Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR-Based Multicolor Melting Curve Analysis

Abstract

α-Thalassemia, which is caused by defective synthesis of the hemoglobin α-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective α-globin gene is challenging because of a variety of large deletions of the α-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error-prone open-tube methods. We report a novel real-time PCR-based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis. The turnaround time of this closed-tube assay was within 3.5 hours, the limit of detection was 5 ng of human genomic DNA per reaction, and as low as 5% mutant DNA could be detected in the mosaic samples. The assay was evaluated using 1213 precharacterized genomic DNA samples in a double-blind manner. All seven α-thalassemia mutations were accurately genotyped, yielding a 99.3% concordance with the comparison assays. The 14 discordant samples contained the HKαα allele that was undetected by the traditional methods. Considering its rapidity, ease of use, and accuracy, we concluded that our real-time PCR assay may be recommended as an alternative screening and diagnostic tool for α-thalassemia.