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Original Studies |
Département de Pédiatrie, Université de Montréal (N.A., G.V.V., L.W., L.L., G.L.), Montréal, Canada H3T 1C5; and Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, Laval University Medical Center (A.-M.M., M.D., J.S.), Québec, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Guy Van Vliet, M.D., Centre de Recherche, Hôpital Sainte-Justine, 3175 côte Sainte-Catherine, Montréal, Canada H3T 1C5. E-mail: gvanvliet{at}justine.umontreal.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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5-4-isomerase
(3ßHSD). In contrast to 21-hydroxylase and 11ß-hydroxylase
deficiencies, in which the defect is restricted to adrenal function,
the severe form of 3ßHSD deficiency impairs steroidogenesis in both
the adrenals and the gonads. In general, this form of CAH does not lead
to masculinization of female fetuses and may therefore remain
unrecognized in girls until a salt-losing crisis occurs. Since this
inborn error of metabolism was first described by Bongiovanni and
Kellenbenz in 1962 (5), considerable progress has been made in its
biochemical and molecular characterization. The conversion of 5-ene to 4-ene steroids is a crucial step in the biosynthesis of all classes of active steroids (progesterone, mineralocorticoids, glucocorticoids, androgens, and estrogens). There are two types of human 3ßHSD isoenzymes, which are 93.5% homologous and are encoded by two genes on chromosome 1p13.1 (6). The structure, tissue-specific expression, and regulation of the two human isoenzymes have been well characterized (6, 7). The type I isoenzyme is predominantly expressed in the placenta and peripheral tissues, whereas the type II isoenzyme is almost exclusively expressed in the adrenals as well as in the testes and ovaries after puberty (8).
In newborns, 3ßHSD deficiency results in incomplete masculinization of genetic males, whereas genetic females have little or no ambiguity of the external genitalia. During childhood, mild signs of androgen excess (such as premature pubarche) develop in both sexes. This observation can be explained by peripheral conversion of the inactive adrenal precursors that accumulate because of the deficient activity of the type II isoenzyme into active androgens by the normal type I 3ßHSD isoenzyme in these patients (6).
In the present report we describe a novel C to A (Ala10Glu) homozygous mutation in the HSD3B2 gene resulting in severe 3ßHSD deficiency with salt-wasting in two French-Canadian patients. The 46,XY individual presented with ambiguous genitalia at birth and subsequently developed normal male secondary sexual characteristics, as described in other 46,XY patients with this condition (9). However, he was azoospermic at 18.5 yr of age. The 46,XX patient developed progressive breast development, followed by menarche and evidence of progesterone secretion. To our knowledge, the only patients with severe salt-wasting 3ßHSD deficiency for whom the evolution after puberty has been reported were a male with proven paternity (10) and a female who was hypogonadal (11, 12). Thus, postpubertal gonadal function in this condition appears to be variable.
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Subjects and Methods |
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Patient 1 is an adopted, French-Canadian child, reportedly the
result of a consanguineous relationship. She was born at term by
vaginal delivery in May 1988 with a birth weight of 3.5 kg. Gestation
and delivery were uneventful. At 3 weeks of age, the infant was
admitted with fever (38.5 C) and dehydration (estimated at 10%). The
external genitalia were those of a normal female. Plasma
Na+ was 122 mmol/L, K+ was
9 mmol/L, HCO3- was 13.5
mmol/L, and glucose was 48 mg/dL (2.6 mmol/L). A preliminary diagnosis
of acute adrenal insufficiency was made, blood was drawn for steroid
hormone measurements, and treatment was started with iv hydration and
gluco- and mineralocorticoids. Pretreatment plasma hormone
concentrations were as follows: cortisol, 28 µg/dL (772.8 nmol/L);
17-hydroxyprogesterone, 10,000 ng/dL (
302.6 nmol/L); and ACTH, 450
pg/mL (99 pmol/L). Dehydroepiandrosterone sulfate (DHEA-S)
was not measured. Ultrasonography revealed enlarged adrenals. The
karyotype was 46,XX. At the age of 4 months, treatment was stopped for
10 days, and the patient was readmitted in acute adrenal crisis. On
admission, plasma Na+ was 129 mmol/L, and
K+ was 6.3 mmol/L. Plasma hormone concentrations
were as follows: cortisol, 8 µg/dL (220.8 nmol/L);
17-hydroxyprogesterone, 4200 ng/dL (127.3 nmol/L); testosterone, 65
ng/dL (2.9 nmol/L); DHEA-S, 519 µg/dL (13.4 µmol/L);
and ACTH, 438 pg/mL (96.4 pmol/L).
Since then, the patient has been treated continuously with cortisone
acetate and 9
-fluorohydrocortisone. At the age of 4 yr, body odor,
comedons, and pubic hair appeared, with a slight acceleration in growth
and bone maturation, but no clitoral enlargement. During childhood,
with good compliance and a dose of cortisone acetate between 14–21
mg/m2, the plasma steroid hormone profiles
consistently showed that DHEA-S was high (313–431
µg/dL; 8.5–11.7 µmol/L; normal, <0.54), whereas
androstenedione and 17-hydroxyprogesterone were only slightly increased
(20–71.6 ng/dL; 0.7–2.5 nmol/L; normal, <0.52; and
267.7–700.7 ng/dL; 8.1–21.2 nmol/L; normal, <1.4 nmol/L,
respectively), and testosterone was at or below the detection limit
(8.7 ng/dL; 0.3 nmol/L). Although 3ßHSD deficiency had been
considered in the neonatal period, this diagnosis had not been
established. As the clinical and biochemical evolution during childhood
was highly suggestive, DNA was obtained from the patient’s peripheral
blood leukocytes for HSD3B2 sequencing (see below).
At 8.3 yr, bone age was 10 yr, and linear growth started to accelerate to a peak of 9.5 cm/yr. Progressive breast and pubic hair development were noted, and menarche occurred at 10.2 yr (Tanner stage M4P3–4) followed by menstrual bleeding about every 30 days for the past 15 months. At 10.9 yr, on day 21 of the cycle, the following studies were performed: plasma LH, 14.1 IU/L; FSH, 5.1 IU/L; estradiol, 246 pmol/L; and progesterone, 1.0 nmol/L. All of these values are within the normal range for girls at Tanner stage 4 (13). A pelvic ultrasound showed bilaterally enlarged ovaries (largest diameter, 6 cm) containing multiple cysts, and a midcycle-type endometrium. At 11.2 yr, bone age was 14 yr, and another hormonal profile on day 18 of the menstrual cycle was essentially unchanged, except for the fact that plasma progesterone level had risen to 7.4 pmol/L.
Patient 2, born in April 1981, is the first child of related
French-Canadian parents (the maternal grandfather and paternal
grandmother are first cousins) with no known relationship to the family
of patient 1. The child’s weight at birth was 4.570 kg after an
uneventful pregnancy followed by a spontaneous vaginal delivery at
term. This child was referred to the endocrinology service at 13 days
of age because of ambiguous genitalia; the genital tubercle measured
2 x 1 cm, the urethral opening was at the perineum, and two
gonads were palpable within a bifid scrotum. The genitalia were
hyperpigmented. The karyotype was 46,XY. Plasma hormone concentrations
on day 14 of life were as follows: cortisol, 26.6 µg/dL (734.1
nmol/L); 17-hydroxyprogesterone, 2263.9 ng/dL (68.5 nmol/L);
DHEA-S, 868.9 µg/dL (23.6 µmol/L); androstenedione,
2552 ng/dL (89.3 nmol/L); and testosterone, 124.3 ng/dL (5.54 nmol/L).
On the same day, plasma Na+ was 125 mmol/L and
K+ was 7.5 mmol/L. A preliminary diagnosis of
3ßHSD deficiency was made, and treatment with iv hydration and with
gluco- and mineralocorticoids was started. The child has since then
been treated continuously with cortisone acetate and
9-fluorohydrocortisone. Hypospadias was surgically corrected in two
steps. Pubic hair was first noted at 10 yr, and testicular volume
started to increase at 10.5 yr. At 17.9 yr, his weight was 96.4 kg, and
his height was 178.0 cm (within the target range of 178.7 ± 8.5
cm). The patient had a well developed musculature and no gynecomastia.
His Tanner stage was G5P5, with a stretched penile length of 10 cm.
Both testes were heterogeneous in consistency and measured 25 mL each.
Testicular ultrasound was compatible with the presence of adrenal
rests. Plasma steroid levels after stopping treatment for 2 days were:
DHEA, 296.4 nmol/L (normal, <22.73 nmol/L),
17-hydroxyprogesterone, 65.1 nmol/L; androstenedione, 33.3 nmol/L; and
cortisol, 24.2 nmol/L; these values did not increase after
administration of 250 µg synthetic ACTH, iv. Plasma testosterone
(23.2 nmol/L), FSH (5.8 mIU/L), and LH (4.8 mIU/) were normal for a
young adult male. At 18.5 yr, while taking gluco- and mineralocorticoid
replacement, a semen analysis revealed azoospermia. Peripheral blood
leukocytes for DNA extraction were obtained from the patient, both
parents, and his younger brother.
Selective PCR amplification of the HSD3B2 gene fragments and direct sequencing of PCR products
Informed consent for molecular analyses was obtained. Selective amplification of the HSD3B2 gene fragments was performed as previously described (10, 14). Six different primer pairs for type II 3ßHSD were used for amplification of the coding region, the exon-intron splicing junction boundaries, the putative promotor region, and the 3'-noncoding region, including the polyadenylation site. The primers used for PCR amplification were the same as those used previously (15). Single stranded DNA was produced, as previously described (10, 14), from both strands, and the PCR DNA fragments were completely sequenced in both orientations. Sequencing was performed by the dideoxy method using the limiting PCR primer or using internal sequence-specific primers and the Thermo Sequenase-radiolabeled terminator cycle sequencing kit from Amersham Pharmacia Biotech (Piscataway, NJ).
Genotyping analysis
Genotyping analysis was performed as previously described (16).
Briefly, microsatellite markers close to the HSD3B loci (17)
D1S252, D1S440, D1S2820, D1S2669, D1S453, D1S2863, D1S514, D1S2344,
D1S2696, and D1S442 were PCR amplified using primers described in the
Généthon human genetic linkage map (18). Standard PCRs were
performed using 100 ng genomic DNA; 10 µM of
each oligonucleotide primer; 80 µM each of
deoxy (d)-CTP, dGTP, and dTTP; 2 µM dATP; 1.5
µCi [-35S]dATP at more than 1200 Ci/mmol;
10 mM Tris (pH 8.3); 50 mM
KCl; and 1.5 mM MgCl2.
Samples were overlaid with mineral oil and denatured for 5 min at 94 C.
Then, at 96 C, 2 U Taq polymerase (Perkin-Elmer Corp., Roche Molecular System, Inc., Laval, Canada)
were added, and the samples were processed through 35 cycles of
amplification consisting of 40 s at 94 C and 30 s at 58 C.
The final extension step was 10 min at 72 C. The marker HSD3B2A was
amplified as previously described (16). Aliquots of the amplified DNA
were mixed with 1 vol formamide loading buffer and electrophoresed on
standard denaturing polyacrylamide DNA-sequencing gels.
Site-directed mutagenesis
The oligo sequences for the C to A mutation were designed such that the desired nucleotide change was in the middle of the primer with 15 bases of correct sequence on either side. Site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit from Stratagene Cloning Systems (La Jolla, CA), according to the manufacturer’s protocol. The DNA sequence of the newly introduced mutation in pcDNA3 type II 3ßHSD was confirmed by double stranded DNA sequence analysis.
Transcription/translation
Transcription/translation was performed using the TNT Quick coupled transcription/translation system from Promega Corp. (Madison, WI) according to the manufacturer’s instructions. Briefly, 0.5 µg DNA was added to 16 µL master mix and 1 µCi [35S]methionine; this was then mixed and incubated at 30 C for 90 min, after which the samples were placed on ice. Translation was assessed by separation on a 12% SDS-PAGE gel, and the gel was dried using a gel dryer followed by exposure to Hyperfilm-MP (Amersham) x-ray film overnight.
Expression of type II 3ßHSD and mutated A10E complementary DNAs (cDNAs)
All media and supplements for cell culture were purchased from Life Technologies, Inc. (Grand Island, NY), except for FCS, which was purchased from Wysent, Inc. (St. Bruno, Canada). Ad293 cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in DMEM/low glucose supplemented with 10% FCS, 1% glutamine, 100 IU/mL penicillin, and 50 µg/mL streptomycin until confluence. Cells were plated in 6-well plates at a density of 400,000 cells/well and were allowed to settle overnight. Medium was changed to DMEM without any supplements just before transfection. Transient transfections were performed using ExGen 500 cationic polymer transfection reagent (MBI Fermentas, Inc., Amherst, NY) according to the manufacturer’s protocol. Mock transfections were carried out with pcDNA3 alone, and transfection efficiency was monitored by cotransfection of a ß-galactosidase expression vector, the activity of which was assessed using the chemiluminescent reporter gene assay system from Tropix (Bedford, MA).
Northern blot analysis
To verify cDNA transcription, total ribonucleic acid (RNA) was
prepared from transfected cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the
manufacturer’s protocol. Total RNA was solubilized in FORMAzol
(Molecular Research Center, Inc.) and stored at -80 C.
For RNA blot analysis, 3 µg total RNA were loaded in each lane and
subjected to electrophoresis in a 1% agarose gel containing 2%
formaldehyde in 1 x 3-[N-morpholino]propanesulfonic
acid (MOPS) buffer. The gel was then transferred by capillarity to
GeneScreen Plus positively charged nylon membrane (Mandel Scientific
Co., Guelph, Canada), and RNA was immobilized by UV cross-linking.
Hybridization with an [-32P]dCTP-labeled
500-bp fragment of type II 3ßHSD generated by PCR was performed in
50% formamide-containing buffer at 42 C, according to the membrane
supplier’s protocol. After hybridization, the membranes were washed in
2 x SSC (1 x SSC = 0.15 mol/L NaCl, 15 mmol/L
Na3citrate·2H2O, and 50
mmol/L Tris-Cl, pH 7.0) at room temperature for 10 min (twice), once in
2 x SSC/1% SDS at room temperature for 10 min, followed by
2 x SSC/1% SDS at 52 C for 10 min. Control hybridization was
performed by cohybridization with a human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA HindIII-XbaI fragment
of 548 bp. Membranes were exposed to Hyperfilm-MP x-ray film at -80 C
for 16 h.
Western blot analysis
Western blot analysis of proteins prepared from transiently transfected Ad293 cells was performed by SDS-PAGE on discontinuous acrylamide gels. Samples were prepared for loading by denaturing at 95 C in 2% SDS, 10% glycerol, 62.5 mmol/L Tris (pH 6.8), and 0.1% dithiothreitol and were electrophoresed at 200 V through 4% stacking and 12% resolving gels using the Mini-Protean II Western apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). Twenty-five micrograms of total protein were loaded per lane, and prestained mol wt markers (Bio-Rad Laboratories, Inc.) were run in parallel lanes. After electrophoresis, proteins were transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech), blocked for nonspecific binding with 5% nonfat milk-phosphate-buffered saline (PBS)-0.1% Tween 20 (0.05 mol/L; pH 7.6) for 16 h at 4 C, then washed briefly in PBS-Tween 20 solution. Membranes were incubated with a polyclonal antibody directed against human placental type 1 3ßHSD at a dilution of 1:2,000 in blocking solution (5% nonfat milk-PBS-0.1% Tween 20) for 2 h at room temperature, washed with PBS-0.1% Tween 20, and incubated with donkey antirabbit IgG peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech) at a dilution of 1:10,000 in blocking solution for 1 h at room temperature. Membranes were washed and proteins were visualized using the Renaissance Plus detection kit (NEN Life Science, Boston, MA) followed by exposure of the membranes to x-ray film for 1–10 min.
3ßHSD enzymatic activity assay
To determine 3ßHSD enzymatic activity in intact transfected Ad293 cells, 10 nM [14C]DHEA (55.2 mCi/mmol; Mandel Scientific Co. Ltd.) was added in the medium at a final concentration of 1% (vol/vol) ethanol 24 h after transfection as previously described (19). After the indicated times, media were collected, and steroids were extracted with 4 vol diethyl ether and chilling the incubation mixture in a dry ice/ethanol bath. The organic phase was evaporated, and steroids were separated by TLC using a mobile phase of toluene-acetone (4:1). Chromatography plates were analyzed using a PhosphorImager imaging system (Molecular Dynamics, Inc., Sunnyvale, CA). All results are expressed as the mean ± SE of at least two separate transfection experiments, performed in triplicate.
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Results |
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To identify the molecular lesion responsible for the severe
salt-wasting and clinical presentations of both patients 1 and 2, we
determined the nucleotide sequence using a set of six primer pairs for
selective PCR amplification (15), including 1) the whole coding region
and exon-intron splicing boundaries; 2) the 5'-noncoding region,
including the putative promoter region; and 3) the 3'-noncoding region,
including the polyadenylation site of the HSD3B2 gene.
Sequencing of exon II of the HSD3B2 gene revealed the
presence of a C to A transversion in both alleles of patients 1 and 2,
converting codon 10 (GCA) encoding an alanine residue to GAA encoding a
glutamic acid residue (Fig. 1
). This
mutation was the only one found in the regions covered by the different
PCR amplicons. Moreover, the C to A transversion was found in the
heterozygous state in both parents of patient 2 as well as his brother
(Fig. 1). DNA samples from the biological parents of patient 1 were
unavailable.
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Genotyping of patients 1 and 2 as well as relatives was carried
out with 11 microsatellite markers spanning about 5.5 centimorgans
(cM), flanking the HSD3B2 locus on chromosomes 1p13.1 to
1q11. Genotyping results are shown diagrammatically as individual
chromosomes inherited by each member of patient 2’s family (Fig. 2). The two affected individuals
(patients 1 and 2) were homozygous at all loci genotyped, whereas the
healthy individuals from family 2 were heterozygous carriers. A
recombination phenomenon can be observed at marker D1S2344 for patient
1 showing that both patients share the same haplotype spanning
approximately 3.3 cM surrounding the HSD3B2 locus. This
observation is consistent with a founder effect for this missense
mutation in French Canadians.
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To assess the influence of the missense A10E mutation on type II 3ßHSD enzymatic activity, we compared the activity of normal type II 3ßHSD with that of mutant type II 3ßHSD A10E protein in vitro in transfected Ad293 cells expressing each corresponding cDNA. This mutation immediately follows the first Gly of the highly conserved fingerprint Gly-X-X-Gly-X-X-Gly domain (where X is any amino acid) found in all members of the 3ßHSD superfamily (for a review, see Ref. 6). It should be noted that the Ala10 is a highly conserved amino acid in the vertebrate 3ßHSDs and is located in the putative NAD-binding domain of the protein. Therefore, it might be expected that the insertion of the bulkier and negatively charged Glu residue, instead of the second smallest amino acid Ala, would disrupt the geometry of the putative NAD-binding site.
We investigated the time course of formation of
[14C]4-androstenedione
from [14C]DHEA in intact Ad293
cells transfected with pcDNA3,
pcDNA3 type II 3ßHSD, or
pcDNA3-A10E plasmid. As illustrated in Fig. 3, no activity could be detected for the
mutant A10E protein. Northern blot analysis demonstrated that both
wild-type and mutant transcripts were expressed at equal levels in
pcDNA3 type II 3ßHSD- and
pcDNA3-A10E-transfected Ad293 cells,
respectively, whereas no significant endogenous type II 3ßHSD
messenger RNA (mRNA; 1.7 kb) was detected in mock
pcDNA3-transfected cells, thus confirming the
efficacy of 3ßHSD transcription from the vector’s adenoviral
promotor.
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support the idea that there is no detectable change in the polyclonal
antibody-binding site as a consequence of this mutation. We then
performed Western blot analysis using the homogenates purified from the
same cells transfected with the indicated expression vector constructs,
which were used for RNA blot analysis illustrated in the Fig. 4A. A 42-kDa band corresponding to the
3ßHSD type II protein was detectable in the homogenate preparation
from Ad293-transfected cells expressing the wild-type protein, whereas
no band was detectable for the mutant recombinant protein (Fig. 4C).
Such a difference in immunoblot signal levels was observed in several
independent experiments and was also recently observed for several
other mutant recombinant 3ßHSD type II proteins (20).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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As in other enzyme deficiencies, the biochemical hallmark of the diagnosis is an elevated ratio of the plasma concentrations of precursor to product (17-hydroxypregnenolone to 17-hydroxyprogesterone or DHEA to androstenedione). However, the interpretation of plasma steroid levels in this condition is sometimes complicated by partial peripheral conversion of the 5-ene to 4-ene steroids by the type I isoenzyme (21, 22). The significant accumulation of precursors may also interfere with the RIAs used in routine laboratories. The biochemical diagnosis is further complicated in early infancy by the rapid age-related changes in the plasma concentrations of various steroids (23). Thus, although the diagnosis of severe 3ßHSD deficiency was suspected in both patients, it was only established through molecular studies that showed a homozygous C to A transversion in the HSD3B2 gene leading to an abolished 3ßHSD activity.
The underrepresentation of 46,XX patients for this autosomal recessive disease suggests that 46,XX individuals, because they do not, in general, present as intersex newborns, are not diagnosed properly and/or may die of adrenal crisis before diagnosis more often than 46,XY individuals. In a 1984 survey, Lafont (24) noted that of 20 patients with the severe salt-wasting form of 3ßHSD deficiency, only 6 were genetic females. Interestingly, the same sex ratio is found in the cases that have now been confirmed by mutation analysis (only 8 females out of 29 patients) (20). This is a mirror image of what occurs in the more common 21-hydroxylase deficiency, in which there is an underrepresentation of 46,XY individuals diagnosed clinically (1).
The finding of a significant peripheral conversion of
5-hydroxysteroids in 3ßHSD-deficient
patients is in accordance with the observation that a large proportion
of androgens (
40% in men) and the majority of estrogens formed in
children and in anovulatory or postmenopausal women arise from
extragonadal steroidogenesis (for review, see Ref. 25). Indeed, 3ßHSD
activity and/or type I 3ßHSD expression have been reported in several
peripheral tissues in humans and primates, including, skin, breast,
endometrium, myometrium, prostate, epididymis, adipose tissue, liver,
and colon (6, 8, 26, 27, 28). On the other hand, it has also been suggested
that although only very low levels of type I 3ßHSD mRNA can be
detected in normal gonads by sensitive ribonuclease protection assay,
4-steroids can originate from gonadal type I
3ßHSD activity, which possesses a roughly 5-fold higher activity than
the type II isoenzyme and could be stimulated after an increase in
gonadotropin secretion resulting from low circulating androgen levels
(8, 10). Indeed, Yoshimoto et al. (29) recently showed that
pubertal levels of gonadotropins may increase intratesticular 3ßHSD
activity despite the low levels of HSD3B1 transcripts.
Our 46,XY patient’s adult height within the target range suggests that the overall control of hyperandrogenism during growth was satisfactory. However, the observation of bilateral testicular masses probably reflects the stimulation of adrenal rests by elevated ACTH. This is observed in about 18% of adult males with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (30), but specific prevalence estimates for 3ßHSD deficiency are lacking because of the small number of patients. Whether these adrenal rests play a role in the azoospermia of our patient remains to be determined.
As stated above, there are very few cases of severe salt-wasting
3ßHSD deficiency that have been diagnosed in genetic females, and
only Zachmann et al. (11, 12) have reported a follow-up at
pubertal age. This patient presented only minimal breast development at
14.7 yr of age. While she was treated with gluco- and
mineralocorticoids, she showed no increase in plasma testosterone,
estradiol, or estrone after 5 days of gonadotropin injections and
required estrogen treatment to undergo complete feminization, including
menses. In the absence of sex steroid replacement, she had continuously
elevated LH and FSH levels and never had signs of ovulation. After
stopping estrogen/progesterone treatment in adulthood, her menses
ceased, and she developed multiple ovarian cysts (Zachmann, M.,
personal communication). In contrast, our patient underwent progressive
feminization starting between 8–9 yr. It is noteworthy that the
patient described by Zachmann et al. (11, 12) has been
demonstrated to be homozygous for the nonsense mutation W171X, which
would lead to a truncated protein of 169 amino acids instead of 371
amino acids in the normal type II isoenzyme (10), thus resulting in a
complete loss of type II 3ßHSD activity. Our patient’s missense
mutation, A10E, affects a highly conserved, glycine-rich sequence of
the NAD-binding domain and also results in complete absence of 3ßHSD
activity. Thus, these two mutations have equally severe functional
consequences in vitro. One possible explanation for the
development of breast and endometrium in patient 1 is local conversion
of inactive adrenal precursors to estrogens. In addition, it should be
mentioned that the adrenal precursor
5-androstenediol exerts by itself a potent
estrogenic effect at nanomolar concentrations in several target tissues
(26, 31, 32, 33). On the other hand, it is possible that, as suggested
above for the testes, pubertal levels of gonadotropins may induce
sufficient 3ßHSD activity by increasing the normally low levels of
type I 3ßHSD expression in the ovary, thereby allowing significant
ovarian production of estradiol; the patient’s very recent rise in
plasma progesterone levels favors the latter hypothesis and raises the
possibility that she may be fertile.
In summary, we describe two French-Canadian patients with severe salt-wasting 3ßHSD deficiency who were homozygous for a novel missense (A10E) mutation in the HSD3B2 gene resulting in the synthesis of an unstable protein and the complete loss of enzymatic activity. The 46,XY patient presented with ambiguous genitalia at birth and underwent normal masculinization at puberty, but was azoospermic. The 46,XX patient presented progressive breast development, menarche, and evidence of progesterone secretion. The only previously reported cases of severe 3ßHSD deficiency with postpubertal follow-up revealed paternity in one male and hypogonadism in one female. These findings demonstrate the complex relationships between genotype and gonadal phenotype and the difficulty in predicting fertility in this condition.
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Acknowledgments |
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Footnotes |
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Received July 29, 1999.
Revised November 10, 1999.
Accepted January 11, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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5-4 isomerase gene
family. J Endocrinol. 150(Suppl):S189–S207.
5-4-isomerase in
human adrenals and gonads. Mol Endocrinol. 5:1147–1157.AA in type II 3ß-hydroxysteroid dehydrogenase gene
(HSD3B2) in three male patients of Afghan/Pakistani origin. Hum Mol
Genet. 3:327–330.5-4-isomerase close
to D1S514 and the centromeric D1Z5 locus. Cytogenet Cell Genet. 69:59–62.[Medline]
5-4 isomerase type
1 gene transcription in human breast cancer cell lines and in normal
mammary epithelial cells by interleukin-4 and interleukin-13. Mol
Endocrinol. 13:66–81.5-4 isomerase
(3ß-HSD), 17ß-HSD 5-reductase and aromatase activities in the
rhesus monkey. Mol Cell Endocrinol. 104:103–111.[CrossRef][Medline]
5-steroids of the human
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E. Codner, C. Okuma, G. Iniguez, M. A. Boric, A. Avila, M. C. Johnson, and F. G. Cassorla Molecular Study of the 3{beta}-Hydroxysteroid Dehydrogenase Gene Type II in Patients with Hypospadias J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 957 - 964. [Abstract] [Full Text] [PDF]
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S. Pang, W. Wang, B. Rich, R. David, Y. T. Chang, G. Carbunaru, S. E. Myers, A. F. Howie, K. J. Smillie, and J. I. Mason A Novel Nonstop Mutation in the Stop Codon and a Novel Missense Mutation in the Type II 3{beta}-Hydroxysteroid Dehydrogenase (3{beta}-HSD) Gene Causing, Respectively, Nonclassic and Classic 3{beta}-HSD Deficiency Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2556 - 2563. [Abstract] [Full Text] [PDF]
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M. Iitaka, S. Miura, K. Yamanaka, S. Kawasaki, S. Kitahama, Y. Kawakami, S. Kakinuma, I. Oosuga, S. Wada, and S. Katayama Increased Serum Vascular Endothelial Growth Factor Levels and Intrathyroidal Vascular Area in Patients with Graves' Disease and Hashimoto's Thyroiditis J. Clin. Endocrinol. Metab., November 1, 1998; 83(11): 3908 - 3912. [Abstract] [Full Text]
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