CHARACTERIZATION OF CAH ALLELES WITH NONRADIOACTIVE DNA SINGLE-STRANDCONFORMATION POLYMORPHISM ANALYSIS OF THE CYP21 GENE

The major cause of congenital adrenal hyperplasia (CAH), a common rece ssive genetic disease, is the deficiency of steroid 21-hydroxylase (21 OH), a microsomal enzyme encoded by the CYP21 gene. Although several C AH causing mutations have been identified in the CYP21 gene of patient s with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of m ultiple mutations on single CAH alleles. In order to perform the compl ete characterisation of the CYP21 gene coding region more simply, we d eveloped a highly sensitive, non-radioactive method allowing DNA singl e strand conformation polymorphism (DNA-SSCP) analysis. This method wa s applied to the characterisation of all the exons and intron-exon jun ctions of the CYP21 gene in five patients affected by the simple viril ising form and one affected by the salt wasting form. In all samples s howing SSCP signals, direct sequence analysis showed the presence of m ore than one single sequence variant. In particular, four mutations wh ich are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A ra ndom sequence analysis, performed on 31 out of 81 exons showing a norm al SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioac tive DNA-SSCP analysis in characterising the CYP21 gene in patients wi th steroid 21OH deficiency. Notwithstanding the complete characterisat ion of all exons and exon/intron junctions of the CYP21 gene, no compl ete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probabl y into the promoter region.