Decreased globin messenger RNA in thalassemia detected by molecular hybridization

In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell free systems reproduces the deficient β chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA instructed DNA polymerase; α and β mRNAs isolated from heavy (β producing) and light (α producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α labeled with [³²P]dCTP and the β cDNA labeled with [³H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA.