, which centered around interferences with work. Participants discussed the physical limitations, SCD-related complications, lack of energy to perform job tasks, and pain crises that would interfere with their work schedule and sometimes keep them out of work for years. Participants described concerns about being forced to retire early or give up their business due to health-related issues. Many described challenges with work as the single-biggest problem they faced as an older adult with SCD. Participants also described obstacles related to a lack of understanding from their employers. They described getting sick and staying in the hospital for days at a time, then being expected to return to work immediately after discharge. One participant described sharing their disease diagnosis with an employer and being discriminated against working that kind of job. Theme 2 was “Strategies to continue working”, which centered around participants knowing their limits and modifying the type of work they did. Participants described learning to listen to their body when it has reached its limit, knowing when to back away and come back to job responsibilities, and knowing when to take breaks. Participants also described the importance of finding accommodating employers who were understanding of when they had to miss work. Some participants described how they had to learn new skills to do alternative, more flexible jobs that were not as physically taxing on their body, such as working from home, starting a business, or becoming a real estate agent. They especially emphasized the importance of being aware of how far they could go with certain jobs to avoid triggering pain crises. Participants also described going through periods of temporary disability or retirement, then getting a new job and slowly working their way up from part-time to full-time. Conclusion Older adults with SCD provided insight into the unique obstacles they faced while working and described work-related challenges as the single biggest problem they faced. Moreover, many described the strategies they developed over time and accommodations that allowed them to continue working or re-enter the workforce. Data provided from these experiences convey the dire need for policymakers and employers to support accommodations needed by adults with SCD to continue working. This information will help guide younger adults with SCD to better navigate the working world and achieve their ambitions as they advance in age.

Excellent Outcomes of HLA Matched Related Donor Transplant for Adults with Severe Sickle Cell Disease Using a Non-Myeloablative Conditioning with Thiotepa and Post-Transplant Cyclophosphamide: Multi-Center International Experience

Introduction: The validated curative treatment for sickle cell disease (SCD) is allogeneic hematopoietic stem-cell transplantation (HSCT). Despite paucity of available matched sibling donors (10-14%), they are preferred due to less immunogenic complications like graft versus host disease (GVHD) and graft failure (GF) using alternative donors. In-vivo T-cell depletion by post-transplant cyclophosphamide (PTCy) has been shown to be most effective in GVHD control and a non-myeloablative regimen minimizes the toxicity associated with HSCT in this vulnerable population. In 2013, the multi-institutional Vanderbilt Haploidentical Learning Collaborative showed the improved outcomes of their non-myeloablative regimen for related haploidentical BMT for SCD (ClinicalTrials.gov identifier NCT01850108). We used a similar non-myeloablative approach to improve donor engraftment rates, while minimizing GVHD. Material and methods: This study was conducted at three international centers using similar conditioning from our experience in multi-institutional Vanderbilt Global Haploidentical Learning Collaborative to optimize haploBMT for SCD. The conditioning regimen included ( Figure3): rabbit anti-thymoglobulin (ATG) 4.5 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and total body irradiation (TBI) 200 cGy. GVHD prophylaxis included PTCy 100 mg/kg, mycophenolate mofetil (MMF), and sirolimus. Stem cell source included bone marrow (BM) mobilized with filgrastim (5-10 μg/kg/d x 3-5 days) and peripheral blood (PB). Primary graft failure (GF) was defined as <5% donor myeloid/lymphoid chimerism or <5% donor cells by whole blood (WB) chimerism by day+28 posttransplant. Secondary graft rejection was defined <5% donor myeloid/lymphoid chimerism or <5% donor cells by whole blood (WB) chimerism with prior documentation of >5% donor cells by day +28. The Event free survival (EFS) was defined by survival with no GF, no death and/or no chronic GVHD. This cohort includes 4/22 patients (18.2%) had prior graft failure from a previous bone marrow transplants. Results: Here we report the outcome of 22 SCD patients (100% HbSS genotype), with a median age of 26.5 (15-41), transplanted from 10/10 MRD 86.4 % (n= 19) or single antigen mismatch related donor (MMRD) (9/10) 13.6% (n=3). The stem cell source was PB in 54.5% (12/22), and BM in 45.5% (10/22). Median CD34 stem cells dose was 6 X 10 6/Kg (1.2 - 10.25). Neutrophils and platelets engrafted at a median of 21.0 (15 - 26) and 25.0 (12 - 45) days, respectively. Demographic and clinical characteristics of participants summarized in Table 1. No primary or secondary graft failure was observed in the participants based on the last follow-up and time of analysis of this report. For participants with durable engraftment, the median WB chimerism values for D+28 and D+365 engraftment was 99.25% (n=14; IQR: 52 - 100) and 96.5% (n=16; IQR: 52 - 100), respectively. The median follow-up time was 14.6 months (n=22; IQR: 0.5-45.44). The overall incidence of acute GVHD grade III-IV was 4.5% (n= 1/22), and no incidence of chronic GVHD. There was no transplant related mortality. A Kaplan-Meier based probability of overall survival (OS) was 100%, Figure 1. All patients were off immunosuppression at 1-year post transplant. EFS as defined in the method was 100%, Figure 2. The EFS of free acute GVHD grade III-IV was 95.5%. Conclusion: This result shows the superiority of Thiotepa based non-myeloablative conditioning and PTCy for MRD transplants in adults with severe SCD. It not only minimizes the toxicity and GVHD but provides optimal donor engraftment mitigating against risk of secondary myeloid neoplasms from mixed chimerism. This needs further validation in a larger cohort.

Sickle Cell Disease and Comorbid Psychiatric Conditions: A Single-Center Retrospective Study

Introduction: It is established that patients with various chronic disease states are predisposed to psychiatric comorbidities. However, in the sickle cell disease (SCD) patient population, psychiatric comorbidities have been uniquely shown to exacerbate disease through a psychosomatic component. A co-existing diagnosis of depression is found in 1 of 3 patients with SCD, and they are more than 5 times as likely to suffer from a mood disorder than the general population. SCD patients with psychiatric illnesses have significantly worse physical and mental health outcomes. Additionally, healthcare costs are more than double for SCD patients with depression than for those without. Thus, SCD patients with psychiatric comorbidities have unique challenges and are an important population to study. Methods: This was a retrospective single-center study of patients with SCD, all genotyped and ≥16 years old, who were treated at UT Physicians Comprehensive Sickle Cell Center during the study period (March-July 2023). The demographics, clinical characteristics, psychiatry history, initial and recent PHQ-9 results, and laboratory findings were collected and analyzed. Descriptive statistics were obtained and analyzed between SCD patients with and without psychiatric disorders and are presented in percentages, medians, and interquartile ranges. Data analysis was completed using a p value < 0.05 to indicate statistical significance, using an independent group unpaired t-test for continuous variables and Chi-square and Fisher's exact tests for categorical data, respectively. Data was analyzed using GraphPad Prism version 9.0.2. Results: Patient sex was 45% male and 55% female; 73% of patients had the HbSS genotype. Of the studied cohort, 33% were affected by at least one psychiatric disorder. The most common psychiatric disorder was depression, which occurred in 26% of patients, followed by anxiety (21%). The most common pharmacotherapy for psychiatric conditions was tricyclic antidepressants (TCAs), followed by SSRIs and SNRIs. There was no significant difference in ED visits or hospitalization rates between patients with known psychiatric disorders versus those without. Common laboratory markers used to assess SCD (LDH, total bilirubin, reticulocyte count) were overall higher in the subset with psychiatric illnesses, but only LDH was statistically significantly higher. Surprisingly, SCD patients with psychiatric disorders used fewer opioids than those without with a lower Morphine Equivalent Daily Dose (although this was not statistically significant). Conclusion: Although it is known that psychiatric illnesses in SCD patients are prevalent, exacerbate disease, and pose a cost burden, in our study there was nosignificant increase in hospitalizations or ED visits. However, it is notable that our study patient population was an outpatient population that is compliant and generally has access to resources including a robust behavioral health team and regular psychotherapy sessions. Due to these factors, ourdata may not be generalizable to the entire SCD patient population. Additionally, althoughlaboratorymarkers were overall poorer in the psychiatric population, they were not statistically significant, perhaps owing to the small sample size. Our study calls for a broader look at SCD patients with psychiatric illness, perhaps also to review these variables in SCD patients in the inpatient setting. A future direction could also be to evaluate the efficacy of psychiatric pharmacotherapy versus cognitive behavioral therapy in this patient population. Word count: 518 words

Knowledge and Perceptions about Curative Therapies for Sickle Cell Disease and Hydroxyurea Among Caregivers and Patients at a Large Tertiary Care Center: A Survey Study

Background: Sickle Cell Disease (SCD) affects approximately 100,000 individuals in the United States. Patients require regular follow-up and monitoring to prevent serious complications. Even with rigorous monitoring, health outcomes can be poor due to pain-seeking stigma and other social determinants of health, namely racial, social, and economic disparities. According to the National Assessment of Health Literacy published in 2006, 36% of adults have limited health literacy in the United States. Among SCD populations, disease-specific knowledge can play a vital role in improving pain control and access to appropriate maintenance therapies. Aims:The purpose of this study is to investigate the knowledge and perceptions of patients with SCD and their caregivers attending The Valerie Fund Pediatric Hematology-Oncology Center at the Children's Hospital of New Jersey in Newark, NJ regarding their disease, complications, genetic impact, general anticipated course of treatment or natural history of disease. Hydroxyurea (HU) and curative therapies include bone marrow transplant (BMT) and gene therapy (GT). Methods: We surveyed 85 participants, accounting for 79 patients as some surveys were completed by each the patient and caregiver. Sixty-six (83.5%) participants were caregivers and 13 (16.5%) were children with sickle cell disease between 12- and 20-years-old. Most patients identified as black, African, or African American (76; 96.2%) with 2 (2.5%) Hispanic patients, and 1 (1.3%) white patient. Forty-three patients (54.4%) were female and 36 (45.6%) were male. The average age of the patients was 10.9 years with a standard deviation of 5.7 years. More than half of patients were HbSS genotype (51; 64.6%). Remaining genotypes were HbSC (21; 26.6%), HbS-β 0-thalassemia (5; 6.3%), and HbS-β +-thalassemia (2; 2.5%). Forty-six (58.2%) of patients had received HU and 33 (41.8%) had not. Results: When assessing background knowledge of sickle cell and heritable risk, most participants could correctly identify the patient's sickle cell genotype (74; 87.1%) and most caregivers could correctly identify their own genotype (49; 74.2%). However, many caregivers exhibited limited understanding of genetic risk with only 18 (27.2%) correctly identifying the one in four chance of having a subsequent child with SCD when both parents are carriers. The results revealed participants could not distinguish between treatment for SCD and sickle cell trait; few participants correctly identified that sickle cell trait is not an indication for treatment with HU (8; 10.1%), BMT (4; 5.1%) or GT (6; 7.6%). Baseline knowledge about BMT and GT was limited. Only 27 (34.1%) participants correctly identified that BMT is indicated for treating patients with HbSS or HbS-b 0-thalassemia, and only 9 (11.4%) participants correctly identified this treatment population for GT. BMT knowledge was slightly greater than GT knowledge, namely that 60.1% (48) correctly identified the benefits of BMT for SCD patients, including decreased pain, decreased need for pain medication, decreased length of hospital stay, and cure, and that either bone marrow, stem cells, or blood could be transplanted materials. Most patients did not correctly identify these benefits of GT (55; 70%), nor could they identify the source of blood or stem cells for GT (65; 82.3%). The ability to correctly identify the benefits of HU for sickle cell disease (48; 60.8%) was positively associated with the ability to correctly identify the benefits of BMT (p-value 0.0091) and GT (p-value 0.0438) using Fisher's exact test with 95% confidence interval. Conclusion: These results suggest that dedicated teaching about HU, a common treatment for SCD, can result in greater patient knowledge about newer and curative treatment options of bone marrow transplant and gene therapy. These findings underscore the necessity of implementation of comprehensive patient teaching that is inclusive of population education levels, literacy rates, health literacy rates, and other cultural and societal factors.

Insights on Sickle Cell Disease Therapies in Sierra Leone

Introduction: Ongoing sickle cell disease (SCD) gene therapy clinical trials in the United States and other high resource countries have shown promising results, and several therapies are under U.S. Food and Drug Administration review in 2023. Despite these advances, the current evidence-based practices are not accessible to much of the world's sickle cell population. As treatment advances are made for SCD, the integration of these therapies in sub-Saharan African countries-which have the highest prevalence of SCD-requires a global partnership. Sierra Leone ranks 9 th in burden in sub-Saharan Africa with annual SCD births between 4000-5000. Efforts are underway in Sierra Leone to improve awareness and healthcare infrastructure for the SCD population; however, the country still lacks a national screening program for diagnosis and faces challenges in providing comprehensive care across the lifespan. This study examines adult SCD patients' knowledge of current evidence-based care and novel gene therapies in Sierra Leone. Methods: In 2021-2022, a cohort (n=123) of adults living with SCD in Sierra Leone were surveyed as part of the INSIGHTS Study (NCT02156102). A mixed-methods recruitment strategy was used, including via public service announcements hosted on local radio stations, information disseminated via a WhatsApp group, and snowball sampling. Recruitment was conducted in two settings, one urban (Freetown) and one rural (Kono). Participants completed this cross-sectional study from September 2021-February 2022. Questions were asked regarding participants' perspectives and knowledge of hydroxyurea, the current standard of care treatment for SCD, and novel experimental gene therapy. Results: The mean age of respondents was 23.6 years (range 18-63). The majority of respondents were female (65.6%), educated at or above the senior secondary school level (78.7%), and students (70.3%). Respondents predominantly had the HbSS genotype (89.4%). Most study participants lacked awareness of hydroxyurea and gene therapy (93.5% had never heard of hydroxyurea; 95.1% had never taken hydroxyurea; 94.8% had never heard of gene therapy for SCD treatment) (Table 1). Despite this gap in knowledge, the majority (95.0%) agreed that genetic research is a necessary way to learn about treatments for SCD (Table 1). Discussion: Our results highlight disparities in current standard of care treatment and knowledge in a country with a high prevalence of SCD. While our cohort indicated a favorable attitude toward SCD research and a willingness to participate, these results highlight a significant barrier for successful implementation of current treatment and the adoption of curative genetic therapies in limited resource settings like Sierra Leone. Given the significant lack of knowledge around hydroxyurea, it is important that public health initiatives and research should be directed toward both established standard of care and creating awareness of experimental therapies.

Puberty Onset and Preservation of Fertility in Male Patients with Sickle Cell Disease Treated with Hydroxyurea: Data from the European Sickle Cell Disease Cohort - Hydroxyurea Extension (ESCORT-HU Extension) Study

Introduction Reproductive health is a major concern in male patients with sickle cell disease (SCD). SCD is associated with pubertal delay (Soliman 2021), however, limited large-scale studies exist on the age of puberty onset in male SCD patients. Additionally, men without hydroxyurea (HU) therapy often experience decreased fertility, with less than 10% having normal semen analysis results according to WHO guidelines (Berthaut 2008). HU treatment may further reduce fertility in many patients (Berthaut 2017). To address these concerns, various fertility preservation strategies have been developed for male SCD patients on HU, including sperm cryopreservation for pubescent boys and adults before starting HU, and temporary HU discontinuation when therapy is initiated in infancy for sperm banking, at best with monthly transfusion during the period without HU. Methods The ongoing European Sickle Cell Disease Cohort - Hydroxyurea Extension (ESCORT-HU Extension) study is a multicentric, prospective, non-interventional study aiming to better document the safety profile of HU. It focuses on poorly understood or documented risks, including puberty and fertility. Around 2000 HU-treated patients are expected to participate over a 5-year duration. The study includes both patients who were previously enrolled in the initial ESCORT-HU study and new patients who meet specific inclusion criteria. Results As of June 28, 2023, a total of 1860 patients were included in the study in France (92% of patients), Germany, Greece and Italy. Median age of patients at inclusion was 24 [IQR: 14-40] years with 44.4% being males, and 87% having the HbSS genotype. At enrollment, 455 (56.5%) males were in the pubescent stage, and 72 males transitioned into the pubescent stage since then. Median duration of HU exposure at the time of analysis was 8.5 [IQR: 6.4-11.6] years. Onset of puberty The mean age at Tanner V stage did not significantly differ between males who reached puberty before starting HU and those who reached puberty after (15.0 [IQR: 14.0-16.0] years vs. 15.2 [IQR: 14.0-16.0] years, respectively). Semen cryopreservation Among the 527 pubescent males, 293 started HU at or after 17 years old with 117/240 (49%) who had a cryopreservation. 234 started HU before the age of 17 years, 23/207 (11%) had a cryopreservation. Unknown data were reported for 80 patients. Median age at cryopreservation was 26.0 [IQR: 21.0-33.0] years. Semen analysis A total of 104 semen analysis were reported in the database with 66 performed before HU treatment initiation and 24 after, with 9 of them without any HU interruption prior the sampling. In 14 cases, the documentation on HU stopping was not available. Among the 66 patients tested before HU treatment initiation, results were not available in 34. They were normal in 25 (78%), abnormal in 7 (22%) (4 qualitative anomalies, 2 azoospermia, 1 both quantitative and qualitative anomalies). For the 24 patients who performed semen analysis after HU initiation, 11 temporarily discontinued the treatment for the procedure, with 3 having normal results, 7 having abnormal results, results not available in 1. One of the patients with oligospermia used successfully its previously cryopreserved semen for his partner's pregnancy. Nine patients did not stop HU before the procedure, with 7 having abnormal results, 1 normal, and result not available in 1. Information on HU interruption during the cryopreservation was not available for 4 patients (Table 1). Four patients have semen analysis results before and after HU treatment (Table 2). Conclusion: This study shows no impact of HU on the age of puberty in males. Data on fertility need to be completed but we show that semen cryopreservation has been well adopted in Europe, since approximately 50% of patients over the age of 17 at HU initiation included in France have undergone this procedure, which is free in France. We confirm a significant proportion of semen abnormalities but not in all cases. The impact of HU on fertility is complex, and cryopreservation appears to be beneficial in mitigating potential adverse effects, with at least one baby in our cohort born after use of cryopreserved semen.

Low Arginine Bioavailability and Clinical Outcomes in Children with Sickle Cell Disease Hospitalized with Vaso-Occlusive Pain Episode

Introduction: Global arginine bioavailability ratio (GABR) (Arginine/[Citrulline+Ornithine]) is associated with clinical outcomes including pain, pulmonary hypertension, and mortality in patients with sickle cell disease (SCD). Arginine replacement therapy has demonstrated opioid-sparing effects, improved blood pressure and cardiovascular function, and shorten length of hospital stay. Arginine is a conditionally essential amino acid synthesized from citrulline in the kidneys. During a vaso-occlusive pain episode (VOE), hemolysis leads to the release of erythrocyte-arginase (arginine-metabolizing enzyme) that metabolizes arginine into ornithine. Arginine-to-ornithine ratio (arg/orn) is a biomarker of arginase activity and GABR incorporates the impact of kidney function on global arginine bioavailability. The association between arginine bioavailability and clinical outcomes has not been sufficiently explored in children with SCD-VOE. Objective: To evaluate associations between arginine bioavailability and clinical outcomes in children with SCD-VOE who received intravenous (IV) arginine replacement therapy. Methods: We conducted a secondary analysis of a prospective, single-center, double-blinded randomized controlled trial of IV arginine therapy (TID, up to 7 days) in children with SCD age 3-21 years hospitalized for VOE requiring IV opioids. Patients with significant liver/renal dysfunction or those previously enrolled were excluded. Subjects were randomized into 1 of 3 arms: 1) 100 mg/kg/dose arginine (standard dose), 2) loading dose: 200 mg/kg followed by standard dose or 3) placebo. Demographics, total parenteral opioid (TPO) use (morphine equivalents, mg/kg) time to crisis resolution (time of study drug delivery to last IV opioid in hours), pain scores, and targeted amino acids were obtained before treatment and at discharge. Mean±SD, paired t-tests, and Pearson correlation analyses between groups were performed. This trial was registered at www.clinicaltrials.goc as #NCT02536170 Results: 1,548 patients were screened, 266 were eligible, 114 consented, and 108 were randomized (54.4% were female, mean age 12.44 ± 3.90). All participants identified as African American and 75% had genotype Hb-SS. 69.1% were on hydroxyurea. Safety results of this RCT have been previously reported ( Reyes et al, Am J Hematol 2022). Plasma arginine levels were low at VOE presentation (mean 50±28 μM), with low arginine levels (<60 μM) found in 74% of patients. Arginine replacement therapy increased arginine concentration by 182% (p<0.0001) compared to 26% (p=0.24) in placebo. Arg/orn correlated to arginase concentration ( r= - 0.38, p<0.0001). Time-to-crisis-resolution strongly correlated with TPO ( r=0.72, p<0.0001). Arginine bioavailability at presentation was inversely correlated to time-to-crisis-resolution for both arg/orn and GABR ( r= -0.37 p=0.02) in the placebo arm only but not in those participants who received arginine therapy (GABR: r= - 0.05, p=0.6; arg/orn: r= - 0.07, p=0.55) (Fig1A,1B). Similar trends were observed for TPO. Pain scores at presentation positively correlated with TPO ( r= 0.33, p=0.04) in the placebo arm only, but not in the arm that received arginine therapy ( r= 0.03, p=0.76). Time-to-crisis-resolution positively correlated with age ( r= 0.2, p=0.03). Conclusion: In the absence of arginine replacement therapy, low arginine bioavailability predicted longer time-to-crisis resolution and a higher use of IV opioids in patients with SCD-VOE. Arginine bioavailability may represent a novel biomarker of SCD-pain severity. This data further confirms arg/orn as a surrogate for arginase activity reflective of hemolytic rate as previously described. Arginine replacement therapy ameliorates the influence of arginine deficiency on clinical outcomes related to pain in SCD. An NHLBI/ Pediatric Emergency Care Applied Research Network (PECARN) supported Phase-3 multi-center trial of arginine replacement therapy enrolling 360 children with SCD (SCD Treatment with Arginine Therapy - STArT) is ongoing.

DNA Methylation Changes in Nucleated Red Blood Cells from Patients with Sickle Cell Disease: A Potential Marker of Engraftment after Stem Cell Transplantation

Hematopoietic cell transplant (HCT) is the only established cure for sickle cell disease (SCD). Engraftment (chimerism) of donor red blood cells (RBCs) determines HCT success but current methods measure only white blood cell chimerism and may be inaccurate or fail to indicate graft rejection in a timely manner. Measurement of differentially methylated regions (DMRs) in nucleated red blood cells (nRBCs) to distinguish individuals with sickle cell anemia (genotype HbSS) nRBCs from HbAA and HbAS nRBCs to quantify RBC chimerism could improve post-HCT monitoring by increasing sensitivity to detect rejection and allow for early intervention. Cell-free DNA derived from nRBCs represents a potential method to specifically measure RBC chimerism. Methylation of cytosine nucleotides that are followed by a guanine (methylated CpG sites) is a major means of cell-specific gene expression, and measuring regions of multiple differentially methylated cytosines (DMRs) enables the identification of cell-of-origin of DNA fragments. Cell-free DNA is released into the circulation from all body tissues and cells, including nRBCs, and retains their cell-specific methylation signatures (figure 1). We hypothesized that DMRs unique to healthy and/or SCD nRBC DNA could be identified and later quantified to assess RBC donor chimerism. We generated genome-wide methylomes of nRBC DNA from pediatric SCD (n=2) and non-SCD control (n=3) patients using enzymatic methyl-sequencing (figure 2). From these 5 novel nRBC methylomes and publicly available methylomes and hydroxymethylomes from 35 different tissues and hematopoietic cells we computationally identified 105 DMRs uniquely found in nRBCs compared to other tissues and cells. There were 34 DMRs that specifically distinguished control nRBCs from SCD nRBCs. The 105 DMRs unique to nRBCs were all relatively hypomethylated. These DMRs were intergenic and not associated with CpG islands or shores. DMRs that distinguished SCD nRBCs from control nRBCs were predominantly hypermethylated (76%) in SCD nRBCs and were commonly (21%-24%) located at promoter, exon or intron sequences while 47% were intergenic. 24% were in CpG islands and 5% were in CpG shores. The most significant gene ontology terms in SCD-specific nRBC DMRs were cardiac-related, including heart contraction and conduction. Other highly significant terms were related to cell signaling, transmembrane transporters, and ion channels. Enriched pathway analysis identified terms related to cell signaling, though these were associated with neuronal and immune signaling pathways. To validate our in silico findings, we amplified 8 DMRs from genomic DNA isolated from 23 different cells and tissues and compared methylation patterns. Four DMRs were relatively hypomethylated in control nRBCs compared to SCD nRBCs and other tissues and cells. Amplification of these DMRs from cell-free DNA in patient plasma before and after HCT will validate them as potential unique markers of successful RBC chimerism. Quantification in recipients with HbAA and HbAS donors will be performed. The successful identification of nRBC-specific DMRs in plasma cell-free DNA will enable novel methodology for the quantification of RBC chimerism after HCT for SCD that can be compared to conventional methods of chimerism measurement in future clinical trials.