Nine unrelated girls and two boys with NCAH and 17 of their parents or relatives were studied. Diagnosis was made at age 4 to16 (8.5  3.6) years at the Department of Endocrinology and Genetics, University Children’s Hospital, Skopje, Republic of Macedonia. The presenting signs were: premature pubarche/adrenarche, hirsutism and/or oligoamenorrhea, and diagnosis of NCAH was based on these and on advanced bone age, abnormally elevated ACTH-stimulated 17OH-P serum levels and molecular gene analysis.

The samples were screened for the pP30L mutation. The samples hetorzygous for pP30L or non carriers for this mutation were also tested for 10 other different mutations (IVS-II-655 C/A>G, 8 bp frameshift deletion at codons 111-113, p.I172N, p.I236N, p.V237E, p.M239K, p.V281L, p.F306+T, p.Q318X and p.R356W) according to the polymerase chain reaction/amplification created restriction site (PCR/ACRS), method. Thus, 90% of all the mutations identified in CAH patients were covered [13,14].

Molecular Analysis. Genomic DNA was extracted from peripheral blood lymphocytes following the standard phenol/chloroform protocol [15]. The primary differential PCR amplification of the active CYP21A2 gene, without contamination from the highly homologous pseudogene sequence, was performed with 20 pmol of each CYP21A2 (21BF/21BR) specific primer (21BF: 5'-TCG GTG GGA GGG TAC CTG AAG-3' and 21BR: 5'-AAT TAA GCC TCA ATC CTC TGC AGC G-3') in a final volume of 100 L containing 1 g of genomic DNA, 200 M of each dNTP, 1.5 mM Mg(OAc)₂ and 4U rTth DNA polymerase, XL (GeneAmp XL PCR kit; Applied Biosystems, Branchburg, NJ, USA). The EcoRI digestion of the 3.2 kb PCR active gene product, with two fragments (1.0 and 2.2 kb) as an end result, ensured that only the gene sequence had been amplified and analyzed (Figure 1).

The primary PCR product was then used as a template for secondary PCR amplification using ACRS with a pair of primers specific for direct mutational detection of p.P30L mutation (C1N: 5'-CTA CAC AGC AGG AGG GAT GGC-3' and C2: 5'-AGC AAG TGC AAG AAG CCC GGG GCA AGc tG-3'). The secondary ACRS PCR was carried out in a final volume of 50 L, containing primary PCR product, 50 pmol of each primer, 200 M of each dNTP, 1.5 mM MgCl₂ and 1.25 U Taq DNA polymerase (Ampli Taq Gold; Applied Biosystems). A 195 bp PCR product was obtained (Figure 2). The amplified fragments were digested with 5-10 U of a PstI restriction enzyme at 37C overnight. The digestion products were run on 2% high resolution agarose gel and visualized under UV light after ethidium bromide staining.

The identification was based on presence of a recognition site for the restriction enzyme PstI. Region specific primers create a PstI restriction site in the mutant allele at codon 30 which yields two fragments (164 and 31 bp). In the normal allele there is no restriction site for PstI (Figure 3). Subsequent restriction analysis also allowed determination of the zygosity of the mutation.