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The MEN-1 Gene Is Rarely Down-Regulated in Pituitary Adenomas¹

Departments of Pathology and Laboratory Medicine (S.L.A., K.S.) and Medicine (S.E.), Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5

Address all correspondence and requests for reprints to: Dr. Sylvia L. Asa, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. E-mail: sasa{at}mtsinai.on.ca

	Abstract

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The gene for multiple endocrine neoplasia type 1 (MEN-1) has recently been cloned and encodes a putative tumor suppressor protein named menin. We have previously reported inactivating MEN-1 gene mutations associated with loss of heterozygosity (LOH) of the normal allele in tumors of patients with MEN-1 and in some sporadic pituitary tumors. These genetic alterations, however, are noted in no more than 10% of sporadic adenomas. To investigate whether other mechanisms may result in down-regulation of menin gene expression in pituitary adenomas, we examined menin gene expression by semiquantitative RT-PCR in 60 sporadic pituitary adenomas. Ribonucleic acid (RNA) was extracted from surgically resected, morphologically characterized tumors. Primers were designed to amplify a 257-bp fragment spanning exons 4–6 of the MEN-1 gene. A product of the predicted size was amplified from normal pituitary samples as well as from adenomas. Competitive PCR was performed with the housekeeping gene PGK-1 to quantitate menin gene expression. A comparable ratio of menin/PGK-1 messenger RNA was identified in all but three samples; in two tumors with LOH, menin expression was weak, and in one tumor, menin messenger RNA was undetectable, associated with LOH and mutation of the other allele.

Reduced expression of menin in some sporadic adenomas is consistent with a putative tumor suppressor role for this gene product. However, lack of menin down-regulation in the majority of these tumors, which exhibit LOH at 11q13 in up to 20% of cases, provides compelling evidence for an additional tumor suppressor gene at this locus, which is more commonly involved in the pathogenesis of pituitary neoplasms.

	Introduction

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THE PATHOGENESIS of pituitary adenomas, despite being the subject of intense investigation, remains enigmatic (1). These tumors form an integral part of the multiple endocrine neoplasia type 1 (MEN-1) syndrome, and the putative tumor suppressor encoded by the MEN-1 gene at 11q13 has been implicated as important in the development of these neoplasms. Although loss of heterozygosity (LOH) at 11q13 was rare (2, 3, 4), accurate analysis was not possible until the gene was cloned (5). This allowed identification of mutations of the transmitted allele in affected members of MEN-1 kindreds and LOH in tumors of patients with familial disease (5), consistent with the Knudsen hypothesis.

The role of menin in the more common sporadic pituitary adenomas remains an important question. We recently showed that LOH is rare (10%) and mutation of the remaining allele is even more rare (5%) in sporadic pituitary adenomas (6). Nevertheless, if menin is an important tumor suppressor in the pituitary, neoplasia may result from decreased gene expression due to epigenetic factors that could result in down-regulation. We therefore investigated the levels of menin messenger ribonucleic acid (mRNA) expression in a series of sporadic pituitary adenomas.

	Materials and Methods

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Human pituitary tissues and tumors

Pituitary adenomas were collected at the time of surgery from patients who had undergone full endocrine preoperative evaluation. Fresh tissue was divided for histological and immunohistochemical studies, electron microscopy, and molecular analysis as described previously (7, 8, 9). Individual tumors were classified based on the clinical and biochemical features, the profile of hormone content by immunohistochemistry, and ultrastructural features.

Normal human pituitaries obtained at autopsy within 8 h of death from patients with no evidence of endocrine abnormality were examined histologically and by immunohistochemistry to exclude incidental pathology. Half of each gland was frozen for molecular analysis.

DNA analysis

Fresh tissue was snap frozen in liquid nitrogen and stored at -70 C. Each specimen was divided into two parts for DNA and RNA analyses. DNA was extracted as described previously (6); the analysis of LOH and mutations in the MEN-1 gene was previously reported (6).

RT-PCR and mRNA analysis

Total RNA was extracted by the guanidinium isothiocyanate method and reverse transcribed. The integrity of RNA from each reverse transcribed sample was verified by RT-PCR for human phosphoglycerate kinase (PGK-1). The primers used to identify PGK-1 were: upstream, 5'-GCT GAC AAG TTT GAT GAG AAT-3'; and downstream, 5'-GCT CCT GGA AGG TAA AGT CCT-3'. These primers span two introns between exons 8 and 10, generating a 338-bp product.

All samples were examined to exclude the possibility of contamination by nontumorous adenohypophysis. Each sample underwent RT-PCR for Pit-1 and for SF-1 mRNA expression as described previously (7, 8). Tumors that expressed GH, PRL, or TSH were considered contaminated if expression of SF-1 was detected, tumors that expressed gonadotropins were considered contaminated if expression of Pit-1 was detected, and ACTH-producing adenomas were considered contaminated if either Pit-1 or SF-1 was detected (9). Any contaminated tumors were excluded from further analysis.

The oligonucleotide primers used to identify menin mRNA were: upstream, 5'-GCT GGC TGT ACC TGA AAG GA-3'; and downstream, 5'-CAC TCA CCC TCT ACC ACA AG-3'. These primers span two introns between exons 4 and 6, and generate a 257-bp product. The conditions for PCR amplification were optimized using normal adenohypophysis. To ensure that the product yield was still in the linear range of the reaction, various conditions were tested. PGK-1 was chosen as the housekeeping gene because it was expressed in a linear range comparable to that of menin. For the final analyses, complementary DNA was denatured at 95 C for 120 s; PCR amplification was carried out through 30 cycles at 95 C for 30 s, 57 C annealing for 30 s, and 72 C extension for 30 s. Amplification was carried out in a final volume of 25 µL, containing 5 µL RT sample, 0.5 µmol/L of each upstream and downstream primer, 2.0 mmol/L MgCl₂, 0.4 mmol/L deoxy-NTP, 2.5 U Ampli-Taq DNA polymerase (Perkin-Elmer, Norwalk, CT), and autoclaved distilled water. Products were visualized on 1.5% agarose gel electrophoresis with ethidium bromide staining. Negative controls omitted reverse transcriptase or replaced template with water.

PCR reactions were performed with each set of primers separately and for semiquantitative assessment using MEN-1 and PGK-1 primers in the same PCR reaction tubes. Each tumor RNA sample was subjected to at least three separate RT-PCR analyses. For analysis of competitive PCR, the intensity of the bands was determined on negative images of the ethidium-stained gels using a computing densitometer (model 300A, Molecular Dynamics, Sunnyvale, CA) and ImageQuant Software. Statistical analysis was performed with paired Student’s t test.

	Results

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Human pituitary tissues and tumors

A total of 60 pituitary adenomas were used for the final analysis based on the integrity of the mRNA and lack of contamination by nontumorous adenohypophysis. Ten nontumorous adenohypophyses had intact mRNA based on PGK-1 mRNA amplification, and all expressed Pit-1 and SF-1.

DNA analysis

Three tumors had evidence of DNA abnormalities in the MEN-1 gene. Two of these, one lactotroph macroadenoma from a 24-yr-old woman with amenorrhea and galactorrhea, and one mammosomatotroph macroadenoma from a 67-yr-old acromegalic woman, exhibited allelic deletion as described previously (6). The third, a 1-cm sparsely granulated somatotroph adenoma from a 55-yr-old acromegalic man, had both allelic deletion and an exon 2 truncating mutation of the retained allele (6).

The remainder of the tumors, which had no evidence of allelic loss or mutation of the MEN-1 gene, included 12 somatotroph adenomas from acromegalic patients, 12 lactotroph adenomas that had given rise to hyperprolactinemia and amenorrhea and/or other sexual dysfunction, 6 mammosomatotroph adenomas associated with acromegaly and variable hyperprolactinemia, 1 thyrotroph adenoma associated with elevated serum TSH, 17 clinically nonfunctioning adenomas with immunohistochemical and ultrastructural evidence of gonadotroph differentiation, 5 corticotroph adenomas associated with Cushing’s disease, and 4 clinically silent adenomas that included 3 plurihormonal silent subtype 3 lesions (10) and 1 silent corticotroph adenoma.

RT-PCR and mRNA analysis

All 10 nontumorous pituitaries and 59 of 60 pituitary adenomas of all types expressed MEN-1 mRNA that was detectable with ethidium bromide staining. The transcripts were all of the expected size (Fig. 1). The ratio of menin/PGK-1 mRNA in 10 normal samples was 0.71 ± 0.07. In 57 tumors of all types with no evidence of allelic deletion or mutation of the MEN-1 gene, the ratio of menin/PGK-1 was 0.69 ± 0.11 (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Figure 1. Representative competitive RT-PCR for menin and PGK-1 mRNA in human adenohypophysis and pituitary adenomas. The samples have bands for menin (257 bp) and PGK-1 (338 bp). Densitometric analysis documented similar levels of menin expression in most tumors (lanes 1–15) and normal glands (lanes 16–18); the tumors in lanes 3 and 7 have reduced menin expression. A negative control (-RT) is shown in the far right lane.

View larger version (57K): [in this window] [in a new window]   Figure 2. Densitometry of RT-PCR for menin and PGK-1 mRNA in human adenohypophysis and 57 pituitary adenomas.

In one tumor, menin mRNA was not detectable (not shown), consistent with LOH and a truncating mutation of the MEN-1 gene in that tumor.

	Discussion

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The recent characterization of the MEN-1 (menin) gene on chromosome 11q13 (5) has facilitated specific identification of molecular defects associated with this condition. Specific germline mutations resulting in predicted loss of function have now been described in patients with MEN-1 (5, 11), and LOH has been confirmed in tumors of those patients (12). Mutations in the MEN-1 gene have also been identified in sporadic endocrine tumors of the pancreas and gut (13, 14, 15), in sporadic parathyroid tumors (16), and in sporadic lipomas (17).

The fact that pituitary adenomas are a common component of this syndrome along with the previously noted LOH at the 11q13 site in pituitary adenomas (2, 3, 4) prompted analysis of this gene in patients with sporadic pituitary neoplasms. Using fluorescent in situ hybridization (FISH) and intragenic as well as flanking microsatellite markers, we recently demonstrated LOH at the menin gene in 4 of 39 adenomas. In 2 of these lesions (1 corticotroph and 1 mammosomatotroph adenoma), we also documented somatic missense mutations in exons 9 and 10 of this gene (6). However, LOH and/or mutation of this gene appears to be more rare in sporadic pituitary tumors than in other sporadic tumors that are associated with MEN-1.

To investigate other possible mechanisms of loss of function of this gene in sporadic pituitary adenomas, such as down-regulation or mutations in the promoter, we pursued characterization of menin mRNA expression in 60 pituitary adenomas and compared it with that in 10 normal pituitary glands. Using semiquantitative RT-PCR, we demonstrate that mRNA expression of this gene is preserved in most lesions, with the exception of 3 adenomas. In 2 tumors, 1 lactotroph adenoma and 1 mammosomatotroph adenoma, mRNA expression was diminished compared with that in other adenomas and the normal gland, and this was associated with LOH. The clinical implications of this reduction of menin mRNA remains to be determined. Both tumors were macroadenomas; the patient with the prolactinoma was apparently cured surgically and has been free of disease for 3 yr, whereas the patient with acromegaly has had persistent disease. The effect of this mRNA alteration on the translation of menin protein remains to be examined. In the third tumor, no menin mRNA was detected, and it therefore seems unlikely that any protein would be present; this acromegalic patient was cured surgically and remains free of disease 8 yr postoperatively.

The quantitative changes in mRNA expression described here are consistent with the theory that altered expression of the MEN-1 gene product plays a role in pituitary tumorigenesis in some sporadic adenomas. These findings are also consistent with the rarity of menin somatic mutations that we have reported previously (6). Previous studies have found LOH at 11q13 in up to 20% of sporadic pituitary adenomas (2, 3, 4). The low incidence of somatic mutations and the now documented intact mRNA expression of the menin gene in most pituitary adenomas provide supportive evidence for an additional tumor suppressor gene at the 11q13 locus, which is more commonly involved in the pathogenesis of these sporadic neoplasms.

	Acknowledgments

 
The authors gratefully acknowledge the contributions of Dr. Harley S. Smyth and of Ms. Lily Ramyar.

	Footnotes

 
¹ This work was supported in part by grants from the Medical Research Council of Canada.

Received April 27, 1998.

Revised May 22, 1998.

Accepted June 15, 1998.

	References

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