| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Service d’Hormonologie (P.P., F.P., C.S.), Hôpital Lapeyronie, Centre Hospitalier Universitaire Montpellier, 34295 Montpellier cedex 5, France; Division of Paediatric Endocrinology and Diabetology (A.B.-L., D.K., E.S.), University Children’s Hospital, 8032 Zurich, Switzerland; Service de Gynécologie-Obstétrique (R.R.), Centre Hospitalier Intercommunal de Creteil, 94010 Creteil, France; Service d’Endocrinologie Pédiatrique (C.P.), Centre Hospitalier Universitaire de Toulouse, 31052 Toulouse, France; and Unité d’Endocrinologie et Gynécologie Pédiatrique (F.P., C.S.), Service de Pédiatrie I, Hôpital Arnaud de Villeneuve, Centre Hospitalier Universitaire Montpellier, 34090 Montpellier, France
Address all correspondence and requests for reprints to: Professor Charles Sultan, Service d’Hormonologie, Hôpital Lapeyronie, Centre Hospitalier Universitaire Montpellier, 34295 Montpellier cedex 5, France. E-mail: c-sultan{at}chu-montpellier.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Objectives: This collaborative work was designed to determine whether the WNT4 mutation could be identified in a group of adolescent girls with Mayer-Rokitansky-Küster-Hauser syndrome.
Results: We analyzed 28 DNA samples from adolescent girls with primary amenorrhea and failure of müllerian duct formation by direct sequencing and identified a new L12P mutation within exon 1 of the WNT4 gene. The substitution of leucine by proline is crucial for the conformation of the expressed protein. This amino acid substitution is unlikely to be a polymorphism because it was not found in 100 DNAs from control subjects. Functional analysis revealed that the mutation induces significantly increased expression of the enzymes involved in androgen biosynthesis (3β-hydroxysteroid dehydrogenase and 17
-hydroxylase). It is interesting to note that the adolescent carrying the mutation was referred to our clinic for primary amenorrhea and hyperandrogenism (severe acne and plasma testosterone: 1.8 vs. 1.2 nmol/liter in controls). She also presented with uterine hypoplasia and follicle depletion.
Conclusions: We suggest that in adolescent girls with primary amenorrhea, müllerian duct abnormalities, and hyperandrogenism, a WNT4 mutation should be sought. Moreover, our data confirm that WNT4 is involved in the regulation of müllerian duct development and ovarian androgen biosynthesis. WNT4 may also contribute to human follicle development and/or maintenance.
|
Introduction |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
In mice, Wnt4 is important for the development of several organs, including the fallopian tubes, uterus, and ovaries, as well as the mammary glands. Wnt4-deficient female mice lack müllerian ducts and show partial female-to-male sex reversal along with the degeneration of follicles (1). The sex-reversal phenotype observed in Wnt4–/– mice is characterized by the presence of wolffian ducts in females (1), a first indication that Wnt4 might be involved in regulating testosterone levels in vivo. Accordingly, analyses on XX Wnt4–/– gonads showed ectopic expression of two steroidogenic enzymes required for testosterone biosynthesis: 3β-hydroxysteroid dehydrogenase (3β-HSD) (HSD3B2), which converts pregnenolone to progesterone, an intermediate in testosterone synthesis; and 17-hydroxylase/C17–20 lyase (P450c17 or Cyp17a1), which catalyzes two reactions converting progesterone to androstenedione (1). Indeed, recent analyses of XX gonads from Wnt4–/– mice revealed that these ovaries produce testosterone (2). In parallel, the use of microarray analyses to compare gene expression between wild-type (WT) and XX Wnt4–/– gonads at 12.5 days post coitum (dpc) revealed that a number of genes involved in testosterone synthesis were elevated in the Wnt4–/– gonad. Testosterone was present in both the gonads and serum of XX Wnt4–/– mice, and was absent from the gonads and serum of WT females. In agreement with these results, the role of Wnt4 in the inhibition of androgen biosynthesis was confirmed by lower testosterone levels in the serum and testis of male mice overexpressing Wnt4 and showing a reduced size of the seminal vesicles, which are androgen sensitive (3). Furthermore, expression of the steroidogenic acute regulatory protein, which shuttles cholesterol into the mitochondria and is, therefore, a rate-limiting step in steroidogenesis, was absent in the testis of these Wnt4-overexpressed transgenic males.
In humans, two female patients carrying a heterozygous mutation in the WNT4 gene were recently identified. Both displayed gonadal phenotypes strikingly similar to that of the Wnt4-knockout mice (4, 5). Examination revealed müllerian duct abnormalities along with clinical and biological evidence of hyperandrogenism. In vitro, the mutant WNT4 protein was unable to repress the expression of 3β-HSD (HSD3B2) and 17-hydroxylase/C17–20 lyase (CYP17A1) in an ovarian cell line. As expected, transfection of the mutant WNT4 plasmids into an ovarian adenocarcinoma cell line did not repress the production of progesterone, hydroxyprogesterone, androstenedione, or testosterone (4, 5).
This study investigated the involvement of the WNT4 gene in the development of genital tracts and gonads. We analyzed a cohort of 28 adolescent girls with primary amenorrhea, XX karyotype, and müllerian duct abnormalities for WNT4 gene mutations.
|
Patients and Methods |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
The 28 adolescent girls were referred for primary amenorrhea along with subnormal female phenotype and varying degrees of müllerian duct abnormality. The study was approved by the institutional review board.
Genomic DNA mutational analysis and expression studies
After obtaining informed consent, genomic DNA was extracted from the peripheral blood leukocytes of these patients. The entire coding region and splice sites of the WNT4 gene were PCR amplified using primers and conditions described previously (4). These PCRs were sequenced directly using BigDye terminator v1.1 (Applied Biosystems, Courtaboeuf, France) and an ABI Prism 310 DNA sequencer (Applera Corp., Courtaboeuf, France). DNAs from 100 normal ethnically matched female controls were simultaneously analyzed.
To study the functional implications of the mutation, we expressed WT and mutant WNT cDNA in human ovarian adenocarcinoma NIH:OVCAR3 (ATCC HTB-161), and analyzed the influence of WNT4 variants on steroidogenic enzyme expression and activity, as previously reported (4).
WNT4 was labeled in vivo with tritiated palmitate, as described previously (6), and the assay was performed in cell extract after immunoprecipitation of the transfected WNT4 using anti-X-press antibodies (4).
The amount of β-catenin in cell lysates was quantified using the Human Total β-catenin DuoSet IC ELISA (R&D Systems, Inc., Minneapolis, MN), as suggested by the provider. The already described E226G mutation (4) was used as an internal control in all assays.
|
Results |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
As mentioned in the Patients section, the 28 adolescent girls presented with primary amenorrhea, in contrast with normal breast and pubic hair development for age. In all cases, normal gonadotropic axes (plasma FSH level) were noted, prompting investigation of müllerian duct development. Only one of the 28 girls presented with clinical (and biological) hyperandrogenism. She had been referred to our pediatric endocrinology clinic for primary amenorrhea at 16 yr, 8 months of age. At clinical examination, she presented with clinical hyperandrogenism with microcystic acne (face, back, chest), and pubertal development was scored as Tanner IV. The WNT4 mutation was found in this girl.
Hormonal analysis showed a high normal testosterone level, 1.8 nmol/liter (0.52 ng/ml) (normal range 0.3–2.0), and slightly elevated androstenedione, 7.15 nmol/liter (2.05 ng/ml) (normal range 1.7–6.9). Levels of dehydroepiandrosterone (DHEA) (4.0 µmol/liter, normal range 1.3–5.4) and 17-hydroxyprogesterone (17OHP) (3.3 nmol/liter, normal range 0.3–3.3) were normal. The LHRH stimulation test showed an increased LH response (peak 42.5 mUI/ml), whereas the FSH response was only slightly elevated (peak 8.1 mUI/ml).
Pelvic ultrasonography revealed a hypoplastic uterus (length: 25 mm). The right ovary was subnormal (37 x 13 mm), whereas the left was normal (29 x 6 mm). No follicles were visible.
At surgery, the ovaries were observed to be whitish and dystrophic. Fallopian tubes were present but covered with fibrous tissue. Left ovarian biopsy revealed the presence of only a very few follicles.
Mutational analysis
Analysis of the WNT4 gene in this patient’s DNA revealed a heterozygous T to C mutation in position 35 of open reading frame, leading to a L12P substitution in the protein sequence. This genetic anomaly was localized within the sequence of the secretion peptide signal (Fig. 1
).
|
Figure 2 shows the analysis of mRNA (real-time quantitative PCR and reverse-transcribed PCR qualitative mRNA assay), which revealed slight inhibition by wild type on the HSD3B2 mRNA but no inhibition of the mutated WNT4 alone or in combination with wild type, indicating that L12P is dominant negative, providing a phenotype/genotype correlation. That L12P is dominant negative, is demonstrated by the dose-response experiments in which the effect of increasing dose of WT WNT4 (1:1; 1:2; and 1:3) was determined by β-catenin measurement. As shown in Fig. 5, at least a 3-fold excess in WNT4 WT construct is necessary to overcome the inhibition by the mutant WNT4, thus demonstrating the negative dominance. This inhibition of mRNA expression by wild type was seen when CYP17A1 was analyzed. In the case of CYP17A1 expression, the mutant WNT4 was unable to inhibit the expression of this androgenic enzyme, as expected (ANOVA, P = 0.0058).
|
|
). WT WNT4 decreased the amount of progesterone, 17OHP, DHEA, androstenedione, and testosterone (ANOVA, P = 0.0006).
|
). Accordingly, the amount of β-catenin was significantly less (P < 0.0005) when the mutant L12P WNT4 was overexpressed in OVCAR3 cells (Fig. 5
). The amount of β-catenin from the nuclear lysates of these cells was decreased in the cells transfected with the mutant WNT4 alone or in combination with an increased amount of WT protein. This suggests that the signaling pathway was weakened because the activation of the canonical WNT signaling cascade results in stabilization of β-catenin. Because the function of WNT proteins is dependent on lipid modification, we checked the palmitoylation of WT and mutant WNT4. As depicted in Fig. 6B, the mutant L12P WNT4 was not lipid modified, and the combination of the two variants led to reduced lipid attachment to WT WNT4 as well, again suggesting a dominant negative type of interaction.
|
). |
Discussion |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
rnusse/wntwindow.html). WNT4 is involved in gonadal development and, therefore, is also expressed in the developing mesonephros (8). Mouse knockout models indicate the crucial role of Wnt4 in female development (1). Mutant females fail to develop müllerian ducts, whereas wolffian ducts are stabilized in the female null mutant because of differentiation of Leydig-like interstitial cells in the ovary. Thus, Wnt4 appears to prevent Leydig cell differentiation in the ovary. In addition, Vainio et al. (1) reported that 3β-HSD, an enzyme strongly expressed in Leydig cell precursors in the testis, is expressed in the ovary of female Wnt4-null mice at 14.5 dpc and that this expression continues until birth. The presence of inappropriate steroidogenic cell markers such as Cyp17a1 and 3β-HSD in the gonad of XX Wnt4–/– mice prompted investigations to understand how these enzymes, usually present in adrenal and testicular Leydig cells, and not in the ovary, were expressed in the extinction of Wnt4 (9).
We present here the third case of Mayer-Rokitansky-Küster-Hauser (MRKH)-like syndrome associated with hyperandrogenism due to a WNT4 mutation. Our patient presented with a lack of uterine development associated with hyperandrogenism and mild follicle depletion. The new mutation in our patient is a C to T substitution in exon 1, leading to an L12P substitution. This mutated leucine in position 12 is localized within the amino-terminal signal sequence (Fig. 1). The secretion signal is highly conserved throughout the mammalian species (Fig. 4), as well as in the WNT family, indicating that all proteins of this family are secreted. However, our current state of knowledge does not allow us to explain the apparent contrast between domain prediction and the actual results, i.e. the lack of secretion problems despite a mutation in the signaling peptide. On the other hand, prediction of the precise functional consequences of a mutation from any structure is never trivial. Lipid modification is essential for the correct activity of WNT4. Although lipid modification was impaired in the mutant WNT4 and secretion was decreased, whereas not as severely as in wild type, the production and secretion of the wild type were not negatively influenced by the coexpression of the mutant L12P WNT4 protein. On the other hand, at least a 3-fold excess of the WT construct is necessary to overcome the effect of the mutant WNT4, i.e. L12P has a demonstrated dominant negative effect. Nevertheless, the activation of the canonical β-catenin/Wnt pathway seemed to be impaired. This suggested that receptor binding, the step downstream of secretion but upstream of signal transduction, was most likely impaired. (Further studies are necessary to prove this hypothesis.)
|
According to the most accepted view, WNT-secreted proteins bind to the frizzled family of receptors and start at least three intracellular pathways, resulting in the regulation of gene expression and/or changes in cell behavior. The canonical pathway involves a coreceptor from the low-density lipoprotein receptor family and results in stabilization of intracellular β-catenin. β-Catenin is a multifunctional protein that can act as a transcriptional regulator upon entering the nucleus. However, it is rapidly degraded by ubiquitination after phosphorylation by a complex consisting of adenomatous polyposis coli and axin, which facilitates the phosphorylation of β-catenin by casein kinase 1 and then glycogen synthase kinase 3. Binding of WNT proteins to the surface receptor(s) leads to activation of the protein, which when disheveled, in turn, inactivates the destruction complex, allowing β-catenin to accumulate in the cytoplasm and enter the nucleus. In the absence of WNT4 binding, the stabilization of β-catenin will not take place, resulting in the alteration of target gene expression (10).
The study of Clement-Ziza et al. (11) showed that a mutation in the WNT4 gene does not cause typical MRKH syndrome without hyperandrogenism. Our work confirms that isolated MRKH syndrome is not due to a WNT4 mutation because 27 out of 28 adolescents presenting with MRKH without hyperandrogenism were without mutation. In our cohort, the only girl with MRKH and a WNT4 mutation also presented with hyperandrogenism. The two other cases of WNT4 mutation were reported to have similar clinical features associating MRKH-like syndrome with hyperandrogenism (Table 1) (4, 5).
|
Moreover, as previously shown in mice, WNT4 appears to play a role in follicle development and maturation. Our patient showed a very low number of follicles in the ovaries, in agreement with the drastic reduction of follicle number in WNT4–/– mice (1). This observation in female null mice suggests that an interaction between oocytes and somatic cells is required to maintain correct follicular differentiation and to prevent Sertoli cell differentiation at later stages (12, 13).
This report confirms the complexity of müllerian duct development, and the involvement of genes in both müllerian duct and ovarian development. It also suggests that the WNT4 gene is implicated in human follicle formation, development, and/or maintenance.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Part of this work was orally presented at the European Society for Pediatric Endocrinology meeting in Rotterdam, The Netherlands, 2006. Abstract in Horm Res 2006; 65(Suppl 4):22 (FC7–73).
First Published Online January 8, 2008
1 P.P. and A.B.-L. contributed equally to this work. 
Abbreviations: DHEA, Dehydroepiandrosterone; dpc, days post coitum; 17OHP, 17-hydroxyprogesterone; 3β-HSD, 3β-hydroxysteroid dehydrogenase; MRKH, Mayer-Rokitansky-Küster-Hauser; WT, wild type.
Received September 10, 2007.
Accepted December 28, 2007.
|
References |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
