Molecular and Genetic Mechanisms of Tumorigenesis in Multiple Endocrine Neoplasia Type-1

Sydney S. Guo and Mark P. Sawicki

Department of Surgery, West Los Angeles Veterans Affairs Medical Center and the University of California Los Angeles School of Medicine, Los Angeles, California 90073

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) is a rare but informative syndrome for endocrine tumorigenesis. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. Mutations of menin have been reported in more than 400 families and tumors, most of which are truncating mutations, thus supporting the function of menin as a tumor suppressor. The exact function of menin is unknown, but overexpression of menin inhibits proliferation of Ras-transformed NIH3T3 cells. Since menin interacts with proteins from both the TGFß and AP-1 signaling pathways, perhaps its tumor suppressor function is related to these key cell growth pathways. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN and sporadic endocrine tumors.

MULTIPLE ENDOCRINE NEOPLASIA type 1 (MEN1, OMIM 131100) is a rare but informative syndrome for endocrine tumorigenesis. Nearly a decade after initial mapping of the disease locus, the responsible gene, called MEN1, was finally cloned by a large NIH collaborative effort. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. In addition, menin has a number of potential roles in normal cell function. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN1 and sporadic endocrine tumors.

MEN1 CLINICAL MANIFESTATIONS AND TUMOR CHARACTERISTICS

The classic clinical manifestation of MEN1 is the triad of parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas (1, 2). Many MEN1 patients, however, do not have all three tumors, even at necropsy. A standard definition of MEN1 has been proposed as an affected individual with at least two of the three above-mentioned endocrine tumors (3). Familial MEN1 requires the above criteria plus at least one first-degree relative with one or more of the three defining endocrine tumors. Sporadic MEN1 describes patients with features of MEN1 syndrome but without a family history of MEN1, indicating de novo germline mutation.

The foremost expression of MEN1 is primary hyperparathyroidism, present in more than 90% of affected patients (4, 5, 6, 7, 8). The parathyroid tumors in these patients are often described as multiglandular hyperplasia, although they may involve the development of polyclonal and clonal tumors (9, 10). Interestingly, whereas parathyroid carcinomas account for 1% of sporadic primary hyperparathyroidism, they are rarely seen in MEN1 patients (11).

Pancreatic endocrine tumors occur less frequently (~60%) than parathyroid tumors in MEN1 (12). Most of these tumors are small, nonfunctional, clinically silent, usually benign, and are commonly located within or around the pancreas (13, 14). The most common functional tumors are gastrinomas and insulinomas. Nonfunctional tumors and insulinomas are located within the pancreas. Gastrinomas, on the other hand, are often found within the soft tissue around the pancreas and within the duodenal submucosa, but not in the mucosa where the gastrin-producing G cells are located. The stem cell responsible for these tumors is unknown, and gastrinoma development is therefore difficult to explain (15). Malignant potential appears to increase with the size of the primary tumor, although small pancreatic endocrine tumors may also become malignant (16). Because there are few prospective studies, it has been difficult to determine whether MEN1-associated pancreatic endocrine tumors have a better or worse prognosis as compared with their sporadic counterparts (17).

Of the three classic tumors in MEN1, pituitary adenomas are the least common and affect approximately 30% of MEN1 patients (12). Usually these are PRL-secreting microadenomas (18, 19). Pituitary carcinomas are rare if they occur at all in MEN1.

When first described by Wermer (2), MEN1 was thought to include only tumors from the classic triad of endocrine sites. However, we now know that MEN1 involves other organs, including carcinoid tumors of the bronchi (20), gastrointestinal tract (21), and thymus (22), and adrenal cortical tumors (23, 24), lipomas, angiofibromas, and collagenomas (25, 26). In most MEN1 families reported, there is considerable interfamily heterogeneity and variable penetrance (27, 28, 29). Within families, on the other hand, there is intrafamily homogeneity as exemplified by MEN1 variants. One variant named for its origin in Newfoundland, called MEN1-Burin, is associated with the development of prolactinomas, carcinoids, and hyperparathyroidism, but rarely pancreatic endocrine tumors (30, 31). Another variant, familial isolated primary hyperparathyroidism (FIHP), is characterized by the occurrence of hyperparathyroidism and the absence of pancreatic and pituitary tumors. While several FIHP families have mutations of MEN1, other FIHP families have linkage to a different locus on chromosome 1 (32, 33, 34, 35, 36, 37). Factors other than the specific MEN1 mutation may influence disease expression and may account for some of the phenotype variants. Monozygotic twins, for example, may have differing phenotypes, indicating that the expression of this disease is affected by environmental factors as well as genetic background (38, 39).

IDENTIFICATION OF THE MEN1 GENE

MEN1 is an autosomal dominant inherited syndrome caused by inactivation of a tumor suppressor gene. The MEN1 gene was first localized to 11q13 by LOH and finally identified by positional cloning (see Ref. 3 for detailed review) (Fig. 1Go). The impetus to pursue this cloning strategy was based upon Knudson’s two-hit hypothesis, an approach that was successful in cloning another tumor suppressor, the retinoblastoma gene (40).



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Figure 1. Positional Cloning of the MEN1 Gene

A chromosome 11 ideogram is shown (left) with a detailed genetic map encompassing a small region of 11q13 containing the MEN1 gene. Linkage analysis of many MEN1 families showed tight linkage to 11q13 polymorphic DNA markers and two recombinants narrowed the region to the interval depicted with the large gray bar (~ 2 Mb). The MEN1 consensus region was further refined to the 600-kb region (small gray bar) by LOH analysis of MEN1 and sporadic endocrine tumors. Large DNA fragments corresponding to the 600-kb region were isolated (e.g., bacterial artificial chromosome BAC137C7) and fully sequenced. Many candidate genes were isolated, and human peripheral blood leukocyte DNA from MEN1 patients was systematically analyzed for mutation of the candidate genes. One of these genes, now called MEN1, was mutated in most of the families studied. The MEN1 gene exon-intron structure is shown (right) as well as the direction of transcription (arrow).

 
Based upon epidemiological studies, Knudson proposed a two-mutation model ("two-hit hypothesis") of tumor development in which familial tumors inherit the first hit or mutation as a germline mutation (Fig. 2Go); frequently, the germline mutation is a point mutation and can be identified by sequencing the germline DNA. The second hit occurs as a somatic mutation in the predisposed endocrine cell, which eventually becomes a tumor. This second mutation may occur either by chromosome loss, chromosome loss with duplication, mitotic recombination, or another localized event such as a point mutation. The first three possibilities are the most commonly identified and are manifested by LOH for polymorphic DNA markers flanking the MEN1 locus. Therefore, tumor LOH can be thought of as a marker for a nearby tumor suppressor gene (41). As a consequence of these two mutations, both alleles of the MEN1 gene are inactivated in the predisposed endocrine cell. Sporadic endocrine tumors undergo a similar sequence of events, except both mutations occur as mitotic events (somatic cell mutation) in the predisposed endocrine cell.



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Figure 2. Knudson Two-Hit Hypothesis

This diagram illustrates the theoretical molecular genetic mechanism underlying inactivation of the MEN1 tumor suppressor gene. In familial cases the first hit is inherited. At the top left a nuclear family is shown with a single affected child. Below the parental and child symbols in the pedigree are allelotypes for two 11q13 polymorphic DNA markers, designated A and B, flanking the MEN1 gene. Each of these markers has two possible alleles numbered 1 and 2. The affected child is heterozygous (A1/A2, B1/B2) for both DNA markers. The normal MEN1 allele is designated "+" whereas the mutated allele is designated MEN1. For the sake of clarity, only one of the potential mechanisms for the second hit is shown. In this particular case, the tumor has undergone chromosome loss as the second hit. The resultant tumor allelotype is shown (A1/-, B1/-). Southern blot analysis with the DNA probes A and B demonstrate that while the normal peripheral blood leukocyte (N) DNA retains a heterozygous (two alleles for each marker) band pattern for both markers A and B, the tumor (T) DNA shows a homozygous (one allele for each marker) band pattern. The change to the homozygous allelotype is called LOH. The lost allele is designated by the arrow. The sporadic tumor arises by a similar mechanism except that both hits arise as somatic mutations.

 
The initial localization of the MEN1 gene to chromosome 11 was determined by LOH analysis of MEN1 pancreatic endocrine tumors (Fig. 1Go). Insulinoma DNA from two related MEN1 patients was analyzed with polymorphic DNA probes representing all human chromosomes (42). These tumors revealed LOH for all informative loci on chromosome 11, but for very few markers on other chromosomes. This suggested that these tumors had lost a chromosome that unmasked a mutated MEN1 gene located on chromosome 11, and genetic linkage analysis indicated the gene was indeed located near the human muscle glycogen phosphorylase gene (PYGM) at 11q13.

Subsequent studies in tumors from other MEN1 patients frequently demonstrated LOH for polymorphic DNA markers located on chromosome 11 near PYGM, suggesting that the MEN1 gene was indeed a tumor suppressor (43, 44, 45). Sporadic tumors of the type associated with MEN1 also demonstrated LOH for DNA markers at 11q13 (43, 44, 45, 46, 47, 48, 49, 50). Additional linkage studies (49, 51, 52, 53, 54, 55) refined the MEN1 locus to a smaller 2 centiMorgan [~2-Megabase (Mb) DNA] interval centered on PYGM (Fig. 1Go). All of the MEN1 families reported to date have tight linkage to the 11q13 locus (42, 49, 51, 52, 53, 54, 55, 56, 57).

Using a positional cloning strategy, the MEN1 gene was localized to a small genomic interval at 11q13 (49, 58) and isolated (59) (Fig. 1Go). Mutation analysis revealed that the gene now called MEN1 was frequently, but not always, mutated in MEN1 families (59). In MEN1-associated tumors, the mutated MEN1 allele is present in all cells at birth. Loss of the remaining wild-type allele gives the predisposed endocrine cell a survival advantage needed for tumor formation. Because MEN1 patients inherit the first hit, MEN1-associated tumors have an earlier age of onset and a much higher rate of postoperative recurrence as compared with their sporadic tumor counterparts. This is consistent with the classic two-hit hypothesis of tumor suppressors described by Knudson (40).

EXPRESSION AND FUNCTION OF THE MEN1 GENE

The MEN1 gene spans 9 kb of the genome, and consists of 10 exons. MEN1 encodes menin, a 610-amino acid protein that is highly conserved (Fig. 3Go) among human, mouse (98%), and rat (97%), and more distantly among zebrafish (75%) and Drosophila (47%) (60, 61, 62, 63, 64), but there is no known homolog in the budding yeast Saccharomyces. Most of the divergence between species is near the carboxy terminus. Database analysis of menin protein sequence reveals no significant homology to known consensus protein motifs, but there are 28 putative phosphorylation sites, two of which are affected by disease-producing missense mutations. There is also a leucine zipper motif within the amino terminus, although there are no data supporting dimerization or DNA binding for menin, suggesting that this motif may not be biologically relevant (59). Since there are few clues to menin’s function by its protein sequence, most of what is known about its function is derived from in vitro studies.



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Figure 3. Alignment of Menin Protein

The human, mouse, zebrafish, and Drosophila menin proteins were aligned with ClustalW program (http://workbench.sdsc.edu/) using the default parameters. Green areas highlight identically conserved amino acids between these species, while yellow and blue areas indicate lesser degrees of conservation. Missense mutations are indicated by red dots above each amino acid affected. Open boxes indicate JunD binding domains. Seventy-one percent of missense mutations occurred on an amino acid that is identically conserved, while 5% of missense mutations occurred on amino acids that were not conserved. This is in contrast with the relatively distant homology between Drosophila and human MEN1 (34% identical and 47% similar to human). Zbfsh, Zebrafish.

 
Menin is variably expressed in both endocrine and nonendocrine tissues. In adult human tissues, menin is widely expressed but is highest in proliferating tissues, such as the uterine endometrium (65). This association suggests that menin expression may be cell cycle regulated, and studies of GH4 (rat pituitary) cells (66) and Chinese hamster ovary (CHO) cells (65) indeed show that menin expression is modestly decreased in mid-G1. Analysis of other cell lines, however, has demonstrated no cell cycle regulation of menin expression (60, 67). Studies of developmental regulation of MEN1 have found that mice express menin as early as embryonic day 7 and later develop a more restricted pattern of expression (60, 68, 69). Similarly, human fetuses also express MEN1 early in development (67). A MEN1 isoform identified in human and mouse is due to an alternate splice site within exon 2 that lengthens menin by five amino acids. The biological relevance of this alternate splice variant is unknown, and no mutations have been mapped to this region.

There is evidence that suggests menin may be regulated, in part, by nuclear localization. Menin localizes predominantly in the nucleus via two C-terminal nuclear localization signals (Fig. 4Go) (70) but may be found in the cytoplasm during cell division in HEK 293 (human embryonal kidney) cells (71). Cytoplasmic localization was also seen in HeLa and NIH3T3 cells, but this was not cell cycle regulated (67). In addition to observations in cell lines, cytoplasmic localization was noted in the developing mouse testes and developing zebrafish embryo proerythroblasts (63, 69). The biological significance of this nuclear shuttling is unknown, but other tumor suppressors, such as von Hippel-Lindau, are regulated in this fashion (72). All of the MEN1 truncating mutations lead to loss of one or both nuclear localization signals. Presumably, this would lead to protein instability or loss of function by displacing menin out of the nucleus. Interestingly, none of the missense mutations reported to date alters either nuclear localization signal (NLS).



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Figure 4. Missense Mutations

Mutation sites and exon lengths were drawn to scale based on the amino acid sequence of MEN1 (GenBank U93236). Several clusters of missense mutations can be seen. Some of these directly involved one of the three JunD interacting regions (amino acids 1–40, 139–242, and 323–428). The two nuclear localization signals, however, were spared (amino acids 479–497 and 588–608). Diseases produced were represented using symbols as follows: solid diamond, parathyroid hyperplasia; arrowhead, neuroendocrine; GI, star, neuroendocrine, lung; and solid square, MEN1 syndrome.

 
Since all cell lines express menin (67, 68), most studies have used overexpression of menin to determine its biological effect on cell growth. In a constitutively activated Ras-transformed NIH3T3 cell model, high levels of menin expression reversed the transformed phenotype (73). These morphological changes were associated with decreased proliferation, suppression of clonogenicity in soft agar, and inhibition of tumor growth in nude mice. Menin was overexpressed 7- to 27-fold in this model, and in vitro growth was suppressed at all levels of menin expression. Since menin overexpression repressed the effects of Ras, it would be expected that suppression of menin expression might increase cell proliferation. However, in a menin antisense model, partial inhibition of menin expression did not increase CHO cell proliferation (65).

There is increasing evidence that menin may function in DNA repair or synthesis. Peripheral blood leukocytes and fibroblasts from MEN1 patients treated with diepoxibutane had an increase in the frequency of spontaneous chromosomal alterations (74). Similar analyses of MEN1 lymphocytes revealed an increased frequency of mitoses with premature centromere division when cells were treated with diepoxibutane (75, 76). More recently, Ikeo et al. (65) demonstrated that CHO cells overexpressing menin had a marked delay in cell cycle progression when exposed to diepoxibutane. The mechanism by which menin regulates DNA synthesis or the cellular response to DNA damage is currently unknown.

The molecular mechanism for menin’s growth suppression is thought to be mediated, in part, through its interactions with transcription factors. The first clue came from yeast two-hybrid protein interaction studies that demonstrated menin binding to JunD (77). JunD is a member of the Jun family of proteins (JunD, JunB, c-Jun) that dimerize with themselves or with Fos family members (c-Fos, FosB, Fra1, Fra2) to form the AP-1 transcriptional factor. Menin binds to JunD, but not to other members of Jun or Fos, and represses JunD-mediated transcriptional activation in an in vitro reporter system. This repression is abrogated by the histone deacetylase inhibitor trichostatin A, suggesting the importance of DNA ultrastructure in this mechanism (78). Agarwal et al. (77) reported three critical regions important for JunD binding based upon interaction in the yeast-two hybrid system and glutathione-S-transferase pull-down assays. In contrast, Gobl et al. (78) suggested primarily a critical region at the extreme C terminus based upon glutathione-S-transferase pull-down assays.

The biological function of JunD is not well characterized. While JunD has significant homology to oncogenic c-Jun, they appear to have opposing effects on cell growth; overexpression of JunD in Ras-transformed NIH 3T3 cells inhibits cell proliferation whereas c-Jun increases cell proliferation (79). Recent studies of JunD-/- fibroblasts reveal that the absence of JunD may result in either early senescence (in primary fibroblasts) or increased proliferation (in immortalized fibroblasts) (80). This would suggest that the menin-JunD interaction must be studied in the proper cellular context. Recently, Kaji et al. (81) have implicated menin in TGFß signaling pathway through its interaction with Smad3. (SMAD = vertebrate homologs to C. elegans Sma and Drosophila Madproteins. Because it is known that JunD interacts with Smad3 (82), it has been proposed by others that TGFß may exert its function, in part, through Smad3, JunD, and menin (81).

An exciting recent development is the creation of a MEN1 knockout mouse through cre/lox recombination that will provide a useful in vivo model in which to study MEN1 function (83). MEN1-/- mice had an embryonic lethal phenotype, while MEN1+/- mice developed tumors of the endocrine pancreas (predominantly ß-cells), parathyroid, and pituitary. LOH analysis revealed that the insulinomas, but not hyperplastic islets, have lost the remaining wild-type allele, thus recapitulating the genetic mechanism seen in human disease. This also suggests that menin has a dosage effect on islet growth regulation. Notably, no gastrinomas were observed. This model, together with the JunD knockout mouse (80), should greatly accelerate our understanding of MEN1 function. In particular, the dependence of JunD in MEN1 tumorigenesis could be explored by crossing these two knockout mouse strains. It would be expected that if JunD is necessary for menin function, then the double knockout mice should not spontaneously develop tumors.

MEN1 MUTATIONS

Since the cloning of MEN1, more than 400 mutations have been reported in families and sporadic tumors. We reviewed a total of 326 MEN1, 7 FIHP, and 92 sporadic tumor mutations, compiled from 51 distinct studies (20, 27, 32, 33, 34, 35, 36, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127). Many mutations were reported more than once in apparently unrelated patients (range, 2–13 times), and the number of unique mutations is 294. There were 169 deletions (40%), 85 missense mutations (20%), 82 nonsense mutations (19%), 56 insertions (13%), 29 splice site defects (7%), and 4 combined deletion/insertion (1%) mutations. Of the 294 unique mutations, 220 of these are truncating mutations resulting from frameshift (deletions, insertions, deletion/insertions, or splice-site defects) and nonsense mutations. These truncating mutations predict a loss-of-function of menin and therefore support menin’s role as a tumor suppressor.

Of the MEN1 missense mutations reported in the literature, 68% occurred on an amino acid that is identical among human, mouse, zebrafish, and Drosophila (Fig. 3Go). A further 27% of missense mutations occurred on amino acids that are not identical among these species but belonged to the same polar/nonpolar side-chain group. Only 5% of missense mutations occurred on amino acids that were not conserved. This is remarkable given the relatively distant homology for the fly (34% identical and 47% similar to human).

Currently, there are five known functional domains of menin (Fig. 4Go): three JunD-interacting domains at codons 1–40, 139–242, and 323–428, and two nuclear localization signals at codons 479–497 and 588–608 (70, 77). Further analysis revealed that there are three clusters of missense mutations that occurred in the three JunD-binding sites (43 of 74 unique missense mutations, or 58%), but several other clusters were scattered outside of all the known functional domains (accounting for 42% of total unique missense mutations). The clusters outside of the JunD binding domains may be important for Smad3 binding or may argue for the existence of as yet unidentified binding partners of menin. It is also conceivable that they may disrupt the three-dimensional conformation and indirectly affect menin’s affinity for JunD or the cellular localization mechanism. Another possibility is that these mutations may alter the protein degradation mechanism, leading to abnormal protein half-life and consequently, loss of function.

MEN1 mutations occur not only in MEN1 patients (both familial and sporadic), but also in a significant percentage of associated sporadic endocrine tumors (Fig. 5Go). Interestingly, the profile of tumors associated with MEN1 differs from the profile of sporadic tumors in which MEN1 has been implicated in the tumorigenesis. For example, approximately 30% of MEN1 patients are affected by pituitary tumors. However, MEN1 mutation is almost never seen in the sporadic form of pituitary tumors (2 of 195, 1%). Among sporadic pancreatic endocrine tumors, the frequency of MEN1 mutation differs between the types of tumor. For example, the frequency of MEN1 mutation in sporadic gastrinomas is 37%, in contrast to the 15% mutation rate in nongastrinoma sporadic pancreatic endocrine tumors. Moreover, even though a recent report found no MEN1 mutation in 27 sporadic insulinomas examined (120), only insulinomas were found in the recently reported MEN1 knockout mouse model (see above) (83). Therefore, the role of MEN1 may be different in familial and sporadic tumors.



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Figure 5. MEN1 Mutation Frequency in Sporadic Tumors of Different Tissues

Mutations were reviewed and categorized according to the site of the sporadic tumor. These numbers were then expressed as a fraction of the total number of each disease type analyzed in each report. Sporadic neuroendocrine tumor (NET)-Pulmonary, 35% (6 of 17); sporadic NET-GI tract (including pancreatic endocrine tumors as well as carcinoids of GI tract), 23% (45 of 196); sporadic parathyroid adenoma, 13.4% (36 of 267); sporadic angiofibromas, 10.5% (2 of 19); sporadic adrenal tumors, 7.1% (1 of 14); sporadic pituitary adenomas, 1% (2 of 195)

 
Although there is not a consistent phenotype-genotype correlation in familial MEN1, there is such an association in sporadic gastrinomas. Primary pancreatic or lymph node sporadic gastrinomas exhibit mutations in exon 2 much more frequently than duodenal sporadic gastrinomas (128). Small (<1 cm) primary tumors were also less likely to have a mutation involving exon 2 of MEN1. However, there was no genotype-phenotype correlation with regard to postoperative disease-free status and overall patient outcome.

Fifty-five MEN1 mutations have been reported repeatedly in the literature. Only mutations that were reported greater than three times in patients who have no apparent relationship with one another are listed in Table 1Go (total, 17). The most frequently repeated mutation, 359del4, occurred in 13 presumably unrelated families and suggests a mutation "hot spot." However, it is possible that some of these patients are related, as demonstrated by a study (129) revealing founder’s effect for two repeat mutations (416delC and 512delC) described in families that were apparently unrelated by both family history and geographical origin. Although this may be the case, analysis of clinical diseases reported with these repeat mutations suggests there is not an emerging phenotype-genotype correlation amongst MEN1 families.


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Table 1. Mutational Hot Spots and Their Clinical Presentations

 
Thirteen polymorphisms have been reported in the literature. Two polymorphisms altered the amino acid sequence of menin (Arg171Gln and Ala541Thr), and both involved amino acid substitutions between different polar side-chain groups. Interestingly, Arg171Gln occurs in the middle of the missense mutation cluster between amino acids 126–184. Missense mutation at almost every other amino acid in this area causes disease. Similarly, Ala541Thr also occurs in the middle of a smaller cluster of disease-producing missense mutations. Of note, there were two reports that categorized the Ala541Thr polymorphism as a disease-producing mutation, leading to a parathyroid adenoma and a neuroendocrine tumor of the foregut, respectively (116, 121). Contrastingly, G894–9A was first reported to be a benign polymorphism by Bassett et al. (86) but later reclassified by several groups to be a disease-causing mutation (96, 99, 102, 111).

In familial MEN1 patients, germline mutation of MEN1 was found in 271 of 351 probands studied (77%). Less frequently, MEN1 germline mutation was found in 55 of 86 cases (64%) of sporadic MEN1 patients. Thus, in total, 326 mutations (271 + 55) were reported in MEN1 patients. Most studies used one of four methods in the detection of mutations: dideoxy fingerprinting, heteroduplex analysis, single strand conformation polymorphism, or direct sequencing of selected regions. Short of sequencing the entire gene and the surrounding areas, none of these methods is completely sensitive. Additionally, mutations in the untranslated regions or regulatory regions may be responsible for the cases where MEN1 mutations have not been detected. Although all MEN1 families tested to date have tight linkage to 11q13, the presence of another tumor suppressor at 11q13 is also a possibility (130). One family with a large germline deletion of MEN1 that would escape detection by PCR-based methods has been reported (103). The existence of phenocopies should also be considered, especially in light of the high frequency of sporadic parathyroid and pituitary adenomas (85). This point is highlighted in sporadic MEN1 patients where the lack of family history makes this diagnosis more susceptible to phenocopies and is reflected in the lower rate of MEN1 mutation in sporadic vs. familial MEN1. Lastly, even when genetic change is identified in MEN1 families, the general slow growth of these tumors and the lack of adequate treatment modalities available make the issue of routine genetic testing, at best, unresolved.

The field eagerly awaits the discovery of a functional assay for menin activity. This assay could not only elucidate mechanisms of menin inactivation other than genetic mutation, but could also lead to the rational design of treatment modalities for both MEN1 and the sporadic endocrine tumors.

ACKNOWLEDGMENTS

We thank Dr. Edward Passaro, Jr., for intellectual guidance and support. We also thank Dr. Greg Brent for invaluable critique of the manuscript, and Alan Shimoide and Karen Duff for technical support.

FOOTNOTES

Address requests for reprints to: Mark P. Sawicki, M.D., Department of Surgery, 11301 Wilshire Boulevard, Los Angeles, California. E-mail: msawicki{at}ucla.edu

{hd1}Note Added in Proof

{smtexf}Menin was recently found to interact with and antagonize the NF-kappaB proteins (131 ).

Abbreviations: CHO, Chinese hamster ovary; FIHP, familial isolated primary hyperparathyroidism; MEN, multiple endocrine neoplasia; Mb, megabase; NLS, nuclear location signal; SMAD, vertebrate homologs to C. elegans Sma and DrosphilaMadproteins.

Received for publication August 31, 2000. Accepted for publication July 10, 2001.

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