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Steroid 17-Hydroxylase Deficiency: Functional Characterization of Four Mutations (A174E, V178D, R440C, L465P) in the CYP17A1 Gene

Centre for Endocrinology, Diabetes and Metabolism (V.D., N.R., C.M.B., W.A., N.K.), School of Clinical and Experimental Medicine, University of Birmingham, Birmingham B15 2TT, United Kingdom; Department of Pediatrics (J.L.), Second Faculty of Medicine, Charles University, 110 00 Prague, Czech Republic; Zentrum für Kinder- und Jugendmedizin (C.K.), Zentrum für Kinderheilkunde I, 60596 Frankfurt, Germany; Department of Endocrinology (H.-P.S.), von Haunersches Kinderspital, University Hospital Munich, 80337 Munich, Germany; Institute of Biochemistry (J.G.), Christian-Albrechts Universität, 24118 Kiel, Germany; and Division of Pediatric Endocrinology and Diabetes (W.G.S., F.G.R.), Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany

Address all correspondence and requests for reprints to: Nils Krone, M.D., Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, Institute of Biomedical Research, Wolfson Drive, Birmingham B15 2TT, United Kingdom. E-mail: n.p.krone{at}bham.ac.uk.

Context: Steroid 17-hydroxylase (CYP17A1, alias P450c17) deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The CYP17A1 enzyme catalyzes two distinct reactions, 17-hydroxylase and 17,20-lyase activities.

Objective: The aim of the study was to analyze the structural and functional consequences of three novel (A174E, V178D, and L465P) and one previously reported (R440C) CYP17A1 mutation found in three patients clinically and biochemically presenting with 17OHD.

Patients and Methods: Two patients suffering from 46,XY disordered sex development presented at ages 5.5 and 8.8 yr, respectively, with tall stature and hypertension. Mutation analysis revealed compound heterozygous CYP17A1 mutations (A174E/K388X; V178D/R440C). The third patient (46,XX) presented with primary amenorrhea and hypertension at age 15 yr. She was homozygous for the novel L465P mutation. Functional studies employing a yeast microsomal expression system compared wild-type and mutant CYP17A1 both with regard to 17-hydroxylase and 17,20-lyase activity. Mutants were examined in a computational three-dimensional model of the CYP17A1 protein.

Results: The activity assays showed that all three mutants retain only 0–7% of both 17-hydroxylase and 17,20-lyase activity relative to CYP17A1 wild-type activity, corresponding to the in vivo situation. Enzyme kinetic studies proved the impairment of both reactions, respectively. Computer-based three-dimensional model analysis of CYP17A1 using CYP2B4 as template showed that three of the mutations had no direct effect on the active center, whereas one affects the heme coordination.

Conclusion: The functional studies revealed that the described missense mutations result in severe 17OHD. Our data are important to predict the phenotypic expressions and provide important information for patient management and genetic counseling.