[Multiplex minisequencing applied in detection of human functional CYP21 gene mutations]

We analyzed seven most common mutations within the CYP21B gene, responsible for congenital adrenal hyperplasia (CAH), using the minisequencing method. Functional CYP21B gene sequences were amplified with the pair of specific primers that pevented amplification of pseudogene CYP21P or pseudogene CYP21P/active CYP21 hybrids. Multiplex minisequencing (SNaPShot PCR) assay was performed with fluorescent dideoxynucleotides ([F]ddNTPs) and originally designed primers, claiming seven most common mutation sites responsible for the CAH symptoms. Using the method we detected five novel substitutions of unknown effect on the CAH course in five out of seven analyzed mutation sites. Compared to classic SNPs analyzing methods, especially single SNP detection, multiplex minisequencing is the same highly specific and sensitive but much faster one. The method is recommended for any population screened for known mutations.