Analysis of four common salt-wasting mutations in CYP21 (steroid 21- hydroxylase) by cleavase fragment length polymorphism analysis and characterization of a frequent polymorphism in intron 6

Background: Congenital adrenal hyperplasia (CAH) owing to steroid 21- hydroxylase deficiency results from mutations in CYP21. The majority of patients with this disorder have salt-wasting because of mineralocorticoid deficiency, and most patients with salt-wasting have one of four common mutations: deletions of CYP21, or nondeletion mutations including a 3' premature splice mutation in intron 2; an 8-bp deletion in exon III; a cluster of mutations in exon VI; and R356W. Methods and Results: The authors analyzed these mutations using a new mutation-detection methodology, cleavase fragment length polymorphism (CFLP) analysis. All of these mutations were detectable using CFLP. The intron 2 splice mutation, exon III 8-bp deletion, and R356W mutation all resulted in novel CFLP bands not seen in subjects without those mutations. The exon VI cluster of mutations resulted in elimination of a CFLP band, which was easily detected in a patient with that mutation and a gene deletion on the homologous chromosome. The authors also report the characterization of a restriction fragment length polymorphism in intron 6 involving the RsaI site around nucleotide 1419. In 48 chromosomes from the parental generation of the Centre d'Etude du Polymorphisme Humain families, the frequency of the polymorphism that destroys the RsaI site was found to be 12.5%. Conclusions: CFLP is a potentially useful analytic method for analyzing frequent CYP21 mutations that cause salt-wasting CAH, although further development of the technique is required to clearly distinguish heterozygotes from homozygotes for mutations that generate novel CFLP bands and to identify heterozygotes for mutations that eliminate a CFLP band. The RsaI polymorphism may be a useful marker for following the segregation of CYP21 alleles in genetic studies and is of use in showing gene deletions of CYP21 in informative families. The polymorphism may also be of use in genetic studies of the human major histocompatibility complex.