0013-7227/02/$15.00/0
`Printed in U.S.A.
`
`The Journal of Clinical Endocrinology & Metabolism 87(2):898 –905
`Copyright © 2002 by The Endocrine Society
`
`Combined 17␣-Hydroxylase/17,20-Lyase Deficiency
`Caused by Phe93Cys Mutation in the CYP17 Gene
`
`ALFREDO DI CERBO, ANNA BIASON-LAUBER, MARIA SAVINO, MARIA ROSARIA PIEMONTESE,
`ANNA DI GIORGIO, MARCO PERONA, AND ANNA SAVOIA
`
`Division and Research Unit of Endocrinology (A.D.C.) and Departments of Medical Genetics (M.S., M.R.P., A.S.) and
`Clinical Pathology (A.D.G.), Istituto di Ricovero e Cura a Carattere Scientifico Ospedale “Casa Sollievo della Sofferenza,”
`71013 San Giovanni Rotondo, Italy; Department of Pediatric Endocrinology/Diabetology (A.B.-L.), University Children’s
`Hospital, CH-8032 Zurich, Switzerland; and Unit of Analitical Chemistry (M.P.), Azienda Ospedaliera O.I.R.M.-S.Anna,
`10126 Turin, Italy
`
`Seventeen ␣-hydroxylase/17,20-lyase deficiency is a rare, au-
`tosomal recessive form of congenital adrenal hyperplasia not
`linked to human leukocyte antigen and characterized by the
`coexistence of hypertension caused by the hyperproduction of
`mineralocorticoid precursors and sexual abnormalities, such
`as male pseudohermaphroditism and sexual infantilism in
`female, due to impaired production of sex hormones. Both
`17␣-hydroxylase and 17,20-lyase reactions are catalyzed by a
`single polypeptide, cytochrome P450c17 (CYP17), which is en-
`coded by the CYP17 gene located on chromosome 10q24-q25.
`Mutations in the CYP17 gene have been recognized to cause the
`17␣-hydroxylase/17,20-lyase deficiency syndrome.
`Here, we describe two phenotypically and hormonally af-
`fected Italian patients with 17␣-hydroxylase/17,20-lyase defi-
`
`ciency. The family history revealed consanguinity of the par-
`ents. Linkage and haplotype analyses using microsatellites on
`chromosome 10q24-q25 demonstrated that the two affected in-
`dividuals were homozygous at these loci. The mutation screen-
`ing of the CYP17 gene identified a new Phe93Cys missense mu-
`tation in exon 1. The amino acid substitution is located in
`a highly conserved region of the protein and is not a poly-
`morphism because it is not present in one hundred normal
`alleles. In vitro functional studies showed that the Phe93Cys
`mutated CYP17 retains only 10% of both 17␣hydroxylase and
`17,20-lyase activities, according to the severe phenotype. Our
`results shed more light on the structure-function relationship of
`the CYP17 protein indicating that Phe 93 is crucial for both
`enzymatic activities. (J Clin Endocrinol Metab 87: 898 –905, 2002)
`
`THE STEROID 17␣-HYDROXYLASE/17,20-LYASE is a
`
`key enzyme required for the production of cortisol and
`sex steroids. Both the 17␣-hydroxylase and17,20-lyase reac-
`tions are known to be catalyzed by a single polypeptide, cy-
`tochrome P450c17 (1–3). P450c17 is expressed in several steroi-
`dogenic tissues (4, 5), including adrenal cortex, ovary, and testis.
`Congenital adrenal hyperplasia resulting from 17␣-
`hydroxylase/17,20-lyase deficiency is a rare autosomal re-
`cessive disease, not linked to human leukocyte antigen (6, 7).
`It is characterized by the presence of hypertension due to an
`excess of mineralocorticoids other than aldosterone associ-
`ated with sexual abnormalities such as male pseudoher-
`maphroditism or sexual infantilism in females (8 –11). The
`affected enzyme is encoded by the CYP17 gene mapped to
`chromosome 10q24-q25 (12–15).
`The present report describes biochemical and molecular
`studies performed in two related individuals affected by
`complete 17␣-hydroxylase/17,20-lyase deficiency syndrome
`(17-OHDS). The molecular analysis of the CYP17 gene al-
`lowed us to identify a novel Phe93Cys missense mutation.
`
`Patient 1
`
`Case Reports
`
`A 22-yr-old female patient born from consanguineous par-
`ents (Fig. 1) presented with primary amenorrhea, sexual
`
`Abbreviations: 17-OHDS, 17␣-Hydroxylase/17,20-lyase deficiency
`syndrome; DHEA, dehydroepiandrosterone; PRA, plasma renin
`activity.
`
`infantilism, and hypertension. On physical examination sex-
`ual hair was completely absent, blood pressure was 170/105
`mm Hg, and infantile genitalia were present. Abdominal
`computed tomography scan showed bilateral adrenal hy-
`perplasia, small uterus measuring 29 and 13 mm in the di-
`ameters, and enlarged ovaries with multiple cysts, the big-
`gest being 4 cm in the diameter. Her karyotype was 46,XX.
`Serum sodium, blood urea nitrogen, and creatinine were
`normal, and her potassium level was 2.87 mmol/liter. Blood
`pH was 7.452. The measurement of plasma and urinary ste-
`roids confirmed the suspicion of combined 17-OHDS (Tables
`1 and 2). Therapy with dexamethasone was started, followed
`by the addition of conjugated estrogens. On dexamethasone
`therapy, plasma renin activity (PRA) and potassium levels
`increased to normal and blood pressure and blood pH fell to
`normal (Table 3).
`
`Patient 2
`
`The family history obtained from patient 1 revealed that
`patient’s sister suffered from primary amenorrhea, sexual
`infantilism, and hypertension. She was an 18-yr-old patient
`who had been raised as a girl. When she was 17 yr old, she
`had been admitted to another hospital because of inguinal
`pain and presence of lumps in inguinal regions bilaterally.
`Both inguinal masses were operated, and pathological ex-
`amination revealed the presence of testes. Based on these
`findings, the absence of pubic and axillary hair and the 46,XY
`karyotype, the diagnosis of androgen resistance syndrome
`was made. The patient was admitted to our hospital 1 yr
`
`898
`
`Amerigen Exhibit 1180
`Amerigen v. Janssen IPR2016-00286
`
`

`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`J Clin Endocrinol Metab, February 2002, 87(2):898 –905 899
`
`Shackleton (16). Appropriate reference steroids were obtained from
`Sigma-Aldrich Corp. (Milan, Italy). Assays were performed in basal
`conditions, after standard ACTH and human CG stimulation tests, dur-
`ing a long-term dexamethasone and dexamethasone plus conjugated
`estrogen therapy, and 2 wk and 4 months after the cessation of glu-
`cocorticoid therapy (Table 3).
`
`Southern blot analysis
`
`Genomic DNA of the four members of family and control DNA were
`prepared from peripheral blood using the standard method and di-
`gested to completion with HindIII restriction enzyme (Roche Molecular
`Biochemicals GmbH, Mannheim, Germany). DNA samples were then
`subjected to electrophoresis on 0.8% agarose gels and blotted onto Hy-
`bond-N nylon membrane (Amersham Pharmacia Biotech, Uppsala,
`Sweden) by the method of Southern blot. The membrane was hybridized
`overnight with a probe containing the CYP17 gene that had been labeled
`with 32P by the random hexanucleotide-primed method. The mem-
`branes were washed at 65 C in 1⫻ SSC (0.15 m sodium chloride and 0.015
`m sodium citrate) and 0.1% SDS, then exposed to x-ray film with in-
`tensifying screens at ⫺70 C for 1–7 d.
`
`Linkage analysis
`
`The polymorphic markers D10S1266, D10S1778, D10S192, D10S1265,
`and D10S587 were amplified for linkage analysis at 10q24-q25 (17).
`Fluorescently labeled PCR amplifications were performed in 25-␮l re-
`action volumes containing 100 ng genomic DNA, 200 ␮m of each dNTP,
`1.5 mm MgCl2, 10 mm Tris-HCl (pH 7.5), 50 mm KCl, 0.01% Tween 20,
`0.01% gelatin, 0.01% NP40, 15 pm of both fluorescently labeled and
`nonlabeled primers, and 1 U Taq DNA polymerase (Roche Molecular
`Biochemicals GmbH). Initial denaturation was for 3 min at 94 C, fol-
`lowed by amplification for 30 cycles with denaturation at 94 C for 30 sec,
`annealing for 30 sec at the required temperatures, and extension at 72
`C for 30 sec. Amplification products were analyzed by GENESCAN
`software in ABI PRISM 377 DNA sequencer (Perkin-Elmer Corp., Foster
`City, CA).
`
`PCR and DNA sequencing
`
`Oligonucleotides were designed spanning all eight exons and intron/
`exon boundaries of the CYP17 gene. PCR amplification of exons was
`carried out in a 25-␮l reaction volumes containing 100 ng genomic DNA,
`15 pm of each primer, 200 ␮m of each dNTP, 10 mm Tris-HCl (pH 7.5),
`50 mm KCl, 1.5 mm MgCl2, and 0.8 U Taq DNA polymerase (Roche
`Molecular Biochemicals GmbH). Initial denaturation was for 3 min at 94
`C, followed by amplification for 30 cycles with denaturation at 94 C for
`30 sec, annealing for 30 sec at the required temperatures, and extension
`at 72 C for 30 sec. PCR samples were purified by use of GFX PCR DNA
`and Gel Band Purification kit (Amersham Pharmacia Biotech) and se-
`quenced in both directions using the Thermo Sequenase dye terminator
`sequencing pre-mix kit (Amersham Pharmacia Biotech, Cleveland, OH).
`Data were analyzed using ABI PRISM 377 DNA sequencer (Perkin-
`Elmer Corp.).
`
`In vitro expression
`
`To study the functional implications of the mutations found, we used
`a RT-PCR method using CYP17 mRNA ectopically expressed in pery-
`pheral blood leukocytes of the patients as described previously (18) The
`mutated cDNAs were subcloned into a pCMV4 vector and transiently
`transfected into confluent COS-1 cells using 50 ␮g Lipofectamine and 10
`␮g DNA on a 10-cm plate (Life Technologies, Inc., Grand Island, NY).
`The correctness of the sequence was proven by sequencing. The trans-
`fection efficiency ranged from 40 – 60%. Forty-eight hours after trans-
`fection, steroidogenic precursors (pregnenolone and progesterone for
`17␣-hydroxylase activity and 17␣-hydroxypregnenolone for 17,20-lyase
`activity) were added at a concentration of 1 ␮mol/liter after suspension
`in 1⫻ phosphate buffer. Six hours after its addition, supernatant was
`removed and kept frozen at ⫺20 C until measured. To standardize the
`steroid production, cells were lysed in 1⫻ PBS, 1.5 mmol/liter MgCl2,
`1 mmol/liter ethylenediamine tetraacetate, 1% Triton-X, and 10% glyc-
`erol in the presence of protease inhibitors (34 ␮g/ml phenylmethane-
`
`FIG. 1. Family pedigree and haplotype reconstruction for informa-
`tive markers close to the CYP17 gene. The at-risk haplotype is boxed.
`
`later. Physical examination showed female infantile external
`genitalia and a complete absence of sexual hair and breast
`development. Blood pressure was 155/110 mm Hg. At ab-
`dominal computed tomography scan there was a bilateral
`adrenal hyperplasia, whereas mu¨ llerian structures were ab-
`sent. Serum sodium, blood urea nitrogen, and creatinine
`were normal. The potassium level was 3.66 mmol/liter.
`Blood pH was 7.424. As in the case of patient 1, the mea-
`surement of plasma and urinary steroids confirmed the sus-
`picion of combined 17-OHDS (Tables 1 and 2). Therapy with
`dexamethasone was started, followed by the addition of
`conjugated estrogens. During therapy with dexamethasone
`serum levels of blood urea nitrogen, creatinine, and potas-
`sium increased. Blood pressure and gas analysis and PRA
`normalized (Table 3).
`
`Family studies
`
`After informed consent was obtained, we also studied the
`family of the two affected sisters. The parents, ages 49 and
`45 yr, were consanguineous (see Fig. 1). Neither had hyper-
`tension or metabolic alkalosis. Both had normal levels of
`serum potassium. Their serum steroid hormone concentra-
`tions are shown in the Table 1.
`
`Materials and Methods
`
`Hormone assays
`
`All serum and urine samples were stored at ⫺30 C until analysis.
`Serum LH, FSH, and steroid levels and plasma ACTH and renin activity
`were measured by commercial kits based on RIAs, immunoradiometric
`assays, electrochemiluminescence immunoassays, and fluoroimmuno-
`metric methods. Urinary steroids were assayed by combined gas chro-
`matography/mass spectrometry following the method described by
`
`

`
`900 J Clin Endocrinol Metab, February 2002, 87(2):898 –905
`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`TABLE 1. Serum steroid hormone concentrations in the patients with 17␣-hydroxylase/17,20-lyase deficiency
`
`Patient 1
`
`Patient 2
`
`Father
`
`Mother
`
`BUN (mmol/liter)
`Creatinine (␮mol/liter)
`Na⫹ (mmol/liter)
`K⫹ (mmol/liter)
`pH
`P (nmol/liter)
`17OH-P (nmol/liter)
`DHEA (nmol/liter)
`DHEAS (nmol/liter)
`4 (nmol/liter)
`⌬
`T (nmol/liter)
`E2 (pmol/liter)
`Cortisol at 0800 h (nmol/liter)
`Cortisol at 1800 h (nmol/liter)
`Aldo supine (pmol/liter)
`Aldo upright (pmol/liter)
`PRA supine (ng/liter䡠sec)
`PRA upright (ng/liter䡠sec)
`ACTH at 0800 h (pmol/liter)
`ACTH at 1800 h (pmol/liter)
`UFC (nmol/24 h)
`LH (IU/liter)
`FSH (IU/liter)
`
`7.9
`69.0
`143
`2.87
`7.45
`38.2
`3.76
`2.15
`0.03
`0.03
`0.03
`0.04
`168
`157
`447
`666
`0.003
`0.003
`23.6
`22.0
`19
`13.60
`8.23
`
`11.1
`86.6
`141
`3.66
`7.42
`34.0
`4.76
`2.98
`3.85
`0.03
`0.03
`12
`218
`281
`583
`804
`0.03
`0.03
`17.0
`28.2
`45
`19.82
`40.11
`
`19.3
`158.2
`148
`4.44
`7.38
`1.5
`4.24
`9.02
`1274
`1.47
`3.88
`57
`66
`NA
`NA
`641
`NA
`0.45
`2.6
`NA
`39
`16.69
`54.84
`
`21.1
`76.9
`144
`4.10
`7.37
`1.3
`2.33
`11.17
`1893
`1.43
`0.49
`93
`179
`NA
`NA
`546
`NA
`0.09
`2.4
`NA
`135
`12.81
`27.76
`
`Normal adult values
`
`Males
`
`Females
`
`3.6 –17.9
`44.2–114.9
`135–153
`3.50 –5.30
`7.35–7.45
`
`0.3–3.8
`1.27–10.61
`4.86 – 43.38
`5065–12834
`1.99 –9.25
`8.32–34.67
`ND–162
`
`0.5– 4.5
`0.61–7.88
`2.78 –36.44
`3409 –9433
`1.64 –9.35
`0.69 –3.12
`37–184
`
`193– 690
`55–248
`21– 416
`97– 832
`0.06 – 0.78
`0.42–1.58
`2.2–13.2
`1.3– 6.6
`22–166
`
`1.7– 8.9
`1.5–12.4
`
`2.4 –12.6
`3.5–12.5
`
`BUN, Blood urea nitrogen; P, progesterone; 17OH-P, 17␣-hydroxyprogesterone; DHEAS, DHEA sulfate; ⌬
`aldosterone; UFC, urinary free cortisol; ND, not detectable; NA, not assayed.
`
`
`
`4, ⌬4-androstenedione; Aldo,
`
`sulfonylfluoride, 0.7 ␮g/ml pepstatin, and 5 ␮g/ml leupeptin; Roche
`Molecular Biochemicals, Rotkreuz, Switzerland), and protein content
`was measured using protein assay reagents obtained from Bio-Rad
`Laboratories, Inc. (Hercules, CA). The secreted steroids [i.e. 17␣-
`hydroxyprogesterone (17␣-hydroxylase activity) and dehydroepiandro-
`sterone (DHEA) (17,20-lyase activity)] were measured in duplicate by
`RIA using Diagnostic Products kits (Los Angeles, CA). All values are
`expressed as the mean ⫾ sd and represent the results of three indepen-
`dent experiments. Western blot analysis was performed using standard
`procedure (19).
`
`Steroid hormones at first admission
`
`Results
`
`Plasma steroids. Both patients had low normal cortisol basal
`values and inadequate response of cortisol to ACTH. Serum
`progesterone was high, and 17␣-hydroxyprogesterone was
`normal. The response of progesterone and that of 17␣-
`hydroxyprogesterone to ACTH were negligible to absent.
`Serum levels of all the C19 steroids were low to undetectable
`before and after ACTH and human CG stimulation (Tables
`1 and 4). In both parents the basal levels of progesterone,
`17␣-hydroxyprogesterone, and DHEA were normal, where-
`as serum levels of androstenedione and T were moderately
`low, predicting their heterozygosity.
`
`Urinary steroids. The basal urinary steroid levels are reported
`in Table 2. In both patients levels of the urinary metabolites
`of corticosterone were largely above the normal range. Levels
`of the urinary metabolites of androstenedione, T, E2, and
`DHEA were low to undetectable according to their serum
`concentrations. Levels of the urinary metabolites of preg-
`nenolone and progesterone were increased, whereas urinary
`metabolites of 17␣-hydroxypregnenolone, 17␣-hydroxypro-
`gesterone, and cortisol were low. By contrast, both parents
`had near-normal levels of the urinary metabolites of preg-
`
`nenolone, progesterone, corticosterone, and cortisol and rel-
`atively low levels of the urinary metabolites of androstenedi-
`one, T, and DHEA.
`
`Clinical course and steroid hormones after treatment
`with dexamethasone
`
`Both patients were initially treated with 0.25 mg dexa-
`methasone. Treatment failed to normalize ACTH and aldo-
`sterone levels and PRA. Moreover, blood pressure was con-
`stantly found above normal values. To achieve a more
`physiological condition, the dose of the drug was doubled.
`After several months of this treatment, in which a normal-
`ization of potassium, aldosterone, renin activity, and blood
`pressure was obtained (Table 3), both patients presented
`claiming the appearance of striae rubrae predominantly lo-
`calized on the abdomen, thighs, and in axillae. Thus, after a
`4-month off-treatment period, the daily dose of dexameth-
`asone was reduced and conjugated estrogens (Premarin)
`were added at a 0.3-mg daily dose for 6 months and 0.625 mg
`afterward (Table 3). Following this regimen, patient 1 expe-
`rienced regular menstrual bleeding and breast development,
`whereas patient 2 experienced breast development also. Hor-
`monal measurements were performed 2 wk and 4 months
`after the cessation of glucocorticoid therapy (early and late
`off-treatment, respectively). As shown in Table 3, progester-
`one and PRA, which had been normalized by treatment,
`rapidly returned to the values seen in the untreated period,
`indicating that both adrenal steroids and renin-angiotensin
`system are ACTH dependent. Moreover, potassium returned
`to the lower limits of the normal range, sodium remained
`essentially unchanged, and hypertension recurred.
`
`

`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`J Clin Endocrinol Metab, February 2002, 87(2):898 –905 901
`
`TABLE 2. Urinary steroid metabolite concentrations
`
`Urinary metabolite
`
`Parent compound
`
`Patient 1
`
`Patient 2
`
`Father
`
`Mother
`
`Normal values
`
`⌬
`
`⌬
`
`Preg, P
`P
`Preg
`17OH-Preg
`16OH-Preg
`17OH-P
`17OH-P
`DHEA
`DHEA
`16OH-DHEA
`16OH-DHEA
`4; T
`⌬
`4; T
`4; T
`4; T
`⌬
`4; T
`⌬
`11OH-⌬
`11OH-⌬
`11OH-⌬
`E2
`E2
`16OH-E2
`A
`A
`A
`A
`B
`B
`B
`B
`S
`21-DO-F
`21-DO-F
`E
`E
`E
`E
`F
`F
`F
`F
`F
`F
`F
`
`⌬
`
`⌬
`
`5␤-Pregnan-3␣-ol-20-one
`5␤-Pregnan-3␣,20␣-diol
`5-Pregnen-3␤,20␣-diol
`⌬
`5-Pregnen-3␤,17␣,20␣-triol
`⌬
`5-Pregnen-3␤,16␣,20␣-triol
`⌬
`5␤-Pregnan-3␣,17␣-diol-20-one
`5␤-Pregnan-3␣,17␣,20␣-triol
`5-Androsten-3␤-ol-17-one
`⌬
`5-Androsten-3␤,17␤-diol
`⌬
`5-Androsten-3␤,16␣-diol-17-one
`⌬
`5-Androsten-3␤,16␣,17␤-triol
`⌬
`5␣-Androstan-3␣-ol-17-one
`5␤-Androstan-3␣-ol-17-one
`5␣-Androstan-3␤-ol-17-one
`5␣-Androstan-3␣,17␤-diol
`5␤-Androstan-3␣,17␤-diol
`5␣-Androstan-3␣-ol-11,17-dione
`5␣-Androstan-3␣,11␤-diol-17-one
`5␤-Androstan-3␣,11␤-diol-17-one
`1,3,5(10)-Estratrien-3-ol-17-one
`1,3,5(10)-Estratrien-3,17␤-diol
`1,3,5(10)-Estratrien-3,16␣,17␤-triol
`5␣-Pregnan-3␣,21-diol-11,20-dione
`5␤-Pregnan-3␣,21-diol-11,20-dione
`5␤-Pregnan-3␣,20␣,21-triol-11-one
`5␣-Pregnan-3␣,20␣,21-triol-11-one
`5␣-Pregnan-3␣,11␤,21-triol-20-one
`5␤-Pregnan-3␣,11␤,21-triol-20-one
`5␣-Pregnan-3␣,11␤,20␣,21-tetrol
`5␤-Pregnan-3␣,11␤,20␣,21-tetrol
`5␤-Pregnan-3␣,17␣,21-triol-20-one
`5␤-Pregnan-3␣,17␣,20␣-triol-11-one
`5␤-Pregnan-3␣,11␤,17␣,20␣-tetrol
`5␣-Pregnan-3␣,17␣,21-triol-11,20-dione
`5␤-Pregnan-3␣,17␣,21-triol-11,20-dione
`5␤-Pregnan-3␣,17␣,20␣,21-tetrol-11-one
`5␤-Pregnan-3␣,17␣,20␤,21-tetrol-11-one
`5␤-Pregnan-3␣,11␤,17␣,21-tetrol-20-one
`5␣-Pregnan-3␣,11␤,17␣,21-tetrol-20-one
`5␤-Pregnan-3␣,11␤,17␣,20␣,21-pentol
`5␤-Pregnan-3␣,11␤,17␣,20␤,21-pentol
`4-Pregnen-11␤,17␣,20␣,21-tetrol-3-one
`⌬
`4-Pregnen-11␤,17␣,20␤,21-tetrol-3-one
`4-Pregnen-11␤,17␣,21-triol-3,20-dione
`Results are expressed as micrograms per 24 h.
`Preg, Pregnenolone; 17OH-Preg, 17␣-hydroxypregnenolone; 16OH-Preg, 16␣-hydroxypregnenolone; P, progesterone; 17OH-P, 17␣-hydroxy-
`progesterone; 16OH-DHEA, 16-hydroxy-DHEA; ⌬
`4, ⌬
`
`4-androstenedione; 11OH-⌬4, 11-hydroxy-⌬
`4-androstenedione; 16OH-E2, 16-hydroxy-E2;
`A, 11-dehydrocorticosterone; B, corticosterone; S, 11-deoxycortisol; 21-DO-F, 21-deoxycortisol; E, cortisone; F, cortisol; ND, not detectable.
`
`4
`
`4
`
`4
`
`210
`2300
`1900
`24
`900
`44
`68
`0
`0
`0
`0
`27
`28
`0
`0
`0
`0
`13
`6
`0
`0
`0
`15000
`2600
`872
`660
`77000
`1400
`672
`150
`5
`1
`34
`0
`225
`40
`24
`17
`257
`21
`45
`0
`0
`0
`
`200
`1067
`2790
`78
`836
`15
`49
`38
`8
`27
`29
`17
`5
`0
`0
`0
`0
`19
`2
`0
`0
`0
`3600
`17000
`1400
`350
`172000
`610
`1900
`206
`10
`7
`31
`52
`172
`12
`18
`60
`631
`26
`14
`0
`0
`0
`
`5
`24
`33
`10
`25
`71
`255
`7
`4
`170
`160
`356
`95
`2
`8
`7
`190
`540
`104
`0
`0
`0
`158
`180
`25
`2
`580
`130
`12
`1
`23
`22
`20
`84
`2600
`580
`280
`900
`1700
`29
`208
`22
`12
`64
`
`4
`98
`26
`14
`27
`42
`125
`70
`7
`110
`17
`317
`220
`11
`2
`5
`94
`177
`120
`0
`0
`0
`36
`146
`19
`9
`320
`140
`6
`2
`11
`5
`3
`30
`1300
`305
`90
`624
`390
`50
`102
`10
`12
`36
`
`ND
`100 –2500
`ND
`40 – 430
`ND
`20 – 600
`100 –1700
`50 –900
`70 – 450
`65–500
`90 – 400
`900 –3500
`700 –3000
`10 –200
`15–230
`15–230
`148 – 650
`318 –1600
`100 –550
`ND
`ND
`ND
`ND
`50 –250
`ND
`ND
`80 –500
`70 –300
`ND
`ND
`10 –100
`10 –100
`20 –140
`ND
`750 –2000
`180 – 850
`100 – 620
`650 –1700
`30 –1500
`10 –500
`50 –300
`ND
`ND
`17–70
`
`Molecular analyses
`
`Microsatellites D10S1266, D10S1778, D10S192, D10S1265,
`and D10S587 located on chromosome 10q24-q25 (17) close to
`the CYP17 gene were used expecting to find a region of
`homozygosity because of the consanguinity in the family
`(Fig. 1). In fact, the two affected individuals shared the same
`allele at all these loci, suggesting that both alleles were af-
`fected by the same germ-line mutation. The maximum LOD
`score for informative markers was 1.29.
`The CYP17 gene was screened for mutations in one
`proband (VI-2) and her father by direct sequencing of the
`coding region, including the eight exons amplified to-
`gether with the flanking donor and acceptor splice sites,
`and the promoter region. Six different nucleotide substi-
`tutions were found in both samples, when each sequence
`was compared with that of the gene in GenBank (accession
`
`no. M63871). They included three silent changes (T138C,
`T195G, and T849C) and two missense mutations, C66G
`and T278G, responsible for Cys22Trp and Phe93Cys amino
`acid substitutions in the coding region of exon 1, respec-
`tively. Cys22Trp is likely to be a polymorphism, as found
`in GenBank (accession no. M14564). Therefore, we be-
`lieved that Phe93Cys was the mutation responsible for the
`syndrome of 17␣-hydroxylase/17,20-lyase deficiency in
`this family. Several aspects of the present work confirmed
`our hypothesis. First, segregation analysis in the family
`demonstrated that both parents were heterozygous
`whereas the two affected daughters were homozygous, as
`expected on the basis of linkage analysis data, for the
`T278G substitution (Fig. 2). Second, the importance of
`Phe93 was supported by the observation that this amino
`acid is conserved in all the P450c17 enzymes characterized
`
`

`
`902 J Clin Endocrinol Metab, February 2002, 87(2):898 –905
`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`to date, including horse, sheep, bovine, guinea pig, rat,
`mouse, rainbow trout, dogfish, chicken, and frog (Fig. 3).
`Third, T278G was absent in 50 unaffected, unrelated con-
`trol individuals.
`The in vitro expression studies demonstrated that the
`Phe93Cys mutation did not affect protein translation and
`stability (data not shown). Transfection of the mutant cDNA
`in COS-1 cells leads to the synthesis of an enzyme retaining
`only about 10% activity compared with the wild type, with
`no significant difference between 17␣-hydroxylase and
`17,20-lyase activities (Table 5).
`
`Discussion
`
`38.2
`22.9
`
`40.9
`20.9
`
`52
`24
`
`52
`24
`
`50
`21.4
`
`8.6
`
`68.5
`
`16.4
`
`109.7
`
`71.3
`
`0.79
`
`0.06
`
`0.05
`
`0.04
`
`0.02
`
`0.73
`
`125
`
`162
`
`185
`
`255
`
`268
`
`NA
`66
`
`0.03
`0.03
`3.07
`3.37
`1.09
`8.0
`
`NA
`0.0455
`0.03
`0.03
`0.03
`0.03
`4.0823.1411.75
`2.88
`2.98
`0.97
`4.76
`
`NA
`
`NA
`
`34
`
`0.03
`0.03
`
`5.45
`1.12
`
`0.04
`0.03
`0.03
`0.03
`2.74
`1.82
`
`11.8
`
`15.3
`
`23.0
`
`22.7
`
`0.625
`0.25
`
`0.3
`0.25
`
`0.3
`0.25
`
`12
`
`6
`
`3
`
`0
`
`0
`
`late
`Off
`
`0
`
`0
`
`early
`Off
`
`0
`
`0.5
`
`5
`
`Patient2
`
`55
`25.3
`
`NA47
`NA21.9
`
`40.1144.6
`19.8223.4
`
`9
`
`16
`
`0.6
`
`1.0
`
`2.2
`
`7.9
`
`17.0
`
`202.6
`
`0.34
`
`0.53
`
`1.92
`
`0.03
`
`1.44
`
`0.46
`
`162
`
`113
`
`160
`
`560
`
`804
`
`1101
`
`NA196
`0.04
`0.03
`0.03
`3.17
`3.30
`0.39
`3.1
`
`NA
`
`NA
`
`NA
`
`14
`
`12
`
`14
`
`NA218
`12
`
`178
`
`0.03
`0.03
`0.03
`4.09
`0.61
`3.4
`
`0
`
`0.5
`
`4
`
`0.03
`0.03
`0.03
`3.47
`0.45
`3.8
`
`0
`
`0.5
`
`2
`
`0.03
`0.03
`4.65
`4.16
`0.97
`5.3
`
`0
`
`0.5
`
`1
`
`0.03
`0.03
`3.85
`2.98
`4.76
`
`0.03
`0.03
`4.21
`2.46
`1.36
`
`34.0
`
`18.1
`
`0
`
`0
`
`0
`
`0.625
`0.25
`
`12
`
`6
`
`Since the original description by Biglieri et al. (20), over
`120 cases of 17␣-hydroxylase deficiency have been re-
`ported (reviewed in Refs. 8 –11). Furthermore, a few cases
`of 17,20-lyase deficiency have been described in which
`17␣-hydroxylase activity was normal (8 –11). Mutations in
`the CYP17 gene have been identified in 28 patients with
`17␣-hydroxylase/17,20-lyase deficiency. The mutations
`are of different natures, including deletions, insertions,
`and single base changes, and are spread throughout the
`gene. Recently, G to A and G to T substitutions have been
`described in Japanese patients at position ⫹5 in the splic-
`ing donor site in introns 2 and 7, respectively (21, 22). Both
`patients were suffering from a combined 17␣-hydroxy-
`lase/17,20-lyase defect.
`Here, we report the cases of two sisters suffering from
`a well documented 17-OHDS. The long-term study of pa-
`tients and obligate heterozygotes (parents) suggests the
`following considerations. The hormonal profile observed
`in basal conditions, characterized by the marked ACTH-
`driven elevation of all compounds above the block, in-
`cluding the mineralocorticoids produced by the zona fas-
`ciculata in the 17-deoxy pathway, the significant reduction
`of cortisol, and the near complete absence of ⌬
`4- and ⌬
`5-
`androgens (Tables 1 and 2), strongly indicates a severe
`form of combined 17␣-hydroxylase/17,20-lyase defi-
`ciency. Serum and urine determinations performed in the
`parents also showed some abnormalities (slight reduction
`of ⌬
`4- and ⌬
`5-compounds below the block, increased ratio
`of C-21,17-deoxy to C-21,17-hydroxy urinary metabolites,
`and suppressed PRA in the mother) (Tables 1 and 2).
`Moreover, the administration of exogenous ACTH, which
`did not evoke any significant rise of 17-hydroxylated ste-
`roids in the patients, elicited a normal production of cor-
`tisol and a near-normal increase of ⌬
`4- and ⌬
`5-androgen
`precursors in both parents. Thus, biochemical data ob-
`tained in this family appear to meet the general criteria
`proposed for the identification of heterozygous siblings
`(23).
`Most patients with 17␣-hydroxylase deficiency have ab-
`sent or subnormal production of aldosterone. It has been
`suggested that the inhibition of aldosterone biosynthesis is
`mediated by the increased levels of mineralocorticoids,
`which lead to suppression of renin-angiotensin system via an
`increased reabsorption of sodium and increased blood vol-
`ume (24). By contrast, in our patients aldosterone levels were
`high in the supine position despite suppressed renin and
`
`SeeTable1forabbreviations.
`
`7.5
`NA12.1
`
`9.16NA
`
`14.8
`
`8.23
`
`13.6
`
`1.4
`
`1.6
`
`1.4
`
`23.6
`
`0.59
`
`0.40
`
`0.08
`
`0.003
`
`67
`
`129
`
`63
`
`NA
`
`NA
`
`60
`
`45
`
`10
`
`0.03
`0.03
`0.03
`3.96
`1.12
`
`0.03
`0.03
`2.73
`0.03
`1.73
`
`0.03
`0.03
`6.71
`3.09
`1.61
`
`666
`
`168
`
`0.04
`0.03
`0.03
`0.03
`2.15
`3.76
`
`23.3
`
`37.5
`
`26.7
`
`38.2
`
`0
`
`0.5
`
`2
`
`0
`
`0.5
`
`1
`
`0
`
`0
`
`0
`
`FSH(IU/liter)
`LH(IU/liter)
`(pmol/liter)
`
`ACTHat0800h
`
`(ng/liter䡠sec)
`
`PRAupright
`(pmol/liter)
`Aldoupright
`(nmol/liter)
`
`Cortisolat0800h
`E2(pmol/liter)
`T(nmol/liter)
`4(nmol/liter)
`
`⌬
`
`DHEAS(nmol/liter)
`DHEA(nmol/liter)
`17OH-P(nmol/liter)
`P(nmol/liter)
`Variable
`
`CE
`Dex
`
`(ng/d)
`
`Treatment
`
`MonthsofRx
`
`TABLE3.Serumsteroidhormoneconcentrationsbeforeandafterdexamethasone(Dex)andconjugatedestrogen(CE)therapy
`
`10.4
`15.8
`
`10.51
`17.25
`
`15.3
`18
`
`10.3
`14
`
`10.4
`13
`
`2.8
`
`3.9
`
`328.1
`
`91.2
`
`1.0
`
`1.37
`
`0.03
`
`0.01
`
`2.20
`
`29
`
`210
`
`316
`
`148
`
`135
`
`NA
`
`168
`
`0.03
`0.03
`12.22
`2.71
`1.09
`
`15.3
`
`NA
`69
`
`0.03
`0.03
`25.87
`3.30
`1.70
`
`25.1
`
`NA
`
`8
`
`0.03
`0.03
`0.03
`1.53
`3.58
`
`44
`29
`
`NA
`
`NA
`
`24
`
`0.03
`0.03
`0.03
`2.12
`1.97
`
`0.03
`0.03
`0.03
`3.19
`1.45
`
`36.3
`
`28.6
`
`18.1
`
`0.3
`0.25
`
`0.3
`0.25
`
`3
`
`0
`
`0
`
`late
`Off
`
`0
`
`0
`
`early
`Off
`
`0
`
`0.5
`
`5
`
`0
`
`0.5
`
`4
`
`Patient1
`
`

`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`J Clin Endocrinol Metab, February 2002, 87(2):898 –905 903
`
`TABLE 4. Basal and peak serum steroid concentrations after ACTH and HCG stimulation tests
`
`P
`(nmol/liter)
`
`17OH-P
`(nmol/liter)
`
`DHEA
`(nmol/liter)
`
`DHEAS
`(nmol/liter)
`
`⌬
`4
`(nmol/liter)
`
`T
`(nmol/liter)
`
`E2
`(pmol/liter)
`
`Cortisol
`(nmol/liter)
`
`Patient 1
`
`Patient 2
`
`Mother
`
`Father
`
`Basal
`ACTH
`HCG
`Basal
`ACTH
`HCG
`Basal
`ACTH
`HCG
`Basal
`ACTH
`HCG
`
`38.2
`51.4
`NA
`34.0
`53.3
`27.7
`1.5
`8.0
`NA
`1.3
`10.6
`NA
`
`See Table 1 for abbreviations.
`
`3.76
`3.79
`NA
`4.76
`6.06
`2.58
`4.24
`20.30
`NA
`2.33
`17.97
`NA
`
`2.15
`2.12
`NA
`2.98
`3.61
`2.32
`9.02
`26.48
`NA
`11.17
`23.94
`NA
`
`0.03
`NA
`NA
`3.85
`NA
`NA
`1274
`NA
`NA
`1893
`NA
`NA
`
`0.03
`0.03
`NA
`0.03
`0.03
`0.03
`1.47
`8.10
`NA
`1.43
`5.17
`NA
`
`0.03
`NA
`NA
`0.03
`NA
`0.03
`3.88
`NA
`NA
`0.49
`NA
`NA
`
`0.04
`NA
`NA
`0.04
`NA
`0.04
`57
`NA
`NA
`93
`NA
`NA
`
`168
`196
`NA
`218
`331
`NA
`66
`770
`NA
`179
`717
`NA
`
`FIG. 2. Partial sequences obtained by a reverse primer
`from individuals of the family carrying the Phe93Cys
`mutation of the CYP17 gene. The father and mother (A
`and B) are heterozygous for T278G. The two 17␣-hy-
`droxylase/17,20-lyase deficiency patients (C and D) are
`homozygous for the nucleotide G at the same position.
`
`sufficiently influenced by upright posture (Table 1). In ad-
`dition, both renin activity and aldosterone returned to nor-
`mal after glucocorticoid replacement therapy (Table 3). These
`
`findings indicate that in these patients aldosterone produc-
`tion is primarily ACTH-mediated rather than dependent on
`renin-angiotensin system. However, we cannot exclude that
`
`

`
`904 J Clin Endocrinol Metab, February 2002, 87(2):898 –905
`
`Di Cerbo et al. • Phe93Cys Mutation in the CYP17 Gene
`
`the
`of
`FIG. 3. ClustalW alignment
`amino acids from position 76–120 of the
`human P450c17 enzyme with those of
`other different species. The accession
`numbers are: Homosapiens, P05093;
`Equus caballus, Q95328; Ovis aries,
`Q29497; Bos taurus, P05185; Sus scrofa,
`P19100; Rattus norvegicus, P11715; Mus
`musculus,
`P27786;
`Oncorhynchus
`mykiss, P30437; Squalus acanthias,
`Q92113; Gallus gallus, P12394; Rana dy-
`bowskii, O57525. Phe93 and Arg96 are
`indicated by boldface and underlined let-
`ters, respectively.
`
`TABLE 5. Percent conversion of 1 ␮mol/liter progesterone (P) to
`17-hydroxyprogesterone (17OH-P) and 17-hydroxypregnenolone
`(17OH-Preg) to DHEA in transfected COS-1 cells
`
`P 3 17OH-P
`
`17OH-Preg 3 DHEA
`
`Mock
`Vector
`WT CYP17
`Phe93Cys
`
`0
`0
`100
`11 ⫾ 1.5
`
`0
`0
`100
`9.8 ⫾ 1.0
`
`WT, Wild type. One hundred percent corresponds to 24 nmol/liter
`17OH-P and 5.5 nmol/liter DHEA. Zero (0) corresponds to 0.1 nmol/
`liter 17OH-P and 0.4 nmol/liter DHEA.
`
`the apparent aldosterone increase is due to cross-reactions
`between aldosterone and the other mineralocorticoid pre-
`cursors that are massively elevated in this disorder.
`Screening of the CYP17 gene identified a new homozygous
`Phe93Cys missense mutation, which represents the fifth mu-
`tation found in the Italian population (18). Other cases de-
`scribed until now include two related patients who were
`homozygous for a 24-bp deletion in exon 1, another indi-
`vidual carrying a homozygous 3-bp deletion with the con-
`sequent loss of a glutamate at position 330, two apparently
`unrelated patients who were found to carry a homozygous
`Arg96Trp missense amino acid substitution, and two sisters
`carrying a 518 nucleotides deletion with an insertion of 469
`nucleotides in exons 2 and 3 (25).
`The finding that the mutant CYP17 protein retains merely
`10% of both 17␣-hydroxylase and 17,20-lyase activities con-
`firms at the molecular level the clinical diagnosis and points
`out the importance of Phe93 for CYP17 function. Phe93 is
`likely to be located in a critical region of the CYP17 protein.
`In fact, the amino acids from 93 to 97 are perfectly conserved
`in the P450c17 enzymes (see Fig. 3). In addition, as also found
`in our patients, the Arg96Trp mutation almost completely
`abolishes both the 17␣-hydroxylase and 17,20-lyase activities
`of CYP17 (18, 26).
`The study of mutations in the CYP17 gene is an important
`tool to better understand the molecular mechanisms of its
`deficiency and to provide further insight into the structure-
`function relationship of the protein. The possibility of ana-
`lyzing patients affected by CYP17 deficiency also provides a
`chance to correlate the mutations to combined and isolated
`deficiencies and define a model to study the influence of
`posttranslational modifications and cofactors on the differ-
`ential regulation of the two activities of CYP17.
`
`Acknowledgments
`
`Received March 8, 2001. Accepted November 8, 2001.
`
`Address all correspondence and requests for reprints to: Dr. Alfredo
`Di Cerbo, Division and Research Unit of Endocrinology, Istituto di
`Ricovero e Cura a Carattere Scientifico Ospedale “Casa Sollievo della
`Sofferenza,” 71013 San Giovanni Rotondo (Foggia), Italy. E-mail:
`adicerb@tin.it.
`This work was supported in part by the Swiss National Science
`Foundation (Grant 3200-052724.97/1 to A.B.-L.).
`
`References
`
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`hydroxylase cytochrome P450 cDNA in nonsteroidogenic (Cos 1) cells. Science
`234:1258 –1261
`2. Zuber MX, John ME, Okamura T, Simpson ER, Waterman MR 1986 Bovine
`adrenocortical cytochrome P45017␣. Regulation of gene expression by ACTH
`and elucidation of primary sequence. J Biol Chem 261:2475–2482
`3. Lin D, Harikrishna JA, Moore CCD, Jones KL, Miller WL 1991 Missense
`mutation Serine106 224 Proline causes 17␣-hydroxylase deficiency. J Biol Chem
`266:15992–15998
`4. Miller WL 1988 Molecular biology of steroid hormone synthesis. Endocr Rev
`9:295–318
`5. Chung B-C, Picado-Leonard J, Haniu M, Bienkowski M, Hall PF, Shively JE,
`Miller WL 1987 Cytochrome P450c17 (steroid 17␣-hydroxylase/17,20 lyase):
`cloning of human adrenal and testis cDNA