Direct Detection of the Common Mediterranean f 3 - Thalassemia Gene with Synthetic DNA Probes AN ALTERNATIVE APPROACH FOR PRENATAL DIAGNOSIS STUART

Address all correspondence to Dr. S. H. Orkin, Children's Hospital Medical Center, Boston, MA 02115. Received for publication 16 November 1982 and in revised form 20 December 1982. INTRODUCTION Recombinant DNA methods have permitted characterization of specific mutations in disorders of fl-globin synthesis, the fi-thalassemias (1). The most common form of fl-thalassemia among Mediterranean patients is due to a single nucleotide change (G A) in the first intervening sequence (IVS-1)' of the fl-globin gene (2, 3). This substitution generates a new splicing signal and leads to the formation of abnormally processed RNA that is unstable in vivo (4, 5). Since some normal messenger (m)RNA is produced as well, albeit in small amounts, this gene has a 8+-thalassemia phenotype (4, 5). On the basis of gene cloning, DNA sequencing, and examination of linked DNA polymorphisms we estimated that about two-thirds of Greek and one-third of Italian #l-thalassemia genes are of this type (1). Independent studies by Fukamaki et al. (5) and Ley et al. (6) are consistent with these findings. As the DNA substitution in this common form of f3-thalassemia does not alter a restriction enzyme site useful for gene mapping, we have explored the direct ' Abbreviations used in this paper: IVS, intervening sequence: kb, kilobase. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/83/03/0775/05 $1.00 Volume 71 March 1983 775-779 775 detection of this gene defect in DNA by small, synthetic DNA fragments (oligonucleotides) as hybridization probes. In pioneering studies Wallace et al. (7) have shown that a single nucleotide mismatch between a cloned gene sequence and an oligonucleotide is sufficient to destabilize the DNA-DNA hybrid relative to a perfect hybrid under carefully controlled conditions. Point mutations in DNA can then be detected by hybridization with appropriate oligonucleotides. In studies presented in abstract form Conner et al. (8) have described the detection of the f3s-gene mutation in uncloned DNA by this strategy. Using synthetic oligonucleotides for the normal and mutant genes, we now report detection of the common Mediterranean #3+-thalassemia gene in cellular DNA samples. Our findings form the basis of an alternate method for the prenatal diagnosis of this and other varieties of fl-thalassemia.