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 11b-hydroxylase deficiency (11b-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 11b-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11b-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 11b-OHD showed a nearly complete loss of 11b-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11b-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 11b-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.

Characterisation of three novel CYP11B1 mutations in classic and non-classic 11β-hydroxylase deficiency

“…Patient 4 showed clinical signs compatible with nonclassic CAH (25). The novel mutation p.Ser150Leu detected in this patient had a residual activity of 19.2% of the WT.…”

“…In vitro transient transfection assay was performed in triplicates using the HEK293 cell line thrice. The cells were transfected with each pcDNA3.1-CYP11B1 mutant constructs with additional transfection of adrenodoxin (pECE-ADX), Adx reductase (pECE-ADR) expression vectors (kindly provided by Prof. W L Miller, Department of Pediatrics, University of California, San Francisco, CA, USA) and pRK-TK (Promega) coding for Renilla luciferase as it was described in our previous paper (25). The kinetic constants of CYP11B1 in intact HEK293 cells were determined 48 h after transfection.…”

“…Each mutation was introduced into a CYP11B1-pcDNA3.1 expression vector construct (kindly provided by Prof. R Bernhardt, Institute of Biochemistry, University des Saarlandes, Saarbrücken, Germany) by site-directed mutagenesis as described previously (24,25). In vitro transient transfection assay was performed in triplicates using the HEK293 cell line thrice.…”

“…The cells were incubated in triplicates for 270 min at 37 8C with 1 ml DMEM medium containing 0.1, 0.25, 0.5, 1, 2, and 4 mmol/l DOC and S in parallel with 10 mM NADPH (Sigma-Aldrich). The steroid hormones DOC, B, S and F were simultaneously determined in the cell culture supernatant using an UPLC-MS/MS method as described previously (25,26,27). Western blot analysis was carried out to ensure the expression and translation of WT and mutant proteins as described previously (24,25,28).…”

“…The steroid hormones DOC, B, S and F were simultaneously determined in the cell culture supernatant using an UPLC-MS/MS method as described previously (25,26,27). Western blot analysis was carried out to ensure the expression and translation of WT and mutant proteins as described previously (24,25,28). All assays were performed in at least three independent triplicate experiments, and data are presented as meanGS.D.…”