Download Multiple Endocrine Neoplasia Type 1

Multiple Endocrine Neoplasia Type 1: Clinical and Genetic Topics
Moderator: Stephen Marx, MD; Discussants: Allen M. Spiegel, MD; Monica C. Skarulis, MD;
John L. Doppman, MD; Francis S. Collins, MD, PhD; and Lance A. Liotta, MD, PhD
Multiple endocrine neoplasia type 1 (MEN1) consists of
benign, and sometimes malignant, tumors (often multiple
in a tissue) of the parathyroids, enteropancreatic neuroendocrine system, anterior pituitary, and other tissues. Skin
angiofibromas and skin collagenomas are common. Typically, MEN1 tumors begin two decades earlier than sporadic tumors. Because of tumor multiplicity and the tendency for postoperative tumor recurrence, specialized
methods have been developed for preoperative and intraoperative localization of many MEN 1-associated tumors.
The MEN1 gene was recently isolated by positional
cloning. This strategy progressively narrows the size of the
candidate MEN1 gene interval on the chromosome and
then finds and tests many or, if needed, all genes within
that interval. The MEN1 gene was finally identified because it was the one gene that contained mutations in
most DNAs from a test panel of MEN1 cases.
It has been suggested that MEN1, like many hereditary
cancer syndromes, is caused by mutation in a tumor suppressor gene that contributes to neoplasia when both
gene copies in a tumor precursor cell have been sequentially inactivated ("two-hit" oncogenesis mechanism).
Germline MEN1 mutations were found in most families
with MEN1 and in most cases of sporadic MEN1. In addition, the MEN1 gene was the gene most likely to show
acquired mutation in several sporadic or nonhereditary
tumors—parathyroid adenomas, gastrinomas, insulinomas, and bronchial carcinoids. Most germline or acquired
MEN1 mutations predicted truncation (and thus likely inactivation) of the encoded protein, supporting expectations for the "first hit" to a tumor suppressor gene. Testing
for MEN1 germline mutation is possible in a research setting. Candidates for MEN1 mutation testing include patients with MEN1 or its phenocopies and first-degree relatives of persons with MEN1.
This paper is also available at http://www.acponline.org.
Ann Intern Med. 1998;129:484-494.
For definitions of terms used, see Glossary at end of text.
An edited summary of a Clinical Staff Conference held on 4 June
1997 at the National Institutes of Health, Bethesda, Maryland.
Authors who wish to cite a section of the conference and
specifically indicate its author may use this example for the form
of the reference:
Skarulis MC. Clinical expressions of multiple endocrine neoplasia type 1 at the National Institutes of Health, pp 486-487. In:
Marx S, moderator. Multiple endocrine neoplasia type 1: clinical
and genetic topics. Ann Intern Med. 1998;129:484-494.
484
Endocrine Neoplasias in Relation to
Oncogenes and Tumor Suppressor Genes
Dr. Allen M. Spiegel (Metabolic Diseases Branch,
National Institute of Diabetes and Digestive and
Kidney Diseases [NIDDK], National Institutes of
Health [NIH], Bethesda, MD): Multiple endocrine
neoplasia type 1 (MEN1) is an autosomal dominantly
inherited disease characterized by variable penetrance
for tumors of the parathyroids, enteropancreatic
neuroendocrine system, and anterior pituitary and,
less commonly, by tumors in other tissues (1). The
"multiple" designation refers both to the occurrence
of multiple tumors in the involved endocrine organ
(for example, multiple pancreatic islet tumors) and
to the occurrence of tumors in multiple endocrine
organs (for example, parathyroid tumor plus pancreatic islet tumor). In contrast, sporadic endocrine
tumors generally occur as a single lesion. Clinical
manifestations of MEN1 include functional effects
of hormone hypersecretion, such as hypercalcemia
or hypoglycemia; mass effects secondary to tumor
growth (particularly in the pituitary); and malignant
neoplasm (particularly with malignant gastrinoma).
Type 1 disease must be distinguished from type 2
disease (MEN2), another autosomal dominantly inherited tumor syndrome. The latter is characterized
by bilateral medullary carcinoma of the thyroid,
pheochromocytomas (often bilateral), and, in the
most common variant, parathyroid tumors (2).
History
The coexistence of tumors of the parathyroids,
pancreatic islets, and pituitary was first noted in
autopsy studies of patients with acromegaly (3). In
1939, Rossier and Dressier (4) described two sisters
with nephrolithiasis in addition to parathyroid and
pancreatic islet tumors. A paternal family history of
ulcers was also noted, but the connection to the
sisters' endocrine tumors was not made until 22
years later (5). Not until 1954, when Wermer (6)
described a family with four daughters and a father
manifesting multiple parathyroid, pancreatic islet,
See related article on pp 472-483.
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and pituitary tumors, was the autosomal dominant
genetic basis of the disease recognized. This condition was later termed the Wermer syndrome or
multiple endocrine adenomatosis, but MENI is now
the preferred term.
Oncogenes and Tumor Suppressor Genes
Two categories of genes contribute to tumorigenesis. First, oncogenes, such as RAS, MYC, and RET,
normally act to stimulate cell proliferation but when
inappropriately activated or overexpressed permit a
cell growth advantage. Second, tumor suppressor
genes, such as P53 (mutation of which causes the
Li-Fraumeni syndrome) or FAP (mutation of which
causes familial adenomatous polyposis of the colon),
normally act as "brakes" on cell proliferation and,
when inactivated, permit a cell growth advantage.
Knudson (7) studied retinoblastoma and recognized
that tumors in familial cases could be explained by
an inherited germline loss of function mutation ("first
hit") of a tumor suppressor gene (termed RBI for
retinoblastoma gene) in all cells (Figure 1). This could
be followed by a somatic loss-of-function mutation of
the second, normal, RBI allele in one cell ("second
hit"). The cell would then be freed from restraint by
both brakes (two normal RBI alleles) and could
proliferate into a clonal neoplasm (Figure 1). The
presence of the first hit in all cells through germline
inheritance and the relative frequency of second hits
in many somatic cells may explain the multiple and
bilateral retinoblastomas and the early age at tumor
onset that are characteristic of the hereditary disease.
Clinical Correlates of Two-Hit Tumorigenesis
Knudson (7) suggested that sporadic retinoblastoma, in contrast to familial retinoblastoma, could
arise after two sequential inactivating somatic mutations of the RBI gene occur in a single cell (Figure 1). The lower frequency with which these two
events occur postnatally in a single cell is reflected
in the unilateral, single nature of the tumor and the
later age at tumor onset in the sporadic form of the
disease.
Although a tumor suppressor gene is recessive at
the tumor level (both alleles in a cell must be inactivated for the cell to proliferate as a tumor clone), a
heterozygous germline mutation combined with the
frequent occurrence of somatic, second hits leads to
dominant inheritance and dominant expression of
tumors. A tumor suppressor gene can contribute to
benign or malignant neoplasia and can synergize with
other tumor suppressor genes or oncogenes.
Second Hits and the Role of Multiple
Endocrine Neoplasia Type 1 in Hereditary
and Sporadic Tumors
Second hits are often large (for example, loss of an
entire copy of a chromosome), and this DNA change
Figure 1. Sequential inactivation of both copies of the MEN1 gene
contributes to hereditary or common variety tumor. The two copies of
chromosome 11 show the inherited DNA pattern (germline nucleus) followed by DNA changes in a tumor precursor cell or a tumor cell (somatic
nucleus). The striped compared with the clear copy of chromosome 11
indicates nonidentity at many loci as they derive from genetically unrelated
parents. The first hit to the MENI gene is generally a small one, such as a
point mutation. Although the hit inactivates one copy of the MEN1 gene, it
generally has no detectable biological effect on the cell. The first hit can be
inherited with the germline, thus being identical in each cell of a case with
multiple endocrine neoplasia type 1 (MEN1). Alternately, the first hit can
occur as a rare event in any somatic cell nucleus. Thus, the same mutation
can contribute to neoplasia on a hereditary or nonhereditary basis. The
second hit in a postzygotic or somatic cell typically involves a larger portion
of the other, normal copy of chromosome 11. The second hit inactivates the
second (or remaining normal) copy of the MEN1 gene by deleting it along
with a large zone of contiguous alleles. Sequential inactivation of both
copies (two mutations, also termed two hits) of the MEN1 gene removes the
brakes to growth in one cell, which may then proliferate to a tumor clone.
is also present in the monoclonal cellular progeny that
grow from this tumor precursor cell. Thus, indirect
evidence for a large second hit is often found when
one copy of a heterozygous DNA marker on either
side of a tumor suppressor gene is lost in tumor DNA
compared with constitutional DNA. This is termed
loss of heterozygosity or allelic loss.
In 1988, pedigree testing first linked the MENI
gene to a section of chromosome 11, llql3 (8). An
analysis of loss of heterozygosity of polymorphic
markers that were mapped near this locus in isletcell tumors from brothers with MENI showed that
the presumed normal allele, inherited from the unaffected parent, had been lost (8). Similar observations were subsequently made for parathyroid tumors
from patients with MENI (9, 10). Furthermore, loss
of heterozygosity at llql3 was also seen in about
one third of sporadic parathyroid adenomas (9).
These observations suggested that 1) MENI is a
tumor suppressor gene; 2) MENI is a familial tumor syndrome caused by a germline inactivating
mutation of the MENI gene, with frequent somatic
second hits leading to multiple endocrine tumors; 3)
the same gene has a similar role in certain sporadic
tumors; and 4) a MENl-related tumor is a monoclonal or oligoclonal expansion after the second hit.
Recent findings have supported these predictions in
considerable molecular detail.
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Table.
Endocrine Tumors Expressed in 130 Patients w i t h
Multiple Endocrine Neoplasia Type 1 Admitted to
the National Institutes of Health*
Tumor
Patients, n (%)
Tumor in principal MEN 1-related organ
Parathyroid tumor
Enteropancreatic neuroendocrine tumor
Gastrinoma
Insulinoma
Nonfunctioning tumort
Other*
Pituitary tumor
Prolactinoma
Nonfunctioning tumor
Corticotropinoma
Somatotropinoma
Tumor in organ possibly related to MEN1
Carcinoid
Bronchial
Gastric
Thymic
Adrenocortical tumor
Nonfunctioning
Functioning
Thyroid tumor
Follicular adenoma
Papillary carcinoma
129(99)
86 (66)
61 (47)
15(12)
5(4)
5(4)
61 (47)
34 (26)
14(11)
9(7)
4(3)
21 (16)
11(8)
9(7)
KD
21 (16)
14(11)
7(5)
16(12)
10(8)
6(5)
* Each tumor type was scored only once per patient, fv1EN1 = multiple endocrine neoplasia type 1.
t Excludes multiple "nonfunctioning" islet tumors thait were encountered incidental to
each pancreatic operation for MEN1.
* Other = glucagonoma, somatostatinoma, and VIP (vasoactivei intestinal peptide)oma.
Clinical Expressions of Multiple Endocrine
Neoplasia Type 1 at the National
Institutes of Health
Dr. Monica C. Skarulis (Division of Intramural
Research, NIDDK, NIH): We reviewed patients with
MEN1 who had been admitted to the NIH at least
once in the past 15 years and had been evaluated by
an NIH endocrine service. Clinical diagnosis of MEN1
was based on the finding of tumors in two or more
of the three principal organs typically affected in the
syndrome. A family history of MENl-related tumor
strengthened the diagnosis. A total of 107 patients
(representing 58 families) had familial MEN1, and
23 other patients were designated as having sporadic MEN1 on the basis of a negative family history
and negative biochemical family screening (where
available).
Overall, the endocrine expressions in the familial
and sporadic cases were indistinguishable from each
other. Furthermore, affected family members varied
widely in endocrine complications from MEN1. Only
one family differed recognizably from the others in
that its members had the prolactinoma variant of
MEN1.
All but one patient had parathyroid tumors
(Table), which were multiple and often asymmetric.
Because of the endocrinology patient referral biases
of the NIH, this population is further enriched for
hyperparathyroidism, the most highly penetrant and
usually the first expression of MEN1 (1).
486
Enteropancreatic neuroendocrine tumors were
identified in 66% of the patients. Gastrinomas of the
duodenum and pancreas (with the Zollinger-Ellison
syndrome) occurred in 47%. Insulinoma, occurring
in 12% of patients, was generally a solitary, benign
lesion. Rarely, a clinical syndrome associated with
glucagonoma, VIP (vasoactive intestinal peptide)oma,
or somatostatinoma was seen (Table). Nonfunctioning islet tumors detected by routine radiographic
screening (computed tomography and magnetic resonance imaging [MRI]) are probably underreported
in MENl because of the variable use and limited
sensitivity of these techniques.
Pituitary tumors were seen in 47% of patients,
and prolactinomas accounted for more than half of
these tumors. The reported prevalence of pituitary
tumors in MENl is 16% to 65% (11). Six members
from one large family with the prolactinoma variant
of MENl, which is characterized by increased expression of prolactinoma and infrequent expression
of gastrinoma (12, 13), were included in this series.
Nonfunctioning pituitary adenomas, adrenocorticotropic hormone (ACTH)-secreting adenomas, and
acromegaly were more rare (Table).
Features with Weaker or Newly Recognized
Association with Multiple Endocrine
Neoplasia Type 1
Bronchial carcinoid was diagnosed in 11 patients
(8%) by the presence of an enlarging chest lesion
on radiography with or without pulmonary symptoms resulting from partial obstruction of a bronchus. Gastric carcinoid was detected incidentally on
endoscopy in 9 patients with the Zollinger-Ellison
syndrome (14). Bronchial or gastric carcinoids were
often multifocal but were not clearly malignant, and
one case of malignant thymic carcinoid was seen.
Neither the carcinoid syndrome nor ectopic ACTH
secretion (15) was seen in any of these NIH cases
with foregut carcinoid tumor.
Tumors that have not been clearly proven to be
a direct result of inactivation of the MENl gene and
that have been only weakly associated with MENl
include thyroid and adrenocortical tumors (16).
Some two thirds of the adrenocortical tumors seen
at the NIH were not associated with Cortisol excess.
In addition, three cases of renal angiomyolipoma
and three cases of leiomyoma of the esophagus
were found (17). One patient had a unilateral pheochromocytoma.
A recent analysis of patients with MENl seen at
the NIH revealed frequent, subtle skin expressions.
Multiple lipomas were found in 34% of patients
with MENl; multiple facial angiofibromas, which
were previously considered pathognomonic for tuberous sclerosis, were found in 88%; and skin collagenomas were seen in 72% (18). The lipomas,
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angiofibromas, and collagenomas showed llql3 allelic loss (similar to loss of heterozygosity), which
suggests that in MEN1, these are clonal neoplasms
caused by inactivation of both MEN1 genes (19).
In addition, clinically apparent malignant transformation of enteropancreatic neuroendocrine tumors occurred, mostly in gastrinomas (nine cases)
or nonfunctional islet-cell tumors (three cases), as
well as in the single case of malignant thymic carcinoid mentioned earlier. Other cancers, probably unrelated to MEN1, included breast cancer (two cases),
colon cancer (two cases), renal papillary cancer
(one case), and uterine leiomyosarcoma (one case).
Six cases of papillary thyroid cancer, all found incidentally on parathyroid exploration, were also noted.
Age at Onset of Tumor Expression
Although we did not prospectively screen for the
development of new endocrinopathy, the peak age
at onset of expression of endocrine tumors in patients with MEN1 (Figure 2) was about 20 years
earlier than the age at onset of sporadic tumors in
patients with hyperparathyroidism (20), gastrinoma
(21), or insulinoma (22). By contrast, the age at
onset of prolactinoma was indistinguishable between
MENl-associated and sporadic tumors (23).
Tumor Localization in Multiple
Endocrine Neoplasia Type 1
Dr. John L. Doppman (Diagnostic Radiology
Department, Clinical Center, NIH): The multiplicity
of endocrine tumors in MEN1 can make surgical
treatment difficult. At the NIH, generally available
noninvasive methods, such as ultrasonography, computed tomography, MRI, and isotope scanning, have
helped localize tumors before surgery in patients
with MEN1. In addition, invasive methods, such as
arteriography and venous sampling, that are specific
to tumor type have been developed at the NIH on
the basis of immunoassay of hormones secreted by
the tumor. Immunoassays have been applied to
MEN1 tumors at the NIH; these assays test blood
from selectively catheterized veins for parathyroid
hormone or ACTH (the latter after systemic infusion of corticotropin-releasing hormone) or from a
vein after infusion of a hormone secretagogue into
a selectively catheterized artery (calcium infusion for
insulin release or secretin infusion for gastrin release).
The following tumor localization guidelines derive
from many institutions but mainly from the NIH.
Parathyroid Tumor
Patients with MEN1 and primary hyperparathyroidism almost always have parathyroid tumors in
four glands. The appropriate first surgical procedure
is a three-and-one-half-gland parathyroidectomy or
Figure 2. Age at onset of endocrine tumor expression in multiple
endocrine neoplasia type 1 (MEN1). Data derived from retrospective
analysis for each endocrine organ hyperfunction in 130 cases of MEN1. Age
at onset is the age at first symptom or, with tumors not causing symptoms,
age at the time of the first abnormal finding on a screening test. The rate of
diagnosis of hyperparathyroidism increased sharply between ages 16 and 20
years.
a four-gland parathyroidectomy with immediate autotransplantation of parathyroid tissue to an accessible
site (11). Because noninvasive studies do not effectively image parathyroid tumors in all four glands
(24), tumor localizing studies are not indicated before initial parathyroid exploration. Sestamibi scanning detects more than 90% of parathyroid adenomas in patients who have not had surgery (25-27),
but this technique images only about 60% of enlarged glands in multigland parathyroid disease.
Successful subtotal parathyroidectomy in MEN1
is often followed by recurrent hyperparathyroidism,
characteristically within 15 years (28). Finding the
recurrent tumor may require not only noninvasive
localization studies (computed tomography, ultrasonography, MRI, and sestamibi scanning) but also
parathyroid arteriography and selective parathyroid
venous sampling for parathyroid hormone. The latter is particularly valuable in predicting multiple
sites of residual parathyroid tissue, as well as the
functional status of parathyroid autografts.
Gastrinoma
Gastrinomas associated with MEN1 are generally
multiple and frequently extrapancreatic; the latter
type occurs in the wall of the duodenum and in
lymph nodes around the pancreatic head (1). Unlike
sporadic gastrinoma, MENl-associated gastrinoma
is generally not cured by localized resection because
of its multiplicity and frequent metastases (29). Because hyperacidity can be controlled medically with
H2-receptor blockers or proton-pump inhibitors, the
principal risk from MENl-associated gastrinoma is
liver metastases, which rarely occur until gastrinomas
reach 3 cm (30). Therefore, our recommendation is
to annually screen the abdomens of patients with gas-
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Figure 3. Computed tomogram of the abdomen in multiple endocrine neoplasia type 1. This patient had symptomatic hypergastrinemia
and hyperinsulinemia. Large tabulated tumors (approximately 4 cm in diameter) were detected in the pancreatic head and tail (arrows). Selective arteriogram with calcium stimulation showed a brisk increase in insulin levels
(threefold step-up, not shown) after injection of calcium into the gastroduodenal artery, suggesting that the insulinoma was in the pancreatic head.
The adenoma, which was removed from the pancreatic head, stained for
insulin. The adenoma in the tail was removed because of its size. It stained
only for gastrin. Hyperinsulinemia remitted, and hypergastrinemia continued
unchanged.
trinoma and MEN1 by using somatostatin-receptor
scintigraphy, the most sensitive test (31), and a crosssectional imaging study (MRI or computed tomography). Pancreatic, duodenal, or peripancreatic tumors larger than 3 cm should be resected (30).
Insulinoma
In patients with MEN1, insulinomas are less
common than gastrinomas (Table), but localization
is a more frequent concern because medical control
of endocrine effects is harder to achieve with insulinoma than with gastrinoma. Although all adults
with MEN1 probably have multiple islet-cell tumors,
MENl-associated hyperinsulinemia, in our experience, is controlled by resecting a single adenoma
(Doppman et al. Unpublished data). In contrast,
hypergastrinemia in MEN1 is almost never controlled by resection of a single adenoma. The most
successful test to localize tumors that oversecrete
insulin is injection of calcium gluconate into selectively catheterized pancreatic arteries with sampling
for insulin gradients in the right hepatic vein (32)
(Figure 3).
Pituitary Tumor
Most pituitary tumors in MEN1 are prolactinomas (Table) that are successfully treated medically,
although macroprolactinomas occasionally require
transsphenoidal surgery because of their mass effect.
The major pituitary tumor localization problem is
ACTH-dependent hypercortisolemia. Patients with
MEN1 and the Cushing syndrome are rare, and
although these patients with MEN1 may have non488
pituitary tumors (pancreatic islet-cell tumors, bronchial carcinoid, or mediastinal carcinoid) that are
theoretically capable of ectopic ACTH production,
they almost always have a pituitary corticotropinoma (15). When gadolinium-enhanced MRI of the
pituitary gland discloses a microadenoma, no further localization is required.
A patient with MEN1 and the Cushing syndrome
whose MRI shows no pituitary tumor is treated like
any patient with ACTH-dependent hypercortisolemia and no demonstrable pituitary mass. Bilateral
petrosal sinus sampling for ACTH, stimulated by
ACTH-releasing hormone, is performed to unequivocally exclude ectopic ACTH production (33) and
to provide lateralizing data that may be needed for
transsphenoidal surgery. Such lateralization is correct in 70% of tumors and is the basis for performing hemihypophysectomy if no tumor is detected.
Recently, intraoperative ultrasonography of the pituitary gland has further increased the accuracy of
pituitary tumor imaging (34).
Several new endocrine tumor localization methods are helpful intraoperatively for MEN1 tumors
of several organs. These include ultrasonography and
rapid (on-line) hormone assay in blood or in tissue
aspirates.
Role of Pedigree Linkage Analysis and DNA
Maps in Localization of the MEN1 Gene
Dr. Francis S. Collins (Genetics and Molecular
Biology Branch, National Human Genome Research Institute, NIH): In 1988, the MEN1 gene was
mapped to the long arm of chromosome 11 (8) by
means of the now-standard method of linkage analysis (35). For a highly penetrant Mendelian disorder
such as MEN1, gene mapping is generally accomplished by collecting pedigrees of families with the
disease and searching for associations with several
heritable DNA sequences that serve as markers for
nearby genes. If a marker is on a different chromosome than the disease gene, no allele of the marker
will be associated with affected family members. With
a large panel of markers (300 to 400), however, there
is a good chance that one or more of them lie near
the gene; coexpression of a marker and the disease
is then to be expected. In that instance, the marker
and the disease gene are on the same chromosome
and are close enough that they are said to be
linked. By observing exceptions (meiotic recombinations between the marker and the MEN1 trait) to
that linkage in occasional members of pedigrees, it
is possible to infer how close the marker lies to the
disease gene. This process is generally iterated until
one or more nearby markers with no recombination
in all of the available families are recognized.
The original localization of MEN1 in 1988 was to
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a very large region of chromosome 11 (8). Over the
past decade, several groups refined the precision of
this mapping by developing new markers and studying more families (13, 36, 37).
Physically Mapping the Candidate Interval
of the MEN1 Gene
Once a candidate interval on chromosome 11
had been defined, it was desirable to obtain a subchromosomal road map (that is, an ordered set of
cloned DNA markers that span this region) in order
to look for candidate genes. Such a map of markers
was made, along with a collection of many large
human DNA fragments cloned into modified yeast
or bacteria to span the entire MEN1 candidate region (38). This cloned human DNA also allowed the
identification of additional polymorphic DNA markers (39), which could then be used with MEN1
pedigrees and endocrine tumors to further narrow
the linkage-derived interval. By early 1997, through
the work of several groups (40, 41), a genetic linkage-based MEN1 candidate interval of about 2 million base pairs had been defined (Figure 4).
Microdissection of Tumor Clones
Dr. Lance A. Liotta (Laboratory of Pathology,
Division of Clinical Sciences, National Cancer Institute, NIH): To go beyond the narrowing that is
obtainable through pedigree linkage analysis, the
MEN1 candidate interval was narrowed further by
analyzing DNA from endocrine tumors.
One possible expression of multiple neoplasia in
MEN1 could be the development of hundreds of
microscopic tumors in a patient with MEN1. Each
of these could reflect an independent clonal second-
hit event, bearing a different loss-of-heterozygosity
zone on the normal copy of chromosome 11. If this
happened, independent tumor clones could show
unique loss-of-heterozygosity boundaries. All loss-ofheterozygosity intervals at chromosome llql3 should
still overlap the MEN1 gene, and a few loss-of-heterozygosity boundaries might establish a narrowed interval for MEN1 gene candidates (Figure 5).
The problem of tumor clone admixture with nontumor cells is particularly notable in parathyroid
tumors. Parathyroid glands in primary hyperparathyroidism show multiple areas of nodular and diffuse
hypercellularity. Consequently, extracting total DNA
from any macroscopic portion of the gland could encompass multiple neoplastic clones with a mixture of
nonidentical second hits. Moreover, the tissues around
and within the neoplasms can be highly vascular; thus,
the neoplastic cells can be interlaced with stromal and
other non-neoplastic elements, as shown by immunohistochemical staining for vessel markers. The heterozygous or unaltered allele from the contaminating normal cells (stromal or vascular) can thus
obscure scoring of the lost allele from a tumor clone.
The use of extremely precise, controlled tissue
microdissection can address the above three problems of adjoining tumor clones, admixture with surrounding normal cells, and admixture with interlaced normal cells. Microdissection was done with
tapered pipettes and with laser-assisted cell capture;
the latter was developed in the NIH Laboratory of
Pathology (43-45).
Minimizing the MEN1 Candidate Interval from
Data on Loss of Heterozygosity
In the late stages of the MEN1 gene discovery
project, loss-of-heterozygosity zones on chromosome
Figure 4. Candidate interval for MEN1 gene. A. A map spanning approximately 2.8 million bases, part of the long arm of chromosome 11. DNA clones
were assembled to span this entire interval. The minimal candidate intervals are shown for the location of the MEN1 gene, as determined separately from
pedigree linkage or from loss of heterozygosity (LOH); the loss-of-heterozygosity-derived interval was far smaller. Genetic markers for chromosome 11, which
by convention begin with D11, are shown above the line. Various expressed genes are shown by their names or abbreviated names below the line. Alpha, beta,
and so forth are anonymous new genes assigned Greek letters as temporary names. B. A higher-resolution map featuring the area of maximum interest. A
DNA sequence was available from three clones (C1, C2, C3 [C = cosmid]). Heavy horizontal bars below the map indicate the location of clones (b137C7,
b79G17, and b18H3 [b = bacterial]), with inserts of human DNA, that were partially sequenced to search for candidate genes. The gene originally designated
Mu turned out to be MEN! (42). Cen = centromere; Tel = telomere.
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489
Searching for Genes within a
Candidate Interval
Figure 5. Method to map from tumors the zone of loss of heterozygosity (LOH) at chromosome 11q13. A. Schematic diagram of
chromosome 11 showing telomeres {Tel), p arm (short arm), centromere
(Cen), and q arm (long arm). The cytochemical banding pattern has been
used for several decades to localize genes and markers. Early studies of
multiple endocrine neoplasia type 1 (MEN1) linked it to the PYGM marker
(not shown) at 11 q 13 (chromosome 11, band q13) (8). B. The two copies of
chromosome 11 from an endocrine tumor and its precursor cell, illustrating
the first hit and the second normal copy of chromosome 11 before and after
the second hit. The tumor could be from a case with MEN1 or from a case
without MEN1 (Figure 1). The second hit caused loss of a large portion of
the q arm (dashed lines). Seven DNA markers from ordered loci on chromosome 11, designated A to G, are illustrated. Each one is highly variable
(polymorphic). The variants or polymorphisms of each marker are numbered
(A1, A2, A3, and so forth). At most markers, the two diagrammed copies of
chromosome 11 are heterozygous; they have two different variants (alleles).
C. This case is not heterozygous in the germline at markers A and F; those
markers are uninformative for this part and therefore are omitted. Markers
B, C, and D have two alleles or retention of heterozygosity; markers E and
G each have lost one allele. This is termed loss of heterozygosity or allelic
loss for those markers. Recognition of loss of heterozygosity could be obscured if the specimen had much DNA from nontumor cells with the germline heterozygous marker patterns.
Ilql3 were determined in DNA from 188 microdissected tumors obtained from 81 patients with
MENl tumors or sporadic endocrine tumors. Tumor types included parathyroid adenomas, pancreatic endocrine tumors, gastrinomas, and lung carcinoids. Six tumors (three MENl-associated and
three sporadic) provided key loss-of-heterozygosity
boundaries (Figure 6). Four exhibited allelic loss
only distal to marker PYGM. One showed loss that
placed the MENl gene proximal (that is, away from
the telomere) to marker D11S4936. This minimal
overlap interval was encompassed by the loss-ofheterozygosity zone in 100% of the microdissected
tumors that showed loss of heterozygosity at llql3.
The minimum overlapping region, bounded centromerically (toward the centromere) by marker
PYGM and telomerically (toward the telomere) by
marker D11S4936 (Figure 4 and Figure 6), became
the new MENl candidate interval; this new interval
was only 0.3 million bases long (45).
490
Dr. Francis S. Collins: Various strategies are
used to find messenger RNA (representing expressed genes) in chromosomal regions of interest
(46). These strategies include consulting already existing maps or using recombinant DNA methods
that trap pieces of genes by their binding to cloned
DNA taken from the candidate region. For regions
consisting of fewer than 1 million base pairs, the
method of choice has recently become direct sequencing of DNA across the entire interval. In
some instances, the region is completely sequenced
and assembled, leaving no gaps; in many others,
however, a partial sequencing strategy is sufficient
because it is likely to uncover sufficient evidence
with which to identify most or all transcribed genes
in the region. Thus, a major effort was undertaken
to sequence three DNA clones that spanned the
minimized MENl candidate interval (Figure 4B).
All of that sequence was assembled and then
used to search the existing database of genomic
sequences from all organisms, looking for any evidence of a match. Many candidate genes were uncovered (Figure 4) (42), some of them known genes
that had not been previously mapped to a specific
chromosomal location. Many, however, were identified only because they matched sequences in the
human database of expressed sequence tags (short
snippets of sequence from RNA). In most instances,
a candidate gene sequence gave no clue about function; thus, the genes were assigned temporary names
corresponding to Greek letters. Each of these genes
then became a potential candidate for MENl. Although some of the known genes seemed potentially
more attractive because of hints of their biological
function (such as a kinase or a zinc finger protein),
none of them turned out to be the MENl gene, and
it was necessary to carefully test most of the MENl
candidate genes for mutations.
Germline and Somatic Mutations of the
MEN1 Gene
Dr. Stephen J. Marx (Genetics and Endocrinology Section, NIDDK, NIH): A strong criterion for
success in identifying a disease-causing gene is the
demonstration of different germline mutations in
different families with the disease. Germline DNA
can be approximated by normal DNA, such as that
from blood cells from lymphocytes, or from a buccal
swab. This approach is particularly promising because most hereditary disorders, with some exceptions (such as MEN2 [2]), are caused by inactivating
mutations that can occur at many different loci in
the gene. Furthermore, most mutations are small
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and are confined to the protein-encoding regions
(the so-called open reading frame).
One MENl candidate gene that had not been
previously cloned showed mutations in germline
DNA from 14 of 15 MENl probands (47), conclusive proof of its identity as the MENl gene. This
was subsequently confirmed in other families with
MENl (48-51). Identifying this gene opened the
possibility of testing for MENl mutations in related
states and tumors as well.
Germline MEN1 Mutation in Several States
Among the 50 index cases from families with
MENl evaluated at the NIH, 32 different MENl
germline mutations were found in 47 cases (47, 48,
52) (Figure 7). No mutation was found in 3 families.
Among the 50 kindreds, there was no correlation of
mutation type with a disease phenotype, although
the analysis included 3 large kindreds with a variant
form of MENl, termed MENl Burin or the prolactinoma variant of MENl (12, 13, 52).
In addition, the NIH analysis included eight cases
of sporadic MENl (48) (defined as endocrine tumor
in two of the three principal endocrine tissues affected by MENl); 7 of 8 showed germline MENl
mutation. This was subsequently supported by the
finding among many familial and nonfamilial cases
in the United Kingdom that 10% of MENl mutations arose de novo (58).
Several possible MENl-like clinical disease
states, or phenocopies, of a sporadic or hereditary
nature might arise from the MENl mutation. Because primary hyperparathyroidism is the most frequent and usually the earliest endocrine expression
of MENl (1), familial isolated hyperparathyroidism
could be an incomplete expression of MENl. Although five kindreds with familial isolated hyperparathyroidism were evaluated at the NIH, none
showed germline MENl mutation; thus, it is more
likely that familial isolated hyperparathyroidism is
often caused by mutations in genes other than
MENl (48). A comparable analysis in Japan of
three kindreds with familial tumor of the pituitary
also showed no MENl germline mutations (51).
Somatic MEN1 Mutations in Endocrine Tumors
Several types of common endocrine tumors were
tested for MENl mutations because of previous evidence from llql3 loss-of-heterozygosity studies (1,
8-10). Studies from the NIH showed that MENl is
the known gene most frequently mutated in many
sporadic tumors, including parathyroid adenomas
(21%) (53), gastrinomas (33%) (54), insulinomas
(17%) (54), and bronchial carcinoid tumors (36%)
(55). MENl is also mutated in a significant but
small subset of sporadic pituitary tumors (5%) (56).
For each sporadic tumor with MENl mutation,
there was no corresponding mutation in paired
germline DNA; the second copy of the MENl gene
was also inactivated for each tumor, as evidenced by
llql3 loss of heterozygosity. These findings reaffirm
the notion that the study of a gene contributing to
rare hereditary tumors often uncovers important
roles for the same gene in more common sporadic
tumors.
Deductions from MEN1 Mutation Patterns
The normal function of the MENl gene is unknown. In fact, its predicted protein ("menin") sequence shows no "signature" stretches that would
predict known functions, such as membrane anchoring or DNA binding (48). Like the proteins encoded
by about half of the known tumor suppressor genes,
menin normally resides in the nucleus (59).
Most germline and somatic MENl mutations
cause premature truncation of the predicted protein
(57) (Figure 7), changes that would probably be
catastrophic for menin bioactivity. The frequency of
inactivating mutations found in germline or somatic
DNA supports the theory of the first of two hits to
a tumor suppressor gene (Figure 1).
Figure 6. Boundaries of loss of heterozygosity in endocrine tumors narrowed the chromosomal interval for the MEN1 gene hunt. Partial map
of chromosome (Chr) 11q13 shows the relative locations of 16 polymorphic DNA probes. Data are diagrammed from 6 tumors (of 188) that provided
loss-of-heterozygosity boundaries, which are helpful for narrowing the candidate interval of the MEN1 gene. Tumors 1 to 4 suggested that the MENl gene
was located toward the telomere (fe/) of the chromosome (telomeric) relative to PYGM. Tumor 6 gave a telomeric boundary at D11S449, and tumor 5 gave
a narrower telomeric boundary at D11S4936. The region between PYGM and D11S4936, the minimal MEN1 candidate interval, is approximately 0.3 million
bases (46). The same major landmarks are shown in Figure 4. Cen = centromere. Black squares = allelic retention; white squares = loss of heterozygosity at
an informative marker.
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491
Clinical Testing for Germline MEN1 Mutation
Although these discoveries at the gene level open
the possibility of clinical genetic testing for MEN1
mutation, several issues will slow the development
of MEN1 gene testing services in clinical laboratories.
1. Many different mutations. MEN1 mutations are
spread across the nine exons of the predicted open
reading frame (Figure 7), with novel mutations still
being uncovered in most newly tested families with
MEN1 (50, 51, 58).
2. No available testing shortcut. Because one
third of mutations predict a change of one amino
acid (missense) without truncation of menin protein, a so-called protein truncation shortcut assay
would not detect any of the latter third.
3. Laboratory regulations. If their results will influence patient management, U.S. laboratories should
comply with the regulations of the Clinical Laboratory Improvement Act (CLIA). Some laboratories
do not yet have CLIA approval.
4. Limited demand. MEN1 is a rare trait, and
testing for MEN1 germline mutation rarely leads to
a major therapeutic intervention.
5. Counseling issues. A physician offering MEN1
gene testing must be prepared to provide or arrange
pretest education, informed consent, and post-test
counseling. A patient education booklet about MEN1
is available on the Internet at http://www.niddk.nih
.gov/health/endo/pubs/fmenl/fmenl.htm.
Testing for MEN1 mutations differs in important
ways from testing in patients with MEN2, for which
RET gene testing can result in a major recommendation: thyroidectomy, often long before 18 years of
age, to prevent or cure cancer (2). Cancer related to
MEN1 has not been reported in patients younger
than 18 years of age, and major morbidity from
other expressions before this age is rare (1). MEN1
gene testing in persons younger than 18 years of age
is rarely indicated because it will not lead to a
major therapeutic intervention and because it would
preclude an independent choice about testing when
the child nears or reaches age 18, when appropriate
understanding of the complex issues involved is
more likely (60). More frequent morbidity before 18
years of age in patients with MEN1 could decrease
the age at which testing is recommended.
Despite the above concerns, MEN1 mutation testing will probably soon be a clinical consideration. It is
already possible, on a research basis, to identify the
MEN1 germline mutation in most MEN1 kindreds.
Clarification is also possible in sporadic (or even
familial) disorders that incompletely resemble MEN1.
Once the specific mutation is identified in an index
case, it becomes technically far easier to test other
relatives because the test can then be selectively
directed at the known mutation. Those who test
positive for the mutation can then be followed more
closely for early signs of tumor expression. Those
without the familial mutation can be spared from
uncertainty and from potentially expensive prospective laboratory testing over many years.
The discovery of the MEN1 gene may promote
future understanding of normal cell growth and of
neoplasia, particularly in endocrine tissues. The gene
will be subjected to intense study with powerful
methods, and new therapies directed at certain sporadic or hereditary neoplasms could be developed
from this knowledge.
Figure 7. Compilation of germline and somatic MEN1 mutations. Locations are shown on the horizontal bar containing MEN1 exons for germline
MEN1 mutation in 56 kindreds (48-51) and for somatic MEN1 mutation in seven parathyroids, nine gastrinomas, two insulinomas, four bronchial carcinoids,
and two pituitary tumors (53-56). Mutations shown above the exons cause protein truncation through stop codon or frameshift (small insertion or deletion),
leading to a premature stop codon; two cause splice error. Those shown below the exons cause missense or one amino acid-codon change, by base
substitution or by deletion of three bases without changing the triplet reading frame. Mutation descriptions follow standard nomenclature (57; see Glossary),
bp = base pair; NIH = National Institutes of Health.
492
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Mutation nomenclature: Amino acids are described by 1
of 20 single letters (not shown) with a number corresponding to their position in the amino acid sequence of
Terms Related to Chromosomes
a protein (Figure 7). The starting letter is the normal
llql3: A subregion of chromosome 11 on the long
amino acid; the ending letter is the changed amino acid;
arm (q arm) at cytologic band 1, subregion 3. The MEN1
thus, A309P changes amino acid 309 from alanine to
gene is within this region.
Centromere: The one major constriction in a chromo- proline. An amino acid change can be harmless (a normal
variation or normal polymorphism) or harmful (a missome. The site of attachment to the mitotic spindle.
Distal and proximal: As applied to chromosomes, each sense mutation). Mutations that create a stop codon are
common and designated by X; thus, W183X changes
chromosomal arm (short = p or long = q) has a distal
amino acid 183 from tryptophan to a stop codon. Preportion (toward the telomere) and a proximal portion
dicted
stop codons are usually deleterious mutations.
(toward the centromere).
Some mutations change the triplet code reading frame
MEN1: The MEN1 gene, whose mutation causes the
(for example, base insertions or deletions that are not
MEN1 disease or trait. Genes and DNA loci (such as
multiples
of three) such that several inappropriate (nonDUS4936) are italicized.
sense)
amino
acids are read before a stop codon is enTelomere: Either end of any chromosome.
countered. These are designated by their nucleotide position in cDNA, followed by the change to the cDNA. For
Terms Related to DNA Zones as They Change
example, 1650insC is insertion of a cytosine nucleotide at
across Generations or through Tumorigenesis
cDNA position 1650. Most MENl mutations are small
Allelic loss: See loss of heterozygosity. Allelic loss has a deletions, insertions, or substitutions, thereby affecting the
broader definition because an allele can be recognized
open reading frame. Related conventions can be used to
even if it does not have a polymorphic marker. Certain
describe large deletions or changes within introns, particmicroscopic methods (for example, fluorescent in situ hyularly because the latter can change splicing sites.
bridization) can detect one compared with two or even
Open reading frame: A portion of messenger RNA that
more copies of a marker in a nucleus.
encodes a protein sequence.
Glossary
Germline DNA: DNA capable of passage to offspring.
It can be present in the haploid state in a gamete or in
the diploid state in gametogenic cells or in the totipotent
cells of zygote. Blood DNA or normal-tissue DNA is
often used to represent germline DNA of an individual
person in studies of families or tumors.
Loss of heterozygosity: Marker loci with two different
variants (alleles) in a germline are heterozygous. If there
is allelic loss of one or more markers from a zone along
one copy of the chromosome, all formerly heterozygous
markers in that zone will have one remaining allele and
thus will have lost the heterozygous state. Loss of heterozygosity is also called allelic loss. It implies loss of one
copy of every gene fully contained in that zone and possible interruption of genes at the borders.
Polymorphic DNA marker: Segment of DNA from a
mapped locus that shows variation from one individual
person to the next and thus allows variants of the marker
to be tracked in a family or during tumor progression. A
synonym is DNA polymorphism. Most polymorphisms
have no biological consequences.
Somatic DNA: DNA in postzygotic tissue, sometimes
modified from the germline, that normally cannot be
transmitted to the next generation.
Terms Related to Genes and Their Encoded
Messenger RNA and Proteins
cDNA: Complementary DNA. DNA sequence copied
from RNA or from DNA.
Exons and introns: DNA stretches in a portion of a
gene that can be transcribed as premessenger RNA. The
introns do not encode protein and are removed by splicing during maturation of messenger RNA. At the same
time, the exons are spliced together as mature messenger
RNA, which has a protein-encoded or open reading
frame surrounded by regulatory elements at either end.
Acknowledgments: The authors thank major collaborators, including Sunita K. Agarwal, Mary Beth Kester, Christina Heppner,
Young S. Kim, Paul K. Goldsmith, and A. Lee Burns (all from
the National Institute of Diabetes and Digestive and Kidney
Disease [NIDDK]); Sirandanahalli C. Guru, Pachiappan Manickam, Shodimu-Emmanuel Olufemi, Judith Crabtree, and Settara C. Chandrasekharappa (all from the National Institute of
Human Genome Research); Larissa V. Debelenko, Zhengping
Zhuang, Irina A. Lubensky, and Michael R. Emmert-Buck (all
from the National Cancer Institute); Mark Boguski and Jane
Weisemann (both from the National Center for Biotechnology
Information); Bruce Roe and Yingping Wang (both from the
University of Oklahoma); and Jane S. Green (from Memorial
University, Newfoundland, Canada). The authors also thank Lee
S. Weinstein, William F. Simonds, and many current and former
staff members and fellows in the NIDDK/National Institute of
Child Health and Development Interinstitute Endocrine Fellowship Program and in the National Cancer Institute Surgery Branch.
Requests for Reprints: Stephen J. Marx, MD, Building 10, Room
9C-101, National Institutes of Health, Bethesda, MD 20892.
Current Author Addresses: Dr. Marx: National Institutes of
Health, Building 10, Room 9C-101, Bethesda, MD 20892.
Dr. Spiegel: National Institutes of Health, Building 10, Room
9N-222, Bethesda, MD 20892.
Dr. Skarulis: National Institutes of Health, Building 10, Room
8S235, Bethesda, MD 20892.
Dr. Doppman: National Institutes of Health, Building 10, Room
10660, Bethesda, MD 20892.
Dr. Collins: National Institute of Human Genome Research,
Building 49, Room 36-13, 49 Convent Drive, Bethesda, MD 20892.
Dr. Liotta: National Institutes of Health, Building 10, Room
2A33, Bethesda, MD 20892.
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15 September 1998 • Annals of Internal Medicine • Volume 129 • Number 6
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