Prenatal diagnosis of β thalassaemia based on restriction endonuclease analysis of amplified fetal DNA

Abstract

In the Mediterranean area, 50% of the β thalassaemia mutations abolish or create a restriction endonuclease site in the β globin gene. This study describes a new procedure for prenatal detection of these β thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus. We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the β globin gene; these are the most frequent β-thalassaemia mutations in this population, accounting for 95% and 2.2% of the β-thalassaemia chromosomes. The main advantages of this procedures are simplicity (no radioactivity), sensitivity (0.2 μg of DNA), and rapidity (12 hours). The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling. By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.