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Complete Sequencing and Messenger Ribonucleic Acid Expression Analysis of the MEN I Gene in Adrenal Cancer¹

Klinik für Allgemeine Chirurgie und Unfallchirurgie (K.-M.S., M.M., M.H., D.S., H.-D.R.) and Biologisch-Medizinisches Forschungszentrum (S.S., K.K.), Heinrich-Heine-University, 40225 Düsseldorf, Germany

Address correspondence and requests for reprints to: Dr. med. Klaus-Martin Schulte, Klinik für Allgemeine Chirurgie und Unfallchirurgie, Medizinische Einrichtungen, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany. E-mail: schultekm{at}med.uniduesseldorf.de

	Abstract

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Adrenal cancer is a rare sporadic disease that has also been observed in the context of multiple endocrine neoplasia type I (MEN I). Adrenal lesions occur in up to 40% of MEN I patients. Loss of heterozygosity of the 11q13 band harboring the menin gene has been reported in more than 50% of patients with adrenal cancer. Despite this high index of suspicion, former screening studies did not reveal mutations of the MEN I gene in 28 patients. We identified loss of heterozygosity of 11q13 microsatellites in five of five patients (100%). In 40%, heterozygosity was retained in codon 418 of the MEN I gene.

Complete direct DNA sequencing data of the entire coding region and adjacent splice sites of the MEN I gene were obtained in 14 patients with sporadic adrenal cancer. In only one of them a heterozygous missense mutation, R176Q (exon 3), was identified. Due to the heterozygous pattern and unknown biological effect of this mutation, it is not clear whether there is a causal relationship with adrenal cancer. The total mutation frequency in sporadic adrenal cancer is 1 of14 (7%). Menin messenger RNA expression was identified in 14 of 14 patients (100%). Transcriptional inactivation of the menin gene is, hence, unlikely to cause loss of its tumor suppressor function in adrenal cancer.

Furthermore, we examined three patients who presented adrenal cancer in the context of sporadic multiglandular endocrine tumor disease previously diagnosed on clinical grounds to be MEN I syndrome. An opal stop codon mutation was identified in codon 126 (exon 2) in the adrenal cancer of one of these patients. Formation of the adrenal cancer in this patient may be rather coincidental because the mutation was present in a heterozygous pattern. There was no mutation of the menin gene in the two other patients. This may mean that formation of adrenal cancer in the context of multiglandular endocrine disease denotes an entity different from MEN I in some patients.

	Introduction

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MALIGNANT ADRENAL tumors occur with an incidence of about 1.7 new cases per million per year (1). The genetic basis of either benign or malignant tumors is not completely understood (2, 3, 4). Lesions involve mutations of such genes as p53 (5), p21 (6), or the ACTH receptor (7). Some adrenal tumors occur in the context of hereditary tumor syndromes such as the Li-Fraumeni syndrome (8), the Beckwith-Wiedemann syndrome (9, 10, 11), or the Carney complex (12). In about 30–40% of patients with multiple endocrine neoplasia type I (MEN I), adenomas or bilateral hyperplasia of the adrenal gland are observed (13, 14, 15, 16). Adrenal cancer has been reported in few patients suffering from a MEN I syndrome (13, 17).

Loss of heterozygosity (LOH) in adrenal tumors frequently involves the loci 2q, 4p, 13p, 18p (18), 17p (19), 11p and 11q (18, 19, 20, 21), and 13q (19). With regard to adrenal cancer only, LOH on chromosome 11q13 is a rather frequent event. In this entity, it was seen in 2 of 2 patients (22), in 1 of 1 (13), 2 of 2 (100%) (21), 4 of 8 (50%) (20), 9 of 19 (47%) (18), 1 of 4 (25%) using a set of 11q13 microsatellites (23), and 6 of 10 (60%) using the MEN I sequence in a fluorescence in situ hybridization assay (24). In total, it was present in 23 of 44 (52%) examined patients in whom different technical approaches were used. In MEN I patients, the frequency of 11q13 LOH in either benign or malignant adrenal lesions was only moderate, however (13, 23). In a study of six patients with clinically overt MEN I syndrome and associated adrenal lesions, only the patient with adrenal cancer had LOH of the 11q13 band (13). The gene responsible for MEN I is located on chromosome 11q13 and has been characterized as a tumor suppressor gene (25). Inactivating mutations of the MEN I gene occur not only in patients with a typical syndrome, but also in sporadic tumors that otherwise are part of the MEN I disease. Examples are parathyroid adenomas (21%) (26), gastrinomas (33%) (27), bronchial carcinoid tumors (36%) (28), and, rarely, pituitary tumors (29, 30).

It is on this background that the MEN I gene has been considered as a candidate gene for the genesis of adrenal neoplasms. Three recent reports described absence of MEN I gene mutation in sporadic adrenocortical neoplasms. These studies used screening techniques for detection of MEN I gene lesions and analyzed a total of 28 patients (18, 23, 24). Our study presents complete direct DNA sequencing of the MEN I gene, including adjacent splice sites in 14 patients with sporadic adrenal cancer and 3 patients with sporadic MEN I-like disease. LOH studies were performed in the 11q13 region harboring the MEN I gene. Messenger RNA (mRNA) expression of menin was studied in 14 patients.

	Subjects and Methods

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Subjects

Tumor tissue was obtained at adrenalectomy (ADX) from the central part of the lesion by an experienced endocrine surgeon, and tissue was immediately frozen at -80 C under tissue tek. Particular care was taken to obtain a part of tumor tissue in which contamination by normal adrenal tissue could be safely excluded already on macroscopical examination. Diagnosis was established by histopathological examination. Malignancy was determined by presence of infiltrative growth including capsule and/or vessels. Clinically, malignancy was defined in presence of metastasis or local recurrence of an infiltrating mass. Patients with hereditary tumor disease were excluded. We included 14 sporadic adrenocortical carcinomas and three patients with adrenal cancer in the setting of sporadic syndrome similar to MEN I disease (patients 5, 14, and 100) (Table 1). Tissue or blood was removed after informed consent of the patients. All parts of the study were conducted according to the Declaration of Helsinki principles.

DNA from blood and tumor was extracted using the Qiagen blood and tissue kits (Qiagen, Hilden, Germany). Tumor tissue was cut to 10-µm slices by a microtome. A hematoxylin and eosin-stained slice adjacent to that used for DNA isolation was obtained to confirm the adrenal identity of the tissue sample.

LOH analysis

We used the polymorphic markers PYGM, D11S480, D11S987, and D11S449. PYGM is located centromeric of the MEN I gene, D11S480 is located centromeric to PYGM, and D11S449 and D11S987 are located telomeric to the MEN I gene (Table 2). PCR conditions and LOH scoring have been formerly published (31). Fluorescence-labeled amplimers were separated by capillary electrophoresis under denaturing conditions with an ABI PRISM 310 Genetic Analyzer A (Perkin-Elmer Biosystems, Branchburg, NJ). GeneScan 350, TAMRA marker, was used as an internal size standard. The allele ratio for the normal sample was calculated by division of the peak area integrals of the two alleles. The LOH ratio was calculated by dividing the LOH ratio of the tumor by that of the corresponding blood control. LOH was defined in presence of an LOH ratio less than 0.4.

Exons 2–10 and the neighboring splice junctions were amplified by PCR using oligonucleotide primers located in the intronic parts of the gene (Table 2). The reaction contained 15 µl TaqMasterMix (Qiagen), 100 ng genomic DNA, and 1 µM oligonucleotide primers and was cycled at the following conditions: 94 C 5 min, 35 cycles of 94 C 30 sec, 55–60 C 30 sec, 72 C 1 min, and 72 C 5 min. Reaction products were purified using the PCR purification kit or the gel extraction kit (Qiagen) after 1% agarose gel electrophoresis. Cycle sequencing by Taq DNA-polymerase was performed with M13-oligonucleotides in a 10-µl volume containing 20–40 ng PCR products and 10 pmol forward and reverse M13 primer using a dye terminator method with 25 cycles of 96 C 10 sec, 50 C 5 sec, and 60 C 4 min (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit; Perkin-Elmer Biosystems). Sequencing products were separated by PAGE with an ABI377 DNA sequencer (Perkin-Elmer Biosystems). Chromatograms were analyzed using the Lasergene Navigator software (GATC, Koustauz, Germany) and blasted against the published MEN I genomic sequence (GenBank accession number U93237). In rare cases where sense- and antisense sequencing did not yield a definite sequence, the exon was reamplified from genomic DNA and resubjected to forward and reverse sequencing. Mutations and polymorphisms were subjected to repeated sequencing from independent tissue samples and independently produced amplimers.

RNA isolation and RT-PCR of the menin gene

A 10-µm microtome slice was used for isolation of RNA using TRIzol (Life Technologies, Inc., Grand Island, NY) by a method modified from Chomczynski and Sacchi (32). RT was done using murine leucemia reverse transcriptase mMuLV using a complementary DNA (cDNA) synthesis kit according to the instructions of the manufacturer (Apbiotech, Freiburg, Germany). PCR of the cDNA was performed using the oligonucleotides menin 4 and menin 6 (Table 2), which span introns 4 and 5 and yield a product of 257 bp. The corresponding DNA amplimer is 668 bp long. PCR conditions were identical to those given above, with an annealing temperature of 57 C. The product was visualized by 1% agarose electrophoresis and ethidium bromide staining. Quality of the cDNA was assured by previous amplification of a house-keeping gene (ß-actin) and a low copy number mRNA (transforming growth factor ß-type II receptor or edg-2-receptor). Only cDNA, which consistently yielded amplimers with these test primers, was used for menin expression analysis. Failure to obtain the mRNA-specific band at 257 bp was controlled by RT-PCR repeated two times.\.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
LOH analysis

Pairs of tumor and leukocyte DNA were available in five patients with sporadic adrenocortical carcinoma. Fig. 1 demonstrates the LOH in informative alleles of 11q13 loci and corresponding sequence information on intragenic polymorphic sites in five adrenal cancers. In all patients with adrenal cancer, LOH could be demonstrated by at least one variable number of tandem repeats-marker. Markers were informative in 75–80% of cases examined. LOH of the 11q13 region did not affect the MEN I gene in two of five patients with 11q13 LOH because these patients had retained heterozygosity at position 7264 (patient 64) or position 2722 (patient 17) in the same DNA sample that had been used for LOH determination. In 12 additional adrenal cancers no patient blood was available. In 5 of these (42%) LOH was excluded by heterozygous sequence determination of the intragenic polymorphic sites 2722 (codon 145) and 7264 (codon 423). In seven patients (58%), sequence information did not allow further discrimination of homo- vs. hemizygosity.

View larger version (34K): [in this window] [in a new window]   Figure 1. LOH analysis in five patients with adrenal cancer.

Unequivocal sequence determination by direct sequencing of strand and antistrand DNA was achieved for 17 patients with adrenal cancer. All coding exons (2, 3, 4, 5, 6, 7, 8, 9, 10) were analyzed. Amplimers typically included the splice junctional positions at the exon/intron borders (Table 2). Analysis of the adjacent intronic sequences of exons 2–10 did not yield any sign of mutations in splice sites. In position 6821 (intron 8) we regularly identified the sequence aca ggcca and not acagggcca, as published previously (GenBank U93237). In position 4243 (intron 2) we regularly identified the sequence tggccccctttc and not tggcccc tttc, as previously published (GenBank U93237).

There were only few mutations and polymorphisms in the entire open reading frame of the MEN I gene in patients with malignant adrenal tumors. Patient 71 had a heterozygous missense mutation R176Q in exon 3 (Fig. 2). Patient 5 had a stop codon mutation in codon 126 by transition of TGG to TGA, forming an OPAL stop codon from tryptophane (Fig. 3). Patients 17 and 64 showed heterozygous transition from AGC to AGT S145S, a previously defined intragenic polymorphism (genomic position 2722).

View larger version (38K): [in this window] [in a new window]   Figure 2. Heterozygosity is present in position 4363 (codon 176) in a patient with adrenal cancer. The missense mutation CAG codes for an exchange from arginine to glutamine in the third exon of the menin gene. Forward and reverse sequence are presented. The above lane shows the generic sequence.

View larger version (31K): [in this window] [in a new window]   Figure 3. Opal stop codon mutation in patient 5. Heterozygosity is present in position 2665 (codon 126). The missense mutation TGA codes for an exchange from tryptophan to an opal stop codon in the second exon of the menin gene. Presented are two reverse and one forward sequence.

High-quality RNA could be obtained from archival tissues of 14 patients with adrenal cancer. Samples with potential contamination by surrounding adrenal tissue were excluded from analysis. Suitability of derived cDNAs for gene expression analysis was ascertained by amplification of a 446-bp ß-actin fragment and a 163-bp fragment of the transforming growth factor ß receptor type II using intron-spanning primers (data not shown). Expression of menin was demonstrated by amplification of a 257-bp fragment including parts of exons 4, 5, and 6. Contaminating DNA yielded a 668-bp amplimer (Fig. 4). The 257-bp menin mRNA amplimer was present in 14 of 14 (100%) of adrenal cancer tissues (Table 3).

View larger version (53K): [in this window] [in a new window]   Figure 4. Menin nRNA expression in adrenal cancer assessed by RT-PCR.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Hitherto, screening data for mutations are available for a total of 28 patients with adrenal cancer from three independent studies. Dideoxyfingerprinting, which was used in 10 patients (26), has a high sensitivity to detect mutations in the MEN I gene (25, 26, 34, 35, 36). Single-strand conformational polymorphism/single-strand conformational analysis/single-strand conformational variation used for the remaining 18 patients is less sensitive and detects about 60–80% of mutations of the MEN I gene when applied after prior LOH analysis (18, 33). Because MEN I mutations in adrenal cancer may have escaped detection due to the relatively small sample size and the limited sensitivity of single-strand conformational polymorphism screening techniques, we here add complete direct sequencing data on 14 additional patients with sporadic isolated adrenal cancer. A single heterozygous missense mutation was observed in exon 3, codon 176, coding for an amino acid exchange from arginin to glutamine in a patient with sporadic adrenal cancer. If the heterozygous amino acid exchange decreases, menin function could not be examined because useful tests for the biological consequences of a mutation are lacking. This 40-yr-old female had neither a family history nor other coincident or metachronic endocrine tumors. No mutation was identified in 13 patients yielding a mutation frequency of 7%, if the above heterozygous mutation is taken into consideration and 0% if it is excluded.

It has long been discussed whether adrenal cancer truly belongs to the clinical spectrum of the MEN I disease. The presentation of two very rare diseases in one patient argues in favor of a mechanistic relation, but molecular proof has not been given yet. A stop codon mutation in exon 2 was identified in patient 5. However, the mutation occurred in a heterozygous pattern not fulfilling Knudson’s theory for the inactivation of tumor suppressor genes (Knudson, 1971 #2192). In this particular patient, admixture of normal adrenal tissue was unlikely because he presented with a bulky, recurrent, infiltrative, and locally inoperable tumor in our institution and the sample was obtained from this tumor mass. This patient fulfills the criteria for diagnosis of sporadic MEN I syndrome by former operations of a pituitary adenoma and multiglandular hyperparathyroidism. This is the first patient with clinical MEN I disease and adrenal cancer in whom molecular proof of an inactivating menin mutation is reported, but the genesis of this adrenal cancer is prone to be accidental or related to a mechanism only indirectly related to his menin gene defect.

No mutation was detected in patient 100. In this 32-yr-old woman, a single parathyroid adenoma had previously been removed at the age 25 yr. One further parathyroid gland was normal, two were not identified. She also presented a follicular thyroid adenoma and a secondary amenorrhea from age of 16 yr until death. Her father had been operated on a double parathyroid adenoma. This constellation can not be described as typical MEN I disease, but may reflect a different entity of multiglandular endocrine disease. We equally did not detect any mutation in patient 14. This 45-yr-old female presented with Cushing’s syndrome caused by bilateral adrenal disease. The patient also suffered from a bicentric prolactinoma. Histopathological examination revealed massive multinodular hyperplasia on the right side and an adrenal cancer of 130 g on the left side. There was no family history.

Analysis of mRNA expression is a suitable method to monitor transcription of the menin gene (37). In our context, we did not search for the gradual decrease observed by deletion of one allele but only for complete loss of menin message that would be necessary to cause loss of menin function in the absence of any mutation in the coding sequence, as had been confirmed in our samples by prior sequencing. All adrenal cancers maintained expression of menin mRNA. Particular care was taken to obtain tumor tissue from the center of the macroscopically identified cancer lesion later histopathologically confirmed. Such samples may be particularly suitable for analysis since by histopathological examination alone it may be virtually impossible to confirm the malignant character of particular areas of adrenal tissue. In presumed absence of contamination by normal adrenal tissue and the well-known monoclonal character of tumorous adrenal lesions (38), the presence of menin mRNA in all samples of adrenal cancer examined renders unlikely that transcriptional inactivation of the MEN I gene is responsible for loss of its tumor suppressor function in such tumors.

In summary, our data identify for the first time a heterozygous missense mutation of the MEN I gene in a patient with sporadic adrenal cancer. However, the biological consequences of the observed heterozygous mutation and its relation to tumor formation are uncertain. The mutation frequency in our group of 14 sporadic adrenal cancers is 7% by full-length direct DNA sequencing, if this mutation is taken into consideration and 0% if it is excluded. We also present the first case where adrenal cancer occurs in a patient in whom MEN I disease is proven by mutational analysis. The heterozygous pattern of this mutation in the tumor tissue argues in favor of a mechanism of tumorigenesis that is accidental or only indirectly related to the MEN I gene defect. Absence of MEN I gene mutations in adrenal cancers of two additional patients with atypical multiglandular endocrine tumor disease argues in favor of the presence of entities that resemble in the phenotype but not the genotype. Presence of menin mRNA expression renders it unlikely that inactivation of menin transcription is responsible for formation of adrenal cancer. The frequent evidence of LOH at 11q13 in adrenal cancer in absence of MEN I gene defects hints toward a role for a different tumor suppressor gene located in this chromosome band.

	Acknowledgments

 
We thank Mrs. K. Alemazkour, Mrs. B. Weller, and Mrs. B. Bosilj for excellent technical assistance.

	Footnotes

 
¹ Supported by Grant 9772041 from the research committee of the Heinrich-Heine-University (Düsseldorf, Germany). K.M.S. is supported by a grant from the German National Research Foundation (Deutsche Forschungsgemeinschaft DFG Schu 1270/1-2).

Received August 27, 1999.

Revised October 4, 1999.

Accepted October 15, 1999.

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