The beta-thalassemias represent a heterogeneous group of diseases, caused by different point mutations on the beta-globin locus. However, very little is known about the mRNA transcriptional level for each of these mutant alleles. To gain insight into this area, we have developed a simple method using the enzyme reverse transcriptase and the polymerase chain reaction to amplify cDNA copies of reticulocyte mRNA so as to quantitate the relative and absolute amounts of alpha-, beta-, and gamma-globin mRNA in nucleated erythroid cells from these individuals. Primers were chosen to optimize resolution and quantitation of the DNA bands separated on agarose gel electrophoresis. Quantitation was performed by scintillation counting or density scans of DNA bands stained with ethidium bromide. In our method normal individuals had a ratio of alpha/beta mRNA of 1. 14 (+/- 0.05) by scinduation counting and 1.33(-+0.04) by the densitometric analysis. Five patients with beta-thalassemia had alpha beta ratios greater than 14.0 by scintillation counting and greater than 100.0 by densitometry. Two patients with beta(+) thalassemia major also had elevated ratios of alpha beta mRNA. Conversely, decreased alpha beta mRNA ratios were found in patients with beta-thalassemia. Patients with beta-thalassemia also had a demonstrable increase in their gamma mRNA as measured in comparison to alpha mRNA. Current studies in progress are aimed at using this PCR techniques to quantitate in absolute terms of the amounts of alpha, beta, and gamma- globin mRNA in nucleated erythroid cells. Having accomplished this goal our efforts will be turned to examining the generality of our approach through the study of beta-thalassemia patients with known mutations, as well as those in whom the mechanisms of the thalassemic phenotype has yet to be determined. Thus, the overall objective is to elucidate the relationship(s) between different mutations and the corresponding level of beta-globin gene expression. On this basis, we will contribute to the specific knowledge of the genetic heterogeneity of beta-thalassemia as well as provide new information on gene structure and function in eukaryotic cells.
