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Institute of Maternal and Child Research (E.C., C.O., G.I., M.A.B., M.C.J., F.C.), School of Medicine, University of Chile; and Hospital Clínico San Borja Arriarán (E.C., A.A.), National Health Service, Santiago, Chile
Address all correspondence and requests for reprints to: Fernando Cassorla, M.D., Casilla 226-3, Santiago, Chile. E-mail: fcassorl{at}machi.med.uchile.cl.
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Introduction |
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One important enzyme involved in the synthesis of mineralocorticoids, glucocorticoids, and sex steroids is the 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which belongs to aldo-keto reductase family and is a membrane-bound protein in the endoplasmic reticulum and mitochondria (7, 8, 9, 10). This enzyme is necessary for the conversion of pregnenolone to progesterone, 17-OH-pregnenolone (17OH Preg) to 17OH progesterone (17OH Prog), and of dehydroepiandrosterone (DHEA) to androstenedione. It catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids.
In humans, there are two isoenzymes, encoded by two genes on chromosome 1p13.1 (11, 12, 13), which are designated type I and type II. Both genes consist in four exons and three introns, and both enzymes have high homology in their amino acid sequence (14, 15). The type 1 gene (HSD3B1) is expressed in placenta, mammary gland, prostate, liver, kidney, and skin. The type II gene (HSD3B2) is expressed in the adrenal gland, ovary, and testis (16).
The clinical presentation of HSD3B2 deficiency is heterogeneous and may range from salt-losing congenital hyperplasia (17) to premature pubarche, and mild hyperandrogenism (18). However, the possibility that isolated abnormalities in male genital development, such as hypospadias, without other clinical manifestations, may be caused by a mutation in the HSD3B2 gene has not been documented up to now. We hypothesized that some cases of apparent idiopathic hypospadias may be consequence of HSD3B2 gene mutations, which may affect androgen production during fetal life. We conducted a prospective endocrine and molecular study in 90 patients with hypospadias with no evidence of salt loss during infancy, to determine the possible presence of molecular abnormalities in the HSD3B2 gene.
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Subjects and Methods |
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Ninety patients (6.0 ± 0.4 yr old) with penile or perineoscrotal hypospadias were studied. Subjects with glandular hypospadias, genetic syndromes, or major anatomical abnormalities of the genitourinary or gastrointestinal tract were excluded. Some patients had additional evidence of ambiguous genitalia, such as cryptorchidism or micropenis. A complete physical examination was performed in each patient. Each parent gave signed informed consent, and the patient gave verbal assent when pertinent. The study was approved by the Ethics Committee of the San Borja Arriarán Hospital.
A standard ACTH stimulation test (Cortrosyn, 0.25 mg iv; Alliance Pharmaceutical, Whitshire, UK) was performed, and we determined the serum levels of 17OH Preg, 17OH Prog, cortisol, DHEA, and androstenedione before and 60 min after ACTH administration. In addition, a standard human chorionic gonadotropin (HCG) stimulation test (Profasi, 100 IU/kg im; Serono, Randolph, MA) was performed, and serum androstenedione, testosterone, and dihydrotestosterone were determined before and 24 h after drug administration. Baseline renin plasma activity was determined in most subjects, provided that they were resting comfortably for at least 30 min. DNA was prepared from peripheral leukocytes. A karyotype was performed in all subjects with hypospadias associated with bilateral cryptorchidism.
Hormone assays
To measure 17OH Preg, the sera were extracted with ethyl acetate:n-hexane (6:4, vol:vol) and purified in LC-18 columns by sequential elution with iso-octane:ethyl acetate (8:2 and 6:4, vol/vol), evaporated, and dissolved in PBS. The recovery was approximately 90%. A competitive binding RIA for 17OH Preg using antibody, tracer, and standards from ICN Pharmaceuticals (Costa Mesa, CA) was used to measure the extracted samples. The interassay coefficient of variation (CV) and intraassay CV were 10.1% and 8.3%, respectively.
Testosterone, dihydrotestosterone, androstenedione, 17OH Prog, cortisol, and DHEA were measured by competitive specific-binding RIAs (Diagnostic System Laboratories, Webster, TX); interassay CVs were 8.1, 6.2, 8.9, 7.3, 5.2, and 7.7%, respectively; the intraassay CVs were 5.3, 5.5, 4.2, 7.7, 3.1, and 5.3%, respectively. Plasma renin activity was measured using 
Coat Plasma Renin Activity 125I RIA Kit (DiaSorin, Stillwater, MN), with inter- and intraassay CVs less than 10%.
The hormonal results in our patients were compared with the normal levels published in Normative Data for Adrenal Steroidogenesis in Healthy Pediatric Population from Lashansky et al. (19).
Molecular methods
Selective PCR amplification of the HSD3B2 gene fragments, denaturing gradient gel electrophoresis (DGGE), and direct sequencing of PCR products.
Genomic DNA of all subjects was extracted from peripheral blood by the method of Lahiri and Nurnberger (20). The coding regions and the exon (ex)-intron (intr) splicing junction boundaries of the four exons of HSD3B2 gene were completely amplified by PCR, using six specific primer pair sets with GC-clamp attached at the 5' end as indicated in Table 1
, according to Marui et al. (21). The existence of a unique band was evidenced in 1% agarose ethidium bromide-stained gel. The mutations were screened by DGGE in the conditions indicated in Table 1, and the bands were evidenced by silver nitrate stain. All the patient samples were run by DGGE in parallel with negative and positive controls when they were available. The positive controls consisted of patients with known mutations in exon 4 (18), kindly provided by Dr. Berenice Mendonca (Hospital das Clinicas, Sao Paulo, Brazil). The PCR fragments with abnormal electrophoretic migration in DGGE gels were again amplified from genomic DNA, purified using QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA), and sequenced with additional internal primers in ABI PRISM model 310 version 3.4 automatic sequencer (Perkin-Elmer Cetus, AT). In addition, in the samples with abnormal migration, an allele-specific PCR was performed.
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Site-directed mutagenesis
We generated vectors carrying the identified mutations using site-directed mutagenesis. (QuikChange Site-Directed Mutagenesis Kit; Stratagene Cloning System, La Jolla, CA) following the manufacturer instructions. Briefly, the point mutations documented by sequencing were included in oligo sequences designed such that the desired nucleotide change was in the middle of the primer with at least 15 bases of correct sequence on each side. The point mutation was introduced into pcDNA3 human type II 3ß-HSD (kindly donated by Drs. Anne-Marie Moisan and Jacques Simard, University of Laval, Quebec, Canada) using Pfu Turbo DNA Polymerase that replicates both plasmid DNA strands. The product was treated with DpnI endonuclease selecting the mutated synthesized DNA. The DNA sequence of the newly introduced mutation in pcDNA3 type II 3ß-HSD was confirmed by double-strand DNA sequence analysis or by PCR amplification followed by DGGE of linearized mutated vector.
Transfection of COS 7 cells and Western immunoblot analysis
COS 7 cells (American Type Culture Collection, Rockville, MD) were cultured in DMEM/high glucose (GibcoBRL LifeTechnology, Bethesda, MD) supplemented with 10% charcoal-stripped fetal bovine serum, 2 mmol/liter glutamax-I (GibcoBRL), 18 mM NaHCO3, 0.25 µg/ml fungizone (GibcoBRL), 100 IU/ml penicillin, and 5 mg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) at 37 C in 5% CO2/air in humidified atmosphere until confluence. Cells were plated in six-well plates at a density of 300,000 cells/well and were allowed to settle for 2 d. Medium was changed to free serum-DMEM media. Transient transfections of wild-type human type II 3ß-HSD and mutated type II 3ß-HSD were performed using Lipofectamine Plus Reagent (GibcoBRL) according to the manufacturer’s instructions.
For heterozygous mutations, the transfection was performed with equimolar concentrations of wild-type human type II 3ß-HSD and mutant vectors. Transfection efficiency was monitored by cotransfection of a ß-galactosidase expression vector, and the activity was assessed by in situ ß-galactosidase staining of transfected cells. To ascertain the amount of translated wild-type human type II 3ß-HSD and mutant recombinant proteins in transfected cells, 50 µg of total proteins was size-separated by electrophoresis on a SDS-12% polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Richmond, CA). Briefly, filters blocked with 5% solution of BLOT-Quick Blocker (Chemicon International, Inc., Temecula, CA) in Tris-buffered saline (TBS)-0.1% Tween 20 (pH 7.6) and washed in TBS-0.1% Tween 20 were incubated with a polyclonal antibody (1:100) directed against human 3ß-HSD (kindly provided by Dr. Van Luu-The, University of Laval) for 2 h at room temperature, washed, and incubated with goat antirabbit IgG peroxidase-conjugated secondary antibody (1:10,000; Chemicon) for 1 h at room temperature. After washes, the positive bands were visualized using the Renaissance Plus Detection kit (NEN Life Science, Boston, MA) followed by exposure of the membranes to x-ray film for 10 sec.
Assay of 3ß-HSD enzymatic activity
The enzymatic activity of 3ß-HSD was determined by the conversion of DHEA to androstenedione by RIA. Briefly, 43 h after transfection, intact transfected COS-7 cells were incubated with 10 nM DHEA (Sigma) for 30, 90, and 360 min in serum-free DMEM media. Subsequently, we determined the concentration of DHEA and androstenedione by RIA in the media as indicated above. Results were normalized by protein concentration determined by BCA protein Assay Kit (Pierce, Rockford, IL).
Statistical analysis
Data are reported as mean ± SEM of serum determinations performed in duplicate, or of culture media of at least two separate transfection experiments in duplicate.
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Results |
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reductase deficiency, or androgen insensitivity in the 90 patients studied. Identification of HSD3B2 gene mutations in patients and control samples
Exons 1–4 were amplified using the primers described in Table 1
. The amplified PCR products were analyzed by DGGE, allowing rapid screening for mutations. With this method, we identified an altered electrophoretic mobility pattern by DGGE in four patients and one control for intron 1, and in five patients for exon 4 (Fig. 1). In addition, an abnormal electrophoretic migration pattern for exon 3 was observed in one control.
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C transition predicts a modification of codon 213 (AGT) encoding Ser into Thr (ACT); whereas in patient 2, the nucleotide transition of CG predicts a change in codon 284 (AGC) encoding Ser into Arg (AGG). These findings were confirmed by allele-specific PCR. In the other three patients, the nucleotide transition did not predict an amino acid change in the primary structure of the enzyme. In patient 3, the substitution of TC at codon 238 (GCT) codes for the same amino acid Ala (GCC). Patient 4 showed an GA substitution in codon 259 (ACG), which encodes for the same amino acid Thr (ACA) and, in patient 5, the nucleotide substitution AC at codon 320 (ACA) codifier for the same amino acid Thr (ACC) (Fig. 2). We observed an abnormal DDGE pattern in patients 6, 7, and 8 consisting of an heterozygous TG substitution in nucleotide 1362 of intron 1. In addition, patient 9 showed a heterozygous AC substitution in nucleotide 1372 of intron 1 (Fig. 2).
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); and the sequencing of the PCR-fragment confirmed the same heterozygous TG substitution change in nucleotide 1362 of intron 1 (Fig. 2). The other control sample (control 2) showed an abnormal DGGE pattern in exon 3 (Fig. 1), and sequencing identified a G A substitution at codon 78 (GTC) that encoded Val into I1e (ATC) in one allele (Fig. 2).
The clinical, hormonal, and molecular characteristics of the nine patients with changes in HSDB2 are shown in Tables 2 and 3. We should note that patient 4 was diagnosed with bilateral Wilms’ tumor at the age of 2 yr 8 months, with no known family history for this condition. The genetic analysis of WT1, kindly performed by Dr. Vicky Huff (Department of Molecular Genetics/Cancer Genetics, M. D. Anderson Cancer Center, Houston, TX), did not detect any mutations of this gene.
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PCR amplification and DGGE for exons 1, 2, 3, and 4 of the HSD3B2 gene were performed in DNA obtained from the parents and siblings in eight of the nine patients (Fig. 3). DGGE analysis was performed in parallel with negative and positive controls, when the relatives were available for study. All samples with an abnormal DGGE pattern were sequenced. The family of patient 1 consists of both parents and a brother, and we documented that the mother and the brother, who had normal external genitalia, were also heterozygous for the S213T mutation, explained by possible phenotypic heterogeneity. The parents of patient 2 did not carry the S284R mutation, suggesting a de novo mutation. The mother of patient 3 did not carry the A238 mutation, and the father was not available for study. Both parents and a brother of patient 4 did not show any abnormality, indicating a de novo T259 mutation in this patient. The study of both parents and three siblings of patient 5 detected the T320 mutation only in the father, who showed a bifid prepuce and a wide urethral meatus. The family of patient 6 consists of both parents, a brother, and a sister, with the father showing the same n1362 TG heterozygous mutation. The father of patient 7 was also heterozygous for this mutation, whereas the mother did not show the nucleotide change, and his sister was not available for study. The family of patient 8 was not available for study. Finally, the family of patient 9 consists of both parents and a sister; the patient and sister carried the n1362 TG substitution in intron 1, whereas the mother was normal, and the father was not available for study.
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To assess the influence of the mutations on type II 3ß-HSD expression and/or activity, COS-7 cells were transfected with the pcDNA3 vector containing wild-type human type II 3ß-HSD or mutant type II 3ß-HSD-S213T, -S284R, or -V78I variants alone or in equimolar concentrations with the wild-type human type II 3ß-HSD. Because an equal amount of each vector was transfected, we assumed that the enzyme expression was 50% from the wild-type vector and the other 50% from the variant. This was not confirmed by other methods, however. In support of this assumption, transfected cells expressed comparable amounts of either normal or mutated type II 3ß-HSD, and COS-7 cells did not express endogenous enzyme, as determined by Western immunoblotting (Fig. 4A). The type II 3ß-HSD activity, studied by the conversion of DHEA to androstenedione, in transfected mammalian nonsteroidogenic cells with the vector expressing heterozygous S213T or S284R mutants, was reduced by 40 and 32%, respectively, compared with wild-type human type II 3ß-HSD (Fig. 4B). The heterozygous V78I mutation detected in exon 3 of the control subject did not affect enzyme activity, as shown in Fig. 4B. In the cells transfected with the vector expressing the homozygous S213T, S284R, or V78I mutants, the enzyme activity was reduced by 96, 80, and 99%, respectively, compared with transfected cells with the wild-type human type II 3ß-HSD vector (Fig. 4B).
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Discussion |
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In this study, we have shown that two patients with hypospadias have an heterozygous missense mutation in the HSD3B2 gene, causing a partial loss of enzymatic activity. These patients showed a normal steroidogenic metabolite response to ACTH and a normal androgen response to HCG. In vitro assays revealed a moderate decrease in 3ß-HSD activity in the heterozygous state, suggesting that this molecular abnormality may affect enzyme function. It is possible that during the first trimester of gestation, a critical period for the development of male external genitalia, this mutation may interfere with the migration of the urethral meatus to the tip of the glans penis. Direct confirmation of this hypothesis, however, cannot be concluded from this study.
We observed an S213T mutation in patient 1, and our in vitro studies demonstrated that this mutation reduced in vitro enzyme activity. The mother and the brother of this patient, however, harbored the same heterozygous mutation but did not exhibit any signs of 3ß-HSD deficiency. Thus, it is unclear whether this mutation caused the hypospadias in our patient. Moisan et al. (18) reported a mutation S213G in this same codon, in a girl with premature pubarche. We should mention that serine in codon 213 is conserved in primates and porcines but not in other species; whereas in nonprimates, species a threonine is not observed in this codon (28, 29).
The S284R mutation harbored by patient 2 is located in a conserved hydrophobic domain localized between amino acids 283 and 310 in the COOH-terminal. The deletion of this region produces a mutant cytosolic enzyme that lacks a targeting sequence that directs the translated protein to specific organelles, so a mutation of this hydrophobic domain may be critical for subcellular localization (28, 29, 30).
Patients 3, 4, and 5 carried heterozygous silent mutations in exon 4, which might be polymorphic variants of the HSD3B2 gene. In our 101 healthy fertile control men, however, no polymorphic changes were observed in exon 4, although we documented a V78I heterozygous mutation in exon 3 in one control individual that displayed normal enzymatic in vitro activity. The number of control individuals we studied, though, is not sufficient to define with certainty the genotype distribution.
The fact that patient 4 developed a bilateral Wilms’ tumor after our study was performed opens the question of whether his hypospadias was caused by a WT1 gene mutation (31, 32, 33). Hypospadias has been observed in patients with Wilms’ syndrome attributable to WT1 gene abnormalities, even without Wilms’ tumor or overt nephropathy. The molecular study of the WT1 gene, however, did not show mutations of this gene in this patient.
An n1362TG substitution was observed in intron 1 in patients 6, 7, and 8 and in one healthy fertile control. The fathers of patients 6 and 7 harbored the same nucleotide substitution but had a normally located urethral meatus. Patient 9 showed heterozygous n1372AC mutation in intron 1, which was also carried by his father and sister, who did not exhibit genital anomalies. These substitutions in intron 1 are likely to represent polymorphisms. Both nucleotide changes in the intron 1 detected in this study are highly conserved through the human 3ß-HSD gene family. Simard et al. (34) reported two sibs with nonsalt-losing form of classic 3ß-HSD, who had a nucleotide mutation n6651GA in intron 3 in one allele, and a missense mutation in exon 4 in the other allele, which reduced enzymatic function. Some intronic nucleotide mutations may affect mRNA processing by modification of splicing sites. Previously, it has been reported that a complex dinucleotide repeat in the HSD3B2 located in the intron 3 constitutes a highly polymorphic region with variable racial/ethnic distribution (35). The present study is the first report of nucleotide changes in intron 1 of this gene.
The hormonal abnormalities observed in patients with classical 3ß-HSD deficiency show increased levels of 17OH Preg and DHEA and increased ratios of 17OH Preg/17OH Prog, 17OH Preg/cortisol, and DHEA/androstenedione (36). We did not observe increased 17OH Preg or DHEA concentrations either in the basal state or after stimulation with ACTH or HCG in our patients with mutations of the HSD3B2 gene, except possibly in patient 3. This is not totally unexpected, however, because there are several difficulties in establishing the criteria for the diagnosis of 3ß-HSD deficiency based on hormonal levels: the levels of 17OH Preg may not be elevated due to normal activity of the 3ß-HSD type I, which is present in other tissues such as skin and liver. In addition, normal hormonal levels vary with chronological age and have broad ranges at different ages (19). Furthermore, Lutfallah et al. (37) studied 12 heterozygous carriers of severe HSD3B2 mutations, and these patients had normal hormonal findings; however, subtle clinical findings or 3ß-HSD in vitro activity were not reported in that study. Recently Lutfallah et al. (38) proposed new hormonal criteria for the diagnosis of 3ß-HSD deficiency. One of the limitations of our study is the lack of normal hormonal values for the ACTH and HCG tests from our own laboratory, which complicates the interpretation of these results.
In summary, we have performed a complete hormonal and molecular study of the HSD3B2 gene in a large group of patients with apparent idiopathic hypospadias. Two patients showed heterozygous missense mutations in exon 4, leading to decreased in vitro enzymatic activity, suggesting a possible cause of reduction in androgen synthesis during the first trimester of gestation. We conclude that subtle molecular abnormalities of the HSD3B2 gene may be observed in some patients with apparent idiopathic hypospadias but that this finding is uncommon.
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Acknowledgments |
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Footnotes |
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E.C. and C.O. contributed equally to this study and should be considered first authors.
Abbreviations: CV, Coefficient of variation; DGGE, denaturing gradient gel electrophoresis; DHEA, dehydroepiandrosterone; HCG, human chorionic gonadotropin; HSD, hydroxysteroid dehydrogenase; 17OH Preg, 17-OH-pregnenolone; 17OH Prog, 17-OH-progesterone; TBS, Tris-buffered saline.
Received June 4, 2002.
Accepted October 29, 2003.
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References |
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5–3 ß-hydroxysteroid dehydrogenase deficiency. J Clin Endocrinol Metab 51:345–353[Medline]
5-4 isomerase are closely linked on mouse chromosome 3. Genomics 16:219–223[CrossRef][Medline]
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4-isomerase. Mol Endocrinol 4:1850–1855[Abstract]
5-4 isomerase (3ß-HSD) gene: adrenal and gonadal specificity. DNA Cell Biol 10:701–711[Medline]
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