Rapid screening method to detect mutations in CYP21, the gene for 21-hydroxylase

To facilitate a rapid and practical molecular diagnosis of 21-hydroxylase deficiency (21-OHD), we developed a polymerase chain reaction (PCR) test in which only the 21-OH gene (CYP21) is amplified. We applied the test to diagnose 23 patients with salt-wasting type of 21-OHD. The upstream and downstream sequences of CYP21 have been specifically amplified by using a primer set containing the 8-bp deletion sequence of exon 3, which is distinct from its pseudogene CYP21P. The amplified PCR products were further subjected to mutation detection by restriction analysis: E1PL by AciI, I2g by PstI, E63a by DraIII, E7VL by ApaLI, E8non by PstI, and E8RW by AciI. To detect delections and/or gene conversions occurring on exon 3, we used the method described by Rumsby and Honour [1990: J Med Genet 27:676-678]. Our method is able to elucidate 8 common CYP21 mutations by using only 3 primer pairs and 4 restriction enzymes. The overall detection ratio of abnormal haplotypes by this method was over 95%, indicating that our method is practical and useful, particularly for carrier detection.