Alterations in the mitochondrial genome integrity, including changes in mitochondrial DNA copy number (mtDNA-CN) and accumulation of mtDNA mutations, are associated with aging and diverse disorders, often linked to underlying systemic inflammation and metabolic stress. In sickle cell disease (SCD), inflammation drives the pathology, resulting in organ damage and early mortality. The prognostic role of mitochondrial genomic variation in SCD is largely unexplored. This study investigated whole blood derived mtDNA alterations, including mtDNACN and mtDNA mutations, in adults with SCD, sickle trait, and healthy controls, and examined their associations with age and mortality in SCD. We also assessed mtDNA heteroplasmy distribution across tissues in a humanized mouse model of SCD. Elevated mtDNA-CN and mtDNA heteroplasmy burden were observed with increasing genotype severity across all cohorts (HbAA, HbAS < HbSB+ < HbSC < SCA (HbSS and HbSβ0)). In sickle cell anemia (SCA) patients, mtDNA mutation burden- including mtDNA heteroplasmy and mtDNA deletions increased with age, whereas mtDNA-CN level declined, indicating progressive deterioration of mtDNA integrity with age. In SCD patients, specific mtDNA variants showed strong positive correlations with mortality risk, lower mtDNA-CN correlated with higher NIH risk scores, and nuclear variants CYB5R3 T117S and PIEZO1 E756del influenced mtDNA mutation burden without affecting NIH risk score. Consistent patterns of mutational load were observed across specific regions in mitochondrial genome in both humans and mice, suggesting potential mtDNA mutational hotspots. We conclude that variations in the mitochondrial genome are potential prognostic markers for SCD.

Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Coinheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering insights into the complex role of PKR deficiency in SCD pathology.

Abstract Sickle cell trait (SCT), the heterozygous state for the hemoglobin S (HbS) mutation, affects roughly 1 in 13 African American individuals and is common among blood donors recruited for antigen‐matched transfusions in sickle cell disease (SCD). While individuals with SCT are typically asymptomatic, it is unclear whether red blood cells (RBCs) from SCT donors have impaired storage quality and transfusion efficacy. Here, we integrate multi‐omics to characterize RBCs from donors SCT and evaluate their performance post‐transfusion. We first profiled RBCs from 174 HbAS and 248 HbAA volunteers, identifying elevated levels of metabolic markers of the storage lesion in SCT RBCs at baseline. We then interrogated the REDS RBC Omics dataset (>13,000 donors), identifying blood donors carrying the HbS E6V variants. SCT RBCs exhibited accelerated metabolic aging, oxidative stress, and proteostatic activation—phenotypes further exacerbated by storage duration and co‐inheritance of G6PD deficiency. Functional assays confirmed decreased osmotic fragility and increased oxidative hemolysis in SCT RBCs by storage Day 42. Pre‐clinically, stored RBCs from Townes mice carrying one allele of human sickle hemoglobin were characterized by a drop in post‐transfusion recovery compared to mice expressing canonical human hemoglobin. Clinically, analysis of 6828 transfusion events revealed that SCT RBCs were associated with lower hemoglobin increments 24 h post‐transfusion. These findings provide mechanistic and clinical evidence that SCT influences RBC quality and transfusion outcomes. Given the overrepresentation of SCT in donor pools serving patients with SCD, our study supports a more personalized approach to inventory management and transfusion strategies in high‐risk populations.

Abstract: The amyloidogenic V122I variant of the transthyretin (TTR) gene is found in ∼3% of African American individuals and increases cardiovascular mortality risk after the age of 65 years. Sickle cell disease (SCD) primarily affects individuals of African descent, leading to multiorgan damage and premature mortality, with cardiopulmonary issues being a major cause of death. We assessed the impact of TTR V122I on cardiac phenotype and survival in a study of 584 patients with SCD (mean age, 35.9 years; 50.7% women). The prevalence was 3.1% (18/584), mainly female (72.2%). Age, blood pressure, body mass index, and liver/renal markers were similar between carriers and noncarriers, except for higher blood urea nitrogen levels in carriers. Echocardiography showed that carriers had increased septal thickness and left ventricular mass index and lower diastolic function indices. Over a median follow-up of 6.5 years, 219 patients died. The coinheritance of TTR V122I with SCD was associated with increased mortality (hazard ratio, 2.82; 95% confidence interval, 1.57-5.06). At 5 years, the cumulative incidence of death was 52.9% among carriers compared with 14.5% among noncarriers, corresponding to an approximate relative risk of 3.6. TTR protein levels were significantly lower in carriers. In conclusion, TTR V122I prevalence in patients with SCD mirrors that of the general African American population but affects cardiovascular function much earlier, and a contributing factor may be the underlying oxidative stress and chronic anemia. Genetic screening for TTR V122I is important and should be considered for patients with SCD. This trial was registered at www.clinicaltrials.gov as NCT00011648.

Abstract Sickle Cell Disease (SCD) is an inherited red cell disorder caused by the presence of the abnormal hemoglobin S (HbS) resulting from a single base substitution in the beta hemoglobin gene. SCD affects approximately 8 million people globally including 100,000 in the United States. HbS polymerizes under low oxygen conditions in the tissues to stiffen and distort red cells (sickling), that underly the 2 key features of the disease – recurrent acute vaso-occlusive crises and hemolytic anemia. The manifestations of SCD are multisystemic with progressive organ damage and premature death. Gene therapy and allogeneic hemopoietic cell transplantion offer potential cures but these are currently only available to few patients, even in well-resourced countries. Hydroxyurea remains the only proven effective pharmacological treatment. There is a huge unmet need to develop more oral drugs accessible to patients worldwide. Since the formation of abnormal HbS fibers initiates the pathology of SCD, understanding how they form and how they impact red cell membrane at high resolution is vital, as this could provide insights in new therapeutic strategies. Extensive research has been conducted ex vivo to solve the structure of the HbS polymer with only partial success. High resolution in situ data offers a direct physiological view on HbS polymers at near atomic resolution, however, it bears a number of technical challenges. Here, we established a cryogenic electron tomography (cryo-ET) pipeline to visualize, for the first time, the intracellular molecular organization of sickled red blood cells (RBCs) from patients with sickle cell disease (SCD; HbSS, HbF <10%) in situ. Using a custom-designed chamber for controlled deoxygenation, we induced sickling in leukodepleted RBCs and vitrified them under fully deoxygenated conditions, preserving their native ultrastructure. Cryo-focused ion beam milling coupled with scanning electron microscopy (cryo-FIB-SEM) was employed to prepare 100–150 nm-thick lamellae from intact vitrified sickled RBCs, maintaining spatial context and avoiding structural disruption. Targeted tomographic data collection was performed on a high-end Titan Krios transmission electron microscope under cryogenic conditions. 3D reconstructions revealed densely bundled HbS polymers, 20 nm in width and tightly aligned within the cytoplasm. The cytoplasm is generally devoid of organelles, with an exception of mitochondria, which we validated to be a mixture of functional and dysfunctional forms. Abnormal mitochondria retention in mature RBCs has also been reported in other haemolytic anemias. We then segmented HbS polymers to train a deep learning model (crYOLO) for automated particle detection, facilitating the extraction of ~20,000 subvolumes. Using subtomogram averaging approaches we obtained a preliminary reconstruction at 20 Å resolution that validates key features learnt from in vitro and in silico models. Ongoing efforts aim to enhance resolution and reveal the molecular architecture of HbS polymers within their native cellular environment to provide mechanistic insights into the pathological transformation underlying sickle cell disease. Efforts are also underway to characterize molecular architecture of the red cell cytoskeleton, retained mitochondria in sickle red cells and how small molecule interventional drugs disrupts formation of HbS fiber formation and impacts distortion of the red cell membrane.

Introduction: Acquired mitochondrial DNA mutations (mtDNA heteroplasmy) and changes in mtDNA copy number (mtDNA-CN) are indicators of aging and inflammation ('inflammaging') in various diseases. In sickle cell disease (SCD), systemic inflammation is a key factor driving the disease progression, and patients with the more severe genotypes (HbSS and HbSb0Thal) display higher levels of inflammation. Our previous research demonstrated that patients with HbSS and HbSβ0Thal exhibit a greater burden of mtDNA heteroplasmy and higher mtDNA-CN levels. We also identified specific 'hotspot' regions in the mitochondrial genome, which are more prone to acquiring mutations. In this longitudinal study, we used the humanized Townes SCD mouse model to explore how dysfunctional mtDNA accumulates over time and if the rate of accumulation is influenced by severity of the SCD genotype. Methods: The study included 60 Townes SCD mice: 20 HbAA (controls), 20 HbAS (sickle cell trait), and 20 HbSS (sickle mice). All groups included an equal number of littermate male and female mice. We collected 100ul- 200ul of blood from anesthetized mice at 8 time points: weeks 16 (T1), 27 (T2), 39 (T3), 58 (T4), 65 (T5), 69 (T6), 71 (T7) and 73 (T8). Genomic DNA extracted from the whole blood, was used to enrich mtDNA and then subjected to deep sequencing. The analysis of mtDNA heteroplasmy utilized LoFreq variant caller (allele frequency filter 2%). SIFT prediction was utilized to identify deleterious mtDNA heteroplasmies. Additionally, the remaining genomic DNA was whole genome sequenced (WGS) to estimate mtDNA-CN. For mtDNA heteroplasmy comparison, changes from the baseline (T1) were derived for each mouse, and the mean percentage change was determined for each genotype at each timepoint. Analysis of deleterious mtDNA mutations and mtDNA-CN were conducted using aggregated group means at each time point, due to the lack of consistently paired samples across the time points. Statistical analyses were conducted with GraphPad Prism. Results: The study is ongoing.At week 73 (T8), 22 mice (11 HbAA, 8 HbAS, 3 HbSS) were alive. HbSS mice had a higher mortality rate compared to HbAA and HbAS mice (p=0.0177). mtDNA mutational burden analysis for the initial 3 time points (T1, T2, T3) containing a total of 176 samples has been completed to date. Over time, the mtDNA heteroplasmy burden increased remarkably, rising by 6.38% at T2 (p=n.s) and 18.38% at T3 (p=0.0368) compared to baseline (T1). HbSS mice displayed an accelerated increase in mtDNA heteroplasmy burden, showing a 42.47% rise at T3 compared to 12.5% at T2 and T1 (p=0.0062 and p=0.0006, respectively) while HbAA and HbAS mice showed minimal changes (p=ns). Deleterious mtDNA heteroplasmy also expanded over time, with increases of 20.83% at T2 and 31.70% at T3 compared to T1, with a dramatic escalation in HbSS mice, 71.43% increase at T2, and 200% at T3 compared to T1. In HbSS mice, the burden of deleterious heteroplasmies at T3 was significantly higher than at baseline (p=0.0015), while that in HbAS and HbAA were relatively stable over time. All identified deleterious heteroplasmies (n=165) were found in mitochondrial genes, ATP8, ATP6, and ND2. This study also observed a decrease in mtDNA-CN at T3 compared to T2 and T1. Of the 176 samples collected, 130 were WGS, 46 were excluded due to insufficient DNA. Analysis of 45 paired samples with two time points showed a significant reduction from T-Young (average of T1 and T2; mean mtDNA-CN=1580) to T-Old (T3; mean mtDNA-CN=1438) (p=0.026). There was an overall declining trend in mtDNA-CN with age, with HbAA mice showing significantly greater reduction compared to HbAS and HbSS mice (p < 0.05). Conclusions: The study reveals that in Townes mice, the mtDNA heteroplasmy burden increases with aging, with HbSS mice showing a faster accumulation of mtDNA mutations and a pronounced rise in deleterious mtDNA mutations, primarily found in genes related to oxidative phosphorylation. Our findings also suggest a potential threshold effect, when the mutational burden escalates dramatically. Completion of the longitudinal mtDNA analysis until death of all HbSS mice in this cohort will provide insights on how mtDNA mutations accumulate and their correlation with sickle cell pathology.

Abstract Sickle cell disease is an inherited red cell disorder that arises from a single nucleotide mutation in the beta hemoglobin gene, yet its effects are far-reaching and multifaceted. Despite its monogenetic origin, the disease leads to a cascade of complications due to the altered shape and rigidity of red blood cells which can cause episodes of severe pain, organ damage, and an increased risk of infections. The variability in clinical presentation and outcome among individuals also highlights the complexity of interactions between genetic, environmental, and lifestyle factors influencing the severity and manifestations of the disease and long-term survival of patients. Clinical risk factors and biomarkers have been extensively studied and have been utilized as decision-making tools, but genetic variants co-inherited with the causal hemoglobin mutation also influence disease progression and mortality. However, identifying prognostic variants is challenging due to the complexity of the genome and the subtle effects of individual markers. Here, we used a combination of machine learning and survival analysis methods to identify and validate genetic variants that can help predict long-term mortality risk in patients with SCD. Methods: We studied a cohort of 673 patients with SCD (554 HbSS and HbSβ⁰, 91 HbSC, 25 HbSβ+, 3 HbSD and HbSO) enrolled at the National Heart, Lung, and Blood Institute (NCT00011648) from 2006 to 2025. Using whole genome sequencing, we identified 75 genetic variants across 50 genes linked to sickle-related complications as reported in the literature. A random survival forest approach ranked the variants by their importance in predicting survival. The top 10 variants were further analyzed using Kaplan-Meier methods, followed by further detailed modeling for the following five significant variants: rs1427407 (BCL11A), a compound APOL1 variant (rs73885319 + rs71785313), rs2235302 (SELP), rs7412 (APOE), and rs76992529 (TTR, V122I). Based on their ranking, patients were then classified into four mutually exclusive genetic risk groups: Group 1 (n=58): Carriers of rs1427407 (BCL11A) or the APOL1 compound variant only; Group 2 (reference group, n=351): Non-carriers of all five variants; Group 3 (n=240): Carriers of rs2235302 (SELP) or rs7412 (APOE) only; Group 4 (n=24): Carriers of rs76992529 TTR V122I only. A Cox proportional hazards model assessed survival differences using Group 2 as reference. Kaplan-Meier curves and 10-year survival estimates were generated, and model performance was evaluated using the concordance (C-index). These analyses were also performed on a subgroup of patients with severe genotypes (HbSS or HbSβ⁰; n=554). Results: Survival rates varied significantly across the four genetic groups (log-rank p < 0.0001). Compared to non-carriers (Group 2), Group 1 had a 57% lower risk of death (HR = 0.43, 95% CI: 0.23–0.80, p = 0.0075). Group 3 had a 57% higher risk of mortality (HR = 1.57, 95% CI: 1.20–2.04, p = 0.0008), while Group 4 had the highest risk with a threefold increase in death risk (HR = 3.05, 95% CI: 1.68–5.55, p = 0.00025). In Kaplan-Meier analysis, ten-year survival rates were 82.7% for Group 1, 64.7% for Group 2, 47.2% for Group 3, and 27.7% for Group 4, indicating significantly better survival in Group 1 and much earlier mortality in Group 4. The model’s C-index was 0.601, indicating modest predictive ability. Despite not being highly discriminative, the model effectively captured a meaningful risk stratification. Similar patterns were observed in the subgroup of patients with severe genotypes (HbSS or HbSβ⁰;), supporting the robustness of these genetic risk classifications. Conclusion: This study demonstrates that combining machine learning with survival analysis is effective in identifying genetic variants associated with SCD mortality. Using random survival forest ranking and Kaplan-Meier analysis, we identified five variants that stratify patients into four distinct genetic risk groups, with different survival outcomes. The findings suggest a protective effect of rs1427407 in BCL11A and the APOL1 compound variant (rs73885319 and rs71785313). The TTR variant was associated with the poorest outcomes, and rs2235302 in SELP or rs7412 in APOE had a modest negative impact. These stratification patterns highlight the potential of genetic profiling to improve personalized risk prediction and guide long-term management in patients with SCD.

Background: The ubiquitin-proteasome system (UPS)is essential for maintaining red blood cell (RBC) membrane homeostasis by degrading damaged and misfolded proteins, thus preventing the accumulation of potentially harmful protein aggregates. Ubiquitin, a small polypeptide, contains seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) that can form various types of ubiquitin chains. Among these, K48-linked chains are the most abundant and are responsible for mediating the proteasomal degradation. Valosin-containing protein (VCP/p97) is crucial in preserving RBC membrane integrity by removing and transporting proteins polyubiquitinated with K48-linked ubiquitin from the membrane to the UPS. In sickle RBCs, oxidative stress and insufficient bioenergetics (ATP) can impair p97 function, leading to the accumulation of dysfunctional p97 on the RBC membrane, elevated levels of K48-polyubiquitinated (K48-Ub) proteins, and compromised RBC membrane integrity. Mitapivat, a pyruvate kinase activator of both PKR and PKM2, is under clinical development for treating sickle cell disease (SCD). A key mechanism by which mitapivat treatment increases hemoglobin levels is through the enhancement of RBC integrity, achieved by boosting ATP production and reducing oxidative stress in patients with SCD. Here, we demonstrate that, in addition to the reduction in tyrosine-phosphorylation of RBC band 3, another underlying mechanism that contributes to improved RBC integrity is the restoration of p97 function and a decrease in the accumulation of ubiquitinated proteins. Methods: RBC ghosts (membranes) were isolated from frozen whole blood samples from 3 individuals each with HbAA, HbAS, and HbSS enrolled under protocol NCT00047996. The levels of K48-Ub and p97 were analyzed by Western blotting and densitometry analysis. To investigate the effect of mitapivat on membrane accumulation of p97 and K48-Ub, RBCs from HbSS patients were treated with varying concentrations (1 µM, 3 µM, 10 µM, and 30 µM) of mitapivat for 1h, 2 h, 4 h, 8 h, and 16 h ex vivo. The levels of ATP and K48-Ub and p97 were measured using a Luminescent ATP Detection Assay Kit (abcam) and Western blotting, respectively. We also utilized frozen whole blood samples from patients in the Phase 1 dose-ascending study (NCT04000165), of which 7 of 16 were accessible. RBC ghosts at baseline, after 2 weeks of 5, 20, 50, and 100 mg twice daily mitapivat, at the end of drug taper, and at the end of study were isolated. The level of membrane K48-Ub was analyzed by Western blotting and quantified by densitometry analysis; p97 was not measured due to shortage of material. The percentage change of K48-Ub from baseline was calculated for each sample at different timepoints, and a one-sample, two-sided t-test was used to assess the significance of the mean change of the 7 patients' samples. Statistical significance was annotated as follows: p < 0.05 (*), p < 0.01 (**), and p ≥ 0.05 (ns). To evaluate the association between K48-Ub and ATP, a Spearman rank correlation analysis was performed using the relative change of K48-Ub and ATP value corrected for hematocrit (ATP/Hct). Results: Significantly higher levels of K48-Ub (p < 0.05) and p97 (p < 0.05) were observed on the RBC membranes of HbSS RBCs compared to HbAA and HbAS RBCs, consistent with previous studies. HbSS RBCs treated withmitapivat ex-vivo showed a dose- and time-dependent increase in ATP production accompanied by a decrease in membrane-associated p97 and K48-Ub levels. ATP/Hct level of the 7 patients in the Phase 1 study was significantly increased after 5-, 20-, 50- and 100-mg mitapivat treatment (p < 0.01, p < 0.05, p < 0.01, and p < 0.05, respectively), while K48-Ub levels of RBC membranes were decreased, suggesting a negative correlation between K48-Ub and ATP/Hct levels, which was confirmed by Spearman rank correlation analysis. The Spearman correlation coefficient was R = –0.38, with a p-value of 0.017, indicating a moderate and statistically significant negative association between K48-Ub and ATP/Hct. Conclusion: The function of p97 was impaired in HbSS RBCs, leading to the accumulation of p97 and K48-Ub on the cell membrane. Mitapivat as a PKR and PKM2 activator, significantly increased ATP production, restoring p97 function and reducing K48-Ub accumulation, further improving RBC membrane integrity.

Background: While the pathology in sickle cell disease (SCD) is initiated by polymerization of deoxygenated hemoglobin S (HbS), the downstream cascade events of vaso-occlusion and hemolysis are caused by changes to the red blood cell (RBC) structure and function (“sickling”). A major source of reactive oxygen species (ROS) and oxidative stress are the contents of the sickle RBCs themselves. Oxidative stress affects the integrity of the RBC membrane; it increases tyrosine phosphorylation of band 3 (tyr-p-bd3) that disrupts its interaction with the ankyrin-spectrin cortical cytoskeleton, leading to membrane destabilization, phosphatidylserine exposure, blebbing of membrane-derived microparticles from the cell surface, and release of hemoglobin into the plasma. Collectively, these processes promote adherence of sickle RBCs to the endothelium, thereby increasing the transit time through the microcirculation with increased probability of vaso-occlusion. All these factors act in a positive feedback-loop driving the pathophysiology of SCD. Restoring integrity of the RBC membrane is thus essential to preventing the vaso-occlusive events that promote SCD. Oxidative stress activates p72 spleen tyrosine kinase (SYK), the major kinase that phosphorylates band 3 tyrosine residues. Fostamatinib is a SYK inhibitor and FDA-approved for treatment in adults with chronic immune thrombocytopenia. To evaluate fostamatinib as an antisickling agent, we incubated HbSS RBCs with fostamatinib and its active metabolite, R406, and showed that both agents reduced tyr-p-bd3 in a dose- and time-dependent manner. We compared the effects of different SYK inhibitors (Imatinib, R112, PRT062607, Endospletinib, Fostamatinib, R406, and cerdulatinib) on the fraction of sickled RBCs vs time following deoxygenation of HbSS RBCs, and showed that R406 slowed the kinetics of sickling while the effect of imatinib on sickling kinetics was minimal. HbSS RBCs treated ex-vivo with fostamatinib and R406 also showed improved deformability. Fostamatinib could have additional benefits in SCD due to its anti-inflammatory effect from inhibition of neutrophil extracellular traps (NETs) as shown in COVID-19. Objectives: The main objectives of this study (NCT05904093) are to assess the clinical safety and tolerability of fostamatinib and to evaluate its anti-sickling effects in patients with SCD. Study Design and Methods: This is an open label, single-center, dose-escalating, phase 1 clinical study of escalating doses of fostamatinib in subjects with stable SCD. Fifteen subjects with SCD will complete two dose levels of fostamatinib, 100 mg twice daily (BID) for 2 weeks escalating to 150 mg BID for four weeks. Timed blood samples for pharmacokinetics will be collected pre-dose and at 1, 2, 4, and 8 hours post-dose on initiation of 100 mg and dose escalation to 150 mg (days 0 and 14). A timed blood sample for pharmacokinetics will also be collected prior to the last dose of fostamatinib on day 42. Eligibility: ≥ 18 years with a documented diagnosis of SCD (HbSS or HbSb0 thalassemia), adequate organ function, baseline Hb ≥ 7 g/dL, and no transfusions or erythropoiesis stimulating agents in the prior 3 months. If taking hydroxyurea (HU), the dose must be stable for ≥ 90 days before starting study drug. Not eligible: history of neutropenia (unrelated to drug suppression), history of poorly controlled blood pressure, currently receiving treatment with SCD therapies (excluding hydroxyurea) or history of gene therapy, bone marrow or stem cell transplantation. The primary objective is to assess the clinical safety and tolerability of a two-dose level dosing regimen of fostamatinib in subjects with stable SCD based on the frequency and severity of adverse events (AEs), and changes in laboratory parameters. Descriptive statistics will include the incidence and severity of AEs with associated confidence intervals for binomial distributions. Conclusion: This study will evaluate the safety and tolerability of fostamatinib across the tested dose levels in adults with SCD. The accompanying changes in the active metabolite of fostamatinib, R406, with changes in tyrosine phosphorylation of RBC membrane band 3, sickling kinetics, deformability, and in micro-vesicle release will determine whether fostamatinib treatment could be beneficial by reducing the duration or severity of pain episodes.

Stable, mixed-donor-recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient-red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

In a phase 1 study (NCT04000165), we established proof of concept for activating pyruvate kinase (PK) in sickle cell disease (SCD) as a viable antisickling therapy. AG-348 (mitapivat), a PK activator, increased adenosine triphosphate (ATP) and decreased 2,3diphosphoglycerate levels while patients were on treatment, in line with the mechanism of the drug. We noted that the increased hemoglobin (Hb) persisted for 4 weeks after stopping AG-348 until the end of study (EOS). Here, we investigated the pathways modulated by activating PK that may contribute to the improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples taken at multiple time points from patients in the phase 1 study, from which RBC ghosts were isolated and analyzed by western blotting for tyrosine phosphorylation of band 3 (Tyr-p-bd3), ankyrin-1, and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the patients, accompanied by an increase in the levels of membrane-associated ankyrin-1 and intact PTP1B, all of which returned to near baseline by EOS. Because PTP1B is cleaved (inactivated) by intracellular Ca2'dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity and the plasma membrane Ca2' ATPase pump-mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activity, and increased Ca2' efflux. Altogether, our data indicate that ATP increase is a key mechanism underlying the increase in hemoglobin levels upon PK activation in SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Introduction: A lower absolute neutrophil count (ANC) is associated with the variant rs2814778 (c.1-67T>C) in the promotor of the ACKR1 gene. Homozygosity for rs2814778 results in the Duffy null phenotype. Duffy null patients have normal bone marrow cellularity and are not at increased risk of infection. Duffy null individuals have malaria survival advantage as the Duffy antigen is the erythrocyte receptor for Plasmodium Vivax, which probably accounts for the allele frequency of 0.81 in African Americans (Afr Am) compared to 0.004 in Caucasians. Among Afr Am, 68% are Duffy null, 23.8% of whom have ANC of <2 k cells/mL and 10% have ANC <1k cells/mL. Sickle cell disease (SCD), a complex multi-systemic disorder, affects ~7-8 million people worldwide, predominantly of African descent and 1 in 365 Afr Am births. Hydroxyurea (HU), a myelosuppressive agent, is standard care in SCD. As HU dosing in SCD is based on achieving maximum tolerated dose (MTD) typically targeting ANC of 1.5-3 x 103 /uL, there is concern about potential underdosing and iatrogenic harm in patients who are Duffy-null. Previous studies on HU dosing in SCD based on Duffy status have focused on children. Here, we investigate dosing differences in adult SCD patients and whether they are at risk of lower ANCs. Method The cohort comprised 673 adult patients with SCD enrolled under protocols NCT00011648 and NCT00081523 at the National Heart, Lung and Blood Institute (NHLBI) between Sept 2006 and Feb 2017. Demographics, HU treatment (dosing based on MTD) status, hematological indices and biochemistry on the day of sampling were recorded in a database with unique identifiers. ACKR1 rs2814778 genotype was derived from whole genome sequence data. Duffy phenotype was established by presence and zygosity of ACKR1 rs2814778. The cohort was stratified by Duffy phenotypic status as well as on/off HU therapy to evaluate differences in ANC, fetal hemoglobin (%HbF), and HU dosing (mg/kg). The comparison was conducted based on Mann-Whitney test using R (v4.3.2). Results: Mean age of the cohort (N=673) was 34.2 (± 12.05 years), 352 female and 321 male. Ethnicity was available for 653 (97.03%) patients of which 630 (96.48%) were Black Afr Am: 522 (82.8%) have sickle cell anemia (SCA) i.e. HbSS or HbSB0 and the remainder have non-SCA SCD (HbSC, HbSB+, HbSD or HbSOArab). Of the 630 Afr Am subjects, 74% were homozygous for ACKR1 rs2814778 and displayed a Duffy null phenotype with an allele frequency of 0.868. The ANC was significantly higher for SCD patients with Duffy non-null compared to those with Duffy null (mean±SD: 6.33±3.17 x 103 /mL vs 5.66±3.05; P=0.011). The elevated ANC in the Duffy non-null subjects sustained when stratifying patients based on HBB genotype (6.55±3.24 vs 5.92±3.19 in SCA patients, P=0.037; 5.41±2.75 vs 4.33±1.67 in non-SCA patients, P=0.041). Only 1% of the SCA patients and 1.23% of the non-SCA SCD patients had ANC of <1.5. There was no statistically significant difference in other markers of disease severity (Platelets, Reticulocytes, ESR, LDH) and no mortality difference between Duffy null and non-null phenotypes in SCA and non-SCA SCD subjects. In the SCA cohort 221 (76.9% Duffy null) were on and 300 (76 % Duffy null) were off HU therapy. Among the 221 on HU, there was no statistically significant difference in ANC, mean %HbF and HU dose between Duffy null and non-null groups (ANC: 5.31±2.77 vs 5.78±2.88, P=0.26; %HbF: 10.4±6.69 vs 10.5±6.23, P=0.74; HU dose: 17.9±8.02 vs 17.1±8.68, P=0.62). Discussion: We confirmed that the allele frequency of ACKR1 rs2814778 in ourSCD cohort is comparable to that in the general Afr Am population with a similar incidence of Duffy null phenotype. Duffy null status confers a lower ANC compared with those who express Duffy antigen, which is most marked in the non-SCA SCD patients. However, the difference is less marked when compared with published studies of healthy black patients. This may be in keeping with the higher ANC associated with the inflammatory milieu of SCD. There is no significant difference in HU dose nor %HbF in patients stratified by Duffy status. The proportion of patients who are on HU is comparable between the Duffy null and Duffy non-null. This important negative finding in adults is comparable to published data on pediatric patients and indicates that Duffy null phenotype does not negatively impact HU dosing.

The amyloidogenic V122I variant (valine to isoleucine substitution at position 122) of the transthyretin (TTR) gene is carried almost exclusively in people of African descent, about 3% African-Americans carry the variant. In the general population, carriers for TTR V122I have a higher risk of heart failure, cardiovascular death and increased mortality after age 65 years compared with non-carriers. Sickle cell disease (SCD), one of the most common genetic blood diseases, predominantly affects individuals of African descent and 1 in 365 African-American births. In adults, SCD manifests as a chronic degenerative illness characterized by progressive multiorgan damage, with cardiopulmonary complications being a leading cause of mortality. The impact of TTR V122I in patients with SCD remains unclear. Objectives: To examine the association between TTR V122I and cardiac phenotype and survival in a cohort of adults with SCD. Methods: We conducted a prospective observational study of 584 adult patients with SCD (mean ± SD age: 35.9 ± 12.9 years, 296 women [50.7%]) enrolled at the National Heart, Lung, and Blood Institute (NCT00011648) between Sept 2006 and Feb 2017. The cohort had previously been utilized to develop a phenotypic risk score for disease severity and prediction of mortality. TTR V122I genotype was derived from whole genome sequence data or targeted DNA sequence analysis. Clinical profiles, laboratory variables, cardiac phenotype as assessed by echocardiography, and the NIH phenotypic risk score were compared between TTR V122I carriers and non-carriers. All-cause mortality was ascertained by online obituary search, reporting by patient's family members, National Death Index, and Social Security Death Index search with a final follow-up in June 2024. The cohort was further stratified by gender to evaluate potential sex differences in genotype and phenotype. All analyses were performed using R 4.3.2 with P<0.05 considered as significant. Results: The prevalence of TTR V122I carriers (Age, 42.2 ± 17.2) was 3.1% (n/N=18/584), and predominantly female at 72.2% (n/N=13/18) compared to males. TTR V122I carriers and non-carriers were similar in age, with no significant differences in blood pressure, BMI, eGFR, creatine and other serologic markers of liver or renal function, except for a higher blood urea nitrogen among carriers (BUN, 16.6 ± 12.2 vs.12.0 ± 12.6 mg/dL; P=0.032). Carriers also exhibited higher baseline septal thickness (10.7 ± 1.4 vs. 9.7 ± 1.7 mm; P=0.004 and higher left ventricular (LV) mass index (110.7 ± 25.3 vs. 99.1 ± 31.0 g/m2; P=0.029). Indices of diastolic function were also lower in carriers (mitral E/A ratio (1.3 ± 0.5 vs. 1.7 ± 0.6; P=0.020) and septal e' velocity (8.8 ± 2.0 vs. 10.3 ± 2.9; P=0.020). TTR V122I carriers had an elevated NIH risk score (2.7 ± 1.9 vs. 1.8 ± 3.0; P=0.002). Overall, 219 patients died during a median follow-up of 6.5 years, with TTR V122I TTR carriers showing a higher risk of all-cause mortality (Hazard Ratio [HR] 2.82, 95% CI 1.57-5.06; P<0.001). The median survival time was 4.4 years for carriers versus 12.0 years for non-carriers. Gender differences were evaluated due to the higher prevalence in women, and besides increased septal thickness and LV mass index, female carriers had significantly higher tricuspid regurgitation velocity (TRV, 2.9 ± 0.6 vs. 2.6 ± 0.5 m/s; P=0.032) and a more pronounced risk of mortality (HR 3.48, 95% CI 1.81 - 6.68; P<0.001) compared to female non-carriers. Conclusion: While prevalence of TTR V122I in patients withSCD is comparable to that in the general African-American population, the variant appears to impact cardiovascular function at a much younger age in SCD patients (mean age ~36 years). SCD patients who are also carriers for TTR V122I have higher septal thickness and LV mass index, lower parameters of diastolic function, a higher NIH risk score, and higher all-cause mortality than non-carriers. It is not clear why females have a higher prevalence of TTR V122I compared to males in our SCD cohort. Female carriers experienced more pronounced cardiac dysfunction with increased mortality compared to female non-carriers. We propose that the high oxidative stress environment and chronic anemia are factors that contribute to the accelerated clinical penetrance of the TTR V122I phenotype in SCD, and that genetic screening for TTR V122I should be included in decision-making in choosing SCD patients for high-risk curative therapies.

The ability of red blood cells (RBCs) to deform is a key factor in the pathophysiology of sickle cell disease (SCD). Deformability measurements of RBCs via ektacytometry are increasingly used as biomarkers of therapeutic efficacy in anti-sickling therapies, including mitapivat, a pyruvate kinase (PK) activator. It has been shown that mitapivat therapy in SCD improves RBC deformability in response to shear stress, osmotic pressure, and decreasing oxygen tension, likely via bolstered membrane integrity. Indeed, analysis of RBC samples from HbSS patients treated with mitapivat in a Phase 1 study showed a significant dose-dependent reduction in tyrosine phosphorylation RBC band 3 (Tyr-p-bd3) with increased membrane-associated ankyrin-1 and intact (active) protein tyrosine phosphatase 1B (PTP1B). Here, we assess sustainability of mitapivat-induced changes in RBC deformability, correlate these changes with those on Tyr-p-bd3 and how they impact the hematologic parameters. METHODS We studied 15 HbSS patients (aged 25-57 years; 10 males) currently enrolled under protocol NCT04610866 evaluating long-term safety and tolerability of mitapivat in SCD patients. All patients started mitapivat at 50 mg twice daily (BID), escalating to 100 mg BID after 4 wks; dose adjustments were performed for safety and tolerability, per PI discretion. Fresh whole blood in EDTA tubes was obtained and processed at baseline (V1, prior to drug initiation) and longitudinal time points up to 2 years (V12). Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, Netherlands) assays were performed on fresh blood following standard procedures. The Elongation Index (EI) measures the cells' ability to deform under different shear stress, continuous osmolality and oxygen pressures. RBC membranes were isolated from frozen whole blood and subjected to Western blotting analyses for Tyr-p-bd3, ankyrin-1 and intact (active) PTP1B and quantified by densitometry. For all assays, we analyzed the %change at each timepoint from baseline for each subject, and then derived the mean %change for each timepoint for all subjects. Significance testing was derived by Wilcox signed rank test. Correlations assessments were based on nonparametric Spearman correlation, with all analyses conducted using R (v4.2.3). RESULTS Mitapivat therapy increased elongation indices (EIs) across all shear stress levels as early as 2 wks, e.g., at shear stress 3 Pa and 30 Pa (mean% ± SD) 30.3 ± 51.7, P=0.001; and 22.2 ± 40.4, P=0.005, respectively. These improvements were sustained with extended therapy although the changes were not significant: 18.3 ± 66.8 at 3 Pa P=0.919, and 12.7 ± 56.9 at 30 Pa, P>0.999, respectively at 2 yrs. The O hyper (Osmoscan) was significantly increased after 12 weeks (3.2 ± 6.9; P=0.036) of treatment and remained significant (3.6 ± 8.3; P= 0.048) after 52 weeks of treatment. Reductions in Point of Sickling (PoS) by Oxygenscan, were observed at 12 (-16.8 ± 13.0; P=0.007) and 52 (-20.4 ± 15.5; P=0.015) wks of treatment. Mitapivat therapy significantly reduced RBC Tyr-p-bd3, accompanied by a significant increase in membrane-associated ankyrin-1 and intact PTP1B, within 2 wks of exposure and sustained improvements throughout 2 yrs of therapy. The %changes in Tyr-p-bd3 and ankyrin-1 and PTP1B were significantly (P<0.0001) negatively correlated and that between ankyrin1 and PTP1B, positively correlated. RBC Deformability, as measured by LORRCA assays, was negatively correlated with LDH (=-0.603 at EI=3 Pa, P<0.0001), and total bilirubin (=-0.412 at EI=3 Pa), P<0.0001. Improved RBC deformability negatively correlated with %change in Tyr-p-bd3, with the association increasing as shear stress increases (= -0.228 at 3 Pa, P = 0.005). Tyr-p-bd3 was also inversely associated with EI max ( = -0.404, P=0.144), and EI at PoS ( = -0.404, P<0.0001) for the Osmoscan and Oxygenscan assays respectively, indicating improved sickling kinetics. CONCLUSION Mitapivat-induced improvements in RBC deformability in SCD patients is rapid and sustained throughout extended therapy, accompanied by decreased hemolysis. Improvements in deformability were accompanied by significant reduction in Tyr-p-bd3, a critical determinant of RBC integrity. Our findings confirm that activating PK in SCD improves RBC deformability, a key mechanism is the reduction in Tyr-p-bd3, leading to an increased interaction with the anchoring protein, ankyrin.

Introduction: Acquired mutations in the mitochondrial genome, referred to as mtDNA heteroplasmy, and alteration in mitochondria DNA (mtDNA) copy number (mtDNA-CN), are emerging markers of aging and inflammation. Given that the pathology in sickle cell disease (SCD) is driven by inflammation leading to premature organ damage, in a process often referred to as “inflammaging”, we hypothesized that these mtDNA dysfunction readouts could have prognostic value in SCD. Here, we investigated if mtDNA heteroplasmy and mtDNA-CN correlate with SCD genotype severity, age and survival in humans. Additionally, we explored the differential tissue distribution of mtDNA heteroplasmy in SCD mice. Methodology: Human subjects included three adult cohorts (≥18 years old) of African descent enrolled under IRB approved protocols NCT00011648, NCT00081523, and NCT03685721. Details of cohorts: Cohort-1 [total 673 SCD patients: 538 HbSS + 16 HbS-Beta-thalassemia0 (554 SCA), 91 HbSC, 25 HbS-Beta-thalassemia+ (SB+), 3 other SCD genotypes]; Cohort-2 [total 171: 113 SCA, 30 HbAS, 16 HbAA, 12 other SCD genotypes]; Cohort-3 [total 199: 96 SCA, 47 HbAS, 44 HbAA, 12 other SCD genotypes]. Whole genome sequence (WGS) was performed on the human DNA samples. Humanized Townes SCD mice comprised of 18 littermates [6 HbSS, 6 HbAS, 6 HbAA; 3 males and 3 females in each genotype]. mtDNA was specifically amplified from genomic DNA from 9 organs (heart, lung, brain, kidney, liver, muscle, blood, bone marrow, and spleen; total 162 samples) at age 16 weeks and subjected to deep sequencing. Sequencing reads mapped to mtDNA genome (mito-genome) were utilized for mtDNA-CN estimation and variant calling (LoFreq). Allele frequency (AF) cutoffs of 1%, 5%, 1%, and 4% were applied for Cohorts-1, 2, 3, and mice, respectively, based on their respective mito-genome coverage. Survival data in Cohort-1 were ascertained by proxy interview, medical records, National Death Index, and Social Security Death Index search. Cox proportional hazards model was applied to analyze the relationship between mtDNA heteroplasmy with all-cause mortality. Results: Overall, considering all 3 human cohorts, mtDNA heteroplasmy burden increased according to severity of SCD genotypes: HbAA < HbAS < SB+ < HBSC < SCA. mtDNA-CN was significantly higher in SCA genotype (p<0.001) compared to all other genotypes across all 3 human cohorts. mtDNA heteroplasmy also increased with age among all SCD patients in cohort-1; mean heteroplasmies: 0.39, 0.39, 0.47, and 0.58 in age (year) groups <40, 41-50, 51-60, and >60, respectively. In mice, a similar pattern of increasing mtDNA heteroplasmy was also observed: HbAA < HbAS < HbSS. Additionally, mtDNA heteroplasmy varied across different tissues within and across genotypes in the mice; with spleen having the highest burden and liver, the lowest. We observed a pattern across the mito-genome with D-loop, RNR1, ND1, COX1, ATP6, ND5, CYT-B regions exhibiting higher heteroplasmic load consistently across HbAA, HbAS and HbSS/SCA genotypes in both humans and mice. In Cohort-1(n=673) where we could ascertain survival data, we observed a positive trend between mtDNA heteroplasmy and all cause-mortality [H.R with 95% CI: Heteroplasmy continuous = 1.01 (0.853 - 1.197); 1 heteroplasmy vs 0 = 1.173 (0.873 - 1.576); 2 hetroplasmy vs 0 = 1.3 (0.806 - 2.096)] that was sustained after correction for age. Seven variants were significantly associated with mortality with HR ranging from 22.09 to 153.98 (P<0.05); of which 6 variants originated from the Complex-I, IV, V genes. Conclusion: mtDNA variants have prognostic value in SCD. mtDNA heteroplasmy burden increased progressively (HbAA

In regions where reads don't align well to a reference, it is generally difficult to characterize structural variation using short read sequencing. Here, we utilize machine learning classifiers and short sequence reads to genotype structural variants in the alpha globin locus on chromosome 16, a medically-relevant region that is challenging to genotype in individuals. Using models trained only with simulated data, we accurately genotype two hard-to-distinguish deletions in two separate human cohorts. Furthermore, population allele frequencies produced by our methods across a wide set of ancestries agree more closely with previously-determined frequencies than those obtained using currently available genotyping software.

BACKGROUND: Mitochondria are important in the pathology of sickle cell disease (SCD). They are abnormally retained in sickle red blood cells (RBCs) and the likely source of cell-free mitochondrial DNA (mtDNA), an erythrocytic-DAMP that triggered formation of neutrophil extracellular traps (Tumburu et al, 2022). Acquired mutations in the mitochondrial genome, referred to mtDNA heteroplasmy, are emerging markers of aging and inflammation. Using whole genome sequence data from peripheral blood DNA, we previously established that patients with SCD not only have increased mtDNA heteroplasmy, but the burden also varied with the sickle genotype [Ahmad et. al, 2021]. The burden of mtDNA heteroplasmy is known to vary across different organs and tissues. Here, we utilized the Townes mouse model of SCD to explore differential mtDNA heteroplasmy burden across the different tissues and sickle genotypes in SCD. METHODS: Genomic DNA was extracted from 9 different organs (heart, brain, muscle, kidney, blood, bone marrow, liver, lungs, spleen) of littermate mice (3 male and 3 female) of three genotypes (HbAA, HbAS, HbSS) in three replica sets (total number of mice = 18, 9 different tissue samples per mouse, total sample number = 162). Mitochondria DNA was enriched from genomic DNA by long range PCR in 2 amplicons of 9kb and 7kb sizes (Misa Hirose et al, 2018). The enriched mtDNA PCR amplicons were mixed together and subjected to library preparation for deep sequencing using Illumina platform. Sequence reads aligned to mtDNA were mapped and called for variants using LoFreq variant calling tool. RESULTS: Of the 162 samples, 1 sample (HbAA, lung) was excluded from analysis due to low coverage. Using LoFreq variant caller tool (VAF cut-off 4% & Coverage Filter 3500X), and after removing the repeating variants, 11 unique variants spanning 16kb mouse mitochondrial genome, were found in 106 of the 161 (65.8%) samples. The variants were differentially distributed across the tissues and genotypes with the variant at mito-position 9820 (MT: 9820) being most frequent, present in 19, 16 and 18 samples in AA, AS and SS mice, respectively (Fig. 1). Two variants at MT: 11866 and MT: 6881 were shared between AA and AS mice. Four variants were unique to SS mice: MT: 6753 in COX1 gene causing Phe476-Leu substitution, MT: 6794 in intergenic region upstream to COX2 gene, MT: 1220 in intergenic region upstream to ND1 gene, and MT425 in regulatory/D-Loop region (Table -1). The SS-unique variants were present in all tissues except for Cox1 Phe476-Leu (MT: 6753), a single variant that was found only in the spleen. Overall, HbSS had the highest concentration of variants distributed among 41/54 (75.9%) followed by AS 38 /54 (70.3 %) and AA 27/53 (50.9%). Female mice had higher mtDNA heteroplasmy burden compared to male mice in all genotypes. Across all genotypes and tissues, spleen had the highest number of variants (16.03%) and liver, the lowest (9.4%). CONCLUSION: In keeping with data in patients with SCD, HbSS mice had the highest mtDNA heteroplasmic burden followed by HbAS and HbAA genotypes. Four mito-variants were unique to HbSS. We also found differentially higher mtDNA heteroplasmic burden in splenic tissues and speculate that this could arise from metabolic stress conditions in the spleen.

Burkitt lymphoma (BL) is an aggressive B-cell lymphoma that significantly contributes to childhood cancer burden in sub-Saharan Africa. Plasmodium falciparum, which causes malaria, is geographically associated with BL, but the evidence remains insufficient for causal inference. Inference could be strengthened by demonstrating that mendelian genes known to protect against malaria-such as the sickle cell trait variant, HBB-rs334(T)-also protect against BL. We investigated this hypothesis among 800 BL cases and 3845 controls in four East African countries using genome-scan data to detect polymorphisms in 22 genes known to affect malaria risk. We fit generalized linear mixed models to estimate odds ratios (OR) and 95% confidence intervals (95% CI), controlling for age, sex, country, and ancestry. The ORs of the loci with BL and P. falciparum infection among controls were correlated (Spearman's ρ = 0.37, p = .039). HBB-rs334(T) was associated with lower P. falciparum infection risk among controls (OR = 0.752, 95% CI 0.628-0.9; p = .00189) and BL risk (OR = 0.687, 95% CI 0.533-0.885; p = .0037). ABO-rs8176703(T) was associated with decreased risk of BL (OR = 0.591, 95% CI 0.379-0.992; p = .00271), but not of P. falciparum infection. Our results increase support for the etiological correlation between P. falciparum and BL risk.

BACKGROUND Red blood cell (RBC) deformability plays a critical role in RBC elongation and viscosity, and when reduced, perfusion of peripheral tissues and oxygen delivery are impaired. Intracellular ATP maintains water and ion homeostasis in RBCs; reduced ATP leads to water and ion loss, dehydration and loss of RBC elasticity. RBC dehydration increases mean corpuscular hemoglobin concentration (MCHC) and also affects distribution of RBC width (RDW). Patients with sickle cell disease (SCD) have reduced RBC ATP, and markedly reduced RBC deformability when compared to healthy individuals. Here, we employed Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics) to evaluate deformability in RBCs from patients with SCD (HbSS) compared to RBCs from sickle cell carriers (HbAS) and ethnic-matched healthy controls (HbAA). We further analyzed how these LORRCA-derived parameters correlate with ATP, MCHC, and RDW. METHODS We enrolled adult subjects (age ≥ 18 years) of African-descent, and not recently transfused (within 8 weeks) under protocol NCT03685721 approved by the NHLBI Institutional Review Board. Fresh whole blood in EDTA was processed on the same day of collection. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing LORRCA assays including deformability, osmoscan, and oxygenscan (HbSS subjects). The deformability assay uses the elongation Index (EI) to measure the cells' ability to undergo deformation with increasing shear stress at 0.95, 3, and 30 Pascals (Pa). The osmoscan assay measures the deformability under continuous osmotic changes (0-600mOsm/kg) and has several parameters: O min (RBC fragility); El max (maximum deformability and membrane flexibility); O max (osmolality where EI max is achieved) and O hyper (intracellular viscosity and maximum deformability in the hypertonic region). The oxygenscan assay measures the oxygen pressure at the time when RBCs start to become rigid (point of sickling). Whole blood levels of ATP were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). Descriptive statistics and correlation (Spearman) of ATP, RDW, and MCHC with each LORRCA parameter were performed using R (v4.2.3) and Prism(v9). RESULTS We studied 156 subjects which comprised of 63 HbSS (33 males), 61 HbAS (23 males) and 32 HbAA (8 males) genotypes. The average age in years (range) in HbSS was 32 (18-58); in HbAS, 43 (18-70), and in HbAA, 38 (19-72). As shear stress and osmotic pressure increased, the mean EI and osmolality were significantly (p<0.001, unpaired two-sample t-test) lower in the HbSS cohort compared to HbAS and HbAA (Figure 1). There was no significant difference in the mean EI and osmolality between HbAA and HbAS with increasing shear stresses and osmotic pressures. As shear stress increased, the correlation of deformability with reduced ATP got significantly stronger in the HbSS cohort (r= -0.351 at 0.95 Pa to r=-0.401 at 30 Pa) but the trend was reversed for the HbAS and HbAA cohort (Table 1). Under increasing osmotic pressure, a significant correlation was found between reduced ATP and maximal deformability (EI Max) in HbSS (p=0.008) compared to HbAS (p=0.896) and HbAA (p=0.114). MCHC was significantly correlated with RBC hydration state (O Hyper) in all 3 genotypic groups (HbSS, HbAS and HbAA). Similarly, RDW correlated with maximal deformability (EI Max) under increasing osmotic pressure in all 3 cohorts. In the HbSS subjects, there was a significant positive correlation of ATP and RDW with point of sickling (oxygenscan); MCHC was negatively correlated but was not significant. CONCLUSION RBC deformability was reduced in HbSS patients compared to HbAS and HbAA under increasing shear stress and osmotic pressure. ATP levels showed significant correlation with RBC deformability as measured by LORRCA, particularly in patients with SCD, suggesting increasing ATP levels as an important therapeutic strategy in patients with SCD. In keeping with published data, our analyses showed that RBC volume and size as well as intracellular hemoglobin concentration can affect deformability.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.