Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0136
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4362-4365
Copyright © 2005 by The Endocrine Society
17
-Hydroxylase/17,20-Lyase Deficiency Caused by a Novel Homozygous Mutation (Y27Stop) in the Cytochrome CYP17 Gene
Karsten Müssig1,
Simone Kaltenbach1,
Fausto Machicao,
Christiane Maser-Gluth,
Michaela F. Hartmann,
Stefan A. Wudy,
Günter Schnauder,
Hans-Ulrich Häring,
Fritz J. Seif and
Baptist Gallwitz
Department of Endocrinology, Metabolism, and Pathobiochemistry (K.M., S.K., F.M., G.S., H.-U.H., F.J.S., B.G.), University Hospital of Internal Medicine, University of Tübingen, D-72076, Tübingen, Germany; Department of Pharmacology (C.M.G.), Steroid Laboratory, University of Heidelberg, D-69120 Heidelberg, Germany; and Steroid Research Unit (M.F.H., S.A.W.), Center of Child and Adolescent Medicine, Justus Liebig University of Giessen, D-35392 Giessen, Germany
Address all correspondence and requests for reprints to: Dr. Karsten Müssig, M.D., Medizinische Klinik IV, Universitätsklinikum Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany. E-mail: karsten.muessig{at}med.uni-tuebingen.de.
 |
Abstract
|
|---|
Context: 17
-Hydroxylase/17,20-lyase deficiency, a rare autosomal recessive form of congenital adrenal hyperplasia, is caused by mutations in the cytochrome P450c17 (CYP17) gene. We report on a case of complete 17-hydroxylase/17,20-lyase deficiency due to a novel homozygous mutation of CYP17.
Design: A 20-yr-old female Turkish patient (46,XX) presented with primary amenorrhea, sexual infantilism, and easy fatigability.
Results: The patient’s steroid metabolism showed increased levels of mineralocorticoid precursors and low or undetectable plasma concentrations of 17-hydroxycorticoids, androgens, and estrogens before and after ACTH stimulation. The gas chromatography-mass spectrometry urinary steroid profile was dominated by metabolites of corticosterone and its precursors, while cortisol and C19-steroid metabolites were lacking. ACTH, FSH, and LH levels were elevated. These hormonal findings were consistent with a combined and total 17-hydroxylase/17,20-lyase deficiency. A therapy with hydrocortisone and a cyclic estrogen/gestagen substitution was initiated.
Conclusion: The CYP17 gene analysis revealed homozygosity of the mutation Y27Stop (TAC
TAA) in exon 1, a mutation that has not been previously described. This novel mutation leads to a stop codon causing a total loss of 17-hydroxlyase/17,20-lyase activity, as reflected biochemically by the detected concentrations of the steroid metabolites.
|
Introduction
|
|---|
17-HYDROXYLASE/17,20-lyase deficiency, a rare autosomal recessive defect in adrenal and gonadal steroidogenesis, results from mutations in the cytochrome CYP17 gene, leading to the insufficiency and failure to synthesize cortisol (17-hydroxylase activity), adrenal androgens (17,20-lyase activity), and gonadal steroids (1, 2). The loss of negative feedback due to the decreased cortisol levels causes increased ACTH secretion, resulting in excessive synthesis of mineralocorticoid precursors, such as deoxycorticosteron, corticosterone, and 18-hydroxycorticosterone (3). At the time of puberty, patients with 17-hydroxylase/17,20-lyase deficiency classically present with hypertension, hypokalemia, female hypogonadism, and male pseudohermaphroditism. Both the 17-hydroxylase and the 17,20-lyase reaction are catalyzed by the same enzyme, CYP17 (4). The encoding gene is located on chromosome 10q24-q25 and is expressed in the adrenals and the gonads but not in the placenta or ovarian granulosa cells (5, 6, 7). The 13-kb-long CYP17 gene comprises eight exons (8).
In this paper, we report the clinical, biochemical, and genetic characterization of a patient with a complete 17-hydroxylase/17,20-lyase deficiency due to a novel homozygous mutation of the cytochrome CYP17 gene.
|
Patients and Methods
|
|---|
Case reports
A 20-yr-old female Turkish patient whose parents were first-grade cousins presented with primary amenorrhea, sexual infantilism, and fatigue. On physical examination, body composition was normal (181 cm, 77 kg), pubic hair was completely absent, and the external genitalia were infantile. Blood pressure (135/85 mm Hg) and pulse rate were normal. Transvaginal ultrasonography showed an extremely hypoplastic uterus and micro-ovaries without follicles. Quantitative computed tomography of the lumbar spine revealed a markedly decreased bone mineral density below –2.5 SD (69.7 mg/ml calcium hydroxyapatite; normal, 159.2), consistent with osteoporosis. The hand-wrist bone age, using the Greulich and Pyle atlas, was retarded by 4–5 yr. On magnetic resonance tomography, the right adrenal gland was of normal size, whereas the left adrenal gland was slightly hyperplastic. The patient’s karyotype was 46,XX. Routine hematological and biochemical findings were normal. The serum potassium was within the normal range. The hormonal findings were consistent with a combined 17-hydroxylase/17,20-lyase deficiency (Table 1
), and therapy with hydrocortisone and a cyclic estrogen/gestagen substitution was initiated. For osteoporosis, a treatment with calcium and cholecalciferol was started. After this regimen, regular menstrual bleeding, breast development, and pubic as well as axillary hair growth occurred.
View this table:
[in this window]
[in a new window]
|
TABLE 1. Plasma steroid and urinary steroid metabolism in our patient with combined 17-hydroxylase/17,20-lyase deficiency
|
|
Hormone assays
RIA.
Serum and urinary aldosterone levels were determined using a commercial RIA kit (Diagnostic Systems Laboratories, Webster, TX). Intraassay coefficients of variation (CVs) were less than 8%, and interassay CVs were less than 10%. Plasma renin activity was measured with a commercial RIA kit (Biochem Immunosystems, Freiburg, Germany). Intraassay and interassay CVs were less than or equal to 6%. Various steroids were determined by RIA in the Steroid Laboratory of the Department of Pharmacology of the University of Heidelberg. Steroids were measured by specific RIA using tritiated steroids (Amersham Biosciences, Freiburg, Germany) and specific antibodies, raised and characterized in this steroid laboratory, as described previously (9). Before RIA, steroids were extracted from plasma or urine (pretreated with ß-glucuronidase) with organic solvents and chromatographically purified using Celite columns (Celite 545 AW; Sigma Aldrich, Taufkirchen, Germany). Intraassay CVs were less than 10%, and interassay CVs were less than 15%.
Gas chromatography-mass spectrometry (GC-MS).
Steroid determinations by GC-MS were performed in the Steroid Research Unit, Center of Child and Adolescent Medicine of Justus Liebig University of Giessen. Plasma steroids were profiled according to a stable isotope dilution/GC-MS method (10). Equilibration of the internal standards (stable isotope-labeled analogs of the analytes) with the plasma sample was followed by solid-phase extraction, purification, derivatization, and GC-MS analysis. Urinary steroids were profiled using GC-MS and selected ion monitoring analysis as described previously (11). In brief, steroids were enzymatically hydrolyzed, extracted by solid-phase extraction, and derivatized before GC-MS analysis.
PCR and DNA sequencing
DNA was extracted from peripheral blood using standard protocols (12). Mutation analysis of the CYP17 gene was performed by direct-sequencing PCR as described previously (13). Oligonucleotides were designed spanning all eight exons and intron/exon boundaries of the CYP17 gene (GenBank accession number NM_000102). PCR amplification of exons was performed in 25-µl reaction volumes containing 100 ng genomic DNA, 1 µM of each primer, 200 µM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 1 U Taq DNA polymerase (Eppendorf, Hamburg, Germany). Initial denaturation was for 5 min at 94 C, followed by amplification for 35 cycles with denaturation at 94 C for 1 min, annealing at 68 C for 1 min, and extension at 72 C for 1 min. PCR products were purified using a multiscreen PCR purification plate (Millipore, Schwalbach, Germany) and sequenced bidirectionally using a dye terminator cycle sequencing ready reaction kit (ABI PRISM 310; Applied Biosystems, Foster City, CA).
The human studies were approved by the Institutional Review Board, and the patient gave written consent for anonymous analysis.
|
Results
|
|---|
Steroid metabolism
Plasma basal and post-ACTH (60 min after iv injection of 250 µg cosyntropin) as well as urinary levels of steroids are summarized in Table 1
.
The plasma concentrations of 17-hydroxycorticoids, androgens, and estrogens were very low or undetectable before and after ACTH stimulation, whereas ACTH (242.7 pmol/liter; normal range, 2.0–11.0), FSH (63 mIU/ml; normal range, 2–10), and LH (27 mIU/ml; normal range, 2–10) levels were increased. The plasma levels of the mineralocorticoid precursors were elevated and increased further after ACTH treatment, whereas the aldosterone concentration was within the lower normal range and plasma renin activity was suppressed (0.07 ng/ml·h angiotensin I; normal range, 0.12–1.59). Urinary excretion of steroid metabolites impressively reflected complete 17-hydroxylase/17,20-lyase deficiency. As shown in Fig. 1, the steroid profile was dominated by metabolites of corticosterone and its precursors, whereas cortisol and C19-steroid metabolites were lacking.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 1. GC-MS urinary steroid profile (scan run) in our patient with 17-hydroxylase/17,20-lyase deficiency. Internal standards are indicated by Arabic numbers (1, 5-androstane-3,17-diol; 2, stigmasterol; 3, 5-cholestene-3ß-ol-butyrate). The steroid profile is dominated by metabolites of pregnenolone (5-PD, pregnenediol), progesterone (PD, pregnanediol), and corticosterone (THB, tetrahydrocorticosterone; 5-THB, 5-tetrahydrocorticosterone; THA, tetrahydro-11-dehydrocorticosterone), whereas 17-oxigenated C21-steroids (cortisol metabolites) and C19-steroids are lacking.
|
|
Diagnostic precursor to product ratios for global 17-hydroxylase to 17,20-lyase activity, as well as for individual 17-hydroxylase and 17,20-lyase activities were all pathological (Table 1). To conclude, our hormonal findings in plasma and urine were consistent with a combined and total 17-hydroxylase/17,20-lyase deficiency.
Molecular analysis
CYP17 gene analysis revealed homozygosity of a single point mutation: a C to A transversion in exon 1 that introduces a stop codon (TAA) in place of tyrosine (TAC) at codon 27. This mutation has not been described previously (Fig. 2).
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 2. CYP17 gene analysis in our patient with complete 17-hydroxylase/17,20-lyase deficiency revealed homozygosity of a single point mutation: a C to A transversion in exon 1 (marked by an arrow) that introduces a stop codon (TAA) in place of tyrosine (TAC) at codon 27.
|
|
|
Discussion
|
|---|
Recently, a number of novel mutations in the 17-hydroxylase gene has been described, indicating that 17-hydroxylase deficiency as a result of corresponding gene mutations is not as rare as anticipated previously (14, 15). Thus, multiple gene mutations in the CYP17 gene may play a role for the clinical picture of 17-hydroxylase deficiency. Rapid, efficient identification of novel mutations in P450c17, and especially the detailed elucidation of their structural, enzymatic, and clinical consequences, is becoming increasingly important (16). Therefore, we want to contribute an additional case of 17-hydroxylase/17,20-lyase deficiency caused by homozygosity of a novel mutation of the cytochrome CYP17 gene.
The pattern of plasma steroids under basal conditions and after ACTH stimulation as well as the GC-MS urinary steroid profile were both characterized by low or undetectable plasma concentrations of 17-hydroxycorticoids, androgens, and estrogens, whereas plasma and urinary levels of the mineralocorticoid precursors were increased. Therefore, the steroid metabolism was consistent with a complete deficiency of 17-hydroxylase/17,20-lyase activity.
CYP17 gene analysis revealed homozygosity of the mutation Y27Stop (TACTAA) in exon 1, leading to a severely truncated enzyme. Homozygosity of the mutation is explained by the consanguinity of the parents. Unfortunately, we were not able to investigate the patient’s parents as well as her siblings, because they all live in Turkey and obtaining blood samples for genetic analysis was a logistic problem. All are reported to be in good general health and free of symptoms.
In our patient, both copies of the 17-hydroxylase gene are defective in the form of a homozygous stop codon in exon 1 severely truncating the protein at amino acid 27 (of 509). The heme-binding site, the substrate binding pocket, and the redox-partner site, all of which have been reported to be essential for catalytic activity (17), are lacking. Therefore, CYP17 in our patient will have no activity, neither as 17-hydroxylase nor as 17,20-lyase. The genetic findings in our patient are in total agreement with the pattern in the steroid metabolism. In a previous study, two heterozygous stop codons in exon 3 and 4 were reported, resulting in combined 17-hydroxylase/17,20-lyase deficiency (18).
Interestingly, when first presented, our patient was neither hypertensive nor hypokalemic, despite high levels of 11-deoxycorticosterone. Whereas most patients with 17-hydroxylase deficiency were reported to be hypertensive and hypokalemic (19), a minority of about 10% was found normotensive and normokalemic at the time of diagnosis (20). However, the underlying mechanism for the latter observation is still unclear.
In summary, we have presented the clinical, metabolic, and genetic findings in an additional female patient with combined and complete 17-hydroxylase/17,20-lyase deficiency. A novel mutation in exon 1 of the CYP17 gene was found. The homozygosity of this Y27Stop codon leads to a total loss of enzyme activity, as reflected by the steroid constellation.
|
Acknowledgments
|
|---|
We thank Melanie Weiss for excellent technical assistance.
|
Footnotes
|
|---|
First Published Online April 5, 2005
1 K.M. and S.K. contributed equally to this study. 
Abbreviations: CV, Coefficient of variation; GC-MS, gas chromatography-mass spectrometry.
Received January 24, 2005.
Accepted March 25, 2005.
|
References
|
|---|
- Zuber MX, Simpson ER, Waterman MR 1986 Expression of bovine 17-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells. Science 234:1258–1261[Abstract/Free Full Text]
- Yanase T 1995 17-Hydroxylase/17,20-lyase defects. J Steroid Biochem Mol Biol 53:153–157[CrossRef][Medline]
- Biglieri EG, Herron MA, Brust N 1966 17-hydroxylation deficiency in man. J Clin Invest 45:1946–1954
- Chung BC, 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 cDNAs indicates the same gene is expressed in both tissues. Proc Natl Acad Sci USA 84:407–411[Abstract/Free Full Text]
- Sparkes RS, Klisak I, Miller WL 1991 Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23–q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24–q25; and P450c17 to 10q24–q25. DNA Cell Biol 10:359–365[Medline]
- Voutilainen R, Miller WL 1986 Developmental expression of genes for the stereoidogenic enzymes P450scc (20,22-desmolase), P450c17 (17-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus. J Clin Endocrinol Metab 63:1145–1150[Abstract]
- Voutilainen R, Tapanainen J, Chung BC, Matteson KJ, Miller WL 1986 Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17-hydroxylase/17,20-lyase) in cultured human granulosa cells. J Clin Endocrinol Metab 63:202–207[Abstract]
- Picado-Leonard J, Miller WL 1987 Cloning and sequence of the human gene for P450c17 (steroid 17-hydroxylase/17,20 lyase): similarity with the gene for P450c21. DNA 6:439–448[Medline]
- Vecsei P, Abdelhamid S, Mittelstädt GV, Lichtwald K, Haack D, Lewicka S 1983 Aldosterone metabolites and possible aldosterone precursors in hypertension. J Steroid Biochem 19:345–351[Medline]
- Wudy SA, Hartmann M, Homoki J 2002 Determination of 11-deoxycortisol (Reichstein’s Compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection. Steroids 67:851–857[CrossRef][Medline]
- Wudy SA, Hartmann MF 2004 Gas chromatography-mass spectrometry profiling of steroids in times of molecular biology. Horm Metab Res 36:415–422[CrossRef][Medline]
- Miller SA, Dykes DD, Polesky HF 1988 A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215[Free Full Text]
- Di Cerbo A, Biason-Lauber A, Savino M, Piemontese MR, Di Giorgio A, Perona M, Savoia A 2002 Combined 17-hydroxylase/17,20-lyase deficiency caused by Phe93Cys mutation in the CYP17 gene. J Clin Endocrinol Metab 87:898–905[Abstract/Free Full Text]
- Costa-Santos M, Kater CE, Dias EP, Auchus RJ 2004 Two intronic mutations cause 17-hydroxylase deficiency by disrupting splice acceptor sites: direct demonstration of aberrant splicing and absent enzyme activity by expression of the entire CYP17 gene in HEK-293 cells. J Clin Endocrinol Metab 89:43–48[Abstract/Free Full Text]
- Costa-Santos M, Kater CE, Auchus RJ 2004 Brazilian Congenital Adrenal Hyperplasia Multicenter Study Group. Two prevalent CYP17 mutations and genotype-phenotype correlations in 24 Brazilian patients with 17-hydroxylase deficiency. J Clin Endocrinol Metab 89:49–60[Abstract/Free Full Text]
- Miller WL 2004 Steroid 17-hydroxylase deficiency—not rare everywhere. J Clin Endocrinol Metab 89:40–42[Free Full Text]
- Auchus RJ, Miller WL 1999 Molecular modeling of human P450c17 (17-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Mol Endocrinol 13:1169–1182[Abstract/Free Full Text]
- Rumsby G, Skinner C, Lee HA, Honour JW 1993 Combined 17-hydroxylase/17,20-lyase deficiency caused by heterozygous stop codons in the cytochrome P450 17-hydroxylase gene. Clin Endocrinol (Oxf) 39:483–485[Medline]
- Goldsmith O, Solomon DH, Horton R 1967 Hypogonadism and mineralocorticoid excess. The 17-hydroxylase deficiency syndrome. N Engl J Med 277:673–677
- Kater CE, Biglieri EG 1994 Disorders of steroid 17-hydroxylase deficiency. Endocrinol Metab Clin North Am 23:341–357[Medline]
Top
Abstract
Introduction
