Relative Quantitation of mRNA in β-Thalassemiamb E Using Real-Time Polymerase Chain Reaction

Abstract

β-Thalassemia and Hb E patients, with seemingly identical genotypes, have a remarkable variability in severity. Reduction in red cell survival in β-thalas-semia is correlated with the amount of intracellular unmatched α-globin chains. However, it was only recently realized that mRNA, whose translation is prematurely terminated, is also unstable. No systematic attempts have been made to investigate mRNA stability in β-thalassemia arising from nonsense mutations located upstream from the normal termination codon. In this study, one-step real-time polymerase chain reaction has been employed to compare the levels of α- and β-globin mRNA in reticulocytes from β-thalassemia/Hb E subjects. The results showed the highest α/β-globin mRNA ratio (median = 5.70, n = 13) in frameshift codons 41/42 (-TTCT)/Hb E individuals compared to normal subjects (median = 1.02, n = 6), or those with Hb E trait (median = 2.15, n = 8). In addition, there was a concomitant increase in the α/β-globin mRNA ratio with decrease in hemoglobin level, ie., increase in severity. The difference in the ratio among β-thalassemia/Hb E patients with the same genotype may be attributed to individual variations of efficiency in βE-globin mRNA splicing and in the destruction of prematurely terminated mRNA.