Premature pubarche/adrenarche was present in six girls, hirsutism and oligoamenorrhea in two, and amenorrhea in one. The boys presented with adrenarche, typical development of genitalia, and increased rate of growth. Bone age was advanced by 3.6  1.2 years (range: 1 to 4.5). The peak of ACTH-stimulated 17OH-P serum level was higher than 60 nmol/L in all patients.

The p.P30L mutation was detected in all NCAH patients: seven patients (63.6%) were homozygous (five girls and two boys) and four girls (36.4%) were heterozygous. One of the heterozygous patients was also heterozygous for a p.Q318X mutation, as previously reported [14].

Patients with the p.P30L allele, although still categorized as nonclassical, tend to have pronounced evidence of androgen excess [16]. A genotype that contains two mild mutant alleles produces clinical symptoms of NCAH, the allele that produces most enzyme activity determining the phenotype [17,18]. Thus, the patient who was heterozygous for p.P30L and p.Q318X presented with a nonclassical phenotype. However, women who have NCAH may give birth to children with a classical form of the disease if the father is heterozygous for a severe mutation. Prenatal diagnosis in the case of such a combination of parents is strongly recommended. However, the correlation of genotype to phenotype that we found in all of our NCAH patients strengthens the concept that the genotype is predictive of phenotype.

Besides the p.P30L mutation which was derived from pseudogene, mutations not found in the pseudogene, p.R339H, p.P453S [19], and p.H62L [20,21] are also associated with partially impaired in vitro activity of 21-hydroxylase but together probably account for no more than 5% of NCAH alleles [22,23]. The p.P30L mutation was found in 11/17 of the family members (64.7%), 10 were heterozygotes (58.8%) and one was a homozygote (5.9%). Only the mother (homozygous for p.P30L) and father (homozygous for p.Q318X) of the compound heterozygous patient, as previously reported, had infertility and oligospermia, respectively [18].

The previously reported genetic analysis confirmed a significant genotype to phenotype correlation and a prevalence of NCAH of 1:7000 in the Macedonian population [24,25]. Although p.P30L is classified as a mild mutation in CYP21A2, its role in increased virilization remains to be elucidated.