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The Journal of Clinical Endocrinology & Metabolism 88(12):5739 –5746
Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2003-030988
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 Clı́nicas 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 Marı́lia
(H.V.), Marı́lia, SP, Brazil; and Unidade de Endocrinologia Pediátrica, Santa Casa de Sao Paulo (C.A.L.), Sao Paulo,
SP, Brazil
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␣-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 defi-
C
YTOCHROME P450C17 (steroid 17␣-hydroxylase/
17,20-lyase; EC 1.14.99.9) is a single microsomal enzyme that sequentially catalyzes two distinct reactions: the
17␣-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
(3–5). 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␣hydroxylase/17,20-lyase deficiency or isolated 17,20-lyase
deficiency (8 –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
Abbreviations: B, Corticosterone; DHEA, dehydroepiandrosterone;
DHEAS, DHEA sulfate; DOC, 11-deoxycorticosterone; E2, estradiol;
17OHP, 17-hydroxyprogesterone; P, progesterone.
ciency 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. (J Clin Endocrinol
Metab 88: 5739 –5746, 2003)
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␣-hydroxylase, is not frequently mentioned in patients
diagnosed with P450c17 deficiency (9, 11, 18 –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.
5739
5740
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746
In the present study we describe the clinical, biochemical,
and hormonal aspects of 11 Brazilian patients with combined
17␣-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
Patients
We studied 11 patients from 6 families presenting with the combined
17␣-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 –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
Martin et al. • P450c17 Deficiency
forward primer 5⬘-TACTCCTCTGTCTGCCATTAAGT-3⬘, and the reverse primer was the same as that used for fragment 5 (Fig. 1). All of the
fragments were amplified under the same conditions in a final volume
of 50 ␮l. 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).
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 ␮l 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 5⫻ sodium chloride, sodium phosphate, EDTA, 5⫻ 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
32
P-end-labeled mutant probe and 250 pmol unlabeled wild-type probe
(Table 1). All of the membranes were rinsed 3 times in 6⫻ standard saline
citrate, then washed in 6⫻ 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.
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.
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.
TABLE 1. Sequence of probes used for allele-specific oligonucleotide hybridization
Screened mutation
Wild-type probe
Mutant probe
Y329D
R362C
W406R
5⬘-AAGAAGCTCTACGAGGAGATT-3⬘
5⬘-GAGAAGGAGTGGCACCAGCCG-3⬘
5⬘-GAGGTGCTTCGCCTCATCAGT-3⬘
5⬘-AAGAAGCTCGACGAGGAGATT-3⬘
5⬘-GAGAAGGAGCGGCACCAGCCG-3⬘
5⬘-GAGGTGCTTTGCCTCATCAGT-3⬘
Martin et al. • P450c17 Deficiency
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746 5741
Results
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 3). Serum cortisol was low or at lower normal levels
with a subnormal elevation after ACTH stimulation. All
cases had elevated basal (ⱖ1.8 ng/ml; ⱖ0.57 pmol/liter) and
ACTH-stimulated (ⱖ2.7 ng/ml; ⱖ0.86 pmol/liter) P levels.
On the other hand, basal 17OHP, ⌬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␣-hydroxylase defect
(Table 4 and see Fig. 4). A human chorionic gonadotropin
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 2).
Biochemical and hormonal measurements
At diagnosis, 100% of the patients had normal levels of
sodium, whereas 82% had hypokalemia, and 18% of them
TABLE 2. Clinical and molecular features of P450c17 deficiency patients
Family
Subject
a
I
II
III
IV
V
VI
1
2a
3
4
5
6a
7a
8a
9
10a
11
Ethnicity
Cons
White
White
White
Mulatto
Mulatto
Mulatto
Mulatto
Mulatto
Mulatto
White
White
Yes
Yes
No
Yes
Yes
No
AD
(yr)
AHD
(yr)
BP
(mm Hg)
19
15
10
35
27
21
28
34
25
23
4
19
150/105
110/70
110/60
150/110
160/120
140/110
140/105
150/100
180/120
160/120
90/60
⬍34
26
21
11
13
5
23
External
genitalia
Karyotype
46,
46,
46,
46,
46,
46,
46,
46,
46,
46,
46,
XY
XY
XY
XX
XX
XY
XY
XY
XY
XY
XY
Female
Ambiguous
Ambiguous
Female
Female
Female
Female
Female
Female
Female
Female
P450c17 genotyping
Homozygous R96W
Homozygous Y329D
Compound
Heterozygous
R362C/W406R
Homozygous P428L
Homozygous P428L
Homozygous P428L
Cons, Consanguinity; AD, age at diagnosis; AHD, age at hypertension diagnosis; BP, blood pressure.
a
Index case.
TABLE 3. Basal laboratorial data of P450c17 deficiency patients
CA
(yr)
ACTH
(pg/ml)
Aldosterone
(ng/dl)
PRA
(ng/ml 䡠 h)
Na
(mEq/liter)
K
(mEq/liter)
LH
(U/liter)
FSH
(U/liter)
T
(ng/dl)
E2
(pg/ml)
1
2
3a
6
7a
8a
9a
10
11
19
15
17
21
28
34
25
23
4
99
209
116
40
33
26
22
4
5.7
8
9.9
6.2
0.3
⬍0.2
⬍0.2
0.4
⬍0.2
1
1
140
140
139
134
141
136
136
136
3.3
3.5
3.4
0.9
2.5
2.8
3.3
3.7
3.5
26
33
15
77
34
24
35
14
⬍0.6
34
24
37
21
102
104
90
15
3
26
33
⬍10
⬍14
⬍14
⬍14
⬍14
⬍14
⬍14
⬍10
⬍10
⬍10
15
⬍13
⬍13
⬍13
Male controls
4
5
136
9.6 –18.6
20
16
1.5–5.7
35
27
135–147
144
146
3.5–5.0
1.4
3.2
1.4 –9.2
53
28
1–12
97
94
200 –950
⬍14
⬍14
⬍35
⬍13
⬍13
⬍60
9.6 –18.6
1.5–5.7
135–147
3.5–5.0
0.9 –9.3b
1.7–9.3b
⬍14 – 86
35–184b
Subject
Female controls
152
68
⬍60
0.2
⬍13
CA, Chronological age; PRA, plasma renin activity. To convert values of ACTH to pmol/liter, multiply by 0.2222; of aldosterone to pmol/liter
multiply by 27.7469; of PRA to ng/liter 䡠 sec, multiply by 0.2778; of T to pmol/liter multiply by 34.6741; of E2 to pmol/liter, multiply by 3.671.
a
Gonadectomized.
b
Normal range for follicular phase.
5742
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746
Martin et al. • P450c17 Deficiency
TABLE 4. Laboratorial data before and after acute ACTH stimulation of P450c17 deficiency patients
Subject
1
2
3
6
7
8
9
10
11
17OHP (ng/ml)
F (ng/ml)
DHEA (ng/ml)
⌬4A (ng/ml)
B
Pk
B
Pk
B
Pk
B
Pk
B
Pk
19
15
17
21
28
34
25
23
4
5.0
14
11
12
1.9
1.8
2.5
5.0
4.9
8.1
19
13
25
3.7
3.9
2.8
5.0
2.7
0.2
0.4
0.3
0.7
0.1
0.3
0.3
0.7
⬍0.1
0.2
0.5
0.4
0.7
0.2
0.4
0.4
0.3
⬍0.1
3
69
64
29
12
34
42
64
53
3
51
58
42
16
52
47
48
46
0.5
1.1
1.0
0.4
⬍0.2
⬍0.2
⬍0.2
0.3
0.3
1.6
1.5
1.2
0.5
⬍0.2
⬍0.2
⬍0.2
0.3
⬍0.2
⬍0.2
⬍0.2
⬍0.2
0.2
0.3
⬍0.2
⬍0.2
0.2
⬍0.2
⬍0.2
⬍0.2
0.5
0.2
0.3
⬍0.2
⬍0.2
0.2
0.2
0.3–1.5
0.3–2.1
0.7–1.5
0.9 –2.2
60 –280
240 – 470
3.0 –5.7
4.3–13
1.1–2.0
1.6 –3.6
38
7.2
0.3– 0.7
41
14
NA
0.5
0.4
0.2–1.3
0.6
0.4
1.4 –5.3
12
34
40 –250
16
57
180 –330
0.3
0.3
2.5– 6.5
0.4
0.3
5.6 –17
0.2
0.2
0.7–2.1
0.3
0.2
1.0 –2.6
Male controls
4
5
Female controlsa
P (ng/ml)
CA
(yr)
35
27
CA, Chronological age; B, basal; Pk, peak; NA, not available; ⌬4A, andosteredione. To convert values of P to pmol/liter, multiply by 0.3180;
of 17OHP to pmol/liter, multiply by 0.3026; of F to nmol/liter, multiply by 2.7586; of DHEA to pmol/liter, multiply by 0.3467; of ⌬4A to pmol/liter,
multiply by 0.3491.
a
Normal range for follicular phase.
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.
stimulation test performed in patients 1, 2, 3, 10, and 11
before gonadectomy did not promote an androgen response.
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. 2). 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).
Martin et al. • P450c17 Deficiency
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 2). 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, ␾X 174-kb ladder.
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. 3). 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. 3).
Discussion
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
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746 5743
amino acids by other ones with different physicochemical
proprieties (R3 W, Y3 D, R3 C, W3 R, and P3 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. 3).
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␣-hydroxylase and 17,20-lyase activities or of isolated
17,20-lyase activity. Although these activities are distinct and
independently regulated (34, 42– 44), in practice the differential diagnosis is difficult because, for unknown reasons,
some individuals present normal 17␣-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␣-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␣-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 –27). It is not clear why P is not
routinely measured for P450c17 deficiency diagnosis, because DOC and B measurements should be performed by
5744
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746
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␣-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, 47–55) 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/⌬4-androstenedione ratio (10).
Besides P levels, the diagnosis of combined 17␣-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. 4).
FIG. 4. The precursor/product ratios
involved in the 17␣-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).
Martin et al. • P450c17 Deficiency
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
Martin et al. • P450c17 Deficiency
J Clin Endocrinol Metab, December 2003, 88(12):5739 –5746 5745
its use can reduce the misdiagnosis of this deficiency
in patients with male pseudohermaphroditism, primary or
secondary amenorrhea, and mineralocorticoid excess
syndrome.
Note
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.
Received June 9, 2003. Accepted September 3, 2003.
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 Clı́nicas da Faculdade
de Medicina da Universidade de Sao Paulo, Sao Paulo, S.P., Brazil.
E-mail: [email protected] or [email protected].
This work was supported by Grant 301246/95-5 from Conselho Nacional de Pesquisa (to B.B.M.).
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