Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency.

Maturation of primary RNA transcripts of eukaryotic genes often involves the removal of introns and joining of exons. The fidelity of RNA splicing is dependent on the identity of the nucleotide (nt) sequences at exon/intron boundaries. Most importantly, the highly conserved intronic 5'GT and 3'AG sequences are essential for correct splicing. Substitution of GT by any other nt leads to incomplete mRNA and a disruption of protein structure. We describe here the results of our transfection experiments in COS-1 cells with a CYP21 genomic construct that contained an IVS 2+1G-->A mutation. Analysis of the transcripts by RT-PCR revealed that two different transcripts were generated by this mutant genome. In all the splicing products, we found that the entire exon 2 was deleted. Surprisingly, 30% of the transcripts from this mutant CYP21 genome were accompanied by an inclusion of 3' intron 2 sequences due to the use of a different splice acceptor site. This is the first report of the molecular characterization of a splice donor site mutation in CYP21 via transcription in COS-1 cells.