Philip Norris

Hexokinase 1 (HK1) deficiency is a rare cause of hereditary nonspherocytic hemolytic anemia characterized by impaired erythrocyte glycolysis, but its broader molecular consequences of HK1 dysfunction remain poorly understood. We integrated population‐scale genetics, hemolytic testing, and multi‐omic data from the REDS RBC‐Omics cohorts with comprehensive clinical, metabolomic, lipidomic, oxylipin, and proteomic profiling of erythrocytes and plasma from an individual carrying the HK1 p.Arg12Ter variant, predicted to cause complete loss‐of‐function of the erythroid‐specific HK1‐R isoform. Population‐scale analyses identified multiple HK1 variants associated with erythrocyte metabolism and susceptibility to osmotic hemolysis. In the affected individual, multi‐omic characterization of HK1 deficiency revealed disruption of glycolysis, the pentose phosphate pathway, and glutathione metabolism, accompanied by ATP depletion, impaired antioxidant defenses, susceptibility to oxidative challenge, and ongoing hemolysis. Lipidomic analyses revealed extensive membrane remodeling with increased oxidized phospholipids, lysophospholipids, oxylipins, and triglycerides, and altered phospholipid remodeling pathways. Proteomics identified signatures of redox imbalance, altered proteostasis, and enrichment of mitochondrial proteins. Reticulocytosis, increased CD235a ⁺ CD71 ⁻ mitochondria‐positive erythrocytes, elevated acylcarnitines, tricarboxylic acid cycle intermediates, and polyamines suggested severe erythropoietic stress and incomplete reticulocyte maturation. Thus, HK1 deficiency extends beyond impaired glycolysis and ATP depletion to encompass altered redox homeostasis, widespread metabolic and membrane remodeling, disrupted proteostasis, and persistence of immature erythroid characteristics. Together, these findings provide the first integrated multi‐omic characterization of HK1 deficiency, define a phenotype of HK1 dysfunction, chronic hemolysis, and compensatory erythropoiesis, and establish a framework linking common and rare HK1 variation to molecular phenotypes in human erythrocytes.

West Nile virus (WNV) is an emerging/re-emerging flavivirus representing one of the major global causes of mosquito-borne encephalitis, yet no approved therapeutics or vaccines currently exist. To characterize the kinetics of WNV-specific immunity, we evaluated the humoral response in a longitudinal cohort of 55 WNV lineage 1-infected blood donors up to 1 year. We demonstrate that WNV infection elicits robust and durable binding and neutralizing antibodies. Using a biphasic decay model, we estimated the half-life of neutralizing antibodies to be 37 days during the rapid decay phase and 472 days during the extended slow decay phase. Notably, the magnitude and durability of these antibodies were largely independent of viral RNA persistence in the whole blood (WB) compartment. To assess the breadth of this response, we evaluated cross-reactivity against an emerging divergent WNV lineage 2 strain and observed that WNV lineage 1-immune sera exhibited reduced neutralizing activity with high variability between donors during both acute and convalescent phases. These deficits in heterologous neutralization were reciprocally confirmed using an independent WNV cohort from Italy. Together, our results establish that lineage-specific divergence results in antibodies with a highly variable reduction in neutralization, indicating that successful WNV vaccine candidates should overcome this variability to confer pan-lineage protection. IMPORTANCE: West Nile virus (WNV) is a growing global health threat spread by mosquitoes that can cause severe disease, yet there are currently no approved vaccines or treatments available. We followed 55 WNV lineage 1-infected blood donors up to 1 year to study their antibody response to infection. We found that natural infection generates a long-lasting antibody response. Interestingly, the magnitude and durability of these antibodies were maintained regardless of viral RNA persistence in the whole blood compartment. However, a potential gap in cross-protective immunity emerged when we examined WNV lineage 1-specific antibodies against WNV lineage 2, a genetically distinct and divergent viral strain with over 25 mutations in the envelope protein. Sera from WNV lineage 1-infected donors showed a highly variable reduction in neutralizing activity against lineage 2 WNV during both the acute and convalescent phases of infection. We corroborated this observation using an independent cohort of patients from Italy, again demonstrating reduced cross-neutralization between the two viral lineages. Together, our results establish that lineage-specific divergence results in reduced cross-neutralization of heterologous strains, indicating that successful WNV vaccine candidates should overcome this barrier to confer pan-lineage protection.

Background: Donated blood is a biological product whose molecular composition is influenced by donor exposures, including environmental toxicants such as heavy metals. Among these, lead is particularly relevant because no exposure level is considered completely without risk. Most circulating lead partitions into red blood cells (RBCs), and transfused RBC units can increase blood lead levels in vulnerable recipients. Whether donor lead burden reflects donor biology and associates with storage phenotypes remains unclear. Study Design and Methods: We analyzed 638 donors from the REDS donor study. Lead was measured by inductively‐coupled plasma mass spectrometry and linked to donor demographics, donation frequency, end‐of‐storage hemolysis phenotypes, and RBC multi‐omics features. Multi‐omics associations were assessed by Spearman correlation and repeated using partial Spearman correlations adjusted for donor age, with false‐discovery‐rate correction across all tested features. Results: Lead levels ranged from 0.14 to 8.56 μg/dL. Donor age was the dominant correlate of lead (rho = 0.51, p < 1 × 10 ⁻⁴ ), whereas BMI showed only a weak inverse association, despite its positive relationship with age in the cohort. Lead did not differ by sex, and frequent donation (>9 donations in the prior 24 months) was not associated with lower lead levels. At day 42, lead associated with 53 and 5 of 3468 multi‐omics features in unadjusted and adjusted analyses, respectively. Conclusion: Low‐level donor lead burden is strongly age‐dependent. These findings identify donor age as a major determinant of the RBC lead exposome and highlight candidate age‐independent features for further mechanistic study.

To understand the impact of blood donor and product characteristics on platelet transfusion outcomes, we linked these variables with recipients of single-unit apheresis-derived platelet products between 1 June 2020 and 31 March 2022. We used multivariable logistic regression to examine associations between donor and product characteristics on posttransfusion platelet counts and 24-hour red blood cell (RBC) transfusion events in 2808 transfusion recipients who received ≥1 platelet transfusions (N = 8207 units). Posttransfusion platelet increments >20 × 109/L and RBC transfusion within 24 hours occurred after 24.9% and 37.7% of platelet transfusion events, respectively. After multivariable adjustment, donor variables, including increasing body mass index (BMI), platelet unit concentration, and elevated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antibody levels, were associated with increased odds of recipient platelet increments of >20 × 109/L; whereas pathogen reduction, storage in platelet additive solution (PAS), platelet splits, and platelet storage between 5 and 7 days were associated with reduced odds. In parallel, increasing donor BMI and elevated SARS-CoV-2 nucleocapsid antibody levels were associated with lower odds of 24-hour RBC transfusion events. In contrast, pathogen reduction and irradiation were associated with increased odds of RBC transfusion, whereas longer storage, storage in PAS, and platelet unit concentration and splits were not associated with changes in RBC transfusion events. In conclusion, blood donor and product characteristics are predictors of changes in platelet counts and RBC requirements after platelet transfusion. Future studies examining the impact of blood donor and product characteristics on platelet function could be used to optimize platelet transfusion practice.

Summary: As the most abundant human cell and the foundation of transfusion medicine, red blood cells (RBCs) offer a unique readout of systemic health, yet they have never been characterized at population scale. We generated a proteome atlas of 13,091 blood donors with multi-omics longitudinal phenotyping, characterizing the influence of demographics and genetic variation on the reproducibility of RBC proteomes across donations. Elastic-net aging clocks captured biological aging with high accuracy and uncovered genetic regulators of ΔAge at FN1, C4/IKZF1, CRAT, PFAS, TRIM58. Across independent cohorts, ΔAge was accelerated in G6PD deficiency, sickle cell trait/disease, and iron deficiency, reversed by iron repletion, and slowed in high-frequency donors, linking molecular aging to brain iron/myelin and cognitive performance. Molecular aging signatures predicted storage, osmotic, and oxidative hemolysis, hemoglobin increments after transfusion, and long-term donor activity over 12-years. These results establish RBC proteomics as a scalable biomarker of aging, donor healthspan, and transfusion outcomes. Abstract Figure: Dzieciatkowska et al. generate the first population-scale atlas of the RBC proteome across 13,000 donors and develop proteomic and metabolomic aging clocks that quantify biological age. Molecular ΔAge is reproducible across donations, genetically encoded and accelerated in G6PD deficiency, sickle cell trait/disease, and iron deficiency - yet reset by iron repletion, tracking with cognitive function and brain iron/myelin. RBC aging clocks predict hemolytic fragility, transfusion efficacy, and donor activity 12 years later. Highlights: RBC proteome atlas of 13,091 donors reveals demographic and genetic programs Genetically encoded RBC aging clocks identify regulators of molecular Δage Molecular aging features predict hemolysis and transfusion response across cohorts RBC molecular age forecasts long-term donor activity over a 12-year follow-up