Rapid differentiation of five common α-thalassemia genotypes by polymerase chain reaction

Abstract

The α-thalassemias are common genetic disorders that arise from reduced synthesis of the α-globin chains. At present, large-scale carrier screening and clinically valuable antenatal detection programs have not been established for the congenital disorder α-thalassemia (α-thal). We have developed a simple nonradioactive polymerase chain reaction (PCR) approach that can detect and differentiate several common α-globin gene deletional α-thals regardless of the break points. When three primer sets were used—two gene-specific sets for the α1- and α2-globin genes and one set for the β-actin gene (serving as an internal control)—PCR products from genomic DNA were simultaneously amplified and analyzed after coamplification and gel electrophoresis. The number of α-globin genes present in the subjects was determined by the intensity of α1 and α2 bands normalized with that of β-actin when using densitometry. Our results demonstrate that five common genotypes of deletional α-thal are differentiated by the ratios of α1/β-actin and α2/β-actin. We also examined the feasibility of coupling this allele-specific amplification to a color-complementary assay. This easy and reproducible PCR assay is suitable for identifying α-thal carriers in screenings of large populations and improving genetic counseling. (J Lab Clin Med 2001;137:290-5)