Clinical Chemistry 45: 29-34, 1999;
(Clinical Chemistry. 1999;45:29-34.)
© 1999 American Association for Clinical Chemistry, Inc.
Identification of MEN1 Mutations in Sporadic Enteropancreatic Neuroendocrine Tumors by Analysis of Paraffin-embedded Tissue
Matthew D. Mailman1,
Peter Muscarella2,
William J. Schirmer2,
E. Christopher Ellison2,
Thomas M. O'Dorisio3 and
Thomas W. Prior1,a
Departments of
1
Pathology,
2
Surgery, and
3
Internal Medicine, The Ohio State University College of Medicine, Columbus, OH 43210.
a Address correspondence to this author at: Department of Pathology, The Ohio State University, 121 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Fax 614-292-7072; e-mail prior-1{at}medctr.osu.edu.
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Abstract
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Gastrinomas and other gastrointestinal neuroendocrine tumors may occur
sporadically or as part of the inherited syndrome multiple endocrine
neoplasia type 1 (MEN1). Mutations in the recently identified
MEN1 gene have been described in sporadic gastrinomas and
insulinomas. This study describes techniques used to identify mutations
in the MEN1 gene in DNA extracted from paraffin-preserved
tissue. Two novel mutations are identified in the MEN1 gene
from nine archived paraffin-embedded neuroendocrine tumors,
demonstrating that retrospective genetic analysis can be used to
identify mutations in the MEN1 gene from preserved tissue.
Conditions are provided by which paraffin-embedded tissue can be used
as a source of genetic material for sequence information of sufficient
quality for mutational studies of the MEN1 gene. It should
also be possible to apply this retrospective genetic analysis of
paraffin-embedded tissue to other disease models.
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Introduction
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Gastrinomas and other gastrointestinal neuroendocrine tumors may
occur sporadically or as part of the autosomal dominant genetic
syndrome multiple endocrine neoplasia type 1 (MEN1), characterized by
parathyroid hyperplasia, pituitary adenomas, and islet cell tumors of
the pancreas. The recently identified MEN1 gene
(1)(2)(3) encodes a putative tumor suppressor protein termed
menin. Germ-line mutations were found in 94% of MEN1 patients
(4).
When a gene responsible for a hereditary cancer is identified, it is
important to next determine if the same gene is also involved in
sporadic forms of the cancer. A gene that affects both familial and
sporadic forms of the cancer is likely to be part of an important
pathway and may provide insight about the mechanism of tumorigenesis.
The breast cancer genes BRCA1 and BRCA2 are
examples of genes that are often mutated in familial breast cancer but
are not mutated in sporadic breast cancer cases. In the case of the
MEN1 gene, evidence is beginning to accumulate that suggests
that its function is likely to be lost in some sporadic cancers of the
same tissues affected in the hereditary MEN1 syndrome. One study showed
that ~21% of sporadic parathyroid tumors had MEN1
mutations (5). Another study reported MEN1
mutations in 5% of sporadic pituitary tumors (6).
Sporadic gastrinomas were first described by Zollinger and Ellison in
1955 (7). Relatively little is understood about the
molecular mechanisms involved in gastrinoma tumorigenesis. A recent
study performed using fresh-frozen tissue has demonstrated that
MEN1 mutations, detected by single-strand conformational
polymorphism analysis, occur in 33% of sporadic gastrinomas and 17%
of insulinomas (8). Other studies have found mutations in 6
of 23, 1 of 4, and 3 of 11 gastroenteropancreatic neuroendocrine
tumors, respectively (9)(10)(11).
We were interested in confirming the role of MEN1 mutations
in sporadic gastrinomas and in determining if mutations in the
MEN1 gene could be identified in DNA extracted from
paraffin-embedded gastrointestinal neuroendocrine tumors. A
retrospective study was performed using nine archived paraffin-embedded
gastrinomas and nonfunctioning neuroendocrine tumors from the tissue
archives at the Ohio State University Medical Center. Heteroduplex
analysis was used to screen 93% of the coding sequences for mutations
of the MEN1 gene. Using this strategy, we identified two
novel frameshift mutations. These inactivating mutations, along with
the finding of a loss of heterozygosity (LOH) in one tumor, are
consistent with the MEN1 gene product acting as a tumor suppressor. We
have confirmed the role of MEN1 mutations in sporadic
enteropancreatic neuroendocrine tumors and have provided conditions for
the use of archival paraffin-embedded tissue as a valuable source of
genetic material for retrospective genetic studies.
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Materials and Methods
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tumor specimens
A total of six gastrinomas and three neuroendocrine tumors, one
from each patient, were obtained at the time of surgical resection at
the Ohio State Medical Center (Table 1
). After resection, the tissues were paraffin-embedded according
to routine protocol. The diagnosis of neuroendocrine tumors was made by
histological analysis, and patients were considered to have gastrinomas
if they had either increased fasting serum gastrin or paradoxical
increases in serum gastrin in response to provocative testing with
secretin. Patients were considered to have nonfunctioning pancreatic
neuroendocrine tumors if they clinically demonstrated no evidence of
hormonally active tumors in the presence of a pancreatic mass with
neuroendocrine characteristics by histopathological analysis. All of
these tumors were sporadic. All procedures performed for handling the
subjects and tissues were in agreement with the ethics standards of the
Ohio State University Human Subjects Committee.
dna extraction
DNA extraction from paraffin-embedded tissue was modified from a
previously described procedure (12). Several 50-µm
sections from a block of paraffin-embedded tumor tissue were placed in
a 1.5-mL microcentrifuge tube. To dissolve the paraffin, the sections
were immersed in 1 mL of xylene, vortex-mixed briefly, and incubated
for 10 min at room temperature. The tube was then centrifuged for 5 min
at 13 800 g. A pipette was used to remove the liquid. This
procedure was repeated twice with 1-mL aliquots of xylene. The tissue
was rehydrated gradually by repeating the above method with 1 mL of
ethanol (twice), then with 800 mL/L ethanol, and then with 500 mL/L
ethanol. One milliliter of H2O was added, and the tube was
refrigerated at 4 °C for 24 h to complete rehydration. To lyse
the cells, the tissue was warmed to room temperature and then
centrifuged for 5 min. The water was removed and replaced with 700 µL
of nuclei lysis buffer (10 mmol/L Tris, 400 mmol/L NaCl, 2 mmol/L
Na2EDTA), and the sample was vortex-mixed. For
solubilization of membranes and degradation of proteins, 70 µL of
proteinase K and 30 µL of 200 g/L sodium dodecyl sulfate were added,
and the sample was vortex-mixed and incubated at 55 °C for 24
h. If the tissue was not completely degraded, an additional 15 µL of
proteinase K was added and incubation was continued for another 24
h. Precipitation of degraded proteins was accomplished by adding 233
µL of saturated NaCl solution. The tube was vortex-mixed for 15
s and spun for 5 min. The supernatant (containing the DNA) was
transferred to a new tube, combined with 1 mL of ethanol, and inverted
to precipitate the DNA.
pcr amplification
Tumor DNA was amplified in the presence of 150 ng of each
oligonucleotide primer and 2.5 U of Taq polymerase (Cetus-Perkin-Elmer)
in a final volume of 100 µL of the following solution: 0.5 mmol/L
deoxynucleotide triphosphates, 1 or 2 mmol/L MgCl2, 67
mmol/L Tris (pH 8.8), 16.6 mmol/L ammonium sulfate, 6.7 µmol/L EDTA,
and 10 mmol/L 2-mercaptoethanol. The primer sequences (Table 2
) were derived directly from the DNA sequence (Genbank accession
no. U93237). Amplification proceeded by a denaturation step of 5 min at
95 °C, followed by 35 cycles (1 min at 95 °C, 2 min at 55 °C,
3 min at 72 °C), and ended with an extension step of 8 min at
72 °C (Ericomp Thermocycler). To achieve optimal amplification, it
was sometimes necessary to increase the number of amplification cycles
to 40. Ninety-three percent of the coding region was amplified;
technical difficulties prevented us from amplifying the other 7% of
the gene.
heteroduplex analysis
The heteroduplex approach relies on the formation of
heteroduplexes between the wild-type and mutant alleles, which are
detected because they migrate differently than the corresponding
homoduplexes in Hydrolink-MDE gels. It has been reported that there is
a 93% rate of LOH in gastrinomas (8). Heteroduplexes would
not form in the DNA of patients with a deleted allele; therefore, PCR
products from different tumors were mixed, thus allowing for
heteroduplex formation. These aliquots of PCR product were heated to
95 °C for 5 min, and then slowly ramped down to 37 °C for 30 min
(13). A 20-µL aliquot of each mixture was then combined
with 4 µL of 6x Triple Dye Loading Buffer (FMC BioProducts) and was
electrophoresed through a 50-cm Hydrolink-MDE gel (AT Biochem)
containing 150 g/L urea in Tris-borate-EDTA buffer (80.3 mmol/L Tris
base, 48.5 mmol/L boric acid, 1.53 mmol/L Na2EDTA) for
15 h at 1000 V. The gel was stained in a solution of
Tris-borate-EDTA buffer containing 1 mg/L of ethidium bromide, and the
DNA was photographed under ultraviolet light. This screening method
would detect mutations in exons of the gene as well as in splice donor
and acceptor sites. Because none of the primer pairs were designed to
amplify promoter or intron sequences, we would not have been able to
detect intronic cryptic splice site mutations, mutations in the
promoter, or mutations in exon 1.
sequence analysis
Sequencing was performed on samples that produced heteroduplexes
on the MDE gel. The Wizard PCR Preps DNA Purification System (Promega)
was used to isolate longer double-stranded DNA strands away from the
degraded DNA, which is abundant in preserved tissue and decreases the
quality of the sequence. Double-stranded sequencing of the purified PCR
product was performed using the double-stranded DNA cycle sequencing
system (Life Technologies). Primers were end-labeled in a volume of 5
µL containing 1 µL of 5x kinase buffer (Life Technologies), 2.5
µL (3 ng/µL) of primer, 1 µL of [
-32P]-ATP,
0.5 µL of T4 polynucleotide kinase (Life Technologies); and were
incubated at 37 °C for 20 min, 55 °C for 10 min; and then placed
on ice. The sequencing reaction proceeded in a volume of 37 µL: 5
µL of end-labeled primer, 26 µL of distilled H2O, 4.5
µL of 10x Taq sequencing buffer (Life Technologies), 1 µL of
template DNA (from PCR amplification), and 0.5 µL (5 U/µL) of
Taq DNA polymerase. The cycle sequencing program consisted of the
following: 6 min at 95 °C, 20 cycles (30 s at 95 °C, 30 s at
55 °C, 30 s at 72 °C), followed by 10 cycles (30 s at
95 °C, 1 min at 68 °C), and 8 min at 72 °C. The product from
the sequencing reaction was analyzed using a 50-cm 5% denaturing
polyacrylamide gel. Tumor DNA was compared with that of a control
patient without MEN1. Mutations were confirmed by repetition and by
separately labeling both forward and reverse primers.
loh
A semiquantitative technique was used in an attempt to determine
the number of MEN1 alleles present in comparison to the
number of alleles of the CFTR gene. Primers M1-7 (see Table 2
) and CF621 (forward: 5'-AGTCACCAAAGCAGTACAGC-3'; reverse:
5'-GGGCCTGTGCAAGGAAGTGTTA-3') were end-labeled in the same reaction
mixture: 1.2 µL of doubly distilled H20, 0.5 µL of
forward primers (15 ng/µL each primer), 0.6 µL of kinase buffer
[350 mmol/L (pH 7.6), 500 mmol/L KCl, 5 mmol/L 2-mercaptoethanol, 50
mmol/L MgCl2], 0.1 µL of [-32P]-ATP,
0.1 µL of T4 polynucleotide kinase (10 U/µL), and 0.2 µL of
Pfu DNA polymerase (2.5 U/µL; Stratagene). The
end-labeling reaction mixture was incubated at 37 °C for 20 min and
55 °C for 10 min, and then transferred to ice. The PCR amplification
protocol was a variation of that described by McAndrew et al.
(14). The PCR reaction mixture consisted of 14.9 µL of
distilled H20, 0.25 µL of dimethyl sulfoxide, 2.5 µL of
10x PE Buffer (Cetus-Perkin-Elmer), 0.75 µL of 100 mmol/L
MgCl2, 0.5 µL of reverse primers (15 ng/µL each
primer), 0.5 µL of 25 mmol/L deoxynucleotide triphosphates, and 0.1
µL of Taq polymerase (5 U/µL). This was added to the end-labeled
primers and to 2 µL of DNA (1 g/L) and amplified as follows: 6 min at
95 °C, 16 cycles (1 min at 95 °C, 2 min at 55 °C, 3 min at
72 °C), and 8 min at 72 °C. The amplified products were analyzed
on a 50-cm 5% denaturing polyacrylamide gel and were quantified by
autoradiography. Densitometry of the bands was performed on a Shimadzu
CS-9000. The genomic CFTR and MEN1 ratio was
determined for all samples. Tumor 5 was used as a positive paraffin
control because its LOH status was determined by sequence analysis.
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Results
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mutation analysis
Nine tumor specimens, including six gastrinomas and three
nonfunctioning neuroendocrine tumors, were evaluated for
MEN1 mutations, using the technique of heteroduplex
analysis. Aberrantly migrating bands were identified in two of nine
tumors at the time of electrophoresis, indicating the presence of
mutations (Fig. 1
). Direct sequencing of tumor 9 revealed a novel one-base pair
insertion at nucleotide 643 (643insC) in exon 3 of the MEN1
gene (Fig. 2
), which produced a shift of the reading frame. The resulting
reading frame was terminated by a new stop codon in the subsequent
triplet codon. Tumor 5 was shown to have a novel seven-base pair
deletion in exon 2 of the MEN1 gene (320del7; Fig. 3
). This mutation also caused a frameshift, which produced a
severe truncation of the message 44 codons downfield from codon 71.
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Figure 1. Heteroduplex analysis of exon 3 of tumor 9
(right) and a healthy control (left).
 indicates heteroduplex.
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Figure 2. Portions of the sequencing gel of DNA from a healthy
control (left) and a patient (right).
Sequencing from primer M1-3R showed the patient to have an insertion of
a single C at nucleotide 643 in exon 3.
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Figure 3. Portions of the sequencing gels of DNA from a patient
(left) and a healthy control (right).
Sequencing is from primer M1-2BR. The patient was found to have a
deletion of seven nucleotides starting at nucleotide 320 in exon 2.
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We observed mutations in one of six (17%) gastrinomas and one of three
(33%) neuroendocrine tumors for an overall frequency of 22%. Although
this frequency is lower than the 33% (9 of 27) reported previously
(8), it is not inconsistent given that we looked at fewer
tumors. Zhuang et al. (8) used the method of single-strand
conformational polymorphism to scan for mutations, whereas we used
heteroduplex analysis, which has been shown to have comparable
sensitivity (15). More than 60% of the MEN1 gene
mutations that were previously identified in sporadic gastrinomas were
located in exons 2 and 3 (8). Both of the mutations found in
this study were also found in exons 2 and 3.
loh
LOH is common in pancreatic endocrine tumors
(16)(17) and was detected in 93% of sporadic
gastrinomas and 50% of sporadic insulinomas investigated by Zhuang et
al (8). LOH was confirmed in one of the two tumors that we
analyzed by sequence analysis. Tumor 5 was shown to have only a single
sequence ladder despite a seven-base pair shift in the sequence of the
homologous allele (Fig. 3
). If both the wild-type allele and the mutant
allele were present, overlapping sequence ladders would be apparent.
The fact that no wild-type sequence was observed indicates that the
wild-type allele had been deleted. In contrast, tumor 9 had a one-base
pair insertion that did demonstrate two overlapping sequence ladders
(Fig. 2
). Because we were dealing with old preserved tumors, we did not
have access to blood samples from the corresponding patients and were,
therefore, not able to perform standard LOH analysis. We attempted to
identify LOH in our tumor samples by semiquantitative coamplification
of exon 7 of the MEN1 gene and exon 10 of the
CFTR gene, which served as an endogenous internal control
known to have a disomic copy number. Any variation between samples or
between the conditions of the PCR reaction would, therefore, not affect
the ratio for each sample, because both amplified products would be
affected equally (18). Amplification proceeded for 16 cycles
so that polymerase enzyme, nucleotides, and primer concentrations were
in excess over the template DNA concentration. This ensured that
amplification was exponential and quantitative. The ratios, however,
were not markedly different for any of the samples including the
positive control, tumor 5. The results are most consistent with
amplification of contaminating stromal tissue from the archival blocks,
which prevented discrimination between the presence of one or two
alleles of the gene.
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Discussion
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During this study, mutations in the MEN1 gene were
identified in two paraffin-preserved sporadic enteropancreatic
neuroendocrine tumors. These mutations in tumors 9 and 5 were
frameshifting mutations in the 5' region of the gene, which would cause
a severe truncation of the menin protein. Tumor 9 was from a
52-year-old woman who presented with multiple gastric ulcers and
diarrhea. Her serum gastrin response to provocation with secretin was
consistent with gastrinoma. The patient's tumor was preoperatively
localized by computer tomography and octreotide scintigraphy. At the
time of surgery, she was found to have a single metastatic lymph node,
which was histologically confirmed to be a gastrinoma. No primary tumor
was identified. Tumor 5 was from a 73-year-old man who was noted to
have multiple liver nodules at the time of a distal pancreatectomy and
splenectomy. These were resected and appeared histologically consistent
with metastatic neuroendocrine tumor. The primary tumor was presumably
in the tail of the pancreas. Because the patient did not exhibit
biochemical evidence of a hormonally active tumor, he was subsequently
diagnosed as having a metastatic nonfunctioning pancreatic
neuroendocrine tumor. Furthermore, the identification of both an
allelic deletion and a one-base pair insertion in tumor 5 is consistent
with the two-hit hypothesis of a tumor suppressor gene (19).
Tissue preserved in paraffin is an important resource for genetic
studies. There is more background and the sequence data are often of
lesser quality than are found from sequenced fresh tissue DNA, because
much of the DNA from paraffin-preserved tissue is degraded, but it is
still quite feasible to find mutations. We established conditions under
which we were able to identify two mutations in DNA extracted from
paraffin in nine embedded blocks. We found that amplification of
relatively small products was effective for DNA extracted from
paraffin-preserved tissue. Because of the highly degraded nature of the
DNA, there is likely to be more short-length DNA that would provide an
intact template. In our study, the ratio of intact to degraded DNA was
increased by using a DNA purification system (see Materials and
Methods). This procedure was also found to increase the quality of
the sequence data.
LOH analysis using microsatellite markers from the MEN1
locus was not feasible because we did not have access to blood samples
from the patients. However, we attempted to apply a semiquantitative
dosage assay by comparing PCR-amplified product from MEN1
exon 7 to amplified CFTR exon 10, which served as our
endogenous internal control. The dosage ratios were not consistent,
which was most likely because of the masking of possible allelic loss
by contaminating stromal cells. Extraction from paraffin-embedded
tissue as described here is sufficient for heteroduplex analysis and
for DNA sequencing; however, gene dosage assays are complicated by the
presence of small amounts of contaminating stromal tissue in preserved
surgical sections. Recently, techniques have been described that might
make a dosage assay on DNA from preserved tissue feasible.
Immunostaining was used by Lubensky et al. (20) to determine
that the endothelial component in MEN1 parathyroid tissue accounted for
the majority of the stromal component of the excised parathyroid
tumors. They used a 30-gauge needle under a light microscope to select
tumor cells and to minimize the amount of contamination by stromal
tissue. They were quite successful at identifying quantitative
differences in MEN1 copy number between DNA from tumor
tissue and from blood. A similar technique may be used for a successful
semiquantitative dosage assay on DNA from paraffin-embedded tissue.
We have performed a retrospective mutation analysis of the
MEN1 gene, using archival paraffin-embedded tissue. This is
important, because enteropancreatic neuroendocrine tumors are rare, and
the collection of tissue for prospective genetic studies may require
long periods of time. Analyzing archival tissue will expedite the
process of understanding the role of the MEN1 gene in
tumorigenesis and would be an aid in understanding the role of other
disease genes. Molecular diagnosis of familial MEN1
mutations from preserved tissue of deceased relatives would provide an
opportunity for accurate family studies and counseling for at-risk
relatives. Finally, by identifying mutations in both alleles of the
gene of one patient, we have presented supporting evidence that the
MEN1 gene is a tumor suppressor gene involved in the
tumorigenesis of some enteropancreatic neuroendocrine tumors.
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Acknowledgments
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We thank Arthur Weeks for photographic assistance.
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The following articles in journals at HighWire Press have cited this article:
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S. S. Guo and M. P. Sawicki
Molecular and Genetic Mechanisms of Tumorigenesis in Multiple Endocrine Neoplasia Type-1
Mol. Endocrinol.,
October 1, 2001;
15(10):
1653 - 1664.
[Abstract]
[Full Text]
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Top
Abstract
Introduction
