Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles

The gene encoding adrenal steroid 21-hydroxylase, CYP21, is located in the MHC class III region. Most cases of congenital adrenal hyperplasia (CAH) ar e caused by mutations in this gene, and most mutations appear to arise from gene conversion-like events involving the transfer of deleterious sequence s from the pseudogene, CYP21P which is located within 30 kb of CYP21. Appro ximately 20-30% of mutations are caused by deletions of CYP21, The second i ntron of CYP21 is polymorphic, and several base substitutions that include nt395, nt453, and nt601 have been reported; however, the frequencies of the se polymorphisms are unknown, Using a combination of cleavase fragment leng th polymorphism analysis and direct sequencing, we examined the sequence of intron 2 in seven wildtype CYP21 genes and determined the frequency of pol ymorphisms at nt395, nt453, and nt601 in 48 chromosomes from the parental g eneration of Centre d'Etude du Polymorphisme Humain families. The observed frequencies of bases at these positions were as follows: 395C, 0.17; 395T; 0.83; 453C, 0.71; 453T: 0.29; 601A, 0.1; and 601C, 0.9. Using a PCR/restric tion digestion approach to examine these intragenic markers, we could follo w the segregation of alleles in informative families with al-hydroxylase de ficiency and identify deletions of CYP21. We emphasize that this method sho uld be used in conjunction with other molecular genetic techniques for diag nosis of CAH. In addition to their potential use in families with CAH, thes e markers may be of use in genetic studies of the MHC in humans.