Use of TaqI digestion may lead to incorrect molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. For reliable and accurate mutation detection in the CYP21 gene it is important to separate the CYP21 gene from the highly homologous CYP21P pseudogene. For this, several different strategies have been developed. In the analysis of the common eight nucleotide deletion at codon 110-112, a strategy using the TaqI restriction enzyme was first applied. In one family, the results showed discordance between parents and offspring. The use of microsatellite markers flanking the genuine CYP21 gene did not lead to a correct assignment. The problem was finally resolved by using differential PCR amplification for generating a CYP21-specific template. It was concluded that incomplete TaqI digestion, although not visible on an agarose gel, allowed the amplification of the CYP21P pseudogene, thus leading to a false positive diagnosis. Therefore, we recommend the use of direct gene-specific primers for the essential step in the molecular diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency.