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Interdisciplinary Research Institute for Human and Molecular Biology, Faculty of Medicine, and Department of Genetics, Erasme Hospital (G.S., G.V.), Free University of Brussels (A.D.L., L.M., J.V.D., G.S., G.V., S.C.), B-1070, Brussels, Belgium; and Department of Obstetrics and Gynecology, University of Ulsan College of Medicine, Asan Medical Center (H.C.), Seoul, 138-736, Korea
Address all correspondence and requests for reprints to: Dr. S. Costagliola, Interdisciplinary Research Institute for Human and Molecular Biology, Free University of Brussels, 808 Lennik Street, B-1070 Brussels, Belgium. E-mail: scostag{at}ulb.ac.be.
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Abstract |
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Objective: The objective of this study was to look for novel FSHr mutations in patients with sOHSS associated with different levels of hCG and TSH to 1) find new residues important for FSHr activation and specificity, and 2) better delineate the pathophysiology of the different presentations of sOHSS.
Design, Intervention, and Patients: After blood sampling, we sequenced the FSHr from genomic leukocytes DNA from eight patients with sOHSS of the first or second trimester of pregnancy with normal or high hCG levels or with high TSH levels associated with severe hypothyroidism.
Setting: This study was performed at a university laboratory.
Main Outcome Measure: The main outcome measure was FSHr sequencing and in vitro evaluation of the variation of cAMP production of FSHr mutants.
Results: A new mutation was found in the patient with sOHSS of the first trimester of pregnancy with a normal hCG level: I5.54545T, in transmembrane helix V of the FSHr. When tested functionally, this mutant displayed promiscuous activation by both hCG and TSH together with detectable constitutive activity. In contrast, no mutations were found in the FSHr from patients with high hCG or TSH levels, indicating that for those seven patients, sOHSS results from the natural promiscuous stimulation of a wild-type FSHr by very high concentrations of hCG or TSH.
Conclusions: sOHSS can occur by at least three different pathophysiological mechanisms.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-subunit and specific ß-subunits that share more than 40% amino acid sequence identities. Although the corresponding glycoprotein hormone receptors [FSHr (1), LH/CG receptor (2, 3), and TSH receptor (TSHr) (4)] also display about 40% sequence identity in their hormone binding domains, coevolution of the hormone-receptor couples resulted in the establishment of tight specificity barriers, preventing promiscuous activation under normal physiological conditions (5). The emergence of CG in primates, which achieves very high concentrations during early pregnancy (6), constituted an evolutionary challenge to this specificity barrier. In humans, in particular during the first trimester of pregnancy, human CG (hCG) reaches concentrations at which it displays some thyrotropic activity, bringing most pregnant women on the fringe of hyperthyroidism (7). Accordingly, overproduction of hCG, as observed in molar or twin pregnancies, may result in overt gestational hyperthyroidism (8). In agreement with these ideas, one would expect mutations affecting the hormone binding surface of the TSHr or FSHr (9) to be responsible for rare cases of gestational hyperthyroidism or spontaneous ovarian hyperstimulation syndrome (sOHSS), presenting with normal hCG concentrations. Although this prediction has been verified in one familial case of gestational hyperthyroidism caused by mutation in the hormone binding domain of the TSHr (10), to date all cases of sOHSS have been associated with mutations in the rhodopsin-like serpentine domain of the FSHr implicated in transduction of the activation signal to the interior of the cell (11, 12, 13, 14, 15).
In the present study we report a novel mutation of the FSHr (I5.54545T; for numbering system, see Patients and Methods) in a patient who presented sOHSS at 12 wk of pregnancy (16). Again, the mutation was located in the serpentine domain of the FSHr affecting a residue located close to the cytoplasmic border of transmembrane helix 5 (TM-V), but outside the hot spots of TM-III or TM-VI, where sOHSS mutations have been described to date (11, 12, 13, 14, 15). When it was explored functionally in transfected cells and structurally by molecular modeling, the mutant pointed to a role of residue I5.54 in stabilizing TM-III, TM-V, and TM-VI in the inactive state.
In addition, since the description of the first sOHSS FSHr mutation in 2003, we also had access to DNA from four other cases of sOHSS related to high hCG levels (17, 18, 19, 20) and to three DNA samples from patients who presented sOHSS in relation to severe hypothyroidism (21, 22) (M. H. Hedayati Omami, unpublished case). To explore the possibility of a relation between these sOHSS presentations and the FSHr, we sequenced the latter in the seven patients; no mutations were found. This absence of FSHr mutation in light of the in vitro demonstration of stimulation of FSHr by high concentrations of hCG and TSH (11, 23, 24, 25, 26) indicates that natural promiscuous activation of the wild-type FSHr by high levels of hCG or TSH is the likely explanation for these sporadic sOHSS presentations.
In conclusion, we propose, for future research and clinical use, a classification based on the three different pathophysiological mechanisms responsible for the occurrence of sOHSS (see Fig. 4
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The standardized numbering system of Ballesteros and Weinstein (27) was used throughout the manuscript. This allows easy identification and comparison of residues in the transmembrane segments of different receptors (27).
Reagent
Recombinant human FSH (rhFSH; purified) was purchased from Organon Belge (Brussels, Belgium), rhCG from was obtained from Sigma-Aldrich Corp. (St. Louis, MO), and rhTSH was purchased from Genzyme (Cambridge, MA).
Patients
The clinical characteristics of the sOHSS patient with the new FSHr mutation have been described previously (16). Briefly, the proband was a 35-yr-old Korean woman, gravida 2, para 1, presenting severe sOHSS at wk 12 of her second pregnancy (first pregnancy was uneventful). Symptoms regressed during the second and third trimesters and resolved in postpartum. Her medical and family histories were unremarkable. All other sOHSS patients for whom no FSHr mutation was found were previously described (Table 1), except the Iranian case referred by Dr. M. H. Hedayati Omami. His 15-yr-old patient presented an association of hypothyroidism and moderate sOHSS symptoms. Her TSH serum levels were greater than 100 µU/ml, and total T4 was less than 2 µg/ml. An autoimmune primary diagnosis was made because of the presence of antithyroid peroxidase antibodies. After a few months of levothyroxine therapy, TSH and T4 levels returned to normal as did the size of her ovaries. Interestingly, her cousin presented exactly the same associations of symptoms as hypothyroidism presentation. Unfortunately, only blood samples from the proband were available for DNA extraction.
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Human DNA was extracted from peripheral blood leukocytes, and the sequences of all the exons of the FSHr gene together with intron-exon boundaries were determined as previously described (13). The patients gave informed consent to participation in this study, which was approved by the ethical committee of Erasme Hospital (Brussels, Belgium). The sequence of the segment harboring the I5.54545T mutation was determined from the product of two independent PCRs. The I5.54545T mutation introduces a BstEII restriction site, thus generating mutation-specific bands at 343 and 190 bp from the PCR product obtained with the proband DNA. A restriction fragment length polymorphism assay using BstEII was used to screen for the presence of the mutation in 167 normal subjects, including 73 ethnically matched individuals.
Construction of human FSHr mutants
Mutations were introduced into the human wild-type FSHr (wt-hFSHr) cloned in the pSVL vector (Stratagene, La Jolla, CA) by site mutagenesis as previously described (25).
Transfection experiments
COS-7 cells were used for all transient expression experiments as previously described (25). For hormone stimulation or binding experiments, a 24-well alternative protocol was used to minimize the amount of hormones needed, as described previously (25). To demonstrate that generation of cAMP by the basal activity of the mutants (when present) is linearly related to the number of receptors present at the cell surface, variable amounts of wt-hFSHr and various I5.54545 mutants were transfected into COS cells (complemented to 1 µg DNA with empty pSVL vector).
Quantification of cell surface expression by FACS
Two days after transfection, cell surface expression was assessed by FACS (FACScan flow cytofluorometer, BD Biosciences, Erembodegem, Belgium) with mouse monoclonal antibodies 5B2 as previously described (25). Cells transfected with pSVL alone (empty vector) and with pSVL-wt-hFSHr (wt sequence) were always used as negative and positive controls, respectively.
Determination of cAMP production
Forty-eight or 72 h after transfection, the intracellular accumulation of cAMP was measured as previously described (25). cAMP concentrations were determined in quadruplicate on extracts from duplicate transfection dishes or wells. Results are expressed as picomoles of cAMP per milliliter or as a percentage of the maximal cAMP response.
Conversion between international units per milliliter and nanograms per milliliters or nanomolar concentrations
A quantity of 1 IU/ml rhCG corresponds to 62 ng/ml, or 2 nM (Sigma-Aldrich Corp.); 1 IU/ml rhFSH corresponds to 100 ng/ml, or 3.3 nM (Puregon, Organon Belge); and 1 mIU/ml rhTSH equals 125 ng/ml, or 4 nM (Thyrogen, Genzyme).
Molecular modeling
The structure of bovine rhodopsin (Protein Data Bank code: 1GZM) (28) was used as template for wt and mutants FSHr models, built with the WHAT IF program (29). All images of the models were generated using the YASARA package (30).
Statistical analysis
Receptor expression at the cell surface, constitutive activities (raw values or normalized), and cAMP production after hCG or TSH stimulation of the mutant FSHrs and wt-hFSHr were compared using the nonparametric paired Wilcoxon test and Instat version 3 (GraphPad, Inc., San Diego, CA), performed with at least four independent experiments. Difference was considered statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A heterozygous substitution of thymidine for cytosine at the second base of codon 545 was identified in exon 10 of the FSHr gene of a patient presenting with sOHSS (Fig. 1, A and B; see case report in Patients and Methods and Ref.16). None of 167 DNA samples from unrelated normal subjects, including 73 ethnically matched individuals, carried the mutation, ruling out the possibility of a common polymorphism. This novel mutation results in the replacement of an isoleucine (I) by a threonine (T; I5.54545T; for numbering system, see Patients and Methods). This Ile residue is located at the cytoplasmic extremity of TM-V (Fig. 1C). It is conserved in human LH receptor, but changed for a valine (V) in human TSHr.
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), the mutant displayed modest, but definite, constitutive activity compared with the totally silent wt-hFSHr (130% of the cAMP level achieved in cells transfected with the wt-hFSHr or empty vector plasmid; P < 0.05; nonparametric paired Wilcoxon test; see Fig. 2, B and C). As expected from results with previously described sOHSS mutations, the I5.54545T mutant showed clear lowering of its specificity toward both rhCG and rhTSH (Fig. 2, E and F). Neither the affinity (IC50, 0.18 ± 0.15 IU/ml for wt-hFSHr and 0.14 ± 0.11 UI/ml for I5.54545T, respectively) nor the sensitivity (EC50, 0.086 ± 0.009 UI/ml for wt-hFSHr and 0.029 ± 0.001 for I5.54545T, respectively) to the natural FSH agonist was affected by the mutation (Fig. 2D).
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Phenotype of other I5.54545 mutants
To explore the molecular mechanisms of the gain of function caused by substitutions at position 5.54, a panel of mutants was engineered in which I5.54545 was replaced by six different amino acids with diverse physicochemical properties (A, F, L, N, T, and V). After transient expression in COS-7 cells, differences were observed in the level of expression at the cell surface of individual mutants, ranging from 22–107% of the wt-hFSHr (Fig. 3A). Unexpectedly, the conservative substitution of I5.54545 by a leucine (L) had the most dramatic effect on both cell surface expression (22% of the wt-hFSHr) and constitutive activity (Fig. 3B). In contrast, the basal activity of the other mutants was barely measurable (I5.54545F and, to a lesser extent, I5.54545N and I5.54545T) or, as for the wt-hFSHr, undetectable (I5.54545A and I5.54545V). To reliably compare the constitutive activities of mutants I5.54545L, F, N, and T despite wide differences in expression at the cell surface, we transfected COS cells with various amounts of the wt-hFSHr and three mutant constructs (I5.54545T, L, and V). cAMP production was plotted as a function of surface expression, measured by FACS (Fig. 3C). No deviation from linearity was observed, demonstrating the validity of the normalization method even when barely measurable cAMP values are divided by FACS results (Fig. 3D). This experiment (Fig. 3C) also demonstrates the very low constitutive activity of wt-hFSHr and I5.54545V mutant.
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). All of them responded readily to rhFSH (Fig. 3D). In addition, except for I5.54545A and I5.54545V (for which no constitutive activity was detected), all mutants displayed an increase in sensitivity to stimulation by rhTSH and rhCG compared with wt-FSHr (P < 0.05, by nonparametric paired Wilcoxon test; Fig. 3E). Mutants I5.54545L and, to a lesser extent, I5.54545F (the two mutants with the strongest basal activity) were particularly sensitive to these two hormones. In particular, stimulation of the I5.54545L mutant by rhCG reached a maximal level similar to that achieved by a saturating concentration of rhFSH (Fig. 3F). Despite the increase in sensitivity of the I5.54545 mutants to hCG and, as previously described for other sOHSS mutants, the affinity for this hormone remained too low to be measured in direct binding experiments with [125I]hCG or by competition for binding of [125I]hFSH (data not shown). Modelization of the environment of I5.54545
I5.54545 resides near the cytoplasmic extremity of TM-V. The model, elaborated on the atomic structure of bovine rhodopsin (34), predicts tight packing via hydrophobic interactions with residues T3.47464 and L3.43460 of TM3 and I6.41579 of TM-VI (Fig. 1C). The rhodopsin structure demonstrates the importance of the cytoplasmic portions of helices V and VI for interaction with the rhodopsin-specific G protein, transducin (28, 35). It is thus conceivable that any structural change disrupting the interactions of helices V and VI with the other helices could facilitate G protein binding and receptor activation.
Substitution of I5.54545 with T (or N) is predicted to interact with T3.44461 and/or T3.47464. This would pull helices III and V toward each other or could cause a reorganization of the local structure around TM-V (Fig. 1D). Introduction of an F (a larger and aromatic residue) or L (with a second 
atom compared with I) at this position would induce a clash with I6.41579 or T3.47464, respectively. The result is that interactions of TM-V and TM-VI with the rest of the molecule would be disrupted.
FSHr mutants with A or V at position 5.54545 do not display measurable basal activity. In agreement with our hypothesis, the models with the I5.54545A and I5.54545V substitutions predict neither atomic clashes nor the possibility of making new interactions (data not shown).
FSHr is not mutated in sOHSS associated with hypothyroidism or high hCG levels
The entire coding sequence and all intro-exon junctions of the FSHr gene were sequenced from leukocyte DNA in four patients with sOHSS and high hCG levels and in three patients with sOHSS symptoms and severe hypothyroidism (Table 1). No mutation was found.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In the present report we show that mutation of isoleucine in position 5.54545 into threonine is also associated with sOHSS and displays a similar in vitro phenotype as T3.32449I/A and D6.30567N/G when assayed in transfected cells. Residue 5.54545 is located at the cytoplasmic border of transmembrane helix V, a position that has previously been found mutated in TSHr (V5.54597F/L) and LH receptor (I5.54542L) in patients presenting severe thyrotoxicosis (36) or male-limited precocious puberty (37, 38), respectively. The present study and previous molecular dynamic analysis predict that even keeping the hydrophobic character at position 5.54, a small change in the physicochemical properties of the amino acid can trigger the release of interhelical interactions stabilizing the wild-type receptor (39), thus leading to its partial activation.
In agreement with previous observations (11, 12, 13, 14), the present results establish a clear relation between constitutive activity and lowering of specificity in FSHr mutants. This is consistent with our suggestion that the gain of sensitivity of the mutants to hCG (or TSH) would be due to the lowering of an intramolecular barrier to activation rather than to an increase in binding affinity (5).
Cases of sOHSS presenting with high levels of hCG have been frequently described in all trimesters of pregnancy (40). To compare their origins to the five mutated cases discussed here above, we sequenced the FSHr in one case of sOHSS of the first trimester of a twin pregnancy (17), two cases of OHSS of the first trimester with idiopathic unexplained high hCG levels (18, 19), and one case of sOHSS of the second trimester with elevated hCG levels on preeclampsia (20). No mutations were found. Hence, we are left with the hypothesis that this situation reflects natural promiscuity of the wt-hFSHr for hCG (11, 25, 26). As such, it would mirror, for the hCG-FSHr couple, the situation in molar or twin pregnancies, in which very high hCG concentrations are able to activate a wt-TSHr (8). However, we cannot exclude that a variant hCG, exhibiting higher biological activity, or granulosa cells in an autocrine environment more sensitive to FSHr stimulation might be implicated.
Finally, again by analogy with the five sOHSS mutated cases discussed above, we reasoned that mutations of the FSHr gene might be responsible for the relatively rare cases of OHSS occurring in profoundly hypothyroid (non) pregnant women (21, 22) (M. H. Hedayati Omami, unpublished case). No mutations were found. Then, as for sOHSS with high hCG levels, we have to postulate that this situation reflects natural promiscuity of the wt-FSHr for TSH (11, 23, 24, 25). It would be for the TSH-FSHr couple, the mirror image of gestational hyperthyroidism and TSHr stimulation by high levels of hCG (8). As in cases with high hCG and considering the rare incidence of sOHSS symptoms in hypothyroid patients, we cannot exclude that a variant TSH, exhibiting higher biological activity, or granulosa cells more sensitive to FSHr stimulation might be implicated.
In all patients in whom no mutation was found in the FSHr gene, we cannot exclude the possibility that a somatic mutation, with manifestation in the ovaries, but absent from leukocytes, could be responsible for the disease. Such a mutation should occur very early in development to account for the bilateral involvement of the ovaries. In the absence of clinical indication for ovarian biopsies, this hypothesis could not be tested.
Although more cases need to be investigated (we welcome, for FSHr sequencing, all well-documented cases), the present results point to genes or mechanisms unrelated to FSHr mutations for the various pathophysiological origins of sOHSS. These different mechanisms have been summarized in Fig. 4 by classification of sOHSS in three types. Type I corresponds to the mutated FSHr cases. Type II corresponds to the sOHSS secondary to high levels of hCG. This type is probably the most frequent one. The third one is related to hypothyroidism. This classification could be useful for clinicians and scientists (see, for example, the propositions in Table 2 of the different clinical tests that could be informative to obtain from the various classes of sOHSS patients). If such propositions are adopted, clinicians will rapidly extent our knowledge on the precise origins of sOHSS, which could result in delineation of new types of sOHSS and certainly help with future counseling and treatment of their patients.
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Acknowledgments |
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), who provided DNA from OHSS patients. We thank Dr. Ho Su-Chin from Singapore who provided ethnically matched DNA. |
Footnotes |
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First Published Online November 8, 2005
Abbreviations: CG, Chorionic gonadotropin; FSHr, FSH receptor; hCG, human CG; OHSS, ovarian hyperstimulation syndrome; rh, recombinant human; sOHSS, spontaneous OHSS; TM-V, transmembrane helix 5; TSHr, TSH receptor; wt, wild type.
Received July 15, 2005.
Accepted November 2, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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