Prenatal Diagnosis of β-Thalassemia: Detection of a Single Nucleotide Mutation in DNA

We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of β-thalassemia due to a single nucleotide mutation. The β 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the β 39 position is mutated to TAG, which results in a stop codon and premature termination of the β-globin chain. We synthesized two oligonucleotides: one homologous to the normal β A gene and the other to the β 0 thalassemia gene at the β 39 location. The oligonucleotides were labeled with 32 P and used as hybridization probes for normal and thalassemic DNA. The β A probe hybridized only to the normal DNA, and the β-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week. (N Engl J Med 1983; 309:284–7.) Prenatal diagnosis of sickle-cell anemia and thalassemia can now be accomplished by Southern's blot analysis 1 of amniocyte DNA. 2 , 3 Abnormal DNA patterns are generated when the globin genes are deleted (as in α-thalassemia) 4 or when single nucleotide mutations affect restriction-endonuclease sites (as in sickle-cell anemia). 5 6 7 Prenatal detection of β-thalassemia is a more complex task because many different mutations can give rise to defective β-globinchain synthesis. 8 , 9 Although a few β-thalassemia lesions alter restriction-enzyme recognition sites directly, 10 most do not. To make a diagnosis in these cases, chromosomes containing the thalassemia mutations are identified by establishing linkages with polymorphic restriction sites that lie . . .