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Analysis of Mutations in Genes of the Follicle-Stimulating Hormone Receptor Signaling Pathway in Ovarian Granulosa Cell Tumors¹

Departments of Human Genetics (M.J.L., M.S., H.G.B.), Pathology (M.J.L., T.G.H.), and Gynaecology (W.W.), University Hospital Nijmegen, 6525 GA Nijmegen, the Netherlands; and Department Endocrinology and Reproduction (A.P.N.T.), Erasmus University, 3015 GE Rotterdam, the Netherlands

Address all correspondence and requests for reprints to: Han. G. Brunner, Department of Human Genetics-417, 6525 GA Nijmegen, the Netherlands. E-mail: h.brunner{at}antrg.azn.nl

	Abstract

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It has been suggested that ovarian granulosa cell tumors may result from unopposed hyperstimulation, either by excessive gonadotropin stimulation, by activating mutations of the FSH receptor gene, or of the G protein subunits, Gs or Gi2. We have examined the entire open reading frame of the FSH receptor gene in ovarian granulosa cell tumors. In addition, these tumors were evaluated for the known oncogenic G protein mutations Gsp and Gip2. Normal results were obtained in all 23 ovarian granulosa cell tumors. We conclude that mutations of the FSH receptor G protein signaling pathway do not play any major role in the genesis of these tumors.

	Introduction

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OVARIAN GRANULOSA cell tumors are rare neoplasms that have not yet been well characterized at the cellular level. The incidence of granulosa cell tumors is approximately 1 per 100,000 women, comprising 10% of all ovarian malignancies. The causes of ovarian granulosa cell tumors are largely unknown. Excessive stimulation by gonadotropins has been suggested, based on a compilation of case reports (1), but this has yet to be confirmed in a strictly controlled study. Activating mutations of the TSH receptor in functioning thyroid adenomas (2) and thyroid carcinomas (3) and of the LH receptor in Leydig cell adenomas (4) have been described, suggesting that chronic stimulation by such mutations may ultimately lead to tumor development. For granulosa cell tumors, the causative mutation would be expected to reside in the FSH receptor gene. However, an initial report of a specific FSH receptor mutation in 9 of a series of 13 sex cord tumors (5) was not confirmed by others (6) and could not be reproduced (7). An early report of an oncogenic mutation (Gip2) in the Gi2 inhibitory G protein in 2 of 3 granulosa cell tumors (8) was not replicated in a study of 13 tumors by another group (9).

To further evaluate the presumed relationship with the FSH signaling pathway, we examined granulosa cell tumors for mutations of the FSH receptor gene and for the presence of known oncogenic G protein mutations (Gsp and Gip2). Finally, we considered the possibility that a naturally occurring polymorphism in the G protein ß3-subunit, which affects signaling (10), might act as a predisposing factor for the subsequent development of a granulosa cell tumor.

	Materials and Methods

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Ovarian granulosa cell tumors (4 from children and 19 from adult women) were obtained from several sources. The steroidogenic activity of the tumors is largely unknown. The histology of each tumor was reexamined by one of us (T. G. Hanselaar). Areas of tumor tissue were marked, and material from these areas of the paraffin-embedded or frozen sections was used for DNA extraction. The entire coding region of the FSH receptor gene was amplified from genomic DNA using PCR primers on either side of the exons, as described (11). For each of the exons 1–9, a single PCR fragment was generated. Exon 10 was amplified using 8 overlapping fragments. The size of the PCR products varied between 147 and 291 bp (primer sequences and PCR conditions are available upon request). Amplified fragments were analyzed for single-strand conformation polymorphisms, on a 12.5% gel, on a Genephore electrophoresis system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and were visualized using silver staining. Electrophoresis was performed at 15 W for about 120 min at 15 C and/or 5 C, depending on the product being tested. PCR fragments generating an aberrant band were analyzed by direct sequencing using the dRhodamine terminator cycle sequencing kit (PE Applied Biosystems and Perkin-Elmer Corp., both at Foster, CA), according to the manufacturers’ instructions. The sequence reactions were run and analyzed using an automatic sequencer (ABI377).

	Results

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In total, 23 ovarian granulosa cell tumors were evaluated for mutations of the FSH receptor gene. In 22 tumors, the entire open reading frame was examined by single-strand conformation analysis. A representative experiment is shown in Fig. 1. In one tumor, a small part of the sequence could not be analyzed because of insufficient quality of the PCR product. In the 23 tumors, two neutral polymorphisms were detected (A to G at nucleotide 2114, and A to G at nucleotide 994). The allele frequencies of the polymorphisms were similar for tumors and for unrelated controls. To address specifically the published FSH receptor mutation (5; T to C at nucleotide 1777), PCR-amplified fragments of exon 10 were also subjected to restriction with Pst I (18 cases) and/or direct sequencing (11 cases). Using these techniques, no FSH receptor mutations were detected in this sample of 23 ovarian granulosa cell tumors. This was not attributable to technical differences, given that each of these techniques readily detected the mutation in a positive control sample that contained the 1777TC mutation.

View larger version (84K): [in this window] [in a new window]   Figure 1. Single-strand conformation polymorphism analysis of PCR products, representing nucleotides 1317–1525 of the FSH receptor gene (genbank reference number M95489), generated from frozen (lanes 1–4) and formalin-fixed paraffin-embedded (lanes 5–10) material.

Oncogenic mutations of the stimulatory -subunit of the G protein (Gs) were examined for codons 201 and 227. Direct sequencing of this region of the gene was completed for 21 tumors. Normal results were obtained for all tumors tested.

The common G protein ß3-subunit polymorphism was analyzed by direct sequencing of the PCR products. The CC, CT, and TT genotypes were found in 9, 10, and 1 tumors, respectively. The frequencies observed in these tumors were not significantly different from those observed in the normal population as described by Siffert and colleagues (Ref. 10 ; 56%, 38%, and 6%, respectively).

	Discussion

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The present results indicate that somatic mutations that activate the FSH receptor are not a major cause of granulosa cell tumors. These results are in accordance with those of Fuller and colleagues (6) and contradict those of a previous study in which a specific FSH receptor gene mutation (Phe591Ser) was found in 5 of 13 sex cord tumors and in 2 of 3 ovarian small cell carcinomas (5). However, these latter results could not be reproduced (7). Our findings also argue against the findings of a previous study that reported a recurrent mutation of the Gi2 gene (8) in ovarian granulosa cell tumors. Normal results in 19 tumors in our study, and in 13 tumors in another series (9), exclude any major role for activating Gi2 mutations in ovarian granulosa cell tumors. In conclusion, mutations of the FSH receptor G protein signaling pathway genes studied here do not play any major role in the genesis of ovarian granulosa cell tumors.

	Acknowledgments

 
The authors thank Dr. L. Looijenga and P. M. J. J. Berns (Daniel den Hoed Kliniek, Rotterdam, The Netherlands), Dr. M. van de Vijver (The Netherlands Cancer Institute, Amsterdam, The Netherlands), Dr. F. J. M. Huikeshoven (Academic Hospital Rotterdam Dijkzigt, Rotterdam), and Dr. B. Tutschek (University Hospital, Düsseldorf, Germany) for patient samples. We also thank Dr. Jameson (Northwestern University Medical School, Chicago, IL) for DNA samples containing a mutation at codon 591 of the FSH receptor gene.

	Footnotes

 
¹ This work was supported by Grant KUN-96–1338 from the Dutch Cancer Society.

Received September 21, 1998.

Revised February 18, 1999.

Accepted February 26, 1999.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ligtenberg, M. J. Articles by Brunner, H. G. Search for Related Content PubMed PubMed Citation Articles by Ligtenberg, M. J. Articles by Brunner, H. G. Pubmed/NCBI databases Medline Plus Health Information Ovarian Cancer