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Pitfalls in Characterizing P450c17 Mutations Associated with Isolated 17,20-Lyase Deficiency

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center (M.K.G., R.J.A.), Dallas, Texas 75390-8857; and Division of Pediatric Endocrinology, Ahmanson Department of Pediatrics, Cedars-Sinai Medical Center (D.H.G.), Los Angeles, California 90048

Address all correspondence and requests for reprints to: Richard J. Auchus, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: richard.auchus{at}utsouthwestern.edu

Abstract

The cytochrome P450c17 enzyme system performs both the 17-hydroxylase and 17,20-lyase reactions in the human adrenal glands and gonads. This 17,20-lyase activity is required for the biosynthesis of dehydroepiandrosterone, the C₁₉ precursor of sex steroids. Considerable evidence supports the idea that the 17,20-lyase activity of this system is particularly sensitive to alterations in the interactions between P450c17 and its cofactor proteins P450-oxidoreductase and cytochrome b₅. We have described two patients with the clinical phenotype of isolated 17,20-lyase deficiency in whom single amino acid replacement mutations in the redox partner binding site of P450c17 (R347H and R358Q) selectively ablate 17,20-lyase activity while preserving most 17-hydroxylase activity. We have shown by computer modeling and detailed biochemical studies that mutations R347H and R358Q impair the interactions of P450c17 with P450-oxidoreductase and cytochrome b₅ (redox partners). Another mutation reported to cause isolated 17,20-lyase deficiency (F417C) does not map within the redox partner binding site, but might nonetheless alter the interaction of the mutant protein with redox partners. To study the interaction of the F417C mutation with P450 oxidoreductase and cytochrome b₅, we expressed the cDNA for this protein in yeast microsomes, a heterologous expression system in which the composition of redox partner proteins can be varied systematically. Although the full-length protein was expressed in quantities comparable to those of wild-type P450c17 in this system, the F417C mutation did not form a classical P450 difference spectrum and was devoid of both 17-hydroxylase and 17,20-lyase activities. To ensure that this result was not unique to the yeast expression system, we also expressed wild-type P450c17 and the F417C mutation in COS-7 cells, and we again found that the F417C mutation was expressed, but was not active. To conclusively demonstrate that a particular mutation in P450c17 causes isolated 17,20-lyase deficiency, accurate enzymatic studies of the mutant protein must reproducibly show activities consistent with the diagnosis. Mutations R347H and R358Q are the only two such mutations found in humans proven to cause isolated 17,20-lyase deficiency.

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