Beta-Thalassemia: Analysis of Mrna Precursors of a Mutant Human Globin Gene with Defective Splicing Using Peripheral Blood Nucleated Red Blood Cells

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Abstract
Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with β-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor 3 -mRNA molecules were observed in a patient with β o-thalassemia, homozygous for a mutation at the 5’ IVS2 splice site of the β-globin gene. Detailed analyses showed that his mature β-mRNA was larger than normal, and that a cryptic 5’ splice site, β50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of β -thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned β-thalassemia genes.