Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Abstract

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

Figure 1

Molecular genetic analysis of the CYP11B1 gene. (a) Schematic localization of the mutations. Upper: known mutations. Down: novel mutations. *previously reported without functional characterization. (b) Pedigrees of the five patients from four unrelated families with electropherograms of the mutations.

Figure 2

Residual 11β-hydroxylase activity of the four CYP11B1 mutants compared with wild type measured in intact HEK293 cells expressing the CYP11B1 enzyme. (a) Conversion of DOC to B. (b) Conversion of 11-S to F. Lineweaver–Burk plots of enzymatic activity of non-classical mutations. (c) Conversion of DOC to B. (d) Conversion of 11-S to F.

Figure 3

Multiple CYP11B1 COBALT (NCBI) alignments. The Arg143, Ala306, Glu310 and Arg332 residues of CYP11B1 and corresponding amino acids of the aligned CYPs are shaded. (a) Alignment of human CYP11B1 with human CYP11B2, the mouse, rat and macaca orthologues. (b) Alignment of different human steroidogenic CYP enzymes (type I enzymes, CYP11B1, CYP11B2, CYP11A1; type II enzymes, CYP21A2, CYP17A1, CYP19A1).

Figure 4

Ribbon representation of the model of the three-dimensional structure of CYP11B1. The central I helix important for a proper haem binding and orientation is coloured in light blue. Side chains of the mutated amino-acid residues p.Arg143, p.Ala306, p.Glu310, p.Arg332 and, in addition, the side chain of p.Glu327, which forms a salt bridge with p.Arg332, are depicted.