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Original Studies |
Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics-Gynecology and Biochemistry (N.M., S.A.), University of Texas Southwestern Medical Center, Dallas, Texas 75235; the Department of Pediatrics (I.A.H.), University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom; and the Department of Medicine (A.D.), Section of Diabetes and Metabolism, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Stefan Andersson, Ph.D., Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: andersson{at}grnctr.swmed.edu
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Abstract |
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AGT). The frequency of the G allele (Gly) was 0.94,
and that of the A allele (Ser) was 0.06. No difference in the
frequencies of the G and A alleles was detected in 32 apparently normal
women and 46 women with polycystic ovary syndrome. Enzymes bearing
either glycine or serine at this position have similar substrate
specificities and kinetic constants. The current findings boost to 16
the number of mutations in the HSD17B3 gene that impair testosterone
synthesis and cause male pseudohermaphroditism, and add 1 apparently
silent polymorphism to this tally. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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17ßHSD type 3 is an integral membrane protein of the endoplasmic reticulum that uses NADPH as cofactor (7). Autosomal recessive mutations affecting its function give rise to a rare form of male pseudohermaphroditism, referred to as 17ßHSD deficiency (7, 9, 10). This inborn error of metabolism was originally described in 1971 (11); the characteristic phenotype is found in 46,XY individuals with testes and male Wolffian duct-derived internal genitalia but female external genitalia and the absence of a prostate. At the expected time of puberty there is a marked increase in plasma levels of androgens, thereby causing virilization. The common endocrine disorder polycystic ovary syndrome also often presents with peripubertal onset of masculinization (12, 13). Thus, we sought to determine whether some cases of the syndrome might be caused by mutations in the HSD17B3 gene.
The HSD17B3 gene is localized on chromosome 9q22 and is comprised of 11 exons (7). To date, 14 mutations in the HSD17B3 gene have been characterized at the molecular level in individuals with 17ßHSD deficiency (7, 9). Of these, 10 are missense mutations, 3 are splice junction abnormalities, and 1 is a small deletion that results in a frame shift. Nine of the missense mutations have been reconstituted by transient expression in cultured cells, and their functional consequences have been determined. Only the R80Q mutation results in a 17ßHSD type 3 with detectable enzymatic activity, albeit with a significantly lower reaction velocity than that of the normal enzyme.
In the current study, we describe two additional missense mutations in the HSD17B3 gene of male pseudohermaphrodites, and a polymorphism (G289S) present in a heterozygous form in apparently normal individuals.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Genomic DNA was obtained from 32 women (15 Caucasians and 17 African Americans) with no apparent abnormality in steroid hormone metabolism and 46 women (45 Caucasians and 1 African American) with polycystic ovary syndrome. Polycystic ovary syndrome was diagnosed by the presence of 6 or fewer menses/yr, a total testosterone level of 85 ± 33 ng/dL, and a nonsex hormone-binding globulin testosterone level of 33 ± 17 ng/dL. The normal control female levels of these androgens are 33 ± 13 and 8 ± 4 ng/dL, respectively (12, 13, 14, 15). Nonclassical adrenal 21-hydroxylase deficiency was excluded by a 1-h ACTH test with basal and 1 h 17-hydroxyprogesterone levels. Circulating PRL levels were within the normal range. All of the PCOS women were from South Central Pennsylvania. This study was approved by the institutional review boards of the Pennsylvania State University College of Medicine, and written informed consent was obtained from all subjects.
17HSD3-Cambridge 1
This girl was evaluated at age 14 yr, because of a 2-yr history of increasing hirsutism, deepening of the voice, and lack of breast development. On physical examination, she showed a male physique, deep voice, extensive body hair, and an enlarged clitoris (4 x 2 cm). The karyotype was 46,XY. Serum testosterone was 14.6 nmol/L, and serum androstenedione was 39.1 nmol/L. Serum LH was 21.5 IU/L, and serum FSH was 40.7 IU/L. A magnetic resonance imaging scan revealed two testes in the inguinal regions. Bilateral gonadectomy was performed. Histological examination of both testes showed thickening of tunica albuginea. The seminiferous tubules were closely packed and lined by Sertoli cells only. No germ cells were identified, and there were large numbers of Leydig cells in the interstitium. The epididymes were normal. DNA was prepared from cultured skin fibroblasts.
17HSD3-Cambridge 2
This subject was born with apparently normal female genitalia. At age 11 yr, increased facial, axillary, and pubic hair; increased body hair; and deepening of her voice and enlargement of the clitoris were noted. The parents are first cousins; one brother and two sisters are apparently normal. On physical examination at age 12.3 yr, she had a deep voice, extensive facial hair growth, and a male pattern of body hair distribution. There was Tanner stage III axillary hair and stage IV pubic hair, and no breast development. The clitoris was enlarged, and inguinal gonads were palpable bilaterally. The karyotype was 46,XY. Endocrine findings included serum testosterone of 10.0 nmol/L and serum androstenedione of 28.5 nmol/L; these values increased to 12.5 and 42.6 nmol/L, respectively, after hCG administration (1500 IU daily for 3 days). Serum LH was 13.9 IU/L, and serum FSH was 15.8 IU/L. Ultrasound examination confirmed two masses anterior of the pubis measuring 3.6 x 1.3 cm. Laparoscopy showed no uterus or Fallopian tubes; there was a normal opening to a short vagina and absent cervix on vaginoscopy. Cytoscopy showed a normal urethra and bladder. Bilateral gonadectomy and clitoral reduction was performed. Histological examination of both testes showed that the seminiferous tubules were lined by Sertoli cells only. There was some thickening of the basement membrane and a marked diffuse increase in Leydig cells. Sections from epididymes and vas deferens were normal. DNA was prepared from cultured skin fibroblasts.
Mutation detection and expression analysis
Genomic DNA was extracted from white blood cells and cultured fibroblasts as described previously (9) or using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Mutations in the HSD17B3 gene were detected by single strand DNA conformation analysis (SSCP) as previously described (9), except that a nondenaturing 0.5 x MDE polyacrylamide gel (J. T. Baker, Phillipsburg, NJ) electrophoresed at 3-watts constant power for 16 h at room temperature was used to separate the PCR products. DNA sequencing, oligonucleotide-directed mutagenesis, and complementary DNA (cDNA) expression in 293 cells were performed as described previously (9). Enzyme activity assays were performed as described previously (7), except that for in vitro assays, freeze-thawing was performed in 100 mmol/L Tris-HCl (pH 7.0) and 1 mmol/L EDTA, and incubations were performed in a buffer containing 100 mmol/L Tris-HCl (pH 7.0), 1 mmol/L EDTA, and 2 mmol/L MgCl2. Where indicated, 20% (vol/vol) glycerol was included in the assay buffer. Kinetic constants (apparent Km and maximum velocity) were determined by a nonlinear regression analysis paradigm for the Michaelis-Menten equation using the K.CAT program (Bio Metallics, Princeton, NJ) (16).
Immunoblotting
A synthetic carboxy-terminal peptide [C]LKLNTKVR,
corresponding to amino acid residues 303–310 in 17ßHSD type 3 (7),
was coupled to keyhole limpet hemocyanin (KLH) and used for
immunization of mice as previously described (17). Hybridomas were
established and screened for antibody production as previously
described (17). Immunoblotting of transfected 293 cell extracts using
the anti-17ßHSD type 3 monoclonal antibody MAb-C3–10 (subclass
IgG1/
) was performed as previously described (17).
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Results |
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The endocrine disorder polycystic ovary syndrome often presents with
peripubertal onset of masculinization due to increased ovarian androgen
secretion. To investigate whether the HSD17B3 gene is mutated in
patients with this disease, we screened the gene in 46 afflicted women.
A single base pair polymorphism was detected by SSCP analysis of a
207-bp PCR-amplified DNA fragment encompassing exon 11 of the gene. The
polymorphic A allele encodes a protein with a serine rather than a
glycine at position 289 (GGTAGT). The
frequency of the G allele (Gly) was 0.94, and that of the A allele
(Ser) was 0.06. The calculated polymorphic information content value
was 0.11. The polymorphism showed a codominant Mendelian inheritance
pattern, and the A allele was only found in heterozygous form.
AciI and BanI restriction sites are present in
codon 289 of the G allele, but are absent in the A allele. The same
frequencies of the G and A alleles were observed when genomic DNAs from
32 women with no apparent abnormality in steroid hormone metabolism
were analyzed. Thus, in this sample size, no difference in the
frequencies of the G and A alleles was detected in apparently normal
women and women with polycystic ovary syndrome.
To determine the functional consequences of these mutations in the
17ßHSD type 3 protein, site-directed mutagenesis and expression of
the altered proteins in cultured cells were performed. Figure 1A
shows the results of a time-course
experiment in which 293 cells were transiently transfected with
expression vectors harboring the normal or mutated cDNAs, or with the
pCMV vector alone. Forty-eight hours after transfection,
[3H]androstenedione (1 µmol/L) was added to the cell
medium, and conversion of this substrate to testosterone was monitored
at the indicated times by thin layer chromatography and radioactivity
scanning. The N130S and A56T mutations severely compromised enzyme
activity. The Q176P mutation, previously described (9) but herein
analyzed for its functional consequences, also severely impaired enzyme
function (Fig. 1A). The polymorphic G289S substitution resulted in an
enzyme that possessed kinetic properties similar to those of the normal
enzyme (Fig. 1A). To confirm that the different enzymes were equally
expressed in the transfected cells, we performed immunoblotting using a
monoclonal antibody directed against the carboxy-terminus of 17ßHSD
type 3 (Fig. 1B).
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), androstanedione to
dihydrotestosterone (Fig. 2B), dehydroepiandrosterone to
5-androstenediol (Fig. 2C), androsterone to androstanediol (Fig. 2D),
and estrone to estradiol (Fig. 2E). Immunoblotting experiments
demonstrated expression levels of the two enzymes in the transfected
cells similar to those shown in Fig. 1B
(lanes 1 and 6),
i.e. slightly higher expression of the S289 enzyme compared
to the G289 enzyme (data not shown). Cells coexpressing the two enzymes
demonstrated the same kinetic profile as those expressing the
individual proteins (data not shown).
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). The A56T mutation resulted in
a 20-fold increase in the apparent Km for the NADPH
cofactor and a 6-fold increase in the apparent Km for
androstenedione. The kinetic constants for enzymes bearing the G289 or
S289 polymorphism were indistinguishable. Accurate kinetic constants
could not be determined for the N130S and Q176P enzymes, even in the
presence of the protein stabilizing agent glycerol. As revealed by
immunoblotting, individual cell extracts contained slightly different
amounts of recombinant enzyme, hence, the maximum velocity values were
normalized for immunoreactive 17ßHSD type 3 protein (data not
shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A total of 16 mutations have now been described in the HSD17B3 gene in subjects with 17ßHSD deficiency. Twelve of these are missense mutations, 3 are splice junction abnormalities, and 1 is a small deletion that results in a premature stop codon. 17ßHSD type 3 belongs to the short chain dehydrogenase/reductase (SDR) gene family, which consists of approximately 60 different enzymes (18). The low protein sequence identity (15–30%) between members of this family implies that they evolved by distant duplications and early divergence. Interestingly, pairwise comparisons of the human 17ßHSD types 1, 2, and 3 reveals a 23% protein sequence identity, surprisingly low in view of the fact that they each catalyze dehydrogenation/hydrogenation at C-17 in the same steroid substrates (7, 19).
A common structure of all members of the SDR family is the cofactor binding fold, termed the Rossmann fold, with a characteristic Gly-X-X-X-Gly-X-Gly motif in the amino-terminal part of the enzyme molecules (20, 21). The corresponding sequence in 17ßHSD type 3 is Gly55-Ala56-Gly57-Asp58-Gly59-Ile60-Gly61. In the 17HSD3-Cambridge 2 allele, the alanine residue at position 56 is substituted with a more hydrophilic threonine, suggesting that this mutation would result in an enzyme with an altered cofactor affinity. In fact, kinetic analysis of the A56T mutation revealed a 20-fold decrease in NADPH cofactor affinity. Interestingly, a 6-fold decrease in affinity for the substrate was also observed. This has been seen elsewhere, in that a missense mutation in the glycine-rich cofactor binding motif of 3ßHSD type 2 was reported to decrease affinities for both cofactor and substrate, thereby causing 3ßHSD deficiency (22).
Asparagine at position 130 of 17ßHSD type 3 is one of the most conserved residues among the members of the SDR family (18). Although the function of this residue is not clear, substitution of this positively charged arginine with a hydrophilic serine in the 17ßHSD type 3 enzyme results in severe attenuation of enzymatic activity. The N130S mutation was only found in a single allele of the 17HSD3-Cambridge 1 patient. With our method of screening, we would have found any mutations that affected the classical exon-intron splice junctions; however, it is conceivable that the other allele contains a mutation that creates a splice donor or acceptor site distant from the exon-intron junctions. This would, in turn, cause the formation of a aberrantly spliced 17ßHSD type 3 messenger ribonucleic acid, a hypothesis that could have been tested by RT-PCR, but gonadal tissue from this patient was not available. This subject presented with the phenotypical and biochemical hallmarks of 17ßHSD deficiency, and this disorder is inherited in an autosomal recessive fashion; therefore, we strongly believe that the other allele contains a mutation in the promoter region, or an intron, of the gene, thus affecting transcription or splicing of the 17ßHSD type 3 messenger ribonucleic acid and bringing forth the disease.
The Q176P mutation, previously described, is shown here to have low
activity in transfected intact cells and no demonstrable activity in
cell lysates. Substitution of a glutamine for a proline may result in
an unstable and/or incorrectly folded protein, as proline residues are
known to cause bends in the 
-helixes of protein structures due to an
inability to form a hydrogen bond from the main chain nitrogen. In an
effort to address the stability issue, the protein-stabilizing agent
glycerol was used in assays with the Q176P as well as the N130S enzymes
under conditions proven successful in reconstituting unstable mutant
3ßHSD type 2 enzymes (23). However, glycerol did not preserve or
restore enzyme activities to these mutant proteins.
The G289S polymorphism showed a Mendelian codominant inheritance pattern and was found in heterozygous form in normal individuals. We investigated the incidence of the G289S allele in women with polycystic ovary syndrome. Screening of 92 chromosomes from these subjects revealed no significant difference in the frequencies of the G289 or S289 alleles compared to those found in the chromosomes of apparently normal subjects. Kinetic analysis of the polymorphic enzymes using five physiological steroid substrates did not show any significant differences in specificity or catalytic efficiency. We conclude that G289S is an apparently silent polymorphism in the HSD17B3 gene. The DNA sequence changes that underlie these two alleles of the HSD17B3 gene may, however, be useful as genetic markers in subjects with other disorders of androgen and estrogen metabolism.
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Acknowledgments |
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Footnotes |
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Received January 14, 1998.
Revised May 6, 1998.
Accepted May 8, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-helixes in dinucleotide
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), or mock (
), were transfected into
human embryonic kidney 293 cells and assayed for activity by adding
tritium labeled steroids (10 nmol/L) to the medium. Aliquots of the
medium were collected at the indicated times, and the conversion of
androstenedione to testosterone (A), androstanedione to
dihydrotestosterone (B), dehydroepiandrosterone to 5-androstenediol
(C), androsterone to androstanediol (D), and estrone to estradiol (E)
was monitored by thin layer chromatography and radioactivity
scanning.