Exon 7 Nco l Restriction Site within CYP21B (Steroid 21-Hydroxylase) Is a Normal Polymorphism (original) (raw)

A point mutation within exon 7 producing an amino acid coding change and a recognition site for the endonuclease A/col has been reported in the HLA-Bw47-linked CYP21A pseudogene and some mutant CYP21B (steroid 21-hydroxylase) genes of patients with congenital adrenal hyperplasia (CAH). Whether this mutation is deleterious was not demonstrated. We analyzed DNA from various subjects for the presence of the exon 7 A/col site: group 1,10 normal subjects; group 2, 11 patients with salt-losing CAH; and group 3, 18 members of an Amish pedigree in which 10 expressed HLA-Bw47 not linked to CAH. Southern blots of A/col-digested genomic DNA which were hybridized with CYP21 cDNA showed that four subjects of group 1 had a heterozygous A/col pattern. In group 2, seven patients had the A/col site; two of them were homozygous for the site and had deletions of both CYP21B genes. The other five were heterozygous for the A/col site, which was linked to a CYP21B deletion and a HLA-Bw47 haplotype. In group 3, no one exhibited the exon 7 A/col site. To map the A/col sites to CYP21A or CYP21B in the normal subjects, DNA from the four A/col heterozygous subjects was double digested with A/col and Mbo\ and hybridized with CYP21 cDNA. Nco\-Mbo\ fragments unique to CYP21A were identified in all four, but the smaller CYP2IB-specific fragments were not detected. Their genomic DNA in the region of exon 7 (bases +1167 to +2058) was then amplified, cloned, and sequenced. Clones that contained both the A/col site and a CYP2IB-specific S/nl fragment were identified in two of the four subjects. Sequence analysis further confirmed them as CYP21B clones. We conclude that the exon 7 A/col site is a normal polymorphism, but that it is also linked to the HLA-Bw47 haplotype in CAH patients but not to that in normal (Amish) subjects. (Molecular Endocrinology 4: 1354-1362,1990) INTRODUCTION Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a relatively common inborn error