Functional studies of two novel and two rare mutations in the 21-hydroxylase gene

Congenital adrenal hyperplasia (CAH) is most commonly due to 21-hydroxylase deficiency and presents with a wide spectrum of clinical manifestations, from prenatal virilization and salt-wasting in the neonatal period to precocious pubarche and late-onset hyperandrogenic symptoms during adulthood. A limited number of mutations account for the majority of all mutated alleles, but a growing number of rare mutations are responsible for the disease in some patients. By sequence analysis of the CYP21A2 gene, we identified two novel (I171N and L446P) and two rare (R341P and R426H) mutations in seven Italian patients with CAH. One of the patients was diagnosed with mild non-classical CAH and was found to be a compound heterozygote (I171N/V281L), while all other patients showed severe phenotypes with latent or manifest salt-wasting. The residual activities measured after expression of the four mutant enzymes in COS-1 cells were all below 1% towards both natural substrates (17-OH-progesterone and progesterone) compared with the wild-type protein. All four mutations are, thus, associated with severe enzyme deficiency and are predicted to cause classic CAH if found in trans with other mutations causing severe enzyme deficiency.