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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 11 3807-3812
Copyright © 1997 by The Endocrine Society

Original Studies

A Single Amino Acid Substitution in the Putative Redox Partner-Binding Site of P450c17 as Cause of Isolated 17,20-Lyase Deficiency1

A. Biason-Lauber, E. Leiberman and M. Zachmann

Department of Pediatrics, Divisions of Pediatric Endocrinology and Clinical Chemistry, Zurich, Switzerland; and Pediatric Endocrine Unit, Soroka Medical Center, Ben-Gurion University of the Negev (E.L.), Beer-Sheva, Israel

Address all correspondence and requests for reprints to: Dr. A. Biason-Lauber, Department of Pediatrics, Divisions of Pediatric Endocrinology and Clinical Chemistry, Steinwiesstrasse 75, 8032 Zurich, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The molecular basis of isolated 17,20-lyase deficiency was clarified in a newborn male patient from Israel with micropenis, undescended testes, and hormonal pattern consistent with isolated 17,20-lyase deficiency. Analysis of the CYP17 gene revealed the presence of a compound heterozygosity. One allele carries a single base pair deletion (T at position 198 in exon 1) leading to a frame shift with the introduction of a premature stop codon, TGA, at residue 74 in place of Val. The other allele bears a missense mutation due to a single base change, T to G, which substitutes Phe417 with Cys. The proof of heterozygosity was possible via amplification and direct sequencing of genomic DNA fragments from the parents and the healthy brother of the index case. We could demonstrate that the mother is the carrier of the nonsense mutation and the father of the missense mutation. The brother carries two normal alleles for the CYP17 gene. The nonsense mutation gives no functional product. The missense mutation causes the synthesis of a protein that retains 17{alpha}-hydroxylase activity but virtually no 17,20-lyase activity. Experiments based on the use of an electron donor independent from enzyme binding (iodosobenzene) demonstrated that the addition of electrons restores, at least in part, in vitro 17,20-lyase activity, with no significant influence on the 17{alpha}-hydroxylase activity. This suggests that the electron transfer system plays a major role in the differential regulation of the two P450c17 activities. This is the first case of mutated CYP17 in which the in vitro model corresponds to the in vivo situation.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
COMPLETE combined 17{alpha}-hydroxylase/17,20-lyase deficiency is a rare cause of sex-insufficient masculinization in 46,XY individuals, and of hypertension, hypokalemia, and lack of sexual maturation in both sexes. Although complete combined 17{alpha}-hydroxylase/17,20-lyase represents the most common scenario, several cases of isolated 17,20-lyase deficiency have been described (1). The clinical picture is characterized by the presence in XY individuals of genital ambiguity of various degree, but no hypertension. The possibility of studying the CYP17 gene of several human knock-outs, i.e. patients affected by 17{alpha}-hydroxylase/17,20-lyase deficiencies, will provide a powerful tool in understanding the molecular bases of the differential regulation of the two activities in physiological and pathological conditions, e.g. adrenarche and functional ovarian hyperandrogenism. Recent studies suggest that the posttranslation modification of P450c17 (i.e. phosphorylation) is crucial for such differential regulation (3). Another major factor in this process is the electron transfer system. Several studies performed in either eukaryotic cells or bacteria gave controversial results concerning the alternative electron donor cytochrome b5 (4, 5, 6, 7). More recent studies using a purified system in bacteria showed that cytochrome b5 indeed enhances the synthesis of androgens, furthermore suggesting that in the testes, where the ratio of cytochrome b5/P450 is high, both {Delta}4 and the {Delta}5 steroidogenic pathways can lead to testosterone production (8). On the other hand, the role of cytochrome b5 in the modulation of action of P450c17 in vivo is still unresolved.

The molecular basis of isolated 17,20-lyase defect was elucidated only in one case where a compound heterozygosity was found. This rearrangement could affect the protein folding, but it does not explain why only the 17,20-lyase is affected, as in vitro both activities are low (9). In the present study, the substitution of a Phe residue in the putative redox partner binding site causes 17,20-lyase deficiency in vitro as well as in vivo. The rearrangement was used as a model to study the differential regulation of the two P450c17 activities.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Case report

A newborn patient with the 46,XY karyotype presented with micropenis and undescended testes at birth. The family history revealed consanguinity of the parents (first degree cousins) and excluded the presence of symptoms in the older brother. The basal hormone values were too low to allow a correct diagnostic interpretation. Stimulation tests demonstrated normal responses of 17-hydroxyprogesterone and cortisol to ACTH, whereas androgen precursors and testosterone did not rise after hCG administration (Table 1Go). Based on these signs and symptoms, we made a diagnosis of complete isolated 17,20-lyase deficiency.


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  		Table 1. Index case hormonal plasma values (nanomoles per L)

 
Mutation analysis

Genomic DNA was extracted from blood leukocytes (5 mL ethylenediamine tetraacetate blood) using the Qiagen DNA blood and cell culture kit (Qiagen, Hilden, Germany) and was used to perform Southern blot analysis and amplification of exon sequences followed by direct sequencing. Ten micrograms of genomic DNA were digested with 10 U EcoRI and PstI at 37 C overnight. The electrophoretic separation of the digested fragments, the capillary transfer on nylon membrane, and the hybridization to a labeled full-length human P450c17 complementary DNA (cDNA) have been carried out using standard procedures (11). The construction of the probe was performed via an amplification-labeling procedure using the following amplification program: 95 C for 1 min, 50 C for 1 min, 72 C for 1.5 min for 30 cycles. Exonic sequences were obtained via direct sequencing of PCR-amplified fragments using the dideoxy method applied to thermal cycling (fmol kit, Promega Corp., Madison, WI). The oligonucleotides were designed to be complementary to the sequence adjacent to exons to be amplified to enable the study of the intron-exon boundaries. The PCR amplification was performed as follows: exons I and II-III: 93 C for 1 min, 58 C for 2 min, 65 C for 1.5 min; exons IV and V–VI: 93 C for 1 min, 65 C for 2 min, 65 C for 1.5 min; exons VII–VIII: 93 C for 1 min, 60 C for 2 min, 65 C for 1.5 min, repeated for 30 cycles. After purification on Microspin S300 HR cartridges (Pharmacia Biotech, Uppsala Sweden), one fifth of the PCR reaction was used for the direct sequencing. The entire procedure was repeated at least three times for each fragment.

In vitro expression

Site-directed mutagenesis according to the mutation found was carried out using the Transformer kit (Clontech, Palo Alto, CA) following the given protocol. The mutagenic primers were designed in the correct orientation (direct) and in the opposite orientation (reverse) to test possible sequence-dependent nonspecific effects. Sequencing was used to test the correctness of the mutagenesis. The mutated cDNAs were then EcoRI/BamHI digested and subcloned in pCMV4 expression vector (12).

Ten micrograms of DNA were transfected into confluent COS-1 cells grown in DMEM medium, using diethylaminoethyl-dextran (50 µg; Sigma Chemical Co., St. Louis, MO) with addition of 100 µmol/L chloroquine (Sigma) (11). The same procedure was used in experiments in which P450c17 cDNA was cotransfected with 1, 2.5, 5, and 10 µg human cytochrome b5 cDNA (provided by Dr. Mason, Edinburgh, UK).

After 48 h, either 1 µmol/L (300 ng/mL) of the steroidogenic precursors was added to the cells, or protein extraction and assay were performed. Six hours later, the medium was removed and assayed for products. Steroid measurements were performed via gas chromatography (Fig. 1Go) and/or RIA (pregnenolone and 17-hydroxypregnenolone: ICN Biochemical, Costa Mesa, CA; progesterone, 17-hydroxyprogesterone, and dehydroepiandrosterone (DHEA): Diagnostic Products Corp., Los Angeles, CA; androstenedione: Diagnostic System Laboratories, Webster, TX). Iodosobenzene (100 nmol/L) was added to the culture medium together with the steroidogenic precursors.



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  		Figure 1. Detection of steroids in medium of transfected COS-1 cells via gas chromatography (representative experiments). An extraction with methylene chloride (1:10) was performed before detection. Peak 1, Pregnenolone; peak 2, unknown; peak 3, DHEA; peak 4, pregnenolone; peak 5, 16-hydroxyprogesterone; peak 6, progesterone; peak 7, 17-hydroxyprogesterone; peaks 8 and 9, 17-hydroxypregnenolone. Vector, Precursor (1 µmol/L pregnenolone). WT, Precursors (1 µmol/L pregnenolone and 1 µmol/L progesterone).

 
Western blot analysis was performed following standard procedures (11) and using polyclonal rabbit antihuman antibodies provided by Prof. Waterman (Nashville, TN). Total protein determination was carried out using the Pierce bicinchoninic acid protein assay kit (Rockford, IL). One hundred and 10 µg total protein were loaded. The same amount of total protein extracted from NCI-H295 human adrenal carcinoma cells was used as a positive control.

Total ribonucleic acid (RNA) was isolated from cell lysate of three 10-cm culture dishes containing confluent COS-1 cells. The isolation of total RNA was performed using a modified standard procedure (13). The probe used for Northern blot analysis was generated via amplification labeling of human full-length P450c17 cDNA, as described for the Southern blot.

The sequence alignments were carried out by the University of Wisconsin Genetics Computer Group suite.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Southern blot analysis of EcoRI/PstI-digested genomic DNA from the index case, his family, and a normal individual as a control showed the absence of gross rearrangements in the CYP17 gene of these subjects (not shown). Direct sequencing of PCR-amplified exonic fragments revealed the presence of a heterozygote deletion of a T on position 198 in exon I, which causes a frame shift with consequent introduction of a premature stop codon TGA on residue 74 (V74X). The proof of heterozygosity was given by the demonstration that the mother is the carrier of the frameshift mutation. The father and the brother bear no change in exon I of the CYP17 gene (Fig. 2Go). The paternal allele carries a T->G transversion that changes the phenylalanine 417 to a cysteine. As shown in Fig. 2Go, the mother and brother carry two normal alleles.



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  		Figure 2. Sequence analysis of PCR products amplified from genomic DNA of the index case and his family. The bases are loaded in the alphabetical order (A, C, G, T). The father and the brother carry two normal alleles. The patient and the father are heterozygotes for the T->G transversion in exon VIII causing the missense F417C mutation, whereas mother and brother are homozygote normal. The results were obtained in at least three independent experiments.

 
The in vitro expression of the mutant proteins demonstrated that the frameshift mutation causes the absence of protein synthesis (data not shown) with consequent inability to catalyze 17{alpha}-hydroxylation and 17,20 cleavage (Fig. 3Go and Table 2Go). Transfection of the F417C-mutated P450c17 showed the presence of a protein synthesized at a normal rate that retains some 17{alpha}-hydroxylase activity (26% of wild type), but lacks virtually any 17,20-lyase activity (activity similar to mock transfection or vector alone; Fig. 3Go and Table 2Go). It is interesting that the F417C mutation prevents the synthesis of 16{alpha}-hydroxyprogesterone (Table 2Go). As expected, no androstenedione was detectable in the culture medium. The transfection of cDNAs mutated with the reverse mutagenic oligonucleotides gave results similar to those with mock vector alone transfection, demonstrating that there is no nonspecific effect on precursor conversion due to the primer sequence (Table 2Go).



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  		Figure 3. Schematic representation of percent conversion of 1 µmol/L pregnenolone or progesterone into their 17{alpha}-hydroxylated products (17{alpha}-hydroxylase activity; filled bars) or DHEA (17,20-lyase activity; hatched bars) in transfected COS-1 cells. All values are expressed as nanograms per mL/mg total protein. For absolute values, see Table 2Go.

 

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  		Table 2. Percent conversion of 1 µmol/L pregnenolone (Preg) or progesterone (Prog) in transfected COS-1 cells

 
As F417 is conserved among all the class II P450 enzymes and is located close to the putative redox partner-binding site in BM3 (14), it may be hypothesized that the electron transfer system plays a role in the onset of this isolated 17,20-lyase deficiency. To test this hypothesis, we applied to the cells carrying the F417C mutation an electron donor independent from binding to the enzyme (15). The addition of iodosobenzene does not affect the protein synthesis rate, but at least in part restores 17,20-lyase activity (from 0.3% to 5.7 ± 0.3% of wild type) without effect on the 17{alpha}-hydroxylase step (from 26 ± 0.4% to 25 ± 0.2 5 of wild type; Fig. 3Go and Table 2Go). The cotransfection experiments using P450c17 and cytochrome b5 cDNAs failed to show an effect of cytochrome b5 on the P450c17 activity (not shown), although the cytochrome b5 transcript was readily detectable by Northern blot analysis (Fig. 4Go). The amount of protein, detected by Western blot, produced by the transfected cDNAs showed comparable amounts of 17{alpha}-hydroxylase protein after transfection with the wild-type cDNA, F417C with or without iodosobenzene (Fig. 5Go), whereas no protein was detectable in cell lysates from mock-, vector-, and {Delta}T198-transfected COS-1 cells (not shown).



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  		Figure 4. Northern blot analysis of 10 µg total RNA extracted from COS-1 cells cotransfected with P450c17 and cytochrome b5 cDNAs, the latter at increasing concentrations (1, 2.5, 5, and 10 µg). The blotted RNA was probed with labeled cytochrome b5 cDNA (Cyt. b5).

 


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  		Figure 5. Western blot analysis of in vivo translated P450c17 normal and mutant proteins: 100 (A) and 10 µg (B) of total protein were loaded, blotted, and probed with rabbit antihuman P450c17 antibodies (1:7000). Comparable amounts of protein are generated upon transfection with wild-type (WT) F417C with or without (+ and -, respectively) 100 nM iodosobenzene (IDB). The addition of 150 nmol/L iodosobenzene does not significantly modify the pattern of expression. Total protein from NCI-H295 human adrenal carcinoma cells was used as a positive control.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Clarification of the genetic rearrangements causing enzymatic defects of steroid biosynthesis is of crucial importance to identify a common pattern of mutations and to allow prenatal diagnosis and treatment. Naturally occurring mutations in the human population permit some more insight into the structure-function relationship of P450c17. In particular, when an isolated 17,20-lyase deficiency is present, the identification of hot spots responsible for the deficit of only one of the activities will furnish clues in the understanding of the still unresolved issue of the differential regulation of 17{alpha}-hydroxylase vs. 17,20-lyase.

The described case represents a minimal percentage of an already rare enzyme defect. The diagnosis of isolated 17,20-lyase deficiency was based on the presence of a micropenis, a normal response of the 17{alpha}-hydroxylated/non-17,20-cleaved steroids, and no response of adrenal or gonadal androgens to stimuli. The molecular analysis of the CYP17 gene of this family revealed a double heterozygosity. Although the maternal allele carries a frameshift mutation leading to a nonsense V74X mutation affecting both catalytic activities, the paternal allele bears a missense F417C mutation in the C-terminal domain of the protein. The latter rearrangement, the first example of P450c17 deficiency where phenotype and genotype match, provides interesting information on the regulation of 17,20-lyase activity. In fact, F417 is conserved among all microsomal P450 enzymes, and it is located in a highly conserved region thought to be involved in redox partner binding. The same Phe is also conserved in P450BM-3, a bacterial P450 whose three-dimensional structure has been recently elucidated and could serve as a better reference than P450cam for three-dimensional modeling of microsomal P450s such as P450c17 (14). In BM-3, in the region thought to form a dock for the redox partner, there is a preponderance of neutral residues. The exchange of a hydrophobic aromatic residue with a neutral polar amino acid could significantly alter the function of the domain. As the patient displays isolated 17,20-lyase deficiency, the F417C mutation lends weight to the hypothesis of the crucial role of the electron donor system in the still unresolved issue of the differential regulation of 17{alpha}-hydroxylase vs. 17,20-lyase activity. Cotransfection experiments using P450 reductase or cytochrome b5 cDNA failed in our hands to show any differential effects on the two P450c17 activities. On the other hand, experiments based on the use of an electron donor independent from enzyme binding (iodosobenzene) (15) in our eukaryotic system demonstrated that the addition of electrons in part restores 17,20-lyase activity with no significant influence on the 17{alpha}-hydroxylase activity, suggesting that the electron transfer system indeed plays a major role in the differential regulation of the two P450c17 activities. At present in our model, it is not clear whether putative alternative electron donor cytochrome b5 is involved in the differential regulation. Although the transcript for cytochrome b5 was detected in transfected COS-1 cells, the lack of antibodies against b5 protein or of a functional assay for b5 activity prevented us from demonstrating that cytochrome b5 is expressed and functional.

The fact that the in vitro 17{alpha}-hydroxylase activity is 26% of the wild type confirms that P450c17 enzyme with only about 25% of its normal activity is adequate to prevent mineralocorticoid hypertension and cortisol insufficiency, but not to determine a normal male phenotype. This mutation model can also help to elucidate whether developmentally regulated factors, such as estrogens and/or insulin-like growth factor I, have any effect on the enzymatic activity in vitro, as it seems to occur in vivo in patients affected by isolated 17,20-lyase deficiency who converted their phenotype to complete combined deficiency in young adulthood (16, 17). Further studies using estrogen receptor-expressing cells are needed to test this hypothesis.


   Acknowledgments
 
We gratefully acknowledge Prof. Claus W. Heizmann’s support. We thank Dr. Markus Lauber for helpful discussion, and Mrs. Brigitte Manella and Mrs. Bianca Kempken for skilled technical assistance. We also thank Prof. Michael Waterman (Nashville, TN) for kindly providing P450c17 antibodies, and Prof J. Ian Mason for providing cytochrome b5 cDNA. We appreciate the help and discussion with Dr. Sandra Graham on the structural implication of the mutations.


   Footnotes
 
1 This work was supported by Swiss National Foundation Grant 32–36275.92, the Sandoz Stiftung, the Helmut Horten Stiftung, the Jubiläumsstiftung of the Swiss Life Insurance and Pension Co., and the Union Bank of Switzerland (on behalf of a client). Back

Received January 23, 1997.

Revised July 22, 1997.

Accepted July 24, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Zachmann M, Prader A. 1980 17,20-desmolase deficiency. In: New MI, ed. Adrenal disease in childhood: pathophysiology and clinical aspects. Pediatric endocrinology. Basel: Karger; 17–21.
  2. Deleted in proof.
  3. Zhang LH, Rodriguez H, Ohno S, Miller WL. 1995 Serine phosphorylation of P450c17 increases 17,20-lyase activity: implications for adrenarche and polycystic ovary syndrome. Proc Natl Acad Sci USA. 92:10619–10623.[Abstract/Free Full Text]
  4. Onoda M, Hall PF. 1982 Cytochrome b5 stimulates purified testicular microsomal cytochrome P450 (c21 side-chain cleavage). Biochem Biophys Res Commun. 108:454–460.[Medline]
  5. Meadus WJ, Mason JI, Squires EJ. 1993 Cytochrome P450c17 from porcine and bovine adrenal catalyses the formation of 5,16-androstandien-3ß-ol from pregnenolone in the presence of cytochrome b5. J Steroid Biochem Mol Biol. 46:565–572.[CrossRef][Medline]
  6. Sakai Y, Yanase T, Hara T, Takayanagi R, Haji M. Nawata H. 1994 In vitro evidence for the regulation of 17,20-lyase activity by cytochrome b5 in adrenocortical adenomas from patients with Cushing’s syndrome. Clin Endocrinol (Oxf). 40:205–209.[Medline]
  7. Lin D, Black SM, Nagahama Y, Miller WL. 1993 Steroid 17{alpha}-hydroxylase and 17,20-lyase activities of P450c17: contribution of serine 106 and P450 reductase. Endocrinology. 132:2498–2506.[Abstract]
  8. Katagiri M, Kagawa N, Waterman MR. 1995 The role of cytochrome b5 in the biosynthesis of androgens by human P450c17. Arch Biochem Biophys. 3174:343–347.
  9. Yanase T, Waterman MR, Zachmann M, Winter JSD, Simpson ER, Kagimoto M. 1992 Molecular basis of apparent isolated 17,20-lyase deficiency: compound heterozygous mutations in the C-terminal region (Arg(496)Cys, Gln(461)Stop) actually cause combined 17{alpha}-hydroxylase/17,20-lyase deficiency. Biochim Biophys Acta. 1139:275–279.[Medline]
  10. Deleted in proof.
  11. Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular cloning, 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory.
  12. Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russel DW. 1989 Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 264:8222–8229.[Abstract/Free Full Text]
  13. Chomczinsky P, Sacchi N. 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 16:156–159.[CrossRef]
  14. Ravichandran KG, Boddupalli SS, Haseman CA, Peterson JA, Deisenhofer J. 1993 Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science. 261:731–736.[Abstract/Free Full Text]
  15. Gustafsson JA, Rondhal L, Bergman J. 1979 Iodosyl benzene derivatives as oxygen donors in cytochrome P450 steroid hydroxylation. Biochemistry. 18:865–870.[CrossRef][Medline]
  16. Zachmann M, Kempken B, Manella B, Navarro E. 1992 Conversion from pure 17,20-desmolase to combined 17,20-desmolase/17{alpha}-hydroxylase deficiency with age. Acta Endocrinol (Copenh). 127:97–99.[Medline]
  17. Yanase T. 1995 17{alpha}-Hydroxylase/17,20-lyase defects. J Steroid Biochem Mol Biol. 53:153–157.[CrossRef][Medline]



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