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Reproductive Endocrinology |
Division of Endocrinology, Metabolism, and Molecular Medicine (T.J.K., C.A., J.L.J.), Northwestern University Medical School, Chicago, Illinois 60611; the Departments of Medicine (W.F.C.) and Pathology (R.H.Y., R.E.S.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15–709, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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C mutation was found at nucleotide 1777 that
converts codon 591 from phenylalanine to serine (F591S). This sixth
transmembrane domain mutation was found in 9 of 13 (69%) sex cord
tumors and 2 of 3 ovarian small cell carcinomas, but it was not present
in control specimens, including 5 normal ovaries, 5 nonsex cord ovarian
tumors, 16 thyroid tumors, or 90 specimens of peripheral blood
leukocyte DNA, suggesting that this nucleotide change is not a
polymorphism. The functional effects of identified mutations were
assessed by expression of the wild-type or the F591S mutant FSH-R in
COS-7 cells. The F591S mutation eliminated FSH-stimulated cAMP
production, and a similar effect was observed when this mutation was
introduced into the homologous location of the LH receptor. The high
prevalence of the F591S mutation in the FSH-R suggests that it plays a
role in the development of ovarian sex cord tumors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Granulosa cell tumors are the most common sex cord tumor and are usually seen in adults, with a peak incidence between 50–55 yr. Granulosa cell tumors are estrogenic, but also produce a variety of other hormones, including the peptides, inhibin (3, 4), and Mullerian inhibiting substance (5). Thecomas occur about one third as often as granulosa cell tumors, typically in postmenopausal women. Thecomas produce androgens as well as estrogens, particularly when they exhibit luteinization. Sertoli and Leydig cell tumors of the ovary are relatively rare, are usually seen in younger women, and often present with virilization.
The functionally differentiated nature of sex cord-stromal tumors makes
them an attractive model for attempting to identify candidate oncogenes
or tumor suppressor genes. In a mouse model in which the inhibin
-subunit gene was disrupted, sex cord tumors developed with nearly
complete penetrance (6). Because the inhibin-deficient mice overexpress
activin (homodimer of inhibin ß-subunits), the uninhibited autocrine
secretion of activin has been postulated to stimulate proliferation and
to predispose to sex cord tumors (6, 7). There is evidence that
gonadotropin stimulation enhances the penetrance of these tumors (8).
Ovarian granulosa cell tumors have also been induced in neonatally
estrogenized mice treated with 7,12-dimethylbenz(a)anthracene (9).
Bilateral granulosa cell tumors were identified in a patient with
leprechaunism (insulin receptor mutation), and it was proposed that
chronic hyperinsulinism may have increased mitogenesis of granulosa
cells by acting via the insulin-like growth factor I receptor (10).
Molecular defects in sex cord tumors have not been well defined. Flow
cytometry indicates that about 38% of granulosa cell tumors are
aneuploid (11), and trisomy 12 is seen commonly in thecoma-fibromas
(90%), but infrequently in granulosa cell tumors (16%) (12). Binding
sites for LH and FSH have also been identified in sex cord-stromal
tumors, suggesting that these tumors might retain responsiveness to
gonadotropins (13). Whether ovarian stimulation by gonadotropins
predisposes to granulosa cell tumors is controversial (14, 15, 16).
The LH and FSH receptors are members of the seven-transmembrane (TM) domain, G protein-coupled superfamily. Recent studies have identified mutations in this family of receptors that can activate or inactivate receptor function (17, 18, 19, 20, 21, 22, 23, 24, 25). For example, inactivating mutations in the FSH receptor (FSH-R) cause autosomal recessive primary ovarian failure (21), and inactivating mutations of the LH receptor (LH-R) cause pseudohermaphroditism in males and ovarian failure in females (22, 23). Activating mutations of the LH-R cause gonadotropin-independent production of testosterone and precocious puberty in males (17). A case report of an activating mutation in the FSH-R was described in a man with preserved spermatogenesis despite hypopituitarism (25). Activating mutations in this family of receptors have been localized within the TM domain, with a particularly high prevalence in a putative G protein coupling region that includes the fifth TM domain, third intracytoplasmic loop, and sixth TM domain (19, 20). As many sex cord-stromal tumors are estrogenic, we hypothesized that the FSH-R was a candidate for mutations that could alter the growth and function of this group of tumors. We examined a group of archival tumor specimens for mutations within the TM domain and tested the functional effects of a mutation that was identified with high frequency in these specimens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA was isolated from paraffin-embedded ovarian specimens as described previously (26, 27). Tissue blocks were cut into 5-µm sections and extracted with xylene followed by washing in absolute ethanol. The dewaxed tissue was suspended in 300 µL lysis buffer [10 mmol/L Tris (pH 7.5), 10 mmol/L NaCl, 10 mmol/L Na2-ethylenediamine tetraacetate, and 0.5% SDS] containing 20 µg/mL proteinase K and incubated at 37 C for 4 days. The solution was then extracted with phenol-chloroform-isoamylalcohol (25:24:1), and DNA was precipitated with ethanol. Leukocyte DNA samples from 90 unrelated individuals were provided by Andrew Arnold (Massachusetts General Hospital, Boston, MA) and Samuel Refetoff (University of Chicago, Chicago, IL).
Analysis of FSH-R DNA sequence
Extracted DNA was subjected to two-stage PCR using nested sets of sense and antisense primers. The primers for first stage PCR were: FSH-R-TM5, 5'-TGA AGG TGA GCA TCT GCC-3' (sense); and FSH-R-TM6, 5'-AAC AGA ACC AGC AGA ATC-3' (antisense). PCR reactions (50 µl) included 300 ng DNA, 10 pmol of each primer, 50 µmol/L deoxynucleoside triphosphates (deoxy-NTPs), and 2 mmol/L MgCl2, in PCR buffer [10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, and 0.1% Triton X-100] containing 2.5 U Taq DNA polymerase (Promega, Madison, WI). Cycle conditions were 1-min hot start at 96 C followed by 12 cycles of 5 s at 96 C, 60 s at 92 C, 45 s at 58 C, 45 s at 72 C, and extension at 72 C for 10 min. An aliquot (2 µl) of this reaction product was used as template for the second stage PCR using two internal primers: FSH-R-TM5I-M13rev, 5'-CAG GAA ACA GCT ATG ACC CAT CTG CCT GCC CAT GGA-3' (sense with 5' M13 sequencing site); and FSH-R-TM6I, 5'-GCA GAA TCT TTG CTT TGG-3'. PCR conditions were the same as described above and were carried out for 25 cycles. Control reactions without the addition of DNA were used to detect contamination of reagents.
The secondary PCR product was prepared for DNA sequencing by treatment with exonuclease (10 U/5 µl PCR product; Amersham, Arlington Heights, IL) and shrimp alkaline phosphatase (2 U/5 µl product; Amersham) for 15 min at 37 C followed by 15 min at 65 C. DNA was purified using Centricon-100 spin columns (Amicon, Beverly, MA). Direct DNA sequencing was performed using the ABI Prism Dye Terminator or Dye Primer Cycle sequencing kits with AmpliTaq DNA Polymerase FS (Perkin-Elmer, Foster City, CA). In some cases, the secondary PCR products were resolved on 1.2% agarose gels, purified with the GeneClean II kit (BIO 101, Vista, CA), and ligated into the TA Cloning Kit PCR 2.1 vector (Invitrogen, San Diego, CA). Plasmid inserts were sequenced as described above.
Screening for the F591S mutation
Two methods (oligonucleotide-specific hybridization and primer-specific PCR) were used to screen DNA specimens for the F591S mutation. For oligonucleotide-specific hybridization, PCR products were attached to membranes and hybridized to 32P-labeled oligonucleotides (26, 27). The sequences of the oligonucleotides were: wild type, 5'-TTC TTT CTT TGC CAT TT-3'; or mutant, SD 5'-TTC TTT CTC TGC CAT TT-3' (mutant base in italics). PCR products from second stage PCR (see above) were fixed to nitrocellulose by baking at 80 C in a vacuum oven for 2 h. Hybridization was carried out at 50 C for 3.5 h in 50 mmol/L Tris-HCl (pH 8.0), 3.0 mol/L tetramethylammonium chloride, 5 x Denhardt’s solution, 0.1% SDS, 33 µg/mL salmon sperm DNA, and 106 cpm/mL labeled oligonucleotide. Membranes were washed twice, for 10 min at room temperature in 2 x SSPE and 0.1% SDS and for 20 min at 50 C in 50 mmol/L Tris-HCl (pH 8.0), 3 mol/L tetramethylammonium chloride, 2 mmol/L ethylenediamine tetraacetate, and 0.1% SDS, before exposure to x-ray film. The stringency of hybridization and washing conditions was verified by the inclusion of control samples with known wild-type or mutant sequence at codon 591 of the FSH-R.
Primer-specific detection of the T to C mutation was based upon the ability to generate a new restriction enzyme site (PstI) by modifying the DNA sequence adjacent to the mutation. The sense primer was the same as FSH-R TM 5I, except that a fluorescent label was attached (5'-Fam-CAT CTG CCT GCC CAT GGA-3'). The antisense primer (5'-ACA CAG TGA TGA GGG GCA CCT TGA GGG AGG CAG AAA CTG CA-3') included two nucleotide substitutions (in italics), which, in conjunction with the T to C mutation, created a new PstI site. After restriction digestion with PstI, samples were analyzed on a 377 DNA Sequencer using GeneScan 2.0.2 software (Perkin-Elmer).
Functional analyses of FSH-R
A full-length complementary DNA (cDNA) construct of the human FSH-R in pSVK3 (28) was used for site-directed mutagenesis to create the T to C mutation in nucleotide 1777 (codon 591, Phe to Ser). The sequence of the PCR product was verified, including the presence of the mutation. An expression construct was prepared by inserting the mutation-containing NcoI-XhoI fragment into the corresponding site of the pSVK3-FSH-R. The wild-type and mutant FSH-R cDNAs were subsequently transferred into the pSVL vector, which resulted in higher levels of expression (Pharmacia Biotech, Uppsala, Sweden).
FSH binding to expressed receptors was examined in transfected COS-7 cells (28). Human FSH (AFP 5720) was labeled with Na125I (New England Nuclear, Boston, MA) using lactoperoxidase and purified by gel electrophoresis (provided by P. Sluss, Massachusetts General Hospital, Boston, MA) (29). Cells were transfected at 50% confluency in 10-cm plates with 60 µg plasmid using a lipid-mediated (DDAB:PtdEtn, 1.0:0.6 mg) protocol (30). After 24 h, cells were split into six-well plates (2 x 105 cells/well) and allowed to recover for 24 h. After rinsing in binding buffer [50 mmol/L HEPES (pH 7.4), 100 mmol/L sucrose, 5 mmol/L MgCl2, and 0.1% ovalbumin], the transfected cells were incubated for 4 h at room temperature in 0.5 mL binding buffer containing 50,000 cpm (34 pmol/L) 125I-labeled FSH with or without excess unlabeled FSH. Cells were rinsed twice in 1.0 mL binding buffer, incubated for 60 min in 0.5 mL 1 mol/L NaOH, and collected for determination of radioactivity.
The function of the expressed FSH-Rs was assessed by measuring the production of cAMP. COS-7 cells (3 x 105 cells/well) were transfected as described above. After 48 h, cells were treated for 60 min with FSH (200 mIU/mL; 23.5 ng/mL) and 200 µmol/L 3-isobutyl-1-methyl-xanthine (Sigma Chemical Co., St. Louis, MO), and the extracellular medium was harvested for cAMP determination by RIA (Biomedical Technologies, Stoughton, MA). Where applicable, empty pSVL expression vector was used to balance the transfection reactions such that the total amount of expression vector was kept constant.
An LH-R mutation (F588S) that is homologous to the FSH-R mutant (F591S) was also created by site-directed mutagenesis. A human LH-R expression vector (pCMX-HLHR; provided by A. Hsueh, Stanford University, Palo Alto, CA) was used as a template. The sequence of the mutant cDNA was verified, and it was subcloned as a BstXI/Bpu1102I fragment into the expression plasmid. The functional properties of the wild-type and mutant LH-R were examined using the same protocol as that used for the FSH-R, except that transfected cells were treated with 500 ng/mL hCG (Serono, Randolph, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A series of 16 archival sex cord and other ovarian tumors were
selected for study by one of the pathologists (R.H.Y.). The
pathological diagnoses were not revealed until after DNA sequence
analyses were completed. Later, an additional 10 ovarian control
specimens, 16 thyroid tumors, and 90 peripheral blood specimens were
examined. DNA was extracted from the paraffin blocks, and PCR was used
to amplify a portion of the FSH-R TM domain, including the fifth TM
domain, the third intracellular loop, and the sixth TM domain (amino
acids 515–611). The DNA sequence was determined by direct cycle
sequencing. A heterozygous mutation (T to C, nucleotide 1777) that
converts Phe591 to Ser (F591S) was found in several of the
specimens (Fig. 1
). No other sequence alterations were
found in this region of the receptor. The sequencing results for
various types of sex cord tumors are summarized in Table 1. The identical mutation was found in 7 of 9 juvenile
granulosa cell tumors and 2 of 3 adult granulosa cell tumors. The
mutation was not present in a luteinized thecoma with sclerosing
peritonitis (31). The mutation was present, however, in 2 of 3 ovarian
small cell tumors of the hypercalcemic type (32). Overall, this
mutation was found in 9 of 13 (69%) sex cord tumors and with similar
prevalence in small cell carcinomas.
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). This assay
confirmed the mutations in all samples tested and excluded the mutation
in the controls.
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). These results
suggest that if this is a polymorphism, it occurs in less than 1 in 232
(0.4%) alleles. Normal tissue was available from only 2 patients (no.
5 and 11), but the sex cord tumors in each of these cases contained
normal FSH-R sequence. Consequently, it was not possible to ascertain
in this study whether the mutation was inherited or somatic in
origin. Functional properties of the F591S mutant
The wild-type and F591S mutant FSH-R were expressed in COS-7 cells
to assess the effect of the mutation on basal and FSH-induced function.
Initially, FSH binding studies were performed to determine whether the
F591S mutation altered receptor expression or ligand binding. Receptor
expression vectors were transfected alone or together, and FSH binding
was determined 48 h later. The specific binding of
[125I]FSH was consistently 2- to 5-fold greater to the
wild-type receptor than to the F591S mutant (Fig. 3). An
intermediate level of binding was seen when equal amounts of the
receptors were coexpressed. There was no alteration in the affinity of
FSH binding to the F591S mutant (data not shown) consistent with its
location in the TM domain rather than the extracellular ligand-binding
domain.
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). The mutant receptor in the antisense orientation was
included as a negative control. Basal cAMP levels were unaffected by
transfecting increasing doses of either the wild-type or the F591S
mutant. Using the wild-type receptor, the addition of FSH induced
progressively greater responses with increasing amounts of receptor.
Maximal responsiveness was 
15-fold. In contrast to the wild-type
receptor, no FSH response was detected using the F591S mutant even at
the highest dose of transfected DNA. These results suggest that the
F591S mutation impairs FSH responsiveness, probably by altering
coupling to the signal transduction apparatus.
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). However, this substitution reduced LH-induced
stimulation of cAMP from 21- to 6-fold. Thus, the effect of this
mutation is similar to that of the F591S mutation in the FSH-R.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because detection of heterozygous mutations from small amounts of DNA is technically challenging, several techniques were used to analyze mutations in the sex cord tumors. All specimens were analyzed by direct DNA sequencing, and mutations were confirmed either using oligonucleotide-specific hybridization or by showing the presence of a new PstI restriction site as a result of the mutation. Surprisingly, the same nucleotide substitution was subsequently identified in the majority of the sex cord-stromal tumors that were available for study. The prevalence of this particular mutation, without identification of other mutations, raised the possibility that it might be a polymorphism. Therefore, we examined a series of control samples for the presence of this mutation. DNA from thyroid tumors was used to control for possible contamination as well as to screen for a polymorphism. Using oligonucleotide-specific hybridization, two of the thyroid tumors were initially suspected of having the mutation. However, restriction analysis excluded a mutation in these cases. In general, mutation detection by hybridization is less reliable because the technique is sensitive to the stringency of hybridization and washing. A large number of specimens of leukocyte DNA (n = 90) also did not contain the F591S mutation, providing further evidence that it is not a common polymorphism. Finally, a series of other ovary specimens was examined without evidence for the mutation. Based upon these results, we conclude that the F591S substitution is a mutation rather than a polymorphism. This concept is supported by the fact that the nonconservative amino acid substitution also alters the function of the receptor.
It is of great interest to consider whether this mutation is acquired somatically, or whether it is a germ-line mutation that somehow predisposes to sex cord tumors. It remains possible that the F591S mutation occurs at a low gene frequency and represents a risk factor for sex cord tumors. For example, a "second hit" may actually cause the tumor, but this mutation may predispose it toward the sex cord phenotype. Unfortunately, we were not able to address this question because no normal tissue was available from this group of patients. These archival specimens were accumulated by referral of specific blocks of tumors over several decades for confirmation of difficult histological diagnoses. We were able to procure only two additional tissue specimens from referring physicians, and both were from patients without a mutation. It should be possible to address this interesting issue in future prospective studies of sex cord tumors.
We initially hypothesized that the F591S mutation might constitutively activate the cAMP pathway, analogous to mutations that have been reported in the structurally related TSH-R (20) and LH-R (19). The activating mutations in TSH-R and LH-R increase the basal activity of adenylyl cyclase and preserve further responsiveness after the addition of hormone. However, rather than exhibit increased basal activity, the F591S mutation caused an apparent loss of receptor function. cAMP production was measured using two different approaches. A RIA was used to measure cAMP production directly, and a cAMP-responsive reporter gene was used as an independent means of detecting the second messenger (data not shown). Basal cAMP levels in the presence of wild-type and mutant receptors were indistinguishable. However, because the basal levels of cAMP are very low, we cannot absolutely exclude the possibility of a low degree of basal activation in these assays. The fact that no cAMP response was seen with the mutant receptor, even after the addition of FSH, provides further evidence that the mutation alters receptor coupling to the cAMP pathway. The homologous mutation was also introduced into the human LH-R. Consistent with the results using the FSH-R, there were no alterations in basal cAMP levels, and responses to LH were blunted. In view of these findings, we conclude that the F591S substitution is a loss of function mutation, at least with respect to the cAMP pathway. It is possible, however, that the mutation alters other signaling pathways. FSH minimally altered the production of phosphoinositides in the transient expression model (data not shown), and previous studies have shown that this response requires relatively high doses of FSH (35).
A number of mutations have now been reported within the third
cytoplasmic loop and sixth TM domain of the glycoprotein hormone
receptors (19, 20). This region is postulated to represent a G protein
interaction domain (36, 37). As depicted in Fig. 6, a
variety of activating mutations have been described in the TSH-R and
the LH-R, and a single activating mutation was reported recently in the
FSH-R (25) (Fig. 6). For the most part, these mutations are clustered
near the end of the third cytoplasmic loop and along one of the
predicted surfaces of the sixth TM domain. On the other hand, the F591S
mutation is located in close proximity to an inactivating mutation in
the LH-R. Ultimately, structural information in combination with
further functional studies will be required to assess the implications
of these mutations. In the interim, these results suggest that
mutations within closely spaced domains can result in divergent effects
on receptor function.
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-subunit gene
has been shown in transgenic mice to strongly predispose to granulosa
cell tumors (6), and mating with p53-deficient mice increased the
penetrance of the inhibin mutation (7). In this model, unopposed
production of activin has been proposed to lead to granulosa cell
tumors. Expression of the FSH-R mutation in transgenic mice or,
ideally, the use of homologous recombination to create the mutation may
ultimately be required to assess its functional role. Although these
mechanistic questions remain unresolved at present, the recognition of
this mutation provides an important first step toward understanding the
genetic basis of this subset of ovarian tumors. It is interesting to speculate about the prevalence of this, or similar mutations in other ovarian disorders. Already it seems that the F591S mutation is found in several different types of sex cord stromal tumors that exhibit distinct histological and functional characteristics. For example, the mutation was found with similar prevalence in juvenile and adult granulosa cell tumors and in a group that we have classified as ovarian small cell carcinomas of the hypercalcemic type (1). The cellular origin of the small cell carcinomas is unclear, but these results suggest that they may fit into the sex cord-stromal tumor category. These observations also raise the possibility that this mutation may occur in other gonadal disorders, including ovarian cysts, polycystic ovarian disease, and Sertoli cell tumors. Further studies will be of interest to address these issues.
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Acknowledgments |
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Footnotes |
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Received August 26, 1996.
Revised November 8, 1996.
Accepted December 5, 1996.
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