Mutation E305G In The Active Site Of CYP17 Causes Isolated 17, 20-Lyase Deficiency By A Novel Mechanism
Daniel Sherbet
Mentor: Dr. Richard Auchus, Division of Endocrinology and Metabolism
 
Introduction: CYP17 catalyzes both the 17-hydroxylase and 17,20-lyase reactions in the human adrenal and gonads. Mutations in the CYP17 gene that destroy both activities of the enzyme result in combined 17-hydroxylase/17,20-lyase deficiency (17OHD). Patients with 17OHD produce low amounts of 17-hydroxysteroids and sex steroids but excess mineralocorticoid, resulting in sexual infantilism and hypertension. In contrast, mutations that destroy only the lyase activity of the enzyme cause isolated 17,20-lyase deficiency (ILD). Genetically male patients with ILD produce low amounts of testosterone but normal amounts of 17-hydroxysteroids and exhibit only ambiguous genitalia. All patients to date with ILD deficiency proven by molecular genetic methods harbor mutations that disrupt interactions between CYP17 and its flavoprotein electron donor CPR but do not alter substrate binding. We report here a novel CYP17 mutation in male pseudohermaphrodites of an inbred Middle-Eastern kindred, presenting with ILD and micropenis, gynecomastia, essentially normal 17-hydroxysteroids, low testosterone levels, and extremely low dehydroepiandrosterone (DHEA) sulfate levels.
Methods: The CYP17 gene was amplified via PCR from the genomic DNA of affected patients and parents, and the exons were sequenced. The mutant cDNA was expressed in HEK-293 cells, which were then incubated with radiolabeled steroids and assayed for activity.
Results: Affected patients were homozygous for a mutation in the active site of CYP17 that replaces a glutamate with a glycine at position 305. When expressed in HEK cells, the E305G mutation, like the wild-type enzyme, 17-hydroxylated both pregnenolone and progesterone. The E305G mutation, relative to the wild-type enzyme, showed increased 17, 20-lyase activity with 17-hydroxyprogesterone substrate. However, E305G did not convert any 17-hydroxypregnenolone to DHEA.
Discussion: E305G is the first mutation located in the active site of CYP17 shown to cause ILD. The enzymology of E305G is unique in that the preferred pathway for human CYP17 (D5-pregnenolone to DHEA) is selectively disrupted, accounting for the very low DHEA sulfate levels seen in the affected patients. Remarkably, mutation E305G exhibits an increased lyase activity for the D4-progesterone-to-androstenedione pathway, which is the dominant pathway for rodent CYP17s. The study of mutation E305G 1) provides genetic evidence that the D4 pathway is insufficient for formation of the male phenotype in humans; 2) shows that mutations in the active site of CYP17 can cause ILD; and 3) provides insight to the D5 preference for the lyase activity of human CYP17.

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