Functional consequences of seven novel mutations in the CYP11B1 gene: four mutations associated with nonclassic and three mutations causing classic 11{beta}-hydroxylase deficiency

Abstract

Context: Steroid 11beta-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency.

Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations.

Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein.

Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11beta-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively.

Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.

Figure 1

Comparison of residual 11β-hydroxylase activity of the CYP11B1 variants. A, The activities of the mild mutants are expressed as percentage of wild-type activity (cortisol synthesis rate, 4.7 ± 0.7 nmol/mg protein/min), which is defined as 100%. Values are depicted for the conversion of 11-deoxycortisol to cortisol at a substrate concentration of 10 μmol/liter of unlabeled steroid. Error bars represent the mean ± sem (%). B, Lineweaver-Burk plots of 11β-hydroxylase activity converting 11-deoxycortisol (S) to cortisol assessed by incubation of transiently transfected COS7 cells coexpressing human wild-type (WT) or mutant CYP11B1, and human Adx reductase and Adx with 2–15 μm 11-deoxycortisol and [³H]-11-deoxycortisol. Error bars represent the mean ± sem (%). The p.P159L mutation is not shown because this mutation did not reach substrate saturation under the employed reaction conditions.

Figure 2

Multiple CYP11B1 clustalW alignments. The M88, W116, P159, A165, R366, and T401 residues of CYP11B1 and corresponding amino acids of the aligned CYPs are shaded and marked by a triangle. A, Alignment of human CYP11B1 with human CYP11B2 and CYP11A1, the mouse and rat orthologs. B, Alignment of different human steroidogenic CYP enzymes. C, Alignment of mammalian CYP11B1 with different cytochrome P450 type 2 enzymes.

Figure 3

Total view on the three-dimensional molecular model of CYP11B1. N-term, Amino terminus.; The B-C loop is colored in dark orange, the D helix in blue, the I helix in red, and the K helix in light blue. Amino acid residues affected by missense mutations are shown in ball representation; the region affected by the six-amino acid deletion in the G helix is depicted in gray.