Relative quantitation of mRNA in beta-thalassemia/Hb E using real-time polymerase chain reaction.
beta-Thalassemia and Hb E patients, with seemingly identical genotypes, have a remarkable variability in severity. Reduction in red cell survival in beta-thalassemia is correlated with the amount of intracellular unmatched alpha-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 beta-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 alpha- and beta-globin mRNA in reticulocytes from beta-thalassemia/Hb E subjects. The results showed the highest alpha/beta-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 alpha/beta-globin mRNA ratio with decrease in hemoglobin level, i.e., increase in severity. The difference in the ratio among beta-thalassemia/Hb E patients with the same genotype may be attributed to individual variations of efficiency in betaE-globin mRNA splicing and in the destruction of prematurely terminated mRNA.
Watanapokasin
Y
Y
Department of Biochemistry, Faculty of Medicine Srinakarinwirot University, Bangkok, Thailand. yuwadee@psm.swu.ac.th
Winichagoon
P
P
Fuchareon
S
S
Wilairat
P
P
D000328:Adult; D016368:Frameshift Mutation; D005914:Globins / Q000235:genetics; D006446:Hemoglobin E / Q000235:genetics*; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods*; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis* / Q000097:blood; D012156:Reticulocytes; D017086:beta-Thalassemia / Q000235:genetics*
D006454:Hemoglobins; D012333:RNA, Messenger; D005914:Globins; D006446:Hemoglobin E