Routine detection of point mutations in non-classic steroid 21-hydroxylase

The most frequent mutations in patients with 21-hydroxylation defects are located in exons 7,3, 8, and 10. Since 1990 the most common method for characterizing mutations on theCYP21B gene has been the polymerase chain reaction, together with dot blot and sequencing procedures. Our aim was to verify point mutations in patients suffering from non-classic congenital hyperplasia with a simplified method substituting restriction enzymes for dot blot. We studied both alleles from 27 subjects, 7 with non-classic congenital hyperplasia and 20 of their first-degree relatives; theCYP21B gene was amplified in two fragments (669-base pair and 1,505-base pair) and digested with AciI and Alw44I restriction enzymes, respectively. The mutation Val-28l → Leu was found in 57.1% of the patients, while the mutation Pro-30 → Leu was not found in any patient. An additional 292-base pair fragment was found on intron 2. We discuss the agreement with other papers and propose the use of this strategy in the clinical laboratory to screen for point mutations in this disorder.