Analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency

A single nonfunctional chimeric gene with its 5' and 3' ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance.

We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3'-specific primer for the CYP21 gene and two different 5' primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel.

After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method.

The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.