"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.

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Abstract
A .beta.+-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 .beta.-globin gene; adenine is substituted for thymine in the 3rd position of codon 24. Codon 24 in both the normal gene (GGT) and the .beta.+-thalassemia gene (GGA) encodes glycine. The function of this .beta.+-thalassemia gene was compared to the function of the normal human .beta.-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 .beta.-globin mRNA, thereby causing the .beta.+-thalassemia phenotype.