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Two Intronic Mutations Cause 17-Hydroxylase Deficiency by Disrupting Splice Acceptor Sites: Direct Demonstration of Aberrant Splicing and Absent Enzyme Activity by Expression of the Entire CYP17 Gene in HEK-293 Cells

Division of Endocrinology and Metabolism, Department of Medicine, Escola Paulista de Medicìna, Federal University of Sao Paulo (M.C.-S., C.E.K.), Sao Paulo, Brazil 04039-034; Felício Rocho Hospital (E.P.D.), Belo Horizonte, Brazil 30130-100; and Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center (R.J.A.), Dallas, Texas 75390-8857

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.

To date, only two among 46 mutations in the CYP17 gene cause 17-hydroxylase deficiency (17OHD) by disrupting mRNA splice donor sites. We studied two subjects with intronic CYP17 mutations: a compound heterozygote for Y329D plus an AG to CG substitution at the 3' end of intron 2, and a homozygote for a TTTT deletion near the 3' end of intron 3. We hypothesized that both mutations caused 17OHD by disrupting splice acceptor sites. To prove this mechanism, the entire CYP17 genes (wild type and both mutations) were amplified, subcloned into pcDNA3, and expressed in HEK-293 cells. The mRNA derived from the wild-type CYP17 gene was correctly spliced and translated into active enzyme, as shown by the correct sequence in the RT-PCR products and by the 17-hydroxylation of progesterone. In contrast, cells expressing the mutant genes had no 17-hydroxylase activity. The mRNA derived from the AG to CG mutation used the first AG in exon 3 as the splice acceptor site, shifting the reading frame and introducing a stop codon. RNA derived from the TTTT deletion skipped exon 4 entirely, deleting 29 amino acids in-frame. Our data show that these are the first two 17OHD cases resulting from mutations that alter splice acceptor sites. These studies also demonstrate the feasibility of expressing the entire CYP17 gene, with simultaneous protein and RNA analysis, as a general methodology for characterizing how intronic CYP17 mutations cause 17OHD.

This work was supported by NIH grants K08-DK-02387 and R03-DK-56641 (to R.J.A.) and by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (96/7449-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (0690/00-7; to C.E.K.).

Results of this work were presented in abstract form at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, June 2002.

Abbreviation: 17OHD, 17-Hydroxylase deficiency.

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