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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1874
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 4 1418-1425
Copyright © 2008 by The Endocrine Society

Carboxyl-Terminal Mutations in 3β-Hydroxysteroid Dehydrogenase Type II Cause Severe Salt-Wasting Congenital Adrenal Hyperplasia

Maik Welzel, Nele Wüstemann, Gunter Simic-Schleicher, Helmuth G. Dörr, Egbert Schulze, Guftar Shaikh, Peter Clayton, Joachim Grötzinger, Paul-Martin Holterhus and Felix G. Riepe

Division of Pediatric Endocrinology (M.W., N.W., P.-M.H., F.G.R.), Department of Pediatrics, Christian-Albrechts Universität zu Kiel, 24105 Kiel, Germany; Children’s Hospital of Bremen-Nord (G.S.-S.), 28755 Bremen, Germany; Division of Pediatric Endocrinology (H.G.D.), Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany; Laboratory of Molecular Genetics (E.S.), 69121 Heidelberg, Germany; University of Manchester (G.S., P.C.), Manchester M13 9PL, United Kingdom; and Institute of Biochemistry (J.G.), Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany

Address all correspondence and requests for reprints to: Felix G. Riepe, M.D., Division of Pediatric Endocrinology, Department of Pediatrics, University Hospital Schleswig-Holstein, Schwanenweg 20, D-24105 Kiel, Germany. E-mail: friepe{at}pediatrics.uni-kiel.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Introduction: 3β-Hydroxysteroid dehydrogenase (3β-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3β-HSD protein is not known.

Objective: We studied the functional consequences of three novel C-terminal mutations in the 3β-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3β-HSD type II deficiency showing different degrees of under-virilization.

Methods and Results: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant’s inactivity predominantly originates from a putative structural alteration of the 3β-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants’ residual in vitro activity.

Conclusions: Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3β-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3β-HSD deficiency cannot be predicted from in vitro 3β-HSD function alone.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Classical 3β-hydroxysteroid dehydrogenase (3β-HSD) deficiency (MIM 201810) is a rare autosomal recessive disorder first described by Bongiovanni in 1962 (1). Affected 46,XY individuals show heterogeneous signs of insufficient virilization ranging from mild hypospadias to genital ambiguity in combination with adrenal insufficiency. In contrast, 46,XX individuals suffer from adrenal insufficiency and no or only moderate signs of virilization (2). Interestingly, 3β-HSD deficiency is rarely found in patients with isolated hypospadias (3).

Two isoenzymes, named type I and type II 3β-HSD and showing 93% homology in the protein sequence, are involved in steroid biosynthesis in humans. These enzymes are encoded by two genes localized on chromosome 1p13.1, HSD3B1 and HSD3B2, respectively (4, 5). Classical 3β-HSD deficiency is caused by inactivating mutations in the HSD3B2 gene. The HSD3B2 gene consists of four exons, three of which are translated. To date, approximately 37 mutations in the HSD3B2 gene have been described (2). In contrast, no mutations in the HSD3B1 gene causing a change in the protein sequence have been found so far, reflecting the importance of the enzyme for prenatal steroid biosynthesis. Type II 3β-HSD belongs to the family of short-chain dehydrogenases and consists of 371 amino acids. Four functional domains, namely cofactor-binding domain, ligand-binding domain, and two membrane-spanning domains, have been described (6, 7, 8, 9).

Herein we describe four patients suffering from classical 3β-HSD deficiency caused by missense or nonsense point mutations situated in the C-terminal part of the protein. Because no known functional domain of 3β-HSD is obviously affected, this gives rise to the question whether these mutations influence enzyme activity. To pin down a possible mechanism for altered 3β-HSD activity with these mutations, we conducted in vitro and in silico studies on these mutations of the HSD3B2 gene.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients, clinical presentation, and hormonal analysis

Patient 1 The term neonate (gestational age, 41 wk; weight, 3960 g) was born to healthy consanguineous parents (second cousins) of Lebanese origin. At birth, he presented with a microphallus (length 1.5 cm), a broad urogenital sinus, and palpable gonads in both groins (Prader stage III, Sinnecker stage III) (10). The subsequent investigations revealed a normal 46,XY karyotype and a lack of Mullerian structures in the ultrasound scan. Steroid analysis yielded a highly elevated basal level of 17-hydroxypregnenolone (17-OHPreg, 101 nmol/liter; normal range for age, 1.2–18.0 nmol/liter) in combination with impaired cortisol synthesis (66.6 nmol/liter after ACTH; normal range for age, >150.0 nmol/liter), indicating 3β-HSD deficiency (11, 12, 13, 14). Hormonal data for all patients is presented in Table 1Go. Hydrocortisone and fludrocortisone substitution was initiated before the clinical onset of adrenal insufficiency. To date, the patient is doing well. He is being reared as a boy and has undergone plastic surgery for correction of hypospadias.


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  		TABLE 1. Hormonal data

 
Patient 2 The infant was born appropriate for gestational age at term (gestational age, 41 wk; weight, 2950 g) by spontaneous vaginal delivery to healthy consanguineous parents of Bangladeshi origin. He had a small phallus (length 2 cm) with a perineal hypospadias and palpable gonads in a bifid scrotum (Prader stage IV, Sinnecker stage II). Initial investigations revealed hyponatremia (127 mmol/liter), mild hyperkalemia (5.9 mmol/liter), and a normal 46,XY karyotype. A subsequent pelvic ultrasound scan identified no Mullerian structures. A standard short-term ACTH stimulation test performed at 3 d of age confirmed adrenal insufficiency (peak cortisol, 200 nmol/liter after ACTH; normal, >550 nmol/liter). A markedly elevated baseline level of dehydroepiandrosterone-sulfate (DHEA-S) was detected (5000 nmol/liter; upper limit for age, <2000 nmol/liter). The urinary steroid profile was in line with 3β-HSD deficiency (DHEA, 154 µmol/d; normal for age, <26 µmol/d; 16{alpha}-hydroxy-DHEA, >1280 µmol/d; normal for age, <205 µmol/d). The gold standard for diagnosing 3β-HSD deficiency is 17-OHPreg; however, it was not measured in patient 2. The diagnosis was made by clinical abnormalities, hormonal data as described, and the molecular analysis. Hydrocortisone and fludrocortisone replacement therapy was initiated in conjunction with sodium chloride supplementation.

Patients 3 and 4 Patients 3 and 4 are siblings. Both were born at term to healthy consanguineous parents of Turkish origin. The first boy showed a normal birth weight and length. He had a small phallus and scrotal hypospadias and a broad urogenital sinus (Prader stage III, Sinnecker stage III). Both testes were palpable in the groin. Initial investigations showed hyponatremia (128 mmol/liter), hyperkalemia (6.1 mmol/liter), and a 46,XY (t1;11) karyotype that was also found in the healthy father. A markedly elevated 17-hydroxyprogesterone (17-OHP) level of 342 nmol/liter (normal range, <40 nmol/liter) was found with newborn screening, raising the suspicion of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. However, detailed hormonal evaluation revealed markedly elevated levels of DHEA (242 nmol/liter; upper limit for age; <34 nmol/liter), DHEA-S (2950 nmol/liter; upper limit for age, <1000 nmol/liter), 17-OHPreg (130 nmol/liter; upper limit for age, <25 nmol/liter), normal testosterone levels (2.7 nmol/liter), and inadequately low cortisol levels (56 nmol/liter; normal lower limit, >25 nmol/liter), suggesting 3β-HSD deficiency. Hydrocortisone and fludrocortisone replacement therapy was started. After a 3-month treatment with daily local application of dihydrotestosterone, he underwent plastic surgery at the age of 15 months with urethroplasty, penile shaft stretching, and correction of the undescended testis.

The second boy was born after his brother had been diagnosed. He, too, had normal birth measurements, a short penis, penoscrotal hypospadias (Prader stage IV, Sinnecker stage II), maldescended testes, and a 46,XY karyotype. Additionally, he had an incomplete cleft lip. Again, a markedly elevated 17-OHP level of 407 nmol/liter (normal range, <40 nmol/liter) was found with newborn screening. Because it was likely that he also suffered from 3β-HSD deficiency, hormonal values were not obtained during the newborn period. The molecular genetic analysis confirmed the diagnosis. When hyponatremia (130 mmol/liter) and hyperkalemia (7.0 mmol/liter) occurred at the postnatal age of 10 d, hydrocortisone and fludrocortisone replacement therapy was started. Plastic surgery was performed at the age of 16 months with urethroplasty, penile shaft stretching, and correction of the undescended testis.

Mutational analysis

Blood samples for DNA analysis were obtained, with informed consent, from all patients and their parents. The study was approved by the ethical review board of the Christian-Albrechts Universität in Kiel. Preparation of genomic DNA from peripheral blood leukocytes followed a standard protocol. Exons II, III, and IV and the exon-intron boundaries of the HSD3B2 gene were amplified by PCR as described previously (15). The PCR products were verified on an agarose gel for DNA size and subsequently purified. The mutational analysis was performed by direct DNA sequencing of the complete coding region of the HSD3B2 gene. The samples were electrophoresed on an automated sequencer (ABI PRISM 310) and analyzed with the ABI SeqScape 3.7 software (Perkin-Elmer, Wellesley, MA). Mutants were designated according to the recommendations of the Nomenclature Working Group (16).

Site-directed mutagenesis

Human full-length HSD3B2 cDNA cloned into a pcDNA3 vector was kindly provided by Anne-Marie Moisan (Cancer Genomics Laboratory, Quebec, Canada). The mutagenesis to introduce the c.1003C->T, c.1022C->T, and c.1064G->A mutations was performed from the pcDNA3-HSD3B2 construct, using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primers for mutagenesis are available on request. The introduction of the mutations was reconfirmed by direct sequencing of the insert between the EcoRI and XbaI restriction sites. The integrity of the expression plasmid was ensured by recloning the mutated HSD3B2 cDNA into the EcoRI and XbaI sites of a newly restricted pcDNA3 expression vector, resulting in the respective pcDNA3 constructs.

In vitro expression and assay of enzyme activity

COS-7 and CHO cells were transiently transfected using Lipofectamine (Invitrogen, Karlsruhe, Germany) with 1 µg of each pcDNA3-HDS3B2 construct and 400 ng pRK-TK (Promega, Mannheim, Germany) coding for renilla luciferase. Posttransfection treatment followed a standard protocol (Invitrogen) using DMEM supplemented with glutamine, antibiotics, and 10% fetal calf serum. The 3β-HSD activity was measured 48 h after transfection in intact COS-7 or CHO cells. Cells were incubated for 180 and 100 min with DHEA and pregnenolone, respectively. The incubation medium contained 1 mmol/liter NAD+ and 0.5 µCi 3H-labeled and 1.0 µmol/liter unlabeled pregnenolone or 0.5 µmol/liter unlabeled DHEA. Subsequently, steroids were extracted from the culture medium with methylene chloride, evaporated to dryness, and dissolved in ethanol. Steroids were separated by thin-layer chromatography (TLC) using a mixture of toluene and acetone in a ratio of 4:1, followed by the direct measurement of radioactivity from the TLC plates using the Rita Star TLC-Scanner (Ray Test, Straubenhardt, Germany) and Rita TLC analysis software version 1.97. Cells were trypsinized and lysed in lysis buffer (Promega). The protein content was determined using the Bradford method. The renilla luciferase activity was measured using a standard renilla luciferase assay (Promega).

To determine apparent kinetic constants, intact COS-7 cells were incubated as described above with 0.1, 0.25, 0.5, 1.0, 2.0, or 4.0 µmol/liter unlabeled steroid. Postincubation treatment and analysis were performed as described above. The 3β-HSD activity was expressed as a percentage of substrate conversion in picomoles per milligram per minute, taking the activity of cells expressing the 3β-HSD wild-type protein as 100% after correction for total protein and renilla luciferase activity and for the 3β-HSD activity of cells transfected with the empty pcDNA3 plasmid. The apparent kinetic constants were calculated from the measurements of 3β-HSD activity in intact COS-7 cells at each of the different substrate concentrations. Calculation of enzymatic activities and kinetic constants was performed using the Graph Pad Prism software (GraphPad Software Inc., San Diego, CA).

To study the relevance of protein degradation for 3β-HSD activity, intact COS-7 and CHO cells were incubated as described above, adding 5, 10, 25, or 50 µM of a proteasome inhibitor (MG132; N-carbobenzoxyl-Leu-Leu-leucinal; Sigma Chemical Co., St. Louis, MO) 8 h before the determination of the enzyme activity.

Western blot analysis using an antihuman 3β-HSD rabbit polyclonal antibody provided by Anne-Marie Moisan (Cancer Genomics Laboratory, Quebec, Canada) was used in a standard protocol to ensure the expression and translation of the intact 3β-HSD wild-type and mutant proteins (17).

Immunofluorescence

Immunofluorescence followed a standard protocol (18). We used the antihuman 3β-HSD rabbit antiserum and a mouse anti-KDEL antibody (Biomol, Hamburg, Germany) as primary antibodies in a 1:200 dilution. The antirabbit-Alexa Fluor488 and antimouse-Alexa Fluor594 (Molecular Probes, Leiden, The Netherlands) antibodies were used as secondary antibodies in 1:500 dilutions. The slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), allowing for nuclear 4',6-diamidino-2-phenylindole staining.

Molecular modeling

A three-dimensional model of 3β-HSD was computed to generate a structural representation of the missense mutation. To substantiate the relatedness of the human 3β-HSD to the overall fold of the short-chain dehydrogenase family revealed by homology analyses, we used a fold recognition algorithm to show that the human 3β-HSD sequence is compatible with the architecture of this family (ProHit package; ProCeryon Biosciences GmbH, Salzburg, Austria). The template structure with the highest score of the pair potential was the x-ray structure of TDP-glucose-4,6-dehydratase (PDB accession code 1R6D), which served as the template for the three-dimensional model of 3β-HSD (Fig. 1Go). According to the alignment obtained by the fold recognition procedure, amino acid residues were exchanged in the template. Finally, these model structures were energy minimized, using the steepest descent algorithm implemented in the GROMOS program package (W. F. van Gunsteren, Laboratory of Physical Chemistry, University of Groningen, The Netherlands; distributed by BIOMOS Biomolecular Software BV). The structural representation was generated with the Deep View/Swiss-PDB Viewer program (19).


Figure 1
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  		FIG. 1. Alignment of the 3β-HSD (3bHSD2) and TDP-glucose-4,6-dehydratase (TDPG4, 6D) enzyme obtained by the fold recognition procedure. This alignment is the basis of the three-dimensional model of 3β-HSD.

 

   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Mutational analysis

Complete DNA sequencing of the HSD3B2 gene revealed a novel homozygous missense mutation (c.1022C->T) in patient 1, predicting an amino acid exchange of proline at codon 341 with leucine (p.P341L). Both parents and a healthy sister were heterozygous for this mutation. The healthy brother was not a carrier of the mutation. The mutational analysis in patient 2 revealed a homozygous mutation (c.1064G->A) in exon 4. The mutation causes the introduction of a stop codon at residue 355 (p.W355X). Both parents were heterozygous carriers of the mutation. Patients 3 and 4 are brothers. Complete sequencing of the HSD3B2 gene revealed a homozygous mutation (c.1003C->T) in exon 4 in both patients. This mutation causes the introduction of a premature stop codon at residue 335 (p.R335X). Both parents were heterozygous for the p.R335X mutation (Fig. 2Go).


Figure 2
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  		FIG. 2. Mutation analysis by direct DNA sequencing. A, The base change from C to T at position bp 1022 leads to the substitution of leucine with proline at amino acid position 341. The consanguineous parents are heterozygous, and the patient is homozygous for the p.P341L mutation. The older sister is heterozygous for the mutation, whereas the older brother shows a wild-type sequence at bp 1022. B, The G at position bp 1064 is changed to A, leading to an amino acid substitution from tryptophan to stop at amino acid position 355 (p.W355X). The mutation was found in the heterozygous state in the consanguineous parents and in the homozygous state in the patient. C, The base change from C to T at position bp 1003 leads to the substitution of arginine to stop at amino acid position 335. The consanguineous parents are heterozygous, and the two affected brothers are homozygous for the p.R335X mutation.

 
In vitro expression and assay of enzyme activity

Transient in vitro expression of all mutant proteins in intact COS-7 cells showed severely reduced 3β-HSD activity (Fig. 3Go). The mutation p.P341L reduced the 3β-HSD activity to 6.0 ± 1.0% (SD) of wild-type activity for the conversion of DHEA to androstenedione and 2.0 ± 0.5% of wild-type activity for the conversion of pregnenolone to progesterone. The activity of the mutant p.R335X-3β-HSD protein was reduced to 2.0 ± 0.5% of wild-type activity for the conversion of DHEA to androstenedione and 2.0 ± 1.5% of wild-type activity for the conversion of pregnenolone to progesterone. The mutation p.W355X reduced the 3β-HSD activity to 2.0 ± 0.5% of wild-type activity for the conversion of DHEA to androstenedione and 1.5 ± 1.0% of wild-type activity for the conversion of pregnenolone to progesterone.


Figure 3
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  		FIG. 3. Residual 3β-HSD activity of the 3β-HSD type II mutants in transiently transfected intact COS-7 cells without proteasome inhibitor and with 5, 10, 25, and 50 µM MG132. The activities of the mutants are expressed as percentage of wild-type (WT) activity, which is defined as 100%. Values are depicted for the conversion of pregnenolone (Preg) to progesterone (Prog) and DHEA to androstenedione (Andro) at a substrate concentration reflecting the wild-type KM (1 µmol/liter pregnenolone and 0.5 µmol/liter DHEA). The bars represent the mean ± 1 SD for six determinations from two different transfections.

 
The addition of the proteasome inhibitor MG132 to the COS-7 cells resulted in significant changes in wild-type 3β-HSD activity (Fig. 3Go). Wild-type activity for the conversion of pregnenolone to progesterone increased to 152 ± 11% with 10 µM MG132 but dropped to 121 ± 7.5% with higher amounts of proteasome inhibitor. No significant changes in 3β-HSD activity were noticed with the truncated mutants p.R335X and p.W355X. 3β-HSD activity of p.P341L was doubled with 10 µM MG132, although still well below wild-type, followed by a comparable decrease in activity with higher concentrations. Comparable results were obtained for the conversion of DHEA to androstenedione. Wild-type activity increased to 163 ± 9% with 10 µM MG132 but decreased to 129 ± 6% with higher amounts of proteasome inhibitor. No significant changes in 3β-HSD activity were noticed with the truncated mutants p.R335X and p.W355X. Activity of p.P341L doubled to 12.0 ± 2% with 10 µM MG132 followed by a comparable decrease in activity with higher concentrations of proteasome inhibitor.

Transient in vitro expression of mutant 3β-HSD proteins in intact CHO cells showed comparable results to the data obtained from COS-7 cells. The mutation p.P341L reduced 3β-HSD activity to 5.0 ± 1.5% of wild-type activity for the conversion of DHEA to androstenedione and to 2.5 ± 1.0% of wild-type activity for the conversion of pregnenolone to progesterone. The activity of the mutant p.R335X-3β-HSD protein was reduced to 1.5 ± 0.5% of wild-type activity for the conversion of DHEA to androstenedione and 1.0 ± 1.0% of wild-type activity for the conversion of pregnenolone to progesterone. The mutation p.W355X reduced the 3β-HSD activity to 1.5 ± 1.0% of wild-type activity for the conversion of DHEA to androstenedione and 1.0 ± 0.5% of wild-type activity for the conversion of pregnenolone to progesterone.

Determination of the apparent kinetic constants in COS-7 cells for the conversion of DHEA to androstenedione revealed that both wild type and p.P341L mutant achieved saturation under experimental conditions. The apparent Michaelis-Menten constant (KM) values were in the same range (0.5 ± 0.10 µM for wild-type 3β-HSD and 0.3 ± 0.15 µM for p.P341L 3β-HSD). The p.P341L mutation showed a remarkably decreased maximal velocity for the synthesis of androstenedione (165 ± 6 pmol/min·mg for wild-type 3β-HSD, 8 ± 1 pmol/min·mg for p.P341L 3β-HSD). The p.P341L mutant did not achieve saturation under the experimental conditions for the conversion of pregnenolone to progesterone. Therefore, no apparent kinetic constants were calculated. In comparison, the p.R335X- and p.W355X-3β-HSD proteins did not achieve saturation either for the conversion of DHEA to androstenedione or for the conversion of pregnenolone to progesterone.

Western blotting of the mutant p.P341L and p.R335X- and p.W355X-3β-HSD proteins derived from COS-7 cells showed a significantly less intense 3β-HSD-specific band compared with the wild-type protein (Fig. 4Go). The addition of MG132 significantly increased the protein abundance of all mutants. Comparable findings have been obtained with 3β-HSD proteins derived from CHO cells (data not shown).


Figure 4
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  		FIG. 4. Western blot analysis of homogenates purified from transiently transfected COS-7 cells with the indicated expression vectors. Arrows indicate the 3β-HSD-specific band. A 42-kDa band corresponding to the type II 3β-HSD protein was detectable in the homogenate preparations from transfected COS-7 cells expressing the wild-type protein. With the addition of MG132, the abundance of the 3β-HSD-specific band increased. No or very little specific protein is detected in cells transfected with the mutant p.P341L, p.R335X-, and p.W355X type II 3β-HSD without proteasome inhibitor. With the addition of the proteasome inhibitor MG132, the full-length band and the truncated bands became clearly visible. The mock pcDNA3 vector alone showed no 3β-HSD-specific band. The nonspecific band observed at 38 kDa can be used as an internal control for protein loading.

 
Immunofluorescence studies on transiently transfected COS-7 cells showed that both wild-type 3β-HSD and mutant p.P341L-3β-HSD protein colocalized with KDEL, a marker of the endoplasmic reticulum (Fig. 5Go). This demonstrates that the p.P341L mutation has no effect on the intracellular localization of the mutant protein.


Figure 5
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  		FIG. 5. Immunofluorescence studies. The left panel shows the localization of the human type II 3β-HSD by using antihuman 3β-HSD rabbit polyclonal antiserum as primary and the antirabbit Alexa Fluor488 antibody as secondary antibody. In the middle panel, the endoplasmic KDEL protein was marked using a mouse anti-KDEL antibody as primary antibody and the antimouse Alexa Fluor594 antibody as secondary antibody. The intracellular endoplasmic colocalization of the 3β-HSD type II protein and the KDEL protein in COS-7 cells is depicted in the right panel.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study, three novel homozygous mutations in the HSD3B2 gene detected in unrelated patients with classic salt-losing 3β-HSD deficiency are characterized. The in vitro findings confirm the basis of salt-wasting 3β-HSD deficiency CAH to be due to these novel mutations.

Patient 1 exhibited typical clinical signs of classical 3β-HSD deficiency. He had a normal 46,XY karyotype, severe under-virilization, and salt-wasting adrenal insufficiency requiring fludrocortisone and hydrocortisone supplementation. In this patient, we detected a novel homozygous missense mutation (p.P341L). In vitro studies showed that the conversion of DHEA to androstenedione was markedly reduced compared with the wild type. The conversion of pregnenolone to progesterone was virtually absent. In 3β-HSD deficiency, the severity of salt wasting usually shows a good correlation with the in vitro data obtained (2, 20). An in vitro activity of 1–2% or less predicts a salt-wasting phenotype. In patients with subclinical salt loss, the predicted mutant enzyme activity ranged from undetectable to 20%. The highest variation was found in patients with non-salt-wasting 3β-HSD deficiency (undetectable to 80%) (2). According to these data, the reduction of enzyme activity for the conversion of pregnenolone to progesterone to 1% is in accordance with the salt-wasting phenotype of the patient described. However, we have to conclude from our data that a residual activity of 6% for the conversion of DHEA to androstenedione is not sufficient for normal gonadal and adrenal androgen production and leads to under-virilized male external genitalia.

To provide a possible explanation for the loss of p.P341L enzyme activity in vitro, we subsequently performed additional experiments. Using Western blot procedures, we were able to show that the in vitro abundance of the mutant p.P341L protein is clearly reduced. Because protein instability was reported to be one factor that may lead to reduced enzyme activity (20, 21), we repeated the expression studies in CHO cells, because these are known for a less active proteasome. However, the mutants’ 3β-HSD activity was comparable to the enzyme activity detected in COS-7 cells. In addition, we incubated COS-7 and CHO cells with a proteasome inhibitor, impeding protein degradation. This led to an accumulation of 3β-HSD protein, supporting the hypothesis that protein instability leads to increased protein degradation. The p.P341L-3β-HSD activity was partially rescued by the inhibitor treatment, although enzyme function was still well below the wild-type activity. Interestingly, high concentrations of proteasome inhibitor reduced enzymatic activity. We have to speculate that this effect is due to the interference with the turnover of other functionally crucial proteins. In conclusion, premature protein degradation can explain the inactivity of the mutant only to a minor extent but not completely.

We therefore performed immunofluorescence studies to find out whether the mutation p.P341L had any influence on the intracellular localization of the protein. We were able to show that the mutant p.P341L protein colocalized with the KDEL protein, indicating a correct localization and membrane anchoring of the mutant protein within the cells. This is in concordance with the theoretical fact that the two known membrane-spanning domains remain unaffected by the mutation (9). Finally, we introduced the p.P341L mutation into a three-dimensional model using the known x-ray structure of mammalian TDP-glucose-4,6-dehydrogenase as template, to gain insight into possible structural consequences of the mutations on interactions with the catalytic center. Proline at position 341 is located in the loop connecting the E-helix and the final C-terminal G-helix of the protein. An exchange of proline with leucine does not obviously interfere with the catalytic center of the 3β-HSD protein (7) (Fig. 6Go). To our knowledge, the function of the C-terminal part of the 3β-HSD protein is unknown to date. However, the alignment of the amino acid sequences of the 3β-HSD protein in different species shows that proline 341 is highly conserved among vertebrates (Fig. 7Go). Proline generally allows for protein stability. Thus, the substitution of proline with the hydrophobic leucine may lead to higher structural flexibility of the protein, which is not predictable by our protein model. Most probably a combination of both, structural alteration of the 3β-HSD protein followed by increased protein degradation, is responsible for the inactivity of the mutant.


Figure 6
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  		FIG. 6. Three-dimensional molecular structure of the type II 3β-HSD protein (template TDP-glucose-4,6-dehydrogenase, PDB accession code 1R60). Residue P341 is located in the loop connecting the E helix with the C-terminal G helix of the protein. NAD+ is shown in ball and stick configuration.

 

Figure 7
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  		FIG. 7. Alignment of the human type II 3β-HSD protein with human type I 3β-HSD protein and different mammalian type II 3β-HSD proteins, including Macaca mulatta, Rattus norvegicus, and Mus musculus. Alignment was constructed with ClustalW. Conserved amino acids corresponding to the described amino acid changes found in classical 3β-HSD deficiency in this study are boxed.

 
Patients 2–4 had small phalluses with various degrees of hypospadias in combination with salt-wasting adrenal insufficiency. We detected two novel homozygous stop mutations (p.R335X and p.W355X) in these patients. Our in vitro experiments show that both mutants have no residual activity, underlining the clinical relevance of these mutations. The enzymatic inactivity of the stop mutations cannot be explained by increased proteolysis, because inhibition of the proteasome did increase the abundance of protein without significant increase in enzymatic activity. Because of this, the less intense band without proteasome inhibitor in the Western blot most likely reflects increased proteolysis as a secondary event in response to an inactive nonsense protein. The inactivation of the p.R335X- and p.W355X-3β-HSD proteins has to be due to the C-terminal truncation.

Two different stop mutations in the C terminus of the 3β-HSD protein have been described in the literature by a Japanese group (22). As with our own patients, the reported 46,XY individuals had a microphallus, hypospadias, and suffered from salt-wasting adrenal insufficiency. Consistent with our data, the two underlying homozygous mutations, R249X and Y308X, had no residual activity in vitro.

Little is known about the function of the C-terminal part of the type II 3β-HSD protein. Tyrosine at position 310 is the last amino acid of the putative second membrane-spanning domain of the type I 3β-HSD protein (9). In vitro data show that this domain of the 3β-HSD protein can be deleted without compromising the enzyme function. The function of the remaining part, a residue of 61 amino acids, remains to be elucidated. 3β-HSD belongs to the family of short-chain dehydrogenases. This protein family has a common protein folding pattern, and crucial functional domains are comparable (23, 24). The C-terminal part of these proteins is highly variable, and hence, a comparison of the different members of this family is difficult and does not provide an explanation for the function in 3β-HSD. Because the C terminus of 3β-HSD is conserved in different mammalian species (Fig. 6Go), it must be important for correct enzymatic function, as shown in our study.

Comparable to the literature, we found no correlation between the virilization deficit and the apparent in vitro enzyme activity. Therefore, the virilization deficit in 3β-HSD deficiency CAH cannot be anticipated and varies in different individuals even with an identical mutation. Interestingly, a comparable observation has been made in 21-hydroxylase deficiency, where functionally identical settings of mutations can lead to quite different degrees of external genital virilization, ranging from Prader stage 1–4 (25). Modifying factors of individual androgen action must therefore be assumed. These factors may even influence extragenital androgen action in 3β-HSD deficiency CAH, e.g. bone maturation, linear growth, final height, and gender aspects. Until now, the molecular background of this variability has been little understood. One potential mechanism may be an interindividual variation of expression and activity of the type I 3β-HSD isoenzyme in the placenta and in peripheral tissues of the fetus, thus converting variable amounts of excess DHEA to testosterone in the periphery (26). Additional potential modifiers include aromatase, 5{alpha}-reductase type 2, and the androgen receptor (27).

We characterized three novel mutations in the C-terminal part of the 3β-HSD protein. All three mutations show markedly reduced residual activity in vitro, explaining the under-virilization and salt-wasting adrenal insufficiency in the 46,XY individuals. The exact genital phenotype cannot be predicted from in vitro data. We conclude that the C-terminal part of the type II 3β-HSD protein is essential for the correct functioning and intracellular processing of the molecule. However, more studies are necessary to clarify the exact role of the C-terminal part of the type II 3β-HSD protein and other short-chain dehydrogenases.


   Acknowledgments
 
We acknowledge an ongoing institutional grant from the Medical Faculty, Christian-Albrechts-Universität zu Kiel. We thank Ann-Marie Moisan (Cancer Genomics Laboratory, Quebec, Canada) for providing the 3β-HSD pcDNA vector and the rabbit-polyclonal 3β-HSD antibody, and Gisela Hohmann and Tanja Dahm for the technical assistance in the laboratory. We acknowledge Mrs. Joanna Voerste’s linguistic help with the manuscript.


   Footnotes
 
Disclosure Statement: M.W., N.W., G.S.-S., H.G.D., E.S., G.S., P.C., J.G., P.-M.H., and F.G.R. have nothing to declare.

First Published Online February 5, 2008

Abbreviations: CAH, Congenital adrenal hyperplasia; DHEA-S, dehydroepiandrosterone-sulfate; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17-OHP, 17-hydroxyprogesterone; 17-OHPreg, 17-hydroxypregnenolone; TLC, thin-layer chromatography.

Received August 21, 2007.

Accepted January 29, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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