A point mutation within exon 7 producing an amino acid coding change and a recognition site for the endonuclease Ncol 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 NcoI 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 Ncol-digested genomic DNA which were hybridized with CYP21 cDNA showed that four subjects of group 1 had a heterozygous Ncol pattern. In group 2, seven patients had the Nco I site; two of them were homozygous for the site and had deletions of both CYP21B genes. The other five were heterozygous for the Nco I site, which was linked to a CYP21B deletion and a HLA-Bw47 haplotype. In group 3, no one exhibited the exon 7 Nco I site. To map the Ncol sites to CYP21A or CYP21B in the normal subjects, DNA from the four Ncol heterozygous subjects was double digested with Ncol and Mbol 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 Ncol site and a CYP2IB-specific Sinl fragment were identified in two of the four subjects. Sequence analysis further confirmed them as CYP21B clones. We conclude that the exon 7 Ncol 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.
ASJC Scopus subject areas
- Molecular Biology