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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.

IN HUMANS, the biosynthesis of C₁₉ sex steroid precursors requires the sequential execution of two chemical transformations by the P450c17 enzyme system (Fig. 1). In the first step, the 17-hydroxylase reaction, pregnenolone is converted to 17-hydroxypregnenolone (17OHpreg). In the second step, the 17,20-lyase reaction, the C₁₇-C₂₀ bond of 17OHpreg is cleaved to form dehydroepiandrosterone (DHEA). The negligible amounts of both 17-hydroxysteroids and C₁₉ steroids produced by patients with severe mutations in the CYP17 gene for P450c17 demonstrates that P450c17 accounts for all of the 17-hydroxylase and 17,20-lyase activities of human steroidogenic tissues (1, 2). Furthermore, expression of the cDNA for P450c17 in nonsteroidogenic cells confers both 17-hydroxylase and 17,20-lyase activities to these cells (3, 4), and both activities are demonstrable in yeast microsomes containing P450c17 (5) as well as in reconstituted systems containing purified recombinant P450c17 protein (6).

View larger version (16K): [in this window] [in a new window]   Figure 1. Biosynthetic reactions catalyzed by the human P450c17 enzyme system. The human enzyme performs the 17-hydroxylation reaction with both pregnenolone and progesterone substrates, whereas the 17,20-lyase reaction is efficient only with 17OH-preg and only in the presence of optimal amounts of b5.

Mutations that selectively impair the 17,20-lyase activity of P450c17 are particularly informative for structure-function studies of P450c17 and may also give insight into the process of adrenarche. We reported two patients whose steroid hormone profiles suggested a preferential, isolated loss of 17,20-lyase activity (18). Both patients were homozygous for mutations that neutralized single positive charges in the redox partner binding site, namely R347H and R358Q. These mutations dramatically impair 17,20-lyase activity in the presence and absence of b₅, and although b₅ does stimulate some DHEA production, the stimulation is not as brisk as with wild-type P450c17 (19). Impaired 17,20-lyase activity with preservation of 17-hydroxylase activity was consistently demonstrated when the mutant cDNAs were expressed in COS-1 cells (18), yeast microsomes (19), and Escherichia coli as a modified, histidine-tagged protein that was purified and reconstituted in vitro (20). In addition, two site-directed mutations [K89N (21) and R449A (22)] that have been studied less intensively also preferentially reduced 17,20-lyase activity, and these residues also map to the redox partner binding site (Fig. 2) (23). Thus, computer modeling studies, genetics, and detailed biochemistry in multiple systems demonstrate that neutralizing key positive charges in the redox partner binding site can cause the isolated loss of 17,20-lyase activity.

View larger version (88K): [in this window] [in a new window]   Figure 2. Mutations in the redox partner binding site of human P450c17 that cause preferential loss of 17,20-lyase activity. These mutations all neutralize positively charged residues (shown in dark color). Mutations R347H and R358Q were found in patients; mutations K89N and R449A were engineered by site-directed mutagenesis. Note that F417, which is also shown in dark color, is not exposed to the protein surface, but lies behind a loop, between the meander and the heme-binding regions. The human P450c17 model used is entry 2c17 from the rcsb repository (www.rcsb.org) (21 ).

Materials and Methods

Site-directed mutagenesis

The cDNA for the P450c17 mutation F417C was constructed by sequential PCR employing overlapping mutagenic oligonucleotides. The first pair of PCR reactions used plasmid pLW01-c17 as template, Pfu Turbo polymerase (Stratagene, La Jolla, CA) with the manufacturer’s buffer and 200 µM deoxy-NTPs in 50 µl total volume and a program of 25 cycles of 95 C for 30 sec, 50 C for 30 sec, and 72 C for 1.5 min. The primer pairs (with restriction sites underlined and mutagenic codon in bold) were C17-S1-Bgl (5'-GGAGATCTATGTGGGAGCTCGTGGCTCTCTTGC-3') plus F417C-AS1 (5'-CGCTGGATTCAAGCAACGCTCAGGC-3') or F417C-S1 (5'-TGCCTGAGCGTTGCTTGAATCCAGC-3') plus C17-AS1-Eco (5'-CCGGAATTCCAGCCTTTAGGTGCTACCCTG-3'). The resulting PCR products (1 µl of each crude amplification mixture) were used as templates for the final amplification of the full-length cDNA. Amplification with Pfu polymerase failed, so instead a 19:1 mixture of Taq (Promega Corp., Madison, WI) and Pfu polymerases in 50 µl 16 mM NH₄SO₄, 0.01% Tween 20, 67 mM Tris-HCl (pH 9), and 2 mM MgCl₂ was used with primers C17-S1-Bgl and C17-AS1-ECo and 25 cycles of 95 C for 30 sec, 55 C for 30 sec, and 72 C for 2 min. The final PCR product was purified on a 1% agarose gel, isolated with the QIAEX-II gel extraction kit (QIAGEN, Valencia, CA), and ligated into the pGEM-T vector using the T-A overhang method according to the manufacturer’s instructions (Promega Corp.). The inserts from several positive colonies were sequenced in their entirety to assure that the only amino acid substitution incorporated was the desired F417C mutation. The T-A cloned product was digested with BglII and EcoRI, gel-purified, and subcloned into the BglII/EcoRI site of the yeast expression vector V10 (5, 25) as well as into the BamHI/EcoRI site of the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The accuracy of one construct was confirmed by sequencing the entire cDNA a second time after subcloning into V10.

Yeast transformation and microsome preparation

Yeast were transformed using a modification of the lithium acetate method (26). Saccharomyces cerevisciae strain W303B (25) was grown in 15 ml YPD medium to an A₆₀₀ of 0.5–0.8, harvested by centrifugation, resuspended in 200 µl 0.1 M lithium acetate plus 10 mM Tris-HCl/1 mM EDTA (pH 8), and incubated for 30 min at 30 C. A pellet containing one third of the yeast cells was mixed first with 240 µl freshly prepared 50% aqueous polyethylene glycol 4000 (BDH Laboratory Supplies, Poole, UK) and then with 36 µl 1 M lithium acetate, 50 µl denatured salmon sperm DNA (2 mg/ml), 31 µl 100 mM Tris-HCl/10 mM EDTA (pH 8), and 1 µg each of plasmids V10 and pYcDE-2 (5). The V10 plasmid contained no insert, the cDNA for wild-type P450c17, or the cDNA for P450c17 mutation F417C; the pYcDE-2 plasmid contained either no insert (with empty V10 vector) or the cDNA for human OR (with V10-P450c17 plasmids). Transformed yeast were selected on minimal medium containing Difco brand yeast nitrogen base (Becton Dickinson and Co., Sparks, MD) with 5 g/liter ammonium sulfate, 2 g/liter glucose, and 40 mg/liter adenine hemisulfate in 2% agar.

Yeast cells were grown in 500 ml liquid minimal medium with adenine and harvested at a density of 3–5 x 10⁷ cells/ml (A₆₀₀ of 1.0–1.8) by centrifugation. Cells were resuspended in 10 ml 0.1 M KCl/50 mM Tris-HCl/1 mM EDTA, kept at ambient temperature for 5 min, and collected by centrifugation. Cells were then resuspended in 5 ml 50 mM Tris-HCl/1 mM EDTA with 71 mM 2-mercaptoethanol, kept at ambient temperature for 5 min, and again collected by centrifugation. Spheroplasts were prepared by resuspending the cells in 4 ml 1.5 M sorbitol/50 mM Tris-HCl/1 mM EDTA and incubating with 2.5 mg Zymolase T100 (Seikagaku Corp., Tokyo, Japan) for 30 min at 30 C. Spheroplasts were resuspended in 9 ml 0.6 M sorbitol/50 mM Tris-HCl/1 mM EDTA and disrupted on ice by sonication at full power with six 5-sec pulses alternating with 15-sec pauses over 2 min. Disrupted cells were centrifuged for 10 min at 10,000 x g twice to remove cell debris, nuclei, and mitochondria; microsomes were collected by ultracentrifugation of the supernatant at 100,000 x g for 50 min. Resulting pellets were homogenized in 50 mM Tris-HCl/1 mM EDTA/20% glycerol by shearing through a 27.5-gauge needle and kept frozen in aliquots at 70 C until needed. Incubation of yeast microsomes with radiolabeled steroids was performed as previously described (5).

COS-7 cell culture, transfection, and assays

All tissue culture reagents were the Cellgro brand purchased from Mediatech, Inc. (Herndon, VA). COS-7 cells were grown in T-75 flasks with DMEM containing 0.584 g/liter glutamine, 10% FBS, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. Trypsinized cells were seeded in six-well plates with 2 ml/well complete medium and grown at least overnight until 60–80% confluent. Fresh complete medium was applied immediately before transfection. The transfection cocktail was prepared by adding 3 µl FuGENE 6 transfection reagent (Roche, Indianapolis, IN) and 2 µg plasmid DNA (pcDNA3 as empty vector or containing the cDNA for either wild-type P450c17 or mutation F417C) sequentially to 100 µl serum-free medium/transfection. After incubation at ambient temperature for 20 min, 100 µl of the transfection cocktail were added to each well and dispersed evenly. After 24 h, the cells were incubated with 2 ml fresh complete medium containing 0.1 µM pregnenolone, 17OHpreg, or progesterone with 60,000 cpm/well ³H-labeled steroid (NEN Life Science Products, Boston, MA). At the specified times, the medium was removed and gently rocked for 1 min with 5 ml ethyl acetate/isooctane (1:1) to avoid emulsion formation (control experiments showed that 90% of the radioactivity was extracted by this method). The organic layer was removed and concentrated under nitrogen, and TLC plus autoradiography were performed as previously described (9). Wells used for immunoblotting received fresh medium only, and cells were harvested after 24 h by scraping with 1 ml PBS and centrifugation.

Miscellaneous

Reagents and chemicals were purchased from either Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO), except as noted. Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). DNA sequencing employed the dye termination method with PE Applied Biosystems instruments at University of Texas Southwestern McDermott Center Sequencing Center. Immunoblotting was performed as previously described (9). Molecular graphics images were generated with an Octane workstation (Silicon Graphics, Inc., Mountain View, CA) using the ribbonjr option of MidasPlus software. Difference spectroscopy for P450 content was performed by centrifuging 70 ml saturated yeast culture and resuspending the cells in 6 ml 0.1 M potassium phosphate (pH 7.4) with 1 mM EDTA. The suspension was divided among two cuvettes, and carbon monoxide gas was bubbled through the sample cuvette for 1 min before recording spectra (Aminco DW2a spectrophotometer modified with the Olis RSM1000 spectroscopy system, Bogart, GA).

Results

As we have previously shown (5), yeast microsomes containing wild-type human P450c17 and human OR sequentially metabolize pregnenolone to 17OHpreg and then to DHEA (Fig. 3). These same microsomes also convert progesterone to 17-hydroxyprogesterone (17OHprog) and 16-hydroxyprogesterone (16OHprog) in a ratio of roughly 4:1 plus a small amount of androstenedione, consistent with the known activities of human P450c17 (Fig. 3A). In contrast, no 17- or 16-hydroxylation of either pregnenolone or progesterone was detected in yeast microsomes containing human OR and the F417C mutation (Fig. 3A). For comparison, a chromatogram of steroids produced by incubations with wild-type P450c17 compared with mutations R347H and R358Q is shown in Fig. 3B. Mutations R347H and R358Q form the same array of products as does wild-type P450c17, except that no DHEA is produced (Fig. 3B), demonstrating severe, selective impairment of only the 17,20-lyase activity in mutations R347H and R358Q. To determine why no 17-hydroxylase activity was observed, we performed immunoblots on the microsomes assayed in Fig. 3. Microsomes from yeast transformed with expression vectors containing the cDNAs for wild-type P450c17 and for mutation F417C contained immunoreactive bands of nearly identical intensity and apparent molecular mass (Fig. 4A). This band was not present in microsomes from yeast transformed with empty vectors. These data indicate that although equivalent amounts of the wild-type and mutant P450c17 proteins are expressed in the yeast microsomes, the F417C mutation is devoid of catalytic activity.

View larger version (48K): [in this window] [in a new window]   Figure 3. Autoradiogram of chromatographed steroids obtained by incubating yeast microsomes with [3H]pregnenolone (5) or [3H]progesterone (4) for 1 h at 37 C. A, Yeast were transformed with empty expression vectors (Mock) or with expression vectors for human OR and either wild-type P450c17 or the F417C mutation. Incubations with Mock microsomes and F417C microsomes used 50 µg microsomal protein, whereas incubations with wild-type microsomes used only 30 µg microsomal protein. Identified steroids are: Preg, pregnenolone; 17Preg, 17OHpreg; Prog, progesterone; AD, androstenedione. B, Comparable incubations with yeast microsomes containing wild-type P450c17 (WT) or mutations R347H or R358Q, demonstrating the selective loss of only the 17,20-lyase activity of these two mutations. Only the wild-type enzyme produces DHEA (arrow), whereas all three enzymes convert pregnenolone to 17OHpreg (17Preg) and convert progesterone to 17OHprog (17Prog) and 16OHprog (16Prog).

View larger version (39K): [in this window] [in a new window]   Figure 4. A, Immunoblot of yeast microsomal proteins with antihuman P450c17 antiserum (9 ). Yeast microsomes described in Fig. 3 (0.3–3 µg protein as indicated) were electrophoresed through an 8% SDS-polyacrylamide gel before immunoblotting. Approximate apparent molecular masses of prestained protein standards, whose electrophoretic mobility is altered by dye coupling (New England Biolabs, Inc., Beverly, MA), are given at the right. Human P450c17 from all sources, including human adrenal glands, consistently migrates adjacent to the 62-kDa marker (open arrow). A prominent P450c17 degradation product observed in yeast is marked by asterisk. B, Carbon monoxide/reduced P450 difference spectra from yeast strain W303B (top) or strain W303B expressing wild-type P450c17 (middle) or F417C mutation (bottom). A peak at 450 nm is observed with wild-type P450c17, but not with mutation F417C. Crosshairs (+) in the bottom and middle spectra lie at 450 nm.

It is possible, however, that the F417C mutation is inactive when expressed in yeast and that the protein might be active when expressed in mammalian cells, as was reported previously (24). To address this possibility, we transfected COS-7 cells with expression vectors containing the cDNAs for wild-type P450c17 and for the F417C mutation. As shown previously, COS-7 cells expressing wild-type P450c17 rapidly metabolize pregnenolone and progesterone to the expected products within 4 h (Fig. 5). Pregnenolone and progesterone metabolism in cells expressing the F417C mutation, however, is limited to by-products produced in a pattern identical to mock-transfected cells, even after 24 h of incubation (Fig. 5). No 17-hydroxysteroid production by the F417C mutation above background was observed in any of five independent transfection experiments using three different cDNA clones, and no [³H]DHEA was produced upon incubation with [³H]17OHpreg (data not shown). Therefore, we found that mutation F417C has no 17,20-lyase activity, but, in contrast to previous reports (24, 28), we found no evidence for preserved 17-hydroxylase activity, a result inconsistent with an isolated deficiency of 17,20-lyase activity. Immunoblotting of transfected cells showed that an F417C mutant protein of the expected size is produced in COS-7 cells in slightly lower amounts than the wild-type P450c17 protein (Fig. 6). Difference spectroscopy was not performed in transfected cells because the amount of protein expressed was too low to observe spectroscopically. These data conclusively demonstrate that the F417C mutation has no 17- or 16-hydroxylase activity when expressed either in yeast or in COS-7 cells. Therefore, mutation F417C cannot be the sole cause of isolated 17,20-lyase deficiency.

View larger version (22K): [in this window] [in a new window]   Figure 5. Autoradiogram of chromatographed steroids obtained by incubating transfected COS-7 cells with [3H]pregnenolone (A) or [3H]progesterone (B). Cells transfected with either empty pcDNA3 expression vector (V) or pcDNA3 containing the cDNA for wild-type (WT) or F417C mutation (M) P450c17 proteins were incubated with radiolabeled steroids for 4 or 24 h as indicated. Extracted steroid substrate from a 0.5-ml aliquot of the incubation medium (--) at time zero was applied to the leftmost lane as a standard. The WT enzyme completely metabolizes pregnenolone to DHEA and progesterone to 17OHprog, 16OHprog, and some androstenedione (AD) after 24 h. In contrast, only background metabolites (<0.5% of radioactivity cochromatographing with 17-hydroxylated products) are observed in cells transfected either with empty vector or with vector for mutation F417C. Abbreviations for steroid substrates and metabolites are the same as in Fig. 3.

View larger version (97K): [in this window] [in a new window]   Figure 6. Immunoblot of transfected COS-7 cells with antihuman P450c17 antiserum. Pelleted cells in one well of a six-well plate from the experiment shown in Fig. 5 were boiled in 100 µl protein sample buffer (New England Biolabs, Inc., Beverly, MA). Aliquots (0.3–3 µl of each sample as indicated) were diluted to 10 µl with sample buffer and loaded onto a 10% SDS-polyacrylamide gel before electrophoresis and immunoblotting. Specific bands of the expected size and comparable intensities are found in the cells expressing wild-type P450c17 and P450c17 with the F417C mutation.

It was not until 1997 that mutations in P450c17 (R347H and R358Q) were conclusively demonstrated to cause isolated 17,20-lyase deficiency (18). To unambiguously confirm the causative role of mutations R347H and R358Q in the biochemical pathogenesis of isolated 17,20-lyase deficiency, we showed that the mutant P450c17 enzymes in the propositi efficiently metabolized [³H]pregnenolone to [³H]17OHpreg (50–65% the rate of the wild-type enzyme), but only produced a trace of [³H]DHEA (<5% the rate of the wild-type enzyme) (18). The isolated loss of 17,20-lyase activity was reproducibly observed when the mutant proteins were expressed either in mammalian COS-1 cells (18) or in yeast microsomes (19), and the same results were independently reported by another group studying purified, recombinant mutant P450c17 proteins expressed in E. coli (22). The critical role of residues 345–360, which lie in the J' and K helixes of the redox partner binding site (21), for 17,20-lyase activity is consistently observed in P450c17 enzymes for other species that have been studied. In rat P450c17, replacements of the homologous arginine residues R346 and R357 with alanine severely impair 17,20-lyase activity, yet preserve most 17-hydroxylase activity, particularly for the R346A mutation (29).

The discrepancies between our results with mutation F417C and those reported previously possibly derive from methodological differences in the two studies. Our studies involved incubations with ³H-labeled steroids, followed by TLC, to study the enzymology of the mutant protein. This approach has been validated in numerous studies over 15 yr (3), and autoradiography of the chromatograms provides the reviewers and readers with graphic, readily interpretable data. In contrast, earlier reports of the F417C mutation used gas chromatography (GC) of unlabeled steroids and RIA. Although GC can be a highly sensitive and reliable method for identifying and quantitating steroid metabolites, GC must be preceded by steroid derivitization and coupled with mass spectrometry to yield reliable results (30), neither of which was employed. In addition, the retention times of peaks attributed to 17OHpreg and 17OHprog in the previous report are inconsistent among their samples (24), precluding reliable identification of the putative metabolites. Furthermore, because the steroid substrates were not isotopically labeled, there is no assurance that the indicated peaks even derive from pregnenolone or progesterone. RIA can also be extremely accurate, but the generation of substances that interfere with these assays under the experimental conditions cannot be excluded (31, 32). Additional trivial explanations, such as contamination of the expression vector for the F417C mutation with a small amount of the expression vector for the wild-type enzyme, could lead to some 17-hydroxysteroid production and relatively little DHEA accumulation. It is also noteworthy that the F417C mutation forms no 16OHprog (either in our hands or in the prior reports), whereas P450c17 mutations R347H and R358Q both form 16OHprog as well as 17OHprog in equivalent proportions, as does wild-type P450c17 (23). The only other P450c17 mutation in the vicinity of F417 associated with clinical disease is mutation P409R, located in the meander eight residues from F417, and this mutation also causes complete loss of both 17-hydroxylase and 17,20-lyase activities (33). Thus, it is not surprising that mutation F417C, located between the meander and the heme-binding domains that are critical for activities of all P450 enzymes, severely impairs all catalytic activities of P450c17.

An additional incongruency with the previous reports of the F417C mutation and our experience concerns the ability of iodosobenzene to restore the 17,20-lyase activity of the F417C mutation in transfected cells. Iodosobenzene is a reagent that can, under certain experimental conditions, directly oxidize the heme center of some purified P450 enzymes, allowing the P450 to oxygenate substrates in vitro without the aid of electron transfer proteins (34). We were not able to reconstitute either the 17-hydroxylase or 17,20-lyase activity of wild-type P450c17 in yeast microsomes with iodosobenzene (an unstable reagent that we obtained from a reliable source, Prof. Paul Ortiz de Montellano in the Pharmaceutical Chemistry Department at the University of California, San Francisco) despite repeated attempts (21). Therefore, it is remarkable that the previous study reported DHEA production in whole cells expressing the F417C mutation when iodosobenzene (source unidentified) was added to the culture medium (24). More likely, the chemical treatment of these cells allowed the production of unknown steroidal or lipid products that were spuriously identified as DHEA using RIA methodology (32).

It is very difficult to conclusively establish the diagnosis of isolated 17,20-lyase deficiency in the newborn, because both normative data and expected ranges for various steroids in affected neonates are scanty. Credible reports of rare diseases must be accompanied by complete clinical and hormonal data and corroborated by molecular genetic and biochemical studies, and the literature describing isolated 17,20-lyase deficiency poignantly illustrates the importance of detailed, internally consistent results. A total of 14 newborns believed to have isolated 17,20-lyase deficiency were reported between 1972 and 1997, but a review of these cases revealed the requisite elevation of the 17OHprog to androstenedione ratio in only 3 subjects (35). Subsequent biochemical and molecular genetic studies revealed that some of these same patients were more accurately characterized as having partial, combined 17-hydroxylase/17,20-lyase deficiency (36); a partial, combined deficiency state may also be the case for the patient bearing the F417C mutation, because a T deletion in exon 1 was not clearly demonstrated in Fig. 2 of the initial report (24).

Even in cases in which the steroid hormone values strongly suggest an isolated deficiency of 17,20-lyase activity, determining the molecular basis of this diagnosis is fraught with pitfalls. Any mutation found in the CYP17 gene, the most obvious candidate gene in this disease, is not necessarily the sole molecular alteration causing isolated 17,20-lyase deficiency, because P450c17 functions not in isolation but, rather, in a catalytic system with the obligate electron donor OR and the allosteric facilitator b₅ (5). Therefore, it is theoretically possible that mutations in at least three different genes could cause isolated 17,20-lyase deficiency: the CYP17 locus encoding P450c17, the CPR locus encoding the flavoprotein OR, and the CYB5 gene for b₅. Indeed, one case of congenital methemoglobinemia due to a CYB5 splice junction mutation was reported to be a male pseudohermaphrodite (37). No steroid hormone values for this patient appeared in this report, but it is quite possible that this patient is an example of isolated 17,20-lyase deficiency due to loss of b₅ function. Consequently, alterations in the CYP17 gene in a patient believed to have isolated 17,20-lyase deficiency cannot be assumed to cause this disease unless the expected biochemistry of the mutant protein, preferably in more than one expression system, is faithfully replicated in detailed biochemical studies with radiolabeled steroids (18, 19).

Another recent report illustrates the complexity of determining the molecular basis of isolated 17,20-lyase deficiency. These same researchers describe two additional cases of isolated 17,20-lyase deficiency in genotypically male patients born with ambiguous genitalia on the basis of the known criteria, yet specific hormone values for the patients are not included in the paper (28). Sequencing of the CYP17 cDNAs from these patients revealed homozygosity for mutation R35L in one and compound heterozygosity for mutations N177D and R496H in the other. All of these mutant proteins, however, demonstrated comparable reductions in both 17-hydroxylase and 17,20-lyase activities (10–38% of wild-type activity by RIA methodology) when expressed in COS-1 cells (28). Thus, mutations N177D, R35L, and R496H do not by themselves cause isolated loss of 17,20-lyase activity for the P450c17 protein. The clinical phenotypes, however, suggest that these patients cannot compensate for the partial reduction in both activities intrinsic to their abnormal P450c17 enzymes. Therefore, although the patients demonstrate undervirilization, the identified P450c17 mutations cannot be the sole reason for their clinical phenotype. Although our biochemical studies show that P450c17 mutations R347H and R358Q are sufficient to account for the isolated loss of 17,20-lyase activity in the adrenals and gonads of the subjects we reported (18), we cannot absolutely exclude the coexistence of additional genetic defects in these individuals as well without further studies.

Two broad classes of additional factors could contribute to undervirilization and/or elevated 17-hydroxysteroid intermediates in patients with mutant P450c17 proteins that exhibit partial reductions in both 17-hydroxylase and 17,20-lyase activities: mechanisms that further impair T production, and mechanisms that impair T action. For example, low expression of OR and/or b₅ in the gonads of these fetuses could exacerbate the reduced level of 17,20-lyase activity caused by these mutations. The researchers suggest that the F417C mutation is not phosphorylated (28), and although our data suggest that this result is not specifically germane, defective P450c17 phosphorylation machinery in the gonads of other patients could also contribute to low sex steroid production (15, 38). Alternatively, partial deficiencies of other terminal enzymes in the androgen biosynthetic pathway, including steroid 5-reductase type 2 activity (39) and the hydroxysteroid dehydrogenases (40), including 17ß-hydroxysteroid dehydrogenases type 3 (41) and type 5 (42), could further impair T production in 46,XY fetuses, but the coexisting deficiency in P450c17 precludes compensation for a blockade in later steps. It is difficult to predict the absolute values of steroid hormones and intermediates in hypothetical patients with deficiencies in both the early and late steps of androgen biosynthesis, yet the precursor to product ratios across these blocks in the pathways should all be elevated. Finally, some patients diagnosed clinically with isolated 17,20-lyase deficiency might also have a partial androgen insensitivity (43), which can be extremely difficult to diagnose (43, 44). The presence of additional defects in androgen action could explain why some of the 46,XY patients reported to have isolated 17,20-lyase deficiency exhibit undervirilization despite basal T concentrations that are within the normal range (24) yet not elevated due to the block at P450c17. In other words, a subset of patients clinically diagnosed with isolated 17,20-lyase deficiency might be more properly, albeit awkwardly, described as having partial, combined 17-hydroxylase/17,20-lyase deficiency with additional defects in androgen production and/or action. This terminology would resolve the discrepancies between the clinical phenotype and the biochemistry and genetics of the P450c17 enzymes from these patients.

In conclusion, all mutations proven to directly and selectively impair the 17,20-lyase activity of human P450c17 involve neutralization of positive charges in the redox partner binding site (18, 21, 22, 23). This property common to mutations R347H, R358Q, R449A, and K89N is wholly consistent with the paradigm that interactions between P450c17 and redox partner proteins are particularly critical for 17,20-lyase activity. Molecular genetics can aid in establishing the diagnosis of isolated 17,20-lyase deficiency, provided that the biochemistry of mutant P450c17 proteins so identified is adequately characterized. Isolated 17,20-lyase deficiency is difficult to diagnose, particularly in newborns, and additional clinical, biochemical, and molecular genetic data from patients affected with partial or selective alterations in P450c17 activities must be accumulated before reliable diagnostic criteria can be established. Finally, additional pathophysiological mechanisms are likely to contribute to what is probably a mixture of genetic maladies that manifest clinically as isolated 17,20-lyase deficiency.

Acknowledgments

We thank Dr. Stephen R. Hammes for COS-7 cells and advice concerning their growth and transfection, Dr. Walter L. Miller for the P450c17 antibody and for plasmid pcDNA3-P450c17 (wild-type), and Dr. Ronald W. Estabrook for use of his spectrophotometer and related equipment.

Footnotes

This work was supported by Grants K08-DK-02387 and R03-DK-56641 from the NIDDK, NIH (to R.J.A.).

Abbreviations: b5, Cytochrome b₅; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; GC, gas chromatography; 17OHpreg, 17-hydroxypregnenolone; 16OHprog, 16-hydroxyprogesterone; 17OHprog, 17-hydroxyprogesterone; OR, oxidoreductase.

Received December 21, 2000.

Accepted May 7, 2001.

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