Blood samples for molecular diagnosis of CYP21 mutations were obtained from the girl and both parents. Genomic DNA was isolated from leukocytes using the standard proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method.

Because of the high homology between the CYP21 and CYP21P genes (98% of nt sequences), a strategy to amplify the CYP21 gene differentially was used, following the polymerase chain reaction (PCR) conditions described in Gene Amp XL (Extra Long) PCR Kit (Applied BioSys tems, Foster City, CA, USA). The primary PCR product was used as a template for secondary PCR amplification. Using the amplification-created restriction site (ACRS) approach for direct detection of mutations [16], a secondary PCR was then performed using a panel of primers specific for 11 known CAH mutations, according to the method previously described [17].

Following the secondary ACRS PCR, 3 µL of the PCR product was incubated, overnight at 37°C, with 5 to10 units of a specific restriction enzyme (Table 1), then analyzed by 2% agarose gel electrophoresis. Subsequent restriction analyses allowed the detection and the determination of the zygosity of the mutation analyzed.