ALTERED MESSENGER-RNA EXPRESSION DUE TO INSERTION OR SUBSTITUTION OF THYMINE AT POSITION--DONOR SITES IN THE ANDROGEN RECEPTOR GENE(3 OF 2 SPLICE)

We have discovered two types of 5' intronic gene mutation that impair androgen receptor (AR) mRNA expression severely, and cause complete an drogen insensitivity, Labium majus skin fibroblasts (LMSF) hemizygous for each mutation had negligible specific androgen binding, and did no t react to an antibody against an N-terminal peptide of the AR. Both m utations were detected by direct sequencing of exons PCR-amplified wit h flanking primers, One mutation is an adenine to thymine transversion at position +3 of the intron 6 splice-donor site, Using LMSF mRNA, RT -PCR of a portion of the AR androgen-binding domain yielded a small am ount of a 302-bp mutant fragment instead of a 433-bp wild-type product , Sequencing established that exon 5 was followed, out of frame, by ex on 7: exon 6 was skipped. The other mutation is a thymine insertion at the +3 position of the intron 1 donor-splice site, RT-PCR and sequenc ing revealed a small amount of normal-size mRNA with normal exon 1-exo n 2 splicing, Quantitative RT-PCR on mutant LMSF showed AR mRNA levels were well below 10% of normal; hence, most of the aberrant AR mRNA re sulting from each mutation is probably unstable, The misbehavior cause d by these two mutations indicates that in the AR the splice-donor sit e +3 adenine is critical; indeed, 57% of eukaryotic introns have adeni ne in the +3 position, while only 2% have thymine.