The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 7 2278-2282
Copyright © 1997 by The Endocrine Society
Endocrinological Oncology |
Allelic Loss in Parathyroid Tumors from Individuals Homozygous for Multiple Endocrine Neoplasia Type 11
Alberto Falchetti,
Annamaria Morelli2,
Andrea Amorosi,
Francesco Tonelli,
Silvia Fabiani,
Valentina Martineti,
Roberto Castello,
Lino Furlani and
Maria Luisa Brandi
Endocrinology (A.F., A.M., S.F., V.M., M.L.B.) and Surgical (F.T.)
Pathology Units, Department of Clinical Physiopathology and Pathology
(A.A.) Institute, University of Florence, 50139 Florence; and Division
of Endocrinology (R.C., L.F.), Hospital, Verona, Italy 50139
Address all correspondence and requests for reprints to: Maria Luisa Brandi, M.D., Ph.D., Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy.
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Abstract
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Homozygosity for the multiple endocrine neoplasia type 1
(MEN1) gene mutation was described in two of three
affected siblings of a kindred in which both parents and the third
daughter were heterozygotes. Surprisingly, in the two homozygotes, the
disease history did not differ from the one of the heterozygotes. In
the attempt to unravel genetic differences in parathyroid tumorigenesis
between homozygotes and heterozygotes, restriction fragment length
polymorphism analysis and microsatellite PCR analysis for loss of
heterozygosity (LOH) at the MEN1 gene region on
chromosome 11q13 was performed in parathyroid tissues removed at
surgery from the mother, her heterozygous sister, and the three
siblings. Allelic losses were evidenced in the larger glands of each
patient, with a similar pattern of chromosome 11q12–13 losses. The
somatic mutation consisted of a large loss of genetic material from
chromosome 11. No gross differences exist in the 11q12–13 LOH observed
between homozygous and heterozygous carriers. Interestingly, one of the
parathyroid tumors from one heterozygote exhibited region of skipped
LOH at the 11q12–13 region. The region in the depth of the critical
interval retained heterozygosity, whereas those flanking it shared LOH.
These findings indicate that inactivation of both copies of the
MEN1 gene are not sufficient for parathyroid tumor
development in MEN 1 patients and that tumor suppressor genes, other
than the MEN1 gene on chromosome 11 or on other
chromosomes, can be involved in the pathogenesis of parathyroid
tumorigenesis in MEN 1 syndrome.
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Introduction
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MEN 1 IS AN autosomal, dominantly inherited
disorder characterized by neoplastic hyperfunction of two or more
endocrine tissues. The most frequent endocrinopathies are represented
by hyperparathyroidism, pancreatic and anterior pituitary neoplasms.
Moreover, adrenocortical and thyroid tumors, carcinoids, lipomas, and
pinealomas are observed more frequently in MEN 1 patients than in the
normal population (1). The predisposing genetic defect previously has
been described to be localized at 11q13 region by physical and genetic
mapping (2, 3, 4, 5, 6). The MEN1 gene would function as a tumor
suppressor gene, being the molecular mechanism that underlies
tumorigenesis in MEN 1 based on loss of function of the wild-type
allele in affected endocrine tissues (2, 3, 4, 5, 6, 7).
We previously described a unique kindred in which both parents and all
their children were affected by MEN 1 syndrome with hyperparathyroidism
manifested around 30 yr of age (8). When the three siblings, their
parents, and relatives were genotyped for polymorphic DNA markers from
chromosome 11q12–13, where the MEN1 locus has been mapped
(2), two of the siblings were found to be homozygotes and one
heterozygote for MEN1, with no differential clinical
features, but infertility in the two homozygotes. Thus, the presence of
two constitutionally mutated MEN1 alleles is not sufficient
for development of hyperparathyroidism early in life. To evaluate the
molecular mechanisms that underlie the development of
hyperparathyroidism in this family, we performed both restriction
fragment length polymorphism (RFLP) analysis and microsatellite-PCR
based analysis for loss of heterozygosity (LOH) in the MEN1
region in parathyroid tissue from either homozygous or heterozygous
patients.
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Materials and Methods
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Patients
Clinical information about this kindred has been provided in a
previous paper (8). Five patients [mother, I; one maternal aunt, II;
and three siblings (homozygous-male, III; homozygous-female, IV; and
heterozygous-female, V)] were affected by MEN 1 syndrome, according to
already described criteria (1). All the patients underwent total
parathyroidectomy, followed by autotransplantation. Patient IV
underwent cephalo-duodeno-pancreatectomy for multiple gastrinomas and
enucleation of a nonfunctional adrenocortical adenoma. Parathyroid
glands were reported as: right superior, A; right inferior, B; left
superior, C; left inferior, D. Three parathyroid glands from patient I
(I-A, I-B, I-C), three from patient II (II-B, II-C, II-D), two from
patient III (III-B, III-C), three from patient IV (IV-A, IV-B, IV-C),
and two from patient V (V-A, V-C) were analyzed.
Pathological evaluation
The mass of each parathyroid gland was estimated from the
dimensions recorded during surgery after removal of the tissue, with
the formula for mass of an ellipsoid (4/3
x r1 x r2 x
r3) x D, where D denotes a water density of 1 g/cm3; and
r1, r2, and r3 are the orthogonal radii (3). Tissues for conventional
histology were obtained for all the excised glands after formalin
fixation and paraffin embedding. Specimens for LOH studies were
collected fresh in at least two glands from a single patient and frozen
in liquid nitrogen. Each gland was cut along the major radium, and one
half has been used for molecular analysis and the other half for the
classical histology.
LOH and mini/microsatellite-PCR analyses
Adequate DNA samples were obtained from all the available
tissues. High molecular-weight DNA was prepared from cryopreserved
specimen and their matched peripheral blood, as already described (9, 10). Five micrograms of DNA were digested to completion according to
the manufacturer’s instructions and size-fractionated by
electrophoresis on 0.8–1.4% agarose gel. Transfer to nylon filters
(GeneScreen Plus, Du Pont, Boston, MA) was performed according to the
manufacturer’s instructions. All the parathyroid DNA was genotyped by
RFLP analysis using five polymorphic probes from region 11q12–13: p3C7
(D11S288, MspI), pmol/L CMP1 (PYGM,
MspI), pmol/L S51 (D11S97, TaqI),
pHBI59 (D11S146, TaqI), and pSS6
(INT2, TaqI). Probes were labeled to a specific
activity of 109 cpm/µg, with the random priming method.
The conditions of prehybridization, hybridization, and washing were
performed using the manufacturer’s specifications (Du Pont). Filters
were autoradiographed at -70 C for 24–72 h.
Mini/microsatellite-PCR-based analysis of the above mentioned DNA was
performed using two CA-repeats from D11S480 and
PYGM loci and two oligonucleotide pairs flanking two highly
informative loci, D11S533 on 11q13.5 and D11S554
on 11p11. The experimental conditions for D11S480,
PYGM, D11S533, and D11S554 were according to the
original paper (11, 12, 13, 14). All the PCR products were then mixed with
formamide gel loading solution, heat denatured at 94 C, separated on a
denaturing 6–8% polyacrilamide, 32% formamide gel (15), and
visualized by autoradiography for 0.5–48 h. Allelic loss was scored as
previously described (15). PYGM locus exhibits recombination
zero with the MEN1 locus (2). Haplotype analysis of patient
II was performed using the same DNA markers here reported, at the same
experimental conditions.
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Results
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Pathological findings
The parathyroid glands from the five patients ranged in mass from
0.06 g (II-C) to 3.76 g (IV-B) (Table
. 1). All
glands from each patient were histologically examined, and pathological
findings were classified as chief cell hyperplasia. The single examined
tumors exhibited a distinct variability in the histological pattern,
with nodular changes prevalent in larger glands and in all glands from
the two homozygous patients (Fig. 1).
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Figure 1. Histological features of representative
parathyroid glands. a, II-C; b, V-A; c, II-B; d, IV-B (see Table 1 for
description). Though the sequence of microphotographs suggests a
morphopathological progression, all these tissues harbored allelic loss
at 11q13. The darker staining of the right
pole of gland V-A is caused by hemorrhage resulting from the
sectioning procedure.
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LOH analysis
Figure 2 depicts the minipedigree of the patients
whose parathyroid tissues were analyzed3).
All five patients were informative for at least one chromosome 11
polymorphism. Similar allelic losses (encompassing D11S554,
D11S288, D11S480, PYGM,
D11S97, D11S146, INT2, and
D11S533 loci) in the MEN1 region were found in
homozygous and heterozygous patients spanning from D11S288
to D11S533 (Figs. 3 and 4).
Allelic losses at 11q13 were exhibited in multinodular glands, in
moderately enlarged glands with a diffuse pattern of hyperplasia, and
in a slightly enlarged gland with subtle histological changes
suggestive of hyperplasia, indicating that monoclonal outgrowths may be
present at an early stage in parathyroid tumorigenesis of MEN 1
patients. Interestingly, all three glands (II-B, II-C and II-D) from a
heterozygous patient showed allelic losses at three 11q12–13 region
loci (D11S288 and D11S480 on the centromeric side
and D11S146 on the telomeric side), whereas the DNA between
these two regions retained both alleles. This patient was not
previously genotyped in the original paper (8). Our haplotyping
revealed that she carries the same mutant allele as that of her sister
(I).
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Figure 2. Minipedigree of the described MEN 1 kindred.
Patients analyzed in this report are indicated with the corresponding
roman number (I-V). The MEN 1 affected husband (F) of patient I also is
represented. Under each familial member, the corresponding chromosome
11q13 genotype is described. The haplotypes deduced to carry the mutant
MEN1 allele in individuals F and I are marked,
respectively, as a hatched line and as a thick
line. *, Patient not previously genotyped (8). Data for markers
at five 11q13 polymorphic DNA markers are shown.
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Figure 3. Representative RFLP analysis. Patients (I,
II, III, and IV) and glands’ codes (A, B, C, and D) are indicated at
the top of the autoradiograms. Analyzed loci and the
used restriction enzyme are reported at the bottom of
autoradiograms. Black arrows indicate the retained
allele in constitutive DNA.
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Figure 4. Subregions of allelic loss in 13 parathyroid
tumors form the 5 MEN 1 patients. Loci are ordered according to the
published data on genetic linkage. For each tumor, loci on chromosome
11 that were informative (e.g., 2 alleles present in
germline DNA represented by peripheral blood leukocytes) are shown.
Solid boxes, Informative loci that showed allelic loss;
open boxes, informative loci with retention of
heterozygosity; hatched boxes; noninformative loci.
Codes for the two homozygotes, III and IV, are framed. Solid
lines between open squares represent presumably
retained subregions (assuming the DNA between two retained loci was
also retained). Dashed lines represent continuous
subregions of presumed loss of alleles from one copy of chromosome 11
(assuming that all the intervening DNA was lost between 2 loci of
allelic loss). *, Locus at recombination zero with the
MEN1 locus. The vertical bar on the
left side indicates the critical interval of the
MEN1 gene region.
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Discussion
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Parathyroid glands in MEN 1 patients are characterized by a
considerable asymmetry in size and a distinct cytoarchitectural
variability, ranging within the same patient from normal to diffuse
and/or nodular hyperplasia and, occasionally, to adenoma-like tumors
(16). It is conceivable that the wide spectrum of histological
abnormalities of the parathyroid glands represents different stages in
the progression from hyperplasia to neoplasia under cumulative effects
of selective extrinsic and/or intrinsic pressures on polyclonal cell
populations (1, 17). Somatic inactivation of the wild-type copy of the
MEN1 gene would be responsible of the development of
monoclonal outgrowths, often present in larger glands. Generally, the
second mutational event is represented by a gross chromosomal deletion
that includes the MEN1 region and that is typical of larger
parathyroid lesions (3). However, conventional histology, failed to
demonstrate any morphopathological difference between polyclonal and
monoclonal parathyroid tissues either in MEN 1 patients or in sporadic
hyperparathyroidism (17, 18, 19), as also observed in the present study. It
is conceivable that multiple clones arise in the setting of diffuse
hyperplasia when this evolves into a multinodular form, where nodules
are, presumably, monoclonal. Therefore, a nodular pattern could be
expected when somatic inactivation of the wild-type copy of the
MEN1 gene occurs, as in patients homozygotes for the
inactivation of the MEN1 gene. Indeed, nodular changes were
prevalent in all glands from homozygous patients when compared with the
findings in heterozygotes.
Based on these observations it was difficult to understand why patients
homozygous for the MEN1 gene inactivation did not develop
hyperparathyroidism early in life (8). Indeed, analysis of allelic
losses in parathyroid tumors from MEN1 homozygotes made
possible to demonstrate a pattern similar to what shown in heterozygous
patients, with gross chromosomal deletions at 11q13, suggesting that
genes, other than the MEN1 gene, may have a role in the
development of endocrine tumors in MEN 1 syndrome. Several disease
genes are localized in the 11q13 region (15, 20, 21, 22, 23, 24) and their possible
role in the pathogenesis of MEN 1 neoplasms cannot be excluded. In
addition, genes localized in other chromosomes could be involved in the
progression of parathyroid tumors in MEN 1 syndrome. In fact,
continuous LOH at 11q13 and 1p loci was described in sporadic adenomas
(25), suggesting the possibility that the inactivation of 1p
anti-oncogene(s) may function either independently or in concert with
the MEN1 gene inactivation in the development of parathyroid
tumors.
Interestingly, patient II showed skipping deletions at 11q in the three
parathyroid glands examined. These deletions could be the result of
recombining events or of a dual composition from two distinct and
dominant clonal cell populations within the multinodular pattern of
parathyroid tissue, supporting the existence of allelic heterogeneity,
as already described in different endocrine tissues from the same MEN 1
patient (26). However, lack of allelic losses does not certainly mean
lack of gene inactivation, if point mutations or small deletions at the
MEN1 gene locus were not revealed by the available molecular
approaches. Alternatively, as discussed above, tumor suppressor genes
other than the MEN1 gene in chromosome 11q may play a role
in the development of parathyroid tumors in MEN 1 syndrome.
Based on the minimal region of overlapping deletions at 11q13 in the
parathyroid tissues here analyzed, the MEN1 gene boundaries
are placed between markers D11S480 (patient IV, IV-C) and
INT2 (patient II, II-B, II-C, II-D), in agreement with
previously published data (27, 28).
In conclusion, the unique opportunity of genotyping allelic losses at
the MEN1 locus in parathyroid tissues from MEN1
homozygotes made possible to demonstrate close similarities of
parathyroid tumor progression between homozygous and heterozygous
patients. These results are in agreement with our previous observations
that constitutive homozygous mutation(s) of the MEN1 gene
are not sufficient for an early onset of hyperparathyroidism and of
other endocrine disorders associated with this syndrome. The cloning of
the MEN1 gene will make possible in the future to
characterize the two MEN1 homozygous germinal mutations and
to evaluate the possibility of complementation at the protein
level.
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Footnotes
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1 This work was supported by grants from the Associazione Italiana per
la Ricerca sul Cancro, from the National Council of Research (PF ACRO
95.00316.PF39 and 94.02563.CTC4), and from the Ministero
dell’Università e della Ricerca Scientifica e Tecnologica (MURST
40% and 60%). 
2 Recipient of a fellowship from the Associazione Italiana per la
Ricerca sul Cancro.
3 For more details of the genotype analysis of
this kindred, see the original manuscript (8), where patients I, III,
IV, V, and F correspond, respectively, to previously codes I:6, II:1,
II:2, II:3, and I:5.
Received October 21, 1996.
Accepted March 20, 1997.
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Abstract
Introduction
