Rapid detection of genetic modifiers of β-thalassemia based on MALDI-TOF MS

Fetal hemoglobin (HbF) levels are influenced by various genetic modifiers, which have clinically beneficial effects on both β-thalassemia and sickle cell disease. HbF-associated genetic variants are distributed throughout the genome, and current detection methods are often costly, time-consuming, and require multiple tests. Therefore, developing rapid and economical methods for the simultaneous detection of HbF-associated variants is essential for improving the accurate diagnosis of β-hemoglobinopathies. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to detect 20 well-documented genetic modifiers in BCL11A, KLF1, HBG2, DNMT1, GATAD2A, and HBS1L-MYB intergenic polymorphism (HMIP). The new assay’s accuracy, repeatability, and lowest detection limit were evaluated. It was subsequently applied to 81 samples, and the clinical effects of the modifiers were further verified in a cohort of 560 β-thalassemia patients. The MALDI-TOF MS assays successfully detected all 20 genetic modifiers simultaneously in a single reaction. Genotyping results from 15 repetitions were consistent and accurate, indicating the stability of this assay. The assay’s lowest detection limit for DNA was as low as 0.2 ng, sufficient for simultaneous genotyping of all loci. A double-blind evaluation of 81 samples showed 100% concordance with traditional genotyping methods. Significant differences were observed in HbF levels, survival time without transfusion, and clinical classification for the detected genetic modifiers. The MALDI-TOF MS detection assay for HbF-related variants is simple, rapid and high throughput. It enables the detection of 20 genetic modifiers in a single test, supporting accurate large-scale detection and enhancing the precise diagnosis and clinical classification of β-thalassemia.