Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive disorders. About 58% of cases are caused by the deficiency of steroid 11β-hydroxylase (CYP11B1) due to mutations in CYP11B1 gene. CYP11B1-inactivating mutations can be found without particular hot-spot spread over the entire gene. A good phenotype-genotype correlation exists for the patients suffering from 11β-hydroxylase deficiency (11OHD). The prediction of disease severity is based on in vitro CYP11B1 activity assays. Here, we present the in vitro analysis of 7 novel CYP11B1 mutations detected during routine genetic analysis. The W116G, A165D and p.K254_A259del mutations were found in classic 11OHD patients. The M88I and P159L mutations were detected in non-classic 11OHD patients, and R366C and T401A were found in individuals who presented with hirsutism and advanced bone age, respectively. The 7 novel mutations were introduced in a pcDNA3-CYP11B1 construct by site directed mutagenesis. Subsequently, COS7 cells were transiently co-transfected with these constructs and with adrenodoxin and adrenodoxin reductase expressing vectors. Transfected cells were incubated with 0.2 μCi 3H-labelled 11-deoxycortisol and 250 nM unlabelled 11-deoxycortisol. The steroids were extracted and separated by thin-layer chromatography. All mutations resulted in a decreased CYP11B1 activity (M88I 72.7±4.2%; P159L 40.4±16.6%; R366C 37.9±9.3%; T401A 60.0±7.9%). The mutant A165D reduced the activity to 2.3±4.5%. No activity was detected for the W116G and p.K254_A259del mutations. All presented CYP11B1 variants are associated with a decreased enzyme activity with M88I, P159L, R366C and T401A being mild CYP11B1 mutations. The W116G, A165D, p.K254_A259del mutations had an activity associated with a classic 11OHD phenotype. A very good correlation between the residual activity of the mutants analyzed and the phenotype of the patients was found. In vitro expression analysis of novel sequence variants allows for the clarification of pathogeneicity and the prediction of the phenotype. Therefore, these assays can provide important additional data for the molecular diagnostic of steroid 11β-hydroxylase deficiency.
05 - 07 Nov 2008
British Society for Paediatric Endocrinology and Diabetes