'Silent' nucleotide substitution in a β⁺-thalassemia globin gene activates splice site in coding sequence RNA

A β⁺-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human β-globin gene-adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the β⁺-thalassemia gene (GGA) encodes glycine. The function of this β⁺-thalassemia gene was compared to the function of the normal human β-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed β-globin mRNA, thereby causing the β⁺-thalassemia phenotype.