Background: Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles.

Objective: Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed.

Design: The study included ten Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone.

Results: Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P and p.R408C mutants classified them as classical CAH (congenital adrenal hyperplasia) mutations, while the p.H62L showed an activity within the range of non-classical mutations. Apparent kinetic constants for p.H62L confirmed the non-classic classification as the substrate binding capacity (K_m) was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination as it caused a significant reduction in the enzymatic activity.

Conclusions: We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R that might not be the case, and the p.H62L is definitely a NC mutation.