This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 92/5/1948    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart 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 Ozawa, A. Articles by Marx, S. J. Search for Related Content PubMed PubMed Citation Articles by Ozawa, A. Articles by Marx, S. J. Related Collections Calcium and Bone Metabolism Endocrine Oncology Neuroendocrinology and Pituitary

The Parathyroid/Pituitary Variant of Multiple Endocrine Neoplasia Type 1 Usually Has Causes Other than p27^Kip1 Mutations

Metabolic Diseases Branch (A.O., S.K.A., C.M.M., A.L.B., T.S.R., P.A.K., C.M.Q., W.F.S., L.S.W., A.M.S., S.J.M.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), Bethesda, Maryland 20892; and Genome Technology Branch (S.C.C., F.S.C.), National Human Genome Research Institute, NIH, Bethesda, Maryland 20892

Address all correspondence and reprint requests to: Atsushi Ozawa, M.D., Ph.D., Building 10, Room 9C-103, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1802. E-mail: ozawaa{at}niddk.nih.gov.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: One variant of multiple endocrine neoplasia type 1 (MEN1) is defined by sporadic tumors of both the parathyroids and pituitary. The prevalence of identified MEN1 mutations in this variant is lower than in familial MEN1 (7% vs. 90%), suggesting different causes. Recently, one case of this variant had a germline mutation of p27^Kip1/CDKN1B.

Objective: The objective was to test p27 in germline DNA from cases with tumors of both the parathyroids and pituitary.

Design: Medical record review and sequence analysis in DNA were performed.

Setting: This study involved an inpatient and outpatient referral program for cases of endocrine tumors.

Patients: Sixteen index cases had sporadic tumors of two organs, both the parathyroids and the pituitary. There were 18 additional index cases with related features of familial tumors. Five subjects were normal controls. No case had an identified MEN1 mutation.

Interventions: Clinical status of endocrine tumors was tabulated. Sequencing of germline DNA from index cases and control cases for the p27 gene was performed by PCR.

Main Outcome Measures: Endocrine tumor types and their expressions were measured, as were sequence changes in the p27 gene.

Results: Tumor features were documented in index cases and families. One p27 germline single nucleotide change was identified. This predicted a silent substitution of Thr142Thr. Furthermore, there was a normal prevalence of heterozygosity for a common p27 polymorphism, making a large p27 deletion unlikely in all or most of these cases.

Conclusions: The MEN1 variant with sporadic parathyroid tumors, sporadic pituitary tumor, and no identified MEN1 mutation is usually not caused by p27 germline mutations. It is usually caused by as yet unknown process(es).

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MULTIPLE ENDOCRINE NEOPLASIA type 1 (MEN1) is a syndrome with predisposition for many types of endocrine and nonendocrine tumors. A working definition of MEN1 came from the Seventh International Congress on Multiple Endocrine Neoplasia (1). MEN1 was defined there as a case with tumors in two of the three main endocrine tissues (parathyroids, pituitary, and enteropancreas), potentially affected by the syndrome. In addition, familial MEN1 was defined there as a case of MEN1 in a subject who had a first-degree relative showing a tumor in at least one of these three tissues. Germline mutations of MEN1, the main gene for MEN1, have been widely assumed to account for all or most cases of sporadic and familial MEN1 (2). In contrast, some states related to MEN1 have MEN1 mutations rarely. These states include familial isolated primary hyperparathyroidism and familial isolated pituitary tumors (3, 4).

The prevalence of identified MEN1 mutations in large series of familial MEN1 is approximately 75% (2, 5, 6). In 2001, Hai et al. (7, 8) reported detecting a MEN1 mutation in only one of nine (11%) cases of an MEN1 variant ¹ with sporadic tumors of both the parathyroids and pituitary. This surprisingly low prevalence of identified MEN1 mutations was confirmed (5, 6). A small fraction of cases with this variant may have undiscovered MEN1 mutations; however, gene(s) different from MEN1 probably explain the majority of cases. Recently, Pellegata et al. (9, 10) reported a germline mutation of the human CDKN1B gene, encoding the cyclin-dependent kinase inhibitor p27, in one index case ² with both pituitary and parathyroid tumors and no identified MEN1 mutations. The prevalence of p27 germline mutations is unknown among cases with the MEN1 variant with sporadic tumors of both parathyroid and pituitary.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical aspects

Each index case was referred to test for MEN1 mutations. Control cases were the unaffected spouses of some patients. Each index case and control case gave written informed consent, and the study was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board.

Clinical data were obtained by interview of the patient and relatives, by interview of their physicians, by review of old records, and by testing in the clinics and/or hospital at the National Institutes of Health (NIH). The minimum criterion to diagnose a parathyroid tumor in an index case was persistently high serum calcium and PTH. The minimum criterion to diagnose pituitary tumor was either an imaged tumor or a state of pituitary hormone excess. Group data were expressed as mean and 1 SD. MEN1 mutation frequencies in reports of MEN1 variants were compared by unpaired t test.

The samples for p27 testing were selected from germline DNA stored at NIH. The first criterion for inclusion was prior testing that identified no MEN1 mutation. The second criterion was a designated clinical expression. This clinical criterion in sporadic index cases was: sporadic tumors of both the parathyroids and pituitary and no relative with endocrine tumor. The clinical criterion in familial index cases was broader: one case with parathyroid and/or pituitary tumors and one first-degree relative with the other in that tumor pair. Enteropancreatic tumor was not allowed in any case. Seven other index cases with related familial tumors were included.

DNA sequencing

Sequence analysis of the MEN1 gene was performed in our laboratory, through the Molecular Diagnosis program of the Children’s Research Institute (Children’s National Medical Center, Washington, DC), or through GeneDx Inc. (Gaithersburg, MD). The PCR conditions and primers were as previously described (3).

Germline DNA sequences of p27, representing the protein coding region and intron-exon boundaries were amplified by PCR. PCR was performed with AmpliTaq Gold with GeneAmp (Applied Biosystems, Foster City, CA). Conditions were 10 min at 94 C for Taq Gold activation, 30 cycles (exon 1), or 35 cycles (exon 2) at 94 C for 30 sec, annealing at 60 C (exon 1) or 55 C (exon 2) for 30 sec, extension at 72 C for 60 sec, and then 5 min at 72 C. Exon 1 (691-bp) was amplified with primers 5'-TCGGGCTGCGTAGGGGCGCTTTG-3' and 5'-CACCTAAGACCAATAAAGTTAGCTC-3'. Exon 2 (543-bp) was amplified with primers 5'-CTCCTGGCTAGGGAAAGAGCTCTG-3' and 5'-GTATCTCTGGGCATAGAAACTCTG-3'. PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) and sequenced in one or both directions. The following primers were used for sequencing exon 1: 5'-TAGGGGCGCTTTGTTTTGTTC-3' and 5'- GCTCTCCCAAAGCTAAATCAG-3'. Primer 5'-GATCCAGGATTGTGGGTGGAGGTAG-3' was used for sequencing exon 2. Sequencing results were both machine-read and manually read by two of the authors (A.O. and S.K.A.), independently.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical findings in the group with sporadic tumor

There were 16 index cases with sporadic tumors of both the parathyroids and pituitary. These were 12 females and four males. Age at first diagnosis for parathyroid tumors was 46 ± 14 yr, and for pituitary tumors 43 ± 15 yr. Both tumors were usually diagnosed together, but in two cases the parathyroids were diagnosed first, and in four the pituitary was first.

For most index cases with the sporadic MEN1 variant, the parathyroid tumors could be classified further (Table 1); parathyroid tumors were single in two cases and multiple in 14. For all index cases with the sporadic MEN1 variant, the pituitary tumors also could be classified further (Table 1). Six tumors oversecreted GH, six oversecreted prolactin, two oversecreted ACTH, and two were nonfunctioning. Six were macroadenoma, and two were microadenoma.

There were 11 index cases for families with tumors of the parathyroids and pituitary. Among these families, there were four lipomas and several other hormonal and nonhormonal tumors (see supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Seven index cases in these 11 families had both parathyroid and pituitary tumors. Among these seven cases, the ages of onset were 46 ± 21 yr for parathyroid tumors and 38 ± 10 yr for pituitary tumors.

In addition, seven index cases with related diagnoses were evaluated. Their diagnoses were familial isolated tumors of the pituitary (five index cases) and familial hyperparathyroidism with renal angiomyolipoma (two index cases). The latter two had been reported (3) and were included because renal angiomyolipoma had occurred in one of six likely carriers of p27 mutation (10).

The p27 gene sequencing in the germline of index cases

Among the 27 index cases for the sporadic or familial MEN1 variant, seven index cases from similar families, and five control cases, only one p27 single nucleotide change was identified (see supplemental data). This predicted a silent change of Thr142Thr (ACG to ACA) (11).

A known common polymorphism in the p27 gene was identified at codon 109 (Val109Gly) (GTC to GGC) (12 ; NCBI Single Nucleotide Polymorphism database). Among all index cases, the rate of homozygosity of allele GTC was 53% (18 of 34), homozygosity of allele GGC was 12% (4 of 34), and heterozygosity (GTC/GGC) was 35% (12 of 34). The genotype distribution was similar among the 16 index cases with sporadic tumors of the parathyroids and pituitary.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Two variants of MEN1

A variant with sporadic tumors of both the parathyroids and pituitary represents half of all sporadic cases of MEN1 (Table 2). The prevalence of MEN1 germline mutations identified in this variant was 7%; this is remarkably lower than the 90% prevalence in familial MEN1 (Table 2) (5, 6, 7, 8, 13, 14, 15, 16, 17). Our own experience has been similar (our unpublished data). No cause of this interesting MEN1 variant other than a rare MEN1 mutation has been shown (9, 10).

A recent report of p27 germline mutation in an index case with this MEN1 variant led to recognition of the likely carrier state for the p27 mutation in five more family members (10). The tumors recognized (and numbers of cases) were GH-secreting pituitary (n = 2), parathyroid (n = 1), renal angiomyolipoma (n = 1), testicular cancer (n = 1), and no tumor (n = 2). Thus, the full clinical expressions from p27 mutations will probably extend beyond parathyroid and pituitary tumors and thereby define a second and new MEN1 variant.

p27 as one cause of an MEN1 variant

p27 belongs to one of two classes of cyclin-dependent kinase inhibitors. Several genes in this cell-cycle-regulating pathway have been implicated in endocrine tumors in humans and, to an even greater degree, in mouse. Mice with homozygous knockout of p27 develop endocrine tumors, but only in the pituitary intermediate lobe. However, mice with various combinations of knockout of p27 and p18 have tumors of the following: pituitary, thyroidal C cells, parathyroids, adrenal medulla, endocrine pancreas, testis, and duodenum. They resemble a combination of MEN1 and MEN2 (18, 19). Furthermore, menin or ret can regulate expression of p27 and p18, suggesting that these cyclin-dependent kinase inhibitors might serve as growth regulators in MEN-like tumors (20).

A spontaneous syndrome termed MENX with features overlapping MEN1 and MEN2 in the rat was recently attributed to a homozygous inactivating mutation of p27. Those authors also reported a heterozygous germline p27 mutation in one human family with MEN1 (see above) (9, 10).

Thus germline mutations of p27 have been implicated in MEN-like tumors in mouse, rat, and one human family. The full spectrum of tumors that can be caused by p27 germline mutations in man will only be defined after much more testing. Other approaches should be made to explore tumors for p27 somatic mutations and for posttranslational changes.

Prevalence of p27 mutations

One index case of parathyroid and pituitary tumors with germline p27 mutation was reported along with identical mutations and other tumors in that family (9, 10). This made further exploration of p27 in index cases with both parathyroid and pituitary tumors important. In the recent report of p27 mutation, the number of index cases without p27 mutation was not mentioned (10). The absence of a pathological p27 germline mutation in any one of 34 carefully chosen index cases herein establishes that germline mutations of p27 cannot be frequent in the MEN1 variant with sporadic tumors of the parathyroids and pituitary.

Furthermore, the distribution of heterozygosity for a common polymorphism was as predicted by the Hardy-Weinberg equation (² test, P = 0.61). This established that a large p27 deletion also was nonexistent or rare in these cases.

Our data do not contradict the recent finding of p27 mutation in one case with parathyroid and pituitary tumors. However, our study indicates that the main cause for this MEN1 variant remains unknown.

	Acknowledgments

 
The authors thank the patients, referring physicians, and the staff of the National Institutes of Health Inter-Institute Endocrine Training Program.

	Footnotes

 
This study was supported by funds from the intramural program of the National Institute of Diabetes and Digestive and Kidney Diseases.

Present address for A.M.S.: Albert Einstein College of Medicine, Bronx, New York 10461.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 13, 2007.

³ A.O. and S.K.A. contributed equally to this work and both should be considered first authors.

¹ Variant denotes a clinical syndrome with typical features of that syndrome but with some consistent variation (such as different tumor frequencies). For example, MEN2a and MEN2b are well-recognized variants of MEN2.

² An index case is the affected case first encountered by investigators and thus chosen to represent its family for testing (herein testing of germline DNA). If a family had only one affected case, then that case could be termed a sporadic case, an index case, or a sporadic index case.

Abbreviation: MEN1, Multiple endocrine neoplasia type 1.

Received November 21, 2006.

Accepted February 5, 2007.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

1. Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, Conte-Devolx B, Falchetti A, Gheri RG, Libroia A, Lips CJ, Lombardi G, Mannelli M, Pacini F, Ponder BA, Raue F, Skogseid B, Tamburrano G, Thakker RV, Thompson NW, Tomassetti P, Tonelli F, Wells Jr SA, Marx SJ 2001 Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab 86:5658–5671[Abstract/Free Full Text]
2. Marx SJ 2002 Multiple endocrine neoplasia type 1. In: Vogelstein B, Kinzler KW, eds. The genetic basis of human cancer. 2nd ed. New York: McGraw Hill; 450–475
3. Simonds WF, James-Newton LA, Agarwal SK, Yang B, Skarulis MC, Hendy GN, Marx SJ 2002 Familial isolated hyperparathyroidism: clinical and genetic characteristics of thirty-six kindreds. Medicine 81:1–26[CrossRef][Medline]
4. Daly AF, Jaffrain-Rea ML, Ciccarelli A, Valdes-Socin H, Rohmer V, Tamburrano G, Borson-Chazot C, Estour B, Ciccarelli E, Brue T, Ferolla P, Emy P, Colao A, De Menis E, Lecomte P, Penfornis F, Delemer B, Bertherat J, Wemeau JL, De Herder W, Archambeaud F, Stevenaert A, Calender A, Murat A, Cavagnini F, Beckers A 2006 Clinical characterization of familial isolated pituitary adenomas. J Clin Endocrinol Metab 91:3316–3323[Abstract/Free Full Text]
5. Ellard S, Hattersley AT, Brewer CM, Vaidya B 2005 Detection of an MEN1gene mutation depends on clinical features and supports current referral criteria for diagnostic molecular genetic testing. Clin Endocrinol (Oxf) 62:169–175[CrossRef][Medline]
6. Klein RD, Salih S, Bessoni J, Bale AE 2005 Clinical testing for multiple endocrine neoplasia type 1 in a DNA diagnostic laboratory. Genet Med 7:131–138[Medline]
7. Hai N, Aoki N, Matsuda A, Mori T, Kosugi S 1999 Germline MEN1 mutations in sixteen Japanese families with multiple endocrine neoplasia type 1 (MEN1). Eur J Endocrinol 141:475–480[Abstract]
8. Hai N, Aoki N, Shimatsu A, Mori T, Kosugi S 2000 Clinical features of multiple endocrine neoplasia type 1 (MEN1) phenocopy without germline MEN1 gene mutations: analysis of 20 Japanese sporadic cases with MEN1. Clin Endocrinol (Oxf) 52:509–518[CrossRef][Medline]
9. Pellegata NS, Quintanilla-Martinez L, Samson E, Siggelkow H, Bink K, Graw J, Hofler H, Atkinson MJ, Germline mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and man. Proc 97th Annual Meeting of The American Association of Cancer Research, Washington, DC, 2006, p 39 (Abstract)
10. Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson E, Bink K, Hofler H, Fend F, Graw J, Atkinson MJ 2006 Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. Proc Natl Acad Sci USA [Erratum (2006) 103:19213] 103:15558–15563
11. Spirin KS, Simpson JF, Takeuchi S, Kawamata N, Miller CW, Koeffler HP 1996 p27/Kip1 mutation found in breast cancer. Cancer Res 56:2400–2404[Abstract/Free Full Text]
12. Li G, Sturgis EM, Wang LE, Chamberlain RM, Spitz MR, El-Naggar AK, Hong WK, Wei Q 2004 Association between the V109G polymorphism of the p27 gene and the risk and progression of oral squamous cell carcinoma. Clin Cancer Res 10:3996–4002[Abstract/Free Full Text]
13. Bergman L, Teh B, Cardinal J, Palmer J, Walters M, Shepherd J, Cameron D, Hayward N 2000 Identification of MEN1 gene mutations in families with MEN 1 and related disorders. Br J Cancer 83:1009–1014[CrossRef][Medline]
14. Dackiw AP, Cote GJ, Fleming JB, Schultz PN, Stanford P, Vassilopoulou-Sellin R, Evans DB, Gagel RF, Lee JE 1999 Screening for MEN1 mutations in patients with atypical endocrine neoplasia. Surgery 126:1097–1104[CrossRef][Medline]
15. Poncin J, Abs R, Velkeniers B, Bonduelle M, Abramowicz M, Legros JJ, Verloes A, Meurisse M, Van Gaal L, Verellen C, Koulischer L, Beckers A 1999 Mutation analysis of the MEN1 gene in Belgian patients with multiple endocrine neoplasia type 1 and related diseases. Hum Mutat 13:54–60[CrossRef][Medline]
16. Tso AW, Rong R, Lo CY, Tan KC, Tiu SC, Wat NM, Xu JY, Villablanca A, Larsson C, Teh BT, Lam KS 2003 Multiple endocrine neoplasia type 1 (MEN1): genetic and clinical analysis in the Southern Chinese. Clin Endocrinol (Oxf) 59:129–135[CrossRef][Medline]
17. Cebrian A, Ruiz-Llorente S, Cascon A, Pollan M, Diez JJ, Pico A, Telleria D, Benitez J, Robledo M 2003 Mutational and gross deletion study of the MEN1 gene and correlation with clinical features in Spanish patients. J Med Genet 40:e72
18. Franklin DS, Godfrey VL, O’Brien DA, Deng C, Xiong Y 2000 Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. Mol Cell Biol 20:6147–6158[Abstract/Free Full Text]
19. Marx SJ 2005 Molecular genetics of multiple endocrine neoplasia types 1 and 2. Nat Rev Cancer 5:367–375[CrossRef][Medline]
20. Karnik SK, Hughes CM, Gu X, Rozenblatt-Rosen O, McLean GW, Xiong Y, Meyerson M, Kim SK 2005 Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c. Proc Natl Acad Sci USA 102:14659–14664[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Beckers and A. F Daly The clinical, pathological, and genetic features of familial isolated pituitary adenomas Eur. J. Endocrinol., October 1, 2007; 157(4): 371 - 382. [Abstract] [Full Text] [PDF]

				L. A Naves, A. F Daly, J.-F. Vanbellinghen, L. A Casulari, C. Spilioti, A. V Magalhaes, M. F Azevedo, L. A Giacomini, P. P Nascimento, R. O Nunes, et al. Variable pathological and clinical features of a large Brazilian family harboring a mutation in the aryl hydrocarbon receptor-interacting protein gene Eur. J. Endocrinol., October 1, 2007; 157(4): 383 - 391. [Abstract] [Full Text] [PDF]

				M. Georgitsi, A. Raitila, A. Karhu, R. B. van der Luijt, C. M. Aalfs, T. Sane, O. Vierimaa, M. J. Makinen, K. Tuppurainen, R. Paschke, et al. Germline CDKN1B/p27Kip1 Mutation in Multiple Endocrine Neoplasia J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3321 - 3325. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 92/5/1948    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart 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 Ozawa, A. Articles by Marx, S. J. Search for Related Content PubMed PubMed Citation Articles by Ozawa, A. Articles by Marx, S. J. Related Collections Calcium and Bone Metabolism Endocrine Oncology Neuroendocrinology and Pituitary