PHENOTYPIC HETEROGENEITY ASSOCIATED WITH THE SPLICING MUTATION IN CONGENITAL ADRENAL-HYPERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY

One mutation frequently identified in 21-hydroxylase deficiency is the intron 2 splicing mutation, in which the normal polymorphic C or A at nucleotide 655 has been converted to G. Using allele-specific oligonu cleotide hybridization, single strand conformational polymorphism anal ysis, and heteroduplex analyses, we identified 38 individuals from 21 different families who had 2 deleterious mutations. All were homozygou s or compound heterozygotes for the splicing mutation. Comparison of t he phenotypic features with the molecular genotypes shows phenotypic h eterogeneity extending from classical salt-losing 21-hydroxylase defic iency to asymptomatic. Single strand conformational polymorphism analy sis followed by DNA sequence analysis revealed numerous sequence varia tions in intron 2, most commonly at nucleotides 601 and 683. Transient transfection experiments show that the 3'-portion of intron 2 is suff icient to transfer the effect of the 655c/a-->g mutation to a chimeric heterologous gene. Clinical correlations and initial transfection stu dies suggest that sequence variations at nucleotides 601 and 683 do no t correlate with clinical severity or substantially affect splicing. I n summary, a single nucleotide change, 655c/a-->g, alters the splice a cceptor site at the intron 2/exon 3 boundary. The molecular basis of t he phenotypic heterogeneity associated with the mutation remains to be elucidated.