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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 5739-5746
Copyright © 2003 by The Endocrine Society

P450c17 Deficiency in Brazilian Patients: Biochemical Diagnosis through Progesterone Levels Confirmed by CYP17 Genotyping

Regina M. Martin, Chin J. Lin, Elaine M. F. Costa, Maria Leocadia de Oliveira, Alexandre Carrilho, Heloisa Villar, Carlos A. Longui and Berenice B. Mendonca

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Divisão de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo (R.M.M., E.M.F.C., C.J.L., B.B.M.), Sao Paulo, S.P., Brazil; Hospital Universitário Regional do Norte do Paraná, Universidade Estadual de Londrina (M.L.d.O., A.C.), Londrina, PR, Brazil; Disciplina de Endocrinologia e Metabologia, Faculdade de Medicina de Marília (H.V.), Marília, SP, Brazil; and Unidade de Endocrinologia Pediátrica, Santa Casa de Sao Paulo (C.A.L.), Sao Paulo, SP, Brazil

Address all correspondence and requests for reprints to: Dr. Regina M. Martin or Dr. Berenice B. Mendonca, Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Divisão de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, S.P., Brazil. E-mail: reginamm@usp.br orberemen{at}usp.br.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 Note
 References
 
P450c17 deficiency is an autosomal recessive disorder and a rare cause of congenital adrenal hyperplasia characterized by hypertension, hypokalemia, and impaired production of sex hormones. We performed a clinical, hormonal, and molecular study of 11 patients from 6 Brazilian families with the combined 17{alpha}-hydroxylase/17,20-lyase deficiency phenotype. All patients had elevated basal serum levels of progesterone (1.8–38 ng/ml; 0.57–12 pmol/liter) and suppressed plasma renin activity. CYP17 genotyping identified 5 missense mutations. The compound heterozygous mutation R362C/W406R was found in 1 family, whereas the homozygous mutations R96W, Y329D, and P428L were seen in the other 5 families. The R96W mutation has been described as the cause of P450c17 deficiency in Caucasian patients. The other mutations were not found in 50 normal subjects screened by allele-specific oligonucleotide hybridization (Y329D, R362C, and W406R) or digestion with HphI (P428L) and were recently found in other Brazilian patients. Therefore, we elucidated the genotype of 11 individuals with P450c17 deficiency and concluded that basal progesterone measurement is a useful marker of P450c17 deficiency and that its use should reduce the misdiagnosis of this deficiency in patients presenting with male pseudohermaphroditism, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 Note
 References
 
CYTOCHROME P450C17 (steroid 17{alpha}-hydroxylase/17,20-lyase; EC 1.14.99.9) is a single microsomal enzyme that sequentially catalyzes two distinct reactions: the 17{alpha}-hydroxylation of steroids and the cleavage of the C17,20 carbon bond, converting C21 compounds to C19 steroids, which are essential for the production of glucocorticoids and sex steroids, respectively (1). There is only one human gene for P450c17 (2), which is located in chromosome 10q24.3 (3, 4, 5). This gene, termed CYP17 (6), consists of eight exons spanning 8673 bp (2) and is expressed in both adrenals and gonads (7).

To date, approximately 40 mutations in CYP17, including missense mutations, insertions, deletions, and splicing defects, have been reported as the cause of combined 17{alpha}-hydroxylase/17,20-lyase deficiency or isolated 17,20-lyase deficiency (8, 9, 10, 11, 12, 13, 14). The CYP17 genotyping and the functional studies of mutant proteins produced by these patients have helped us understand the phenotypic spectrum shown by affected individuals.

Patients with P450c17 deficiency have an impaired production of cortisol with secondary ACTH hypersecretion, resulting in the production of large amounts of 11-deoxycorticosterone (DOC) by the adrenal cortex, leading to a state of mineralocorticoid excess characterized by hypertension, hypokalemia, suppressed plasma renin activity, and low aldosterone concentrations (15). Because of abnormally high corticosterone (B) production (a weak glucocorticoid), differently from other causes of congenital adrenal hyperplasia with impaired glucocorticoid production, an adrenal crisis is rarely the initial presentation of P450c17 deficiency. For these reasons this condition usually remains unsuspected until adolescence or early adulthood, when the patients are evaluated because of hypokalemia, hypertension, or delayed puberty (10). Another key feature is the impaired production of gonadal sexual steroids due to the absence of 17,20-lyase activity. This enzymatic defect leads to male pseudohermaphroditism (16), primary or, seldom, secondary amenorrhea in 46,XX patients (17, 18), and the absence of pubertal development and hypergonadotropic hypogonadism in both genetic sexes. In this situation, patients with ambiguous genitalia have been misdiagnosed with other defects in testosterone biosynthesis or action, because mineralocorticoids are not routinely measured if hypertension is not present (10).

It is remarkable that progesterone (P), which is a substrate of 17{alpha}-hydroxylase, is not frequently mentioned in patients diagnosed with P450c17 deficiency (9, 11, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). Besides P450c17 deficiency, P levels are elevated in other enzymatic defects, such as 21-hydroxylase and 11-hydroxylase deficiencies, but the absence of virilized phenotype and the high 17-hydroxyprogesterone (17OHP) or 11-deoxycortisol levels exclude the latter conditions.

In the present study we describe the clinical, biochemical, and hormonal aspects of 11 Brazilian patients with combined 17{alpha}-hydroxylase/17,20-lyase deficiency in whom the diagnosis was confirmed by CYP17 genotyping, and we point out the usefulness of basal P measurements for the diagnosis of P450c17 deficiency.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 Note
 References
 
Patients

We studied 11 patients from 6 families presenting with the combined 17{alpha}-hydroxylase/17,20-lyase deficiency phenotype after obtaining their informed written consent. They came from different regions of Brazil, and the ethics committees of the four hospitals, where the patients were followed-up, approved this study.

Biochemical and hormonal measurements

Serum 17OHP, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS) and androstenedione were measured by RIA using Abraham’s method without previous chromatography after demonstration that the antisera used were adequately specific (28). Tritiated steroids were purchased from NEN Life Science (Boston, MA), and the antisera were obtained from Radioassay Systems Laboratories, Inc. (Carson, CA). Serum aldosterone and cortisol were measured with Gamma Coat I RIA kits (Diagnostic Products, Los Angeles, CA). Plasma renin activity was evaluated by RIA of generated angiotensin I with the kit from CIS BioInternational (Gif-Sur-Yvette, France). Plasma ACTH was measured by an immunoradiometric kit from Nichols Institute Diagnostics, Inc. (San Juan Capistrano, CA). Serum LH, FSH, P, estradiol, and testosterone were measured by fluoroimmunoassay methods (AutoDelfia, Wallac Oy, Turku, Finland). Intra- and interassay variations were less than 10% and 14%, respectively, in all steroid assays. Assays were performed under basal conditions and after standard ACTH and human chorionic gonadotropin stimulation tests. The results were compared with normal values established in our laboratory (29, 30, 31).

Molecular analysis

Patients’ genomic DNA was obtained from peripheral blood leukocytes using the salt precipitation method (32). The entire coding region of CYP17 was PCR-amplified into five fragments using intronic primers previously described by Lin et al. (25). Except for fragments 1 and 3, which include, respectively, exons I and IV, all amplified fragments comprise two consecutive exons and their interspersed introns. Exon VII was also amplified alone using the forward primer 5'-TGATCTGGCAGAAGCTGAGG-3' and the reverse primer 5'-GCGTCAACAGGTCGGTATA-3' (fragment 6) as well as exon VIII (fragment 7) with the forward primer 5'-TACTCCTCTGTCTGCCATTAAGT-3', and the reverse primer was the same as that used for fragment 5 (Fig. 1Go). All of the fragments were amplified under the same conditions in a final volume of 50 µl. After initial denaturation (94 C for 5 min), PCR was performed for 35 cycles of denaturation (94 C for 60 sec), annealing (58 C for 30 sec), and extension (72 C for 90 sec) in a thermal cycler (PE 9700, PerkinElmer, Foster City, CA). A final extension step at 72 C for 7 min was added after the last cycle. PCR products were directly sequenced using the automated fluorescence-based dideoxynucleotide termination method (ABI 310, PE Applied Biosystems, Foster City, CA).



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  		FIG. 1. Schematic representation of the genomic structure of human CYP17 and PCR amplification strategy of this coding region (represented by open boxes). The lines between open boxes correspond to the introns (not represented in scale), and the gray boxes correspond to the 5'- and 3'-untranslated regions (left and right boxes, respectively). The five missense mutations found in the patients are also represented as translated into the P450c17.

 
Allele-specific oligonucleotide hybridization. To determine whether Y329D, R362C, and W406R mutations were present in the normal population, we screened for these alleles in 50 normal subjects. Fragment 4 was PCR-amplified using as template the genomic DNA from 50 subjects from our population and patients from families II and III. Fragment 6 was PCR-amplified using as template the genomic DNA from the same 50 subjects from our population and patients from family III. Ten microliters of PCR products containing either exon VI (fragment 4) or exon VII (fragment 6) were mixed with 200 µl 0.4 M NaOH and 25 mM EDTA, heated to 95 C for 2 min, and vacuum-transferred onto a nylon membrane. Two replicates for each mutation were prepared, resulting in 6 membranes. The membranes were prehybridized in 5x sodium chloride, sodium phosphate, EDTA, 5x Denhardt’s solution, and 0.5% sodium dodecyl sulfate for 1 h. The hybridization was carried out overnight at 37 C in the same solution after addition of the probes. Three membranes were hybridized with 10 pmol 32P end-labeled oligonucleotide wild-type probe in the presence of 250 pmol unlabeled mutant probe, whereas their twin membranes were hybridized with 10 pmol 32P-end-labeled mutant probe and 250 pmol unlabeled wild-type probe (Table 1Go). All of the membranes were rinsed 3 times in 6x standard saline citrate, then washed in 6x standard saline citrate for 30 min at room temperature. After rinsing in a tetramethylammonium chloride solution [3 M tetramethylammonium chloride, 50 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0), and 0.1% sodium dodecyl sulfate], the membranes were washed separately at different temperatures ranging from 58–66 C. The membranes were exposed at -80 C for 1–7 d before autoradiographies.


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  		TABLE 1. Sequence of probes used for allele-specific oligonucleotide hybridization

 
Restriction digestion with HphI. The P428L mutation was screened in 50 normal subjects. Initially, fragment 7 was PCR-amplified using as template the genomic DNA of 50 subjects from our population and patients from families IV, V, and VI. Five microliters of PCR products were digested with HphI (New England Biolabs, Beverly, MA) according to the manufacturer’s specifications. The digestion of this fragment in normal individuals results in 3 fragments, but 1 restriction site is eliminated in the mutants, producing 2 fragments. Reaction products were resolved by 3% agarose gel electrophoresis.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 Note
 References
 
Patients

All patients with female external genitalia were diagnosed because of sexual infantilism in adulthood, except for patient 11, who was diagnosed during childhood due to the index case diagnosis and the presence of bilateral nodules in the inguinal region. Ambiguous genitalia was the main complaint of patients from family II. They had clitoral enlargement, single perineal opening, and blind-ending vagina. The nine 46,XY patients were registered and raised as females and maintained the female social sex. At diagnosis, the three younger (4- to 15-yr-old) patients had normal blood pressure, whereas the remaining eight had arterial hypertension, although three of them had been diagnosed as being hypertensive since childhood and adolescence (patients 7–9). Consanguinity or other affected members were present in all families, and their ethnic backgrounds were Caucasian or Brazilian mullato (Table 2Go).


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  		TABLE 2. Clinical and molecular features of P450c17 deficiency patients

 
Biochemical and hormonal measurements

At diagnosis, 100% of the patients had normal levels of sodium, whereas 82% had hypokalemia, and 18% of them had borderline potassium levels. Patient 6 presented the lowest potassium level (0.9 mEq/liter), which was responsible for rhabdomyolysis and reversible acute renal failure. Plasma ACTH levels were slightly high in the six patients evaluated. Basal DHEAS levels were below the sensitivity of the method in all patients, except cases 2 and 3, in whom DHEAS levels were measurable, but low for age. Basal aldosterone levels were suppressed in patients 7, 8, 9, and 11 and were normal or slightly elevated in the remaining patients, in contrast with the suppressed levels of plasma renin activity presented in all cases. All adult patients had high gonadotropin levels, with very low testosterone and estradiol (E2) levels, whereas the prepubertal patient had LH, FSH, testosterone and E2 levels compatible with her age (Table 3Go). Serum cortisol was low or at lower normal levels with a subnormal elevation after ACTH stimulation. All cases had elevated basal (>=1.8 ng/ml; >=0.57 pmol/liter) and ACTH-stimulated (>=2.7 ng/ml; >=0.86 pmol/liter) P levels. On the other hand, basal 17OHP, {Delta}4-androstenedione, and DHEA levels were low and did not rise after ACTH stimulation. The P/17OHP and P/cortisol ratios (before or after ACTH stimulation) confirmed the 17{alpha}-hydroxylase defect (Table 4Go and see Fig. 4Go). A human chorionic gonadotropin stimulation test performed in patients 1, 2, 3, 10, and 11 before gonadectomy did not promote an androgen response.


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  		TABLE 3. Basal laboratorial data of P450c17 deficiency patients

 

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  		TABLE 4. Laboratorial data before and after acute ACTH stimulation of P450c17 deficiency patients

 


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  		FIG. 4. The precursor/product ratios involved in the 17{alpha}-hydroxylation and 17,20-lyase activities before and after ACTH stimulation. The black lines represent the 46,XY patients, the dashed lines represent the 46,XX patients, and the normal values are expressed as a range (gray areas).

 
Genetic analysis of CYP17

After CYP17 genotyping of the 11 patients, 5 missense mutations were identified. A compound heterozygous R362C/W406R mutation was found in family III, whereas homozygous R96W, Y329D, and P428L mutations were seen in the other families (Fig. 2Go). The R96W mutation was not screened in normal alleles, because it was previously described in Caucasian patients with P450c17 deficiency, and its functional study has proved to nearly abolish both P450c17 activities (33). Y329D, R362C, and W406R mutations were screened by allele-specific oligonucleotide hybridization, and they were not found in 50 normal subjects (data not shown).



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  		FIG. 2. Direct sequencing of CYP17 PCR products showing the five missense mutations (arrows) found in the patients on their respective exons. The bold nucleotides represent the codons where the substitutions occurred, and they are also represented as expected in the P450c17. *, The sequence is shown in the antisense direction.

 
The strategy used to screen P428L was PCR amplification of fragment 7, followed by HphI digestion. This fragment contains part of intron 7, the entire exon 8, where the mutation is located, and part of the 3'-untranslated region, resulting in a 609-bp fragment (7822–8430 nucleotides according to the GenBank sequence, accession no. M63871). After HphI digestion, we expected 3 fragments of 317, 221, and 71 bp (normal individuals) or 2 fragments of 388 and 221 bp (P428L mutant). Surprisingly, when the PCR products from 50 normal subjects were submitted to electrophoresis on a 3% agarose gel, 3 different patterns were observed before and after digestion with HphI (Fig. 3Go). The direct sequencing of these fragments identified an allelic variant that lacked nucleotides 7983–8016 from intron 7 (data not shown). This short allele was found in 22 of 100 alleles of the Brazilian population. Nevertheless, the P428L screening was performed, and all normal subjects presented the HphI restriction site abolished in P428L mutants (Fig. 3Go).



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  		FIG. 3. Screening of the P428L mutation with HphI digestion. First set (left), Fragment 7 (see Subjects and Methods) was obtained by PCR amplification from three normal controls (A, B, and C) and patients 7–11 with the P428L mutation (Table 2Go). Depending on the presence or absence of a 34-bp sequence in intron 7, the expected sizes of the amplicon can be either 575 bp (short allele) or 609 bp (long allele). Subject A is homozygous for short alleles, individual B is homozygous for long alleles, and individual C presents both short and long alleles, whereas all P428L patients are homozygous for short alleles. Second set (right), Digestion of PCR products with HphI. Digestion with HphI yields two fragments of 221 and 71 bp, and a third of either 317 or 283 bp (long or short allele, respectively). P428L mutation eliminates one of the HphI sites. Thus, only fragments of 354 and 221 bp (black arrows) were obtained. White arrows represent bands of 317, 283, 221, and 71 bp. M1, 1-kb ladder; M2, {phi}X 174-kb ladder.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
Note
 References
 
In our cohort, all patients suspected to have P450c17 deficiency were screened through clinical features (undervirilized genitalia in males, primary amenorrhea, sexual infantilism, and hypertension), biochemical and hormonal data (hypokalemia, high P levels, and low androgen or estrogen levels).

All missense mutations lead to replacement of the original amino acids by other ones with different physicochemical proprieties (R->W, Y->D, R->C, W->R, and P->L). The R96W mutation has been shown to almost completely abolish the enzymatic activity of P450c17 (33). Despite the absence of functional studies, there is evidence allowing us to predict that the remaining mutations also severely impair its enzymatic activity. Amino acids R362, W406, and P428 are conserved among the P450c17s from different vertebrates (monkey, guinea pig, rat, and trout), as shown by sequence alignment, which suggests that these residues are critical. Furthermore, according to a published structure model of human P450c17 (34), these mutations would eliminate the invariant EXXR motif (R362C) (35), grossly disrupt the coordination of heme group (W406R and P428L), or affect the integrity of core P450 structure (Y329D).

Mutations in CYP17 due to substitutions, microdeletions, microinsertions, or, rarely, splice site changes result in P450c17 deficiency; only one case had a partial deletion of the CYP17 gene associated with insertion of a foreign DNA fragment (24).

A founder effect has been suggested for some mutations: the microinsertion of four nucleotides in codon 480 described in Mennonite descendants (8, 36, 37), the deletion of a phenylalanine in codon 53 or 54 described in Japanese subjects (18, 19, 27), and the microdeletion of nine nucleotides covering codons 487–489 in Asian individuals (20, 38, 39). A founder effect can be evoked for the R96W mutation, considering that it was found in four Caucasian families: two French-Canadian patients (33), one Italian patient (40), and one patient from our series whose ancestors came from Europe. The association of the P428L mutation and the short allelic variant for intron 7, which is present in only 22% of Brazilian normal alleles and is seen in all five patients from three different families, also suggests a founder effect (Fig. 3Go).

In a recent study of 24 Brazilian patients with P450c17 deficiency (41), the Y329D, R362C, W406R, and P428L mutations were also identified in other families, indicating the presence of common ancestors.

Theoretically, P450c17 defects can induce impairment of both 17{alpha}-hydroxylase and 17,20-lyase activities or of isolated 17,20-lyase activity. Although these activities are distinct and independently regulated (34, 42, 43, 44), in practice the differential diagnosis is difficult because, for unknown reasons, some individuals present normal 17{alpha}-hydroxylase activity throughout childhood and adolescence that decreases in adulthood (23, 45, 46).

As for all steroidogenic enzyme deficiencies, the biochemical diagnosis of P450c17 deficiency is established by measuring precursor to product ratios before and after an ACTH stimulation test. The combined 17{alpha}-hydroxylase/17,20-lyase deficiency laboratorial diagnosis is based on the increase in the 17-deoxysteroid P, B, and DOC, which rise to 5–10 times the normal levels after ACTH administration (10).

In literature, it is surprising to observe that the diagnosis of combined 17{alpha}-hydroxylase/17,20-lyase deficiency is based mainly on DOC and B measurements, instead of P levels, which are not frequently mentioned in patients with P450c17 deficiency (9, 11, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). It is not clear why P is not routinely measured for P450c17 deficiency diagnosis, because DOC and B measurements should be performed by RIA after liquid chromatography or gas chromatography/mass spectrometry, which is only available in a few centers, whereas P measurement is easily measured by RIA, a reliable, available, and less expensive method. A review of the literature and our data indicated that all patients with combined 17{alpha}-hydroxylase/17,20-lyase deficiency, confirmed by CYP17 genotyping, had high basal P levels (0.7–14 ng/ml; 0.22–4.45 pmol/liter) (8, 12, 33, 39, 40, 47, 48, 49, 50, 51, 52, 53, 54, 55) compared with the respective normal values. Moreover, our patients were correctly diagnosed, even without DOC and B measurements.

In patients with isolated 17,20-lyase deficiency (8, 56), P levels are also elevated, mostly after ACTH stimulation, but the best parameter used to demonstrate this defect is to establish the 17OHP/{Delta}4-androstenedione ratio (10).

Besides P levels, the diagnosis of combined 17{alpha}-hydroxylase/17,20-lyase deficiency can be accomplished by the P/17OHP, P/cortisol, P/androstenedione, and P/DHEA ratios (before or after ACTH stimulation), because there is an impairment of 17-hydroxysteroid production (Fig. 4Go).

Aldosterone levels are another intriguing aspect of this disorder. Classically, P450c17-deficient patients present low levels of aldosterone secondary to suppressed plasma renin activity due to the high levels of DOC and B from the zona fasciculata (14, 15, 57). On the other hand, some patients, particularly those of Asian origin, present hyperaldosteronism (14, 48, 58). The possibility that P450c17 mutant protein could synthesize aldosterone from DOC was ruled out by the experiments performed by Monno et al. (58). Although Suzuki et al. (48) believe that this disorder might be ethnically linked to steroid genesis and regulation, other researchers (39, 40, 47) attribute this phenomenon to cross-reactions between aldosterone and other mineralocorticoid precursors that are elevated in this disorder. The latter seems more likely, because the elevated aldosterone levels in cases 2 and 3 were actually low when remeasured by RIA after liquid chromatography by Dr. Edward Biglieri (data not shown).

Therefore, we elucidated the genotype of 11 individuals with P450c17 deficiency and concluded that basal P measurement is a useful marker of P450c17 deficiency and that its use can reduce the misdiagnosis of this deficiency in patients with male pseudohermaphroditism, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.


   Note
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note
References
 
When this manuscript was near completion we became aware of an in-press manuscript from Dr. Richard J. Auchus’s laboratory reporting that missense mutants of P450c17 (R362C, W406R, and P428L) were found to be functionally inactive, and Y329D mutant presents around 5% of enzymatic activity.


   Acknowledgments
 
We thank the colleagues from Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42-HCFMUSP, for the follow-up of the patients; Cristina Rossi, Maria Aparecida Medeiros, Mirian Nishi, Emilia P. Modolo, and Ana Elisa C. Billerbeck for technical assistance; and Sonia Strong for help with the manuscript.


   Footnotes
 
This work was supported by Grant 301246/95-5 from Conselho Nacional de Pesquisa (to B.B.M.).

Abbreviations: B, Corticosterone; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; DOC, 11-deoxycorticosterone; E2, estradiol; 17OHP, 17-hydroxyprogesterone; P, progesterone.

Received June 9, 2003.

Accepted September 3, 2003.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note
 References
 

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