Two Autonomous Nodules of a Patient with Multinodular Goiter Harbor Different Activating Mutations of the Thyrotropin Receptor Gene1
Laurence Duprez2,
Jacques Hermans,
Jacqueline Van Sande,
Jacques E. Dumont,
Gilbert Vassart and
Jasmine Parma
Institut de Recherche Interdisciplinaire, Faculty of Medicine,
University of Brussels (L.D., J.V.S., J.E.D., G.V.), and Service de
Génétique Médicale, Hôpital Erasme, University
of Brussels, Campus Erasme (G.V., J.P.), Brussels; and Centre
Hospitalier de Jolimont, Haine-Saint-Paul (J.H.), Belgium
Address all correspondence and requests for reprints to: Dr. G. Vassart, IRIBHN, Faculty of Medicine, University of Brussels, Campus Erasme, Route de Lennik 808, 1070 Brussels, Belgium. E-mail:
gvassart{at}ulb.ac.be
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Introduction
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Exon 10 of the TSH receptor gene was sequenced on
DNA extracted from two different autonomous nodules in a patient with
toxic multinodular goiter. Each nodule was shown to harbor a different
mutation, leading to the amino acid substitutions Met453Thr
and Thr632Ile in the second and sixth transmembrane
segments, respectively. Each mutation was found only in the nodules;
the adjacent quiescent tissue was negative. Both mutations have been
previously described in blood genomic DNA from a patient with
congenital hyperthyroidism and in somatic DNA of solitary toxic
adenomas, respectively. They were shown to increase the constitutive
activity of the TSH receptor toward stimulation of adenylyl cyclase.
Our data show that a similar pathogenic mechanism can be responsible
for the autonomy of solitary toxic adenomas and multiple adenomas in a
toxic multinodular goiter.
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Clinical presentation
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In April 1995, a 58-yr-old woman consulted for the recent
development of a thyroid nodule in the right lobe. Upon physical
examination, two nodules were identified, one in each lobe. No sign of
autoimmunity was detected. Laboratory data were compatible with
subclinical hyperthyroidism: TSH, less than 0.1 µU/ml (normal range,
0.23); free T3, 9.2 pmol/L [normal range, 2.26.8; 5.98
pg/mL (normal range, 1.44.4)]; and free T4, 27.6 pmol/L
[normal range, 923; 21.42 pg/mL (normal range, 718)].
Tc99m scintigraphy demonstrated several hypercaptant areas
in both lobes (Fig. 1
). Thallium scintigraphy revealed
heterogeneous captation in both lobes (not shown). In December 1995,
she underwent thyroidectomy.

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Figure 1. A, Illustration of the Tc99m
scintigraphy performed before surgery. Captation is heterogeneous in
both lobes, with multiple hypercaptant nodules. B, Direct sequencing of
PCR product from the right lobe shows substitution of a threonine for a
methionine. Coding strands are shown for quiescent tissue harboring
normal allele (N) and for nodule harboring mutated allele at
heterozygote status (M). The mutated vs. normal allele
ratio is less than 1. C, Direct sequencing of PCR product from the
left lobe shows substitution of a isoleucine for a threonine. Coding
strands are shown for quiescent tissue harboring normal allele (N) and
for nodule harboring mutated allele at heterozygote status (M). The
mutated vs. normal allele ratio is approximately 1.
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Histology confirmed the presence of adenomatous tissues in both lobes
without any sign of malignancy. The right lobe was occupied by a well
individualized nodule measuring 2.5 cm in diameter and three secondary
nodules. Microscopic examination of the main nodule revealed
adenomatous hyperplasia. The epithelium was cubical or cylindrical;
many resorption vesicles were observed in the colloid. The left lobe
displayed a well individualized nodule as well as several secondary
nodules. Microscopic examination revealed the same characteristics as
in the right lobe nodule. The adjacent tissue had a quiescent aspect
and displayed some compression features. Neither the right nor the left
nodule was encapsulated. These thyroid lesions were histologically
classified as adenomatous nodules.
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Differential diagnosis
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Autonomous activity of thyroid tissue occurs in a spectrum of
different pathological situations, ranging from the solitary
encapsulated adenoma on a background of normal tissue to multiple, more
or less well delimited, autonomous regions in a context of multinodular
goiters. The prevalence of these different forms of thyroid autonomy is
higher in areas with limited iodine supply or in regions with endemic
goiter that have been recently supplemented (1, 2). In areas with mild
iodine deficiency such as Belgium, a negative correlation has been
found between thyroid size, plasma thyroglobulin, and age, on the one
hand, and plasma TSH, on the other (3). This suggests that autonomous
tissue appears over time in a significant proportion of the
multinodular goiters of these patients. Our patient clearly belongs to
the group in which multiple autonomous hypercaptant nodules develop on
a background of a multinodular goiter.
Molecular mechanisms leading to the development of autonomous thyroid
tissue have recently been elucidated in a proportion of solitary
hyperfunctioning adenomas (Ref. 4 and references therein), in autosomal
dominant toxic thyroid hyperplasia (5), and in the McCune-Albright
syndrome (6). They involve activating mutations of the TSH receptor
gene occurring at the somatic or germline level and of the
Gs
gene, respectively. The prevalence of TSH receptor
and Gs
mutations in solitary toxic adenomas is still
controversial. In our series, TSH receptor mutations account for more
than two thirds of the cases (6) (our unpublished data), whereas in
others, it is much lower (7). It is likely that differences in
methodology, recruitment, and geographical origin of patients explain
these discrepancies.
Whether the same pathophysiological mechanisms can account for the
development of tissue autonomy on a background of multinodular goiter
is presently unknown, but can easily be explored by standard molecular
genetics studies.
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Diagnostic procedures
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Genomic DNA was extracted from two nodules corresponding to the
main hot zones (one in each lobe, giving 285 and 110 µg DNA for right
and left nodules, respectively) and from nonnodular quiescent tissue
(250 µg DNA). The entire exon 10 was sequenced using five different
overlapping fragments. The two fragments that were found to harbor the
mutations identified in the present study were amplified by PCR using
the following primers: fragment A including the right nodule mutation
(position 12671630): forward primer,
5'-aatacgactcactatagggttcgttagtctgctggctc-3'(a);
reverse primer,
5'-caggaaacagctatgacccaaccatgatggcacatg-3'(b);
and fragment B including the left nodule mutation (position
15671960): forward primer,
5'-tgtaaaacgacggccagtctggtatgccatcaccttc-3'(c);
reverse primer,
5'-caggaaacagctatgacctgagaggcttgttcagaatt-3'(b).
The italicized sequences correspond to universal sequencing
primers (T7a, M13RP1b, and -21
m13c).
PCR was performed as previously described (5), and sequencing of both
strands was realized with the Thermosequenase sequencing kit (RPN 2436,
Amersham Corp., Arlington Heights, IL) according to the
manufacturers recommendations. Samples were loaded onto an Applied
Biosystems 373 Stretch Sequencing Instrument (Foster City, CA), and
mutations were identified using the Factura and Sequence Navigator
Software (Applied Biosystems).
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Diagnosis
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Direct sequencing of genomic DNA extracted from the
two hot nodules revealed the presence of a different mutation in the
heterozygous state in each of them. In the nodule from the right lobe,
a T to C transition caused substitution of threonine for methionine at
position 453 (ATG
ACG; Fig. 1b
); the nodule in the left lobe harbored
a C to T transition that substitutes isoleucine for threonine at
position 632 (ACC
ATC; Fig. 1c
). No mutation was found in the
juxtanodular tissue. In the right nodule, the picture of direct
sequencing indicated a ratio of the mutated vs. wild-type
allele less than 1 (Fig. 1b
). This suggests that the nodular tissue
dissected from the surgical specimen was heterogeneous or contaminated
by normal cells harboring the wild-type genotype (fibroblasts,
endothelial cells, blood cells, etc.).
The presence of both mutations was confirmed by restriction
endonuclease analysis; the M453T and T632I mutations created a
PmlI and a TaqI site, respectively (data not
shown).
The functional characteristics of these two mutants have been
described. Both display an increase in constitutive activity towards
the cAMP regulatory pathway when expressed transiently in COS cells (8, 9).
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Discussion
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After the demonstration that solitary toxic adenomas are mainly
caused by activating mutations in the TSH receptor and to a lesser
extent by Gs
mutations (4) (our manuscript in
preparation), the present study demonstrates that a similar pathogenic
mechanism can account for the development of multiple zones of autonomy
taking place during the evolution of a multinodular goiter.
At first, the occurrence of two independent mutagenic events activating
the cAMP regulatory cascade in the same thyroid sounds improbable.
However, since the original description of 2 gain of function mutations
in the TSH receptor gene (10), 15 different amino acids of the receptor
have been recognized as targets for activating mutations, and there is
no indication that this list is exhaustive (4) (our manuscript in
preparation). In addition, when mutated, 2 residues of the
Gs
protein are also able to cause adenylyl cyclase
activation (6). This abundance of targets makes it less unlikely to
observe independent activating mutations within the same goiter.
Incidentally, the larger number of effective targets in the TSH
receptor compared to Gs
(15:2) may explain the higher
proportion of TSH receptor over Gs
mutations observed in
solitary toxic adenomas.
An open question is whether the occurrence of TSH receptor or
Gs
mutations in the various presentations of thyroid
autonomy reflects a general increase in the frequency of mutations in
the thyroid gland. Solitary adenomas on a homogeneous thyroid
background would constitute one extreme of the spectrum where the
probability of mutation is low and a single "lucky" hit would cause
the phenotype. Multiple autonomous zones on a multinodular background
would reflect a higher propensity for mutations in the TSH receptor or
Gs
, to account for autonomy, and in other still
undefined targets, to account for tissue nodularity. The coexistence of
monoclonal and polyclonal nodules in multinodular goiters is compatible
with a scenario where clonality arises from a hyperplastic polyclonal
environment (11, 12, 13). TSH and/or other undefined mitogenic stimuli
would promote hyperplasia; in iodine-deficient areas or in the presence
of goitrogens, this effect could be amplified by a decrease in
iodine-dependent inhibition of growth (14). Only somatic mutations with
a positive effect on both growth and function (TSHr and
Gs
) would cause clonal expansion of autonomous tissue.
The high frequency of thyroid nodularity and autonomy in old age,
especially in iodine-deficient areas, suggests that a mutagenic
agent(s) may be involved in addition to a constant mitogenic stimulus.
Indeed, cell renewal in normal or even goitrous thyroid is too low to
account for a high frequency of somatic mutations. It is tempting to
follow the hypothesis that the generation of reactive oxidative
substances by the H2O2-generating system and
thyroperoxidase may contribute to an increase in mutagenesis within the
stimulated thyroid.
The case described here demonstrates that the pathogenic mechanism
responsible for solitary toxic adenoma can account for at least some of
the classical forms of Plummers disease. As iodine supplementation is
thought to act as a revelator of preexisting autonomous tissue, it will
be of interest to look for TSH receptor and Gs
mutations
in populations of severe endemic goiter areas that show a burst of
thyrotoxicosis after iodine supplementation (3).
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Footnotes
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1 This work was supported by the Belgian Program on University Poles
of Attraction initiated by the Belgian State, Prime Ministers office,
Service for Sciences, Technology, and Culture, and by grants from the
Fonds de la Recherche Scientifque Médicale, the FNRS,
Télévie, the European Union (Biomed), the Association Belge
contre le Cancer, and the Association de Recherche Biomédicale et
de Diagnostic. The scientific responsibility is assumed by the
authors. 
2 Aspirant of the Fonds National de la Recherche Scientifique. 
Received July 9, 1996.
Accepted August 30, 1996.
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