The beta-thalassemias are a group of inherited blood disorders characterized by decreased (beta-plus) or absent (beta-zero) beta-globin synthesis. Recent studies have revealed that the disease results from a heterogeneity of point mutations on the beta-globin gene locus. However, little is known about the mRNA expression for each of the mutant beta- globin genes. Recently, mutations at IVS-2 region of the beta globin gene have been of general interest as it is evident that this intervening sequence plays an important role in regulation and expression of the gene. Beta-IVS-2 nt. 654 cytosine-to-thymine is a common Chinese beta-thalassemia mutation. In order to investigate the mRNA expression and the splicing defect of this specific mutation, we used our newly developed method, i.e., enzyme reverse transcription and polymerase chain reaction (RT-PCR) to amplify cDNA copies of reticulocyte mRNA followed by direct sequencing of the amplified cDNA, to analyze the mutant mRNA transcript. The result showed that, in the homozygous patient, two principle unusually long amplified fragments of 254-bp and about 190-bp were observed. Direct sequencing of the patient's amplified cDNA revealed that the 181-bp fragment had a normal sequence, but in the 254-bp fragment, the new donor- like site due to the IVS-2 nt. 654 cytosine-to-thymine substitution, was exactly spliced to the normal IVS-2 3' acceptor site and an internal cryptic acceptor site at position of IVS-2 nt. 654 cytosine-to-thymine mutant gene does not abolish the normal beta-globin mRNA processing entirely but does produce a small amount of normal spliced beta-globin mRNA. Hence IVS-2 nt. 654 mutation should be classified as beta-plus thalassemia at mRNA level. Current studies in progress are aimed at: (1) quantitation in absolute term of globin mRNA amount in erythroid cells from the patients with IVS-2 nt. 654 cytosine-to-thymine mutation as well as other known beta-thalassemia mutations using PCR techniques; (2) investigation of the expression of IVS-2 nt. 654 and other known mutant genes at translational level using globin chain biosynthesis with HPLC techniques. Thus, the overall objective is to elucidate the relationship between specific mutations and beta-globin gene expression. On this basis, we will be able to contribute to the knowledge of the genetic heterogeneity of beta-thalassemia as well as provide new information on gene structure and function in eukaryotic cells.
