A silent DNA mutation that may cause anomalous results in antenatal diagnosis

Abstract

The β thalassaemia mutation βIVS^1-6 can be detected by restriction enzyme digestion of DNA amplified by the polymerase chain reaction (PCR), since the nucleotide substitution involved creates an additional cutting site for the enzyme SfaNI. Depending on the particular fragment of the β globin gene that is amplified, however, problems of interpretation can arise. This may occur if, in addition to the βIVS^1-6 thalassaemia mutation, a common silent mutation at codon 2 of the β globin gene is also present. With the primers routinely used in this laboratory the following fragment sizes are obtained.MutationFragment Sizes (bp.)Normal β globin gene-542,127β thal geneβIVS^1-6364,179,127β thal gene + codon 2 mutationβIVS^1-6 + codon 2274,179,127,90(not seen)Normal β Globin Gene + codon 2 mutationcodon 2274,268,127 If the fragments of 274 bp and 268 bp are poorly resolved by electrophoresis, then misdiagnosis of homozygous β thalassaemia may occur. Confirmation of the presence of the silent codon 2 mutation, may however be obtained by using a different set of primers to amplify just the region of the β globin gene surrounding codon 2 but excluding βIVS^1-6 Using this strategy we were able to identify one patient with a silent codon 2 mutation alone and another with this mutation in association with the βIVS^1-16 thalassaemia mutation. These results indicate that care must be taken in designing primers to amplify the β globin gene by PCR in order to avoid anomalous results in antenatal diagnosis.