Integrated Multi‐Omic Characterization of Hexokinase‐1 Deficiency in Red Blood Cells Reveals Altered Redox Homeostasis, Membrane Remodeling, and Features of Incomplete Erythroid Maturation

ABSTRACT 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.