Congenital adrenal hyperplasia due to 11-hydroxylase deficiency: functional characterization of two novel point mutations and a three-base pair deletion in the CYP11B1 gene

Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion (DeltaF438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 +/- 0.9% (sd) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 +/- 0.9%, whereas the DeltaF438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the DeltaF438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.