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Activating Mutation of the Stimulatory G Protein (gsp) as a Putative Cause of Ovarian and Testicular Human Stromal Leydig Cell Tumors¹

Division of Endocrinology, Developmental Endocrinology Unit and Hormone and Molecular Genetics Laboratory (M.C.B.V.F., A.C.L., J.A.M.M., V.S.L., E.T.F., B.B.M., S.M.F.V.), Department of Pathology (F.M.C., M.C.N.Z.), Hospital das Clínicas, São Paulo University School of Medicine, São Paulo; Unit of Endocrinology, Hospital Universitário Professor Edgard Santos, Bahia University School of Medicine (L.M.B.A.), Salvador, Brazil

Address all correspondence and requests for reprints to: Dr. Sandra M. F. Villares, Hospital das Clínicas, Endocrinologia, Caixa Postal 3671, CEP 01060–970, Sao Paulo, Brazil. E-mail: smvillar{at}usp.br

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Activating mutations of the G protein genes have been associated with the development of several endocrine neoplasms. Such activating mutations, gip2, affecting the -subunit of the G_i2 protein were previously described by a single group in 30% of ovarian sex cord stromal tumors. Other activating mutations of the -subunit of the G_s (gsp) have been identified in GH-secreting and nonfunctioning pituitary tumors, autonomous thyroid adenomas, and all affected McCune-Albright tissues, but not in sex cord stromal tumors. In the present study, we investigated the presence of gip2 and gsp mutations in 14 human sex cord stromal tumors. Six Leydig cell tumors (4 ovaries and 2 testes), 2 thecomas, 2 granulosa cell tumors, 3 androblastomas, and 1 gonadoblastoma (sex cord and germ cell) were included in this study. Genomic DNA was obtained from either fresh-frozen tumor tissues or paraffin-embedded sections and in some cases from blood samples. Using PCR, denaturing gradient gel electrophoresis, and direct sequencing, we detected 4 tumors (66.6%) with the gsp mutation (R201C) in our series of ovarian and testicular Leydig cell tumors. In contrast, no gip2 mutations were found in any of the sex cord stromal tumors studied. In conclusion, our findings suggest that the putative oncogene gsp may play a significant role in the molecular mechanism of these tumors.

	Introduction

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SEX CORD stromal tumors are neoplasms that contain granulosa cells, thecal cells, Sertoli cells, Leydig cells, fibroblasts of specialized stromal origin, or the precursors of these cells, either separately or in some combination (1). These tumors are notable for their endocrine activity and account for 3–10% of all gonadal tumors (2). The pathogenetic mechanisms underlying the development of these tumors as well as the exact origin of the proliferating cell are still unknown.

The heterotrimeric G proteins, composed of -, ß-, and -subunits, are a family of proteins that link cell surface receptors for a wide variety of extracellular signals to either enzymes or ion channels, resulting in the generation of an intracellular second messenger (3). Mutations of the -subunit genes that lead to constitutive activation (_s and _i2) are associated with human disease (4). The _s mutations (gsp) were found in several somatotroph and nonfunctioning pituitary adenomas, thyroid tumors, and McCune-Albright syndrome (5, 6, 7). These mutations result in decreased intrinsic guanosine triphosphatase activity and accelerated cAMP production in the absence of stimulatory hormone (8, 9, 10, 11, 12).

An activating mutation at codon 179 of the G_i2 gene (gip2) was identified in 2 granulosa cell tumors and 1 thecoma of 10 ovarian tumors (4). However, Shen et al. were unable to confirm gip2 mutations in any of 13 granulosa cell tumors examined (13). The mechanism by which gip2 transmits mitogenic signals remains unclear. The most readily detectable effect of gip2 expression is a sustained inhibition of adenylyl cyclase activity, resulting in a decreased basal level of cAMP (14, 15). In the present study, we investigated a group of sex cord stromal tumors for activating mutations at codons 179 and 205 of G_i2 and at codons 201 and 227 of G_s to establish the molecular pathogenesis of these tumors.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This study was approved by the ethics committee of our Department. Informed consent was obtained from patients.

Subjects

Fourteen patients, 6–60 yr old, 12 females and 2 males, were included in this study (Table 1). Nine women presented with clinical virilization features, 2 with menstrual irregularities, and 1 with an abdominal mass. The boy presented with isosexual pseudoprecocious puberty and an enlarged right testis. The man presented with bilateral Tanner V gynecomastia, a 1.9-cm testicular nodule, and azoospermia. None had clinical or radiological findings suggestive of McCune-Albright syndrome. Hormonal data were available for 11 subjects (Table 2). Patients 2, 13, and 14 were treated many years before, and their hormonal data were not available. Eight female patients with virilization signs had elevated serum testosterone levels. The testosterone levels of subject 3 were slight elevated. The man (subject 10) with feminization had low levels of testosterone associated with slightly elevated estradiol levels. Estradiol levels were elevated for chronological age in the 3 females with the highest testosterone values, suggesting peripheral conversion from androgen to estrogen. Serum dehydroepiandrosterone sulfate levels were normal for chronological age in 9 patients studied. All cases were reviewed by a pathologist specialized in gynecology. They were classified as Leydig cell tumors (4 in the ovary and 2 in the testes), thecoma (2 cases), granulosa cell tumors (2 cases), androblastomas (3 cases), and gonadoblastoma (1 case).

Genomic DNA was isolated from fresh-frozen tissue or archival paraffin-embedded gonadal tumors and in some cases also from peripheral blood by standard procedures. Three 5-µm sections of the paraffin-embedded tumors were cut from each block and mounted on glass slides. The middle section was stained with hematoxylin-eosin and reviewed by the pathologist to confirm the normality of the controls and to demarcate areas of tumor and nontumors in each case. The excess paraffin was removed, and the tissue was scraped into sterile 1.5-mL tubes. Isolation of genomic DNA from these tumor tissues was performed according to previously described procedures (16).

PCR was used to amplify DNA fragments including codons 201 and 227 of G_s, as previously described (7). Exon 8 of the G_s gene was amplified using a pair of oligonucleotides: forward primer with GC clamp (M), 5'-CAGAAACCATGATCTCTGTTA-3'; and reverse primer (J), 5'-TCGGTTGGCTTTGGTGAGATCCAT-3'. Primers for exon 9 of the G_s gene were: forward (P), 5'-AACTGCAGCCAGTCCCTCTGGAATAACCAGG-3'; and reverse with GC clamp (S), 5'-CAGCGACCCTGATCCCTAACA-3'. Genomic DNA was amplified in a 100-µL PCR mixture containing 0.2 mmol/L of each deoxynucleotide triphosphate, 50 mmol/L KCl, 20 mmol/L Tris-HCl (pH 8.3), 1.5–2.5 mmol/L MgCl2, 50 pmol of each primer, and 1.25 U Taq DNA polymerase (Pharmacia Biotech, Uppsala, Sweden). Thermal cycling was performed in a Gene Amp PCR system (Perkin-Elmer/Cetus, Norwalk, CT). The reaction included an initial 94 C denaturation step for 5 min, followed by 30 cycles of denaturation at 94 C (30 s), annealing at 60 C (45 s), and extension at 72 C (45 s), with a final extension step of 10 min at 72 C. Seven PCR products (204 bp) from paraffin-embedded DNA were subsequently used as a template in a second PCR, using the forward primer M and a nested reverse primer (Y), 5'-GTTGGCTTACTGGAAGTTGAC-3', for the amplification of a fragment of 170 bp in exon 8 of the G_s that contains R201. These PCR products were directly sequenced. Exons 5 and 6 of the G_i2 gene were amplified using a pair of oligonucleotide primers: forward, 5'-CCCAGCTACCTGAACGACCTGG-3'; and reverse with GC clamp, 5'-GCTCACTTGAAGTGTAGGTC-3', as previously described (5). The cycling protocol was identical for both exons and consisted of an initial denaturation step at 94 C (5 min), followed by 40–45 cycles of annealing at 61 C (45 s), extension at 72 C (45 s), and denaturation at 94 C (30 s), with a final extension step of 5 min at 72 C (5). All PCR products were analyzed on 2% agarose gel, followed by ethidium bromide stain.

Denaturing gradient gel electrophoresis (DGGE)

The theoretical melting profiles of the amplified fragments were generated using the computer algorithm of Lerman et al. (17). Melting maps predicted that the coding regions of the G_s and G_i2 genes analyzed would all be contained in a single, low melting domain. Hence, any mutations that occur within those regions are likely to be detected. Forty-five microliters of the PCR products were required for the DGGE analysis. Electrophoresis was performed for 15 h at 80 V at 60 C on a 10% acrylamide gel with increasing denaturant concentrations (45–85% for exons 5 and 6 of PG_i2, 37–67% for exon 8 and 40–90% for exon 9 of PG_s). Gels were stained with ethidium bromide. For comparative purposes, samples of normal ovaries and testes and mutant controls (R201C and Q227R) of patients with GH-secreting pituitary tumors were included. No mutant controls for G_i2 were available. However, three randomly chosen samples from each exon of G_i2 that appeared to have a normal migration pattern on DGGE were sequenced to serve as the controls.

Direct sequencing

DNA sequencing was performed on all PCR products displaying abnormal migration patterns on DGGE or when screening by DGGE was not possible. PCR products were directly sequenced by the dideoxy nucleotide chain termination method, using modified T7 DNA polymerase (Sequenase, U.S. Biochemical Corp., Cleveland, OH) in the presence of [-³⁵S]deoxy-ATP. All reaction products were run on an 8% polyacrylamide gel.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The entire exons 5 and 6 of G_i2 and exons 8 and 9 of G_s were amplified by PCR from tumoral tissues of all patients and from blood samples of five patients. All fragments showed the expected size on a 2% agarose gel. DGGE analysis of PCR products from blood samples were normal in all patients analyzed. Four Leydig cell tumors (three ovaries and one testis) displayed an abnormal migration pattern on DGGE, suggestive of a heterozygous mutation in exon 8 of the G_s gene (Fig. 1). No gsp mutations (codons 201 and 227) were detected in the other patients. Both mutant controls in exons 8 and 9 of G_s displayed abnormal migration patterns, as expected (Figs. 1 and 2, respectively). Sequencing analysis of these four PCR products revealed a transversion from C to T in the first position at codon 201 of exon 8 of the G_s gene, which resulted in the encoded amino acid changing from arginine (CGT) to cysteine (TGT); (Fig. 3). PCR products generated with primers targeted at exons 5 and 6 of G_i2 gave a single sharp band corresponding to the wild-type band on the DGGE analysis in all samples examined. Three of the 14 samples from each exon presented only wild-type sequences, confirming the negative results of the DGGE.

View larger version (95K): [in this window] [in a new window]   Figure 1. Analysis of tissue samples from patients with Leydig cell tumors, a positive control, and a normal control. PCR-amplified genomic fragments encompassing exon 8 from control tissues and tissues of patients 9 and 10 were analyzed by DGGE. Paraffin- embedded normal testis and ovarian tissues were used as a normal control, and a pituitary tumor known to contain the R201C mutation was used as a positive control. In the samples analyzed by DGGE, two additional upper heteroduplex bands and the mutant homoduplex band were detected in the positive control and in patients 9 and 10.

View larger version (73K): [in this window] [in a new window]   Figure 2. DGGE analysis of sex cord stromal tumors show no mutation in exon 9 of PGs. Paraffin-embedded normal testis and ovarian tissues were used as a normal control, and a pituitary tumor known to contain the Q227R mutation was used as a positive control. In the samples analyzed by DGGE, two additional upper heteroduplex bands and a mutant homoduplex band were detected only in the positive control.

View larger version (86K): [in this window] [in a new window]   Figure 3. Direct sequencing of amplified DNA from normal control tissue and from Leydig cell tumors. The mutation and normal controls are marked by arrows; the nucleotide and amino acid sequence of the mutated codon are given on the right (exon 8, codon 201), and the normal control is on the left of the sequencing gel.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In this study, we investigated gsp and gip2 mutations in patients with sex cord stromal tumors. We identified the R201C of the G_s protein in 66.6% of Leydig cell tumors studied (one testicular and three ovarian tumors). No gip2 mutations were demonstrated in any of the patients studied.

Molecular defects in sex cord stromal tumors have rarely been reported. The -inhibin gene has been shown to be a suppressor of granulosa tumorigenesis in knockout mouse models (18). Watson et al. detected a low frequency of loss of heterozygosity in the -inhibin gene on chromosome 2q in 17 human granulosa cell tumors (19). Trisomy 12 was detected in a large proportion in a series of benign sex cord stromal tumors, mainly in thecomas, but the role of this in the genesis of these tumors is unknown (20, 21).

The fact that many sex cord stromal tumors are hormonally active led to the assumption that alterations in the signaling cell pathway might play a role in the development of these tumors (22). Binding sites for LH and FSH have also been identified in sex cord-stromal tumors, suggesting that these tumors might retain responsiveness to gonadotropins (23). Recently, Kotlar et al. identified a FSH receptor mutation in granulosa cell tumors that impairs FSH responsiveness, probably by altering coupling to the signal transduction apparatus. This mutation, F591S, is located in the region postulated to represent a G protein interaction domain (24). In a previous study of archival tissues from a wide variety of tumors, Lyons et al. identified the presence of a mutation in the G_i2 gene in 3 of 10 ovarian sex cord stromal tumors (4). These 10 tumors consisted of 7 granulosa cell tumors, 2 thecomas, and 1 androblastoma. Two granulosa cell tumors and 1 thecoma were positive for the gip2 oncogene, showing a histidine for arginine substitution at codon 179. No mutations on the G_s gene were identified in any of those samples (4). Lately, Shen et al. examined 13 granulosa cell tumors, and no gip2 mutations were found using both direct sequencing and allele-specific oligonucleotide hybridization in any of the tumors studied (13). No Leydig cell tumor was analyzed in either study.

We investigated whether the known gsp- and gip2- activating mutations of the G protein genes are involved in the pathogenesis of sex cord stromal tumors. We analyzed activating mutations of G proteins in a subset of Leydig cell tumors. At this time, gsp mutations have only been described in gonadal tumors associated with McCune-Albright syndrome. Using DGGE and direct sequencing, we found the gsp oncogene in 4 of 14 stromal sex cord tumors. All tumors with the gsp oncogene were Leydig cell tumors. All women with gsp mutations had virilization, with markedly elevated levels of testosterone. Interestingly, patient 9 had decreased testosterone levels after 2 doses of agonist LHRH depot administration (25). One male patient with feminization and infertility had elevated estradiol levels associated with decreased testosterone levels. All patients showed normal clinical and hormonal profiles after surgery, and no recurrence was observed. No systematic clinical and hormonal differences between patients with Leydig cell tumors with and without mutations could be found in our series. This may be due to the small number of patients with Leydig cell tumors and gsp mutations as well as to the clinical and hormonal heterogeneity of the group studied.

In conclusion, the gsp mutation (R201C) was found in 66.6% of ovarian and testicular Leydig cell tumors, suggesting that the putative oncogene gsp may play a significant role in the pathogenesis of these tumors.

	Acknowledgments

 
The authors thank Dr. Andrew Shenker for providing the gsp mutant controls. We thank the staff of the Laboratorio de Pesquisa da Clínica Médica I. We also thank Dr. Ana Elisa Correia Billerbeck, Dr. Marcelo C. Batista, and Dr. Luiza Villa, for excellent support, and Drs. João Norberto Stavvle and Maria Odete Ribeiro Leite for presenting the patients with sex cord stromal tumors.

	Footnotes

 
¹ This work was supported in part by a grant from the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP 950370–2 to S.M.F.V.).

Received December 16, 1997.

Revised February 12, 1997.

Accepted February 20, 1998.

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