Multi-omics-based analysis of the N6-methyladenosine regulatory network in hemoglobin H-constant spring disease and functional prediction of IGF2BP2 target genes

Hemoglobin H-Constant Spring (HbH-CS) disease is a severe subtype of α-thalassemia characterized by unstable hemoglobin, causing hemolytic anemia, hepatosplenomegaly, and skeletal deformities, yet its molecular pathogenesis remains incompletely understood. This study aimed to elucidate the role of N6-methyladenosine (m 6 A) RNA modification in HbH-CS and identify key pathogenic genes. Using bioinformatics analysis of m 6 A microarray data, we screened 20 m 6 A-related genes, of which 10 were differentially expressed in HbH-CS compared to controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed significant involvement in RNA processing and immune response pathways. Protein-protein interaction network construction via Cytoscape identified CBLL1, RBM15, and IGF2BP2 as hub genes within the predicted network. These findings were corroborated by reverse transcription-quantitative polymerase chain reaction in 30 clinical samples, which validated their differential expression patters. Furthermore, multi-database intersection analysis identified IKBKB, HDLBP, and HSPD1 as high-cofidence IGF2BP2 target genes. These findings suggest that aberrant m 6 A regulation, particularly the antagonistic relationship between writer and reader proteins, may contribute to HbH-CS pathogenesis through disrupted mRNA metabolism. Targeting the m 6 A regulatory axis may represent a novel therapeutic strategy for HbH-CS.