Characterization of a spontaneous mutation to a β-thalassemia allele

We have studied a nuclear family containing a single child with severe β-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of β-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the β-globin gene, it was possible to clone and sequence the β-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA→TAA) was found in the β-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 β-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the β-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10⁸:1. The data provide evidence that the G→T change in codon 121 of the β-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.