Multiomics analysis of red blood cells reveals thalassemia severity beyond globin gene mutations.

Hemoglobinopathies are the most common monogenic genetic disorders, primarily managed through blood transfusions or bone marrow transplantation. Clinical severity other than mutational effect is not well investigated and still unknown. This study aimed to identify dysregulated molecular pathways in red blood cells (RBCs) contributing to thalassemia severity. From a cohort of 285 patients with hemoglobinopathy, 10 age-matched individuals with identical compound heterozygous mutations (IVS 1-5 G>C and CD 26 G>A) were screened. Five had severe thalassemia requiring regular transfusions, whereas 5 had a nonsevere form requiring fewer transfusions. RNA sequencing and proteome analysis were conducted on isolated RBCs using NovaSeq and Orbitrap mass spectrometry platforms, respectively. Bioconductor R and different bioinformatics tools were used subsequently. Integrated transcriptome-proteome analysis revealed a global loss of messenger RNA-protein concordance. CDK11A and MCTS1 lost positive correlation, whereas RLP38 and H3C1 showed compensatory overtranslation, linked to transcription factors regulating erythropoiesis. In transfusion-dependent thalassemia (TDT), WNK3, HNRNPUL1, COPS7A, and HTATSF1 displayed discordant expression, indicating posttranscriptional aberrations. Protein-to-transcript ratio analysis showed reduced cytoskeletal (ankyrin, spectrin) expression, elevated chaperone activity, elevated ferroptosis markers (ferritin heavy chain 1, ferritin light chain, heme oxygenase-1), and suppressed autophagy. Collectively, these multilayered alterations, including splicing dysfunction, posttranscriptional deregulation, ferroptosis, autophagy suppression, oxidative stress, and cytoskeletal fragility, underlie the greater disease severity observed in TDT than non-transfusion-dependent thalassemia.