Steroid 17α-hydroxylase (CYP17A1) deficiency (17OHD) is a rare form of congenital adrenal hyperplasia (CAH). Both, 17-hydroxylase and 17,20-lyase reactions are catalysed by CYP17A1. Here, we analysed the structural and functional consequences of 3 novel CYP17A1 mutations found in 3 patients suffering from 17OHD. Two individuals with 46,XY DSD, presented with tall stature and one with arterial hypertension. Mutation screening of the CYP17A1 gene revealed compound heterozygosity for a novel mutation in exon 3 in each patient (A174E/K388X; V178D/R440C). The third patient (46, XX) presented with primary amenorrhea. She was homozygous for a novel L465P mutation. The in vitro functional studies were performed using a yeast microsome assay co-expressing human P450 oxidoreductase, the electron donor to CYP17A1, and CYP17A1 either wild-type or mutant. Microsomes were incubated with 1 μM cold and H3-labelled steroid precursors (progesterone for 17-hydroxylase activity, 17-pregnenolone for 17,20-lyase activity). Steroids were separated by TLC and quantified using a TCL scanner. The activity assays showed that all three mutants had 08% of both 17-hydroxylase and 17,20-lyase activity relative to CYP17A1 wildtype activity correlating with the in vivo phenotype. Enzyme kinetic studies proved the impairment of both reactions, respectively. Surprisingly, the L465P mutation allowed for significant breast development (B4) most likely due to low residual 17,20-lyase activity in combination with obesity. Furthermore, computer-based three-dimensional model analysis of CYP17A1 using CYP2B4 as template was performed. None of the mutations had a direct effect on the active centre. A174 and V178 are located in the E helix. Introducing a longer polar side chain as in A174E interferes with E149 and changes the charge in a small cleft on the surface. Substituting D178 for valine disrupts a stabilising hydrophobic interaction with I145 and I146 in helix D. Modelling suggested that L465P might have an indirect effect on the orientation of the L helix, which is involved in heme binding. In conclusion, these findings provide evidence for the clinical significance of structural and functional analysis of CYP17A1 sequence variants.
05 - 07 Nov 2008
British Society for Paediatric Endocrinology and Diabetes