Multi-omics 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 not well investigated and still unknown. This study aims to identify dysregulated molecular pathways in red blood cells contributing to thalassemia severity. From a cohort of 285 hemoglobinopathy patients, 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, while five had a non-severe form requiring fewer transfusions. RNA sequencing and proteome analysis were conducted on isolated RBCs, through Novaseq and Orbitrap MS platform respectively. Bioconductor-R and different bioinformatics tools were utilized subsequently. Integrated transcriptome-proteome analysis revealed a global loss of mRNA-protein concordance. CDK11A and MCTS1 lost positive correlation, whereas RLP38 and H3C1 showed compensatory overtranslation, linked to transcription factors regulating erythropoiesis. In TDT, WNK3, HNRNPUL1, COPS7A, and HTATSF1 displayed discordant expression, indicating post-transcriptional aberrations. PTR analysis showed reduced cytoskeletal (ankyrin, spectrin) expression, elevated chaperone activity, ferroptosis markers (FTH1, FTL, HMOX1), and suppressed autophagy. Collectively, these multilayered alterations-splicing dysfunction, post-transcriptional deregulation, ferroptosis, autophagy suppression, oxidative stress, and cytoskeletal fragility-underlie the greater disease severity observed in TDT compared with NTDT.