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Original Studies |
Division of Endocrinology and Department of Surgery, Mayo Clinic and Medical School, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. John C. Morris, Division of Endocrinology, Mayo Clinic and Medical School, Rochester, Minnesota 55905.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recently, it has become apparent that patients with solitarily hyperfunctioning thyroid nodules frequently harbor somatic mutations of the human TSH receptor (hTSHR) within the nodules that cause the receptor to express constitutive activity (7, 8). Found mostly within the transmembrane domain of hTSHR (although involvement of the extracellular domain is less commonly seen) (6), these mutations confer a growth and functional advantage to the cells in which they arise, leading to production of a neoplasm that is visualized histologically as a thyroid adenoma (9). The frequency with which these activating mutations are found in AFTN is variable, however, ranging from as high as 80% (10) to as low as less than 10% (11). One potential reason suggested for this apparent discrepancy is iodine exposure, in that patients from iodine-deficient areas appear to have a higher frequency of hTSHR mutations, whereas those from iodine-replete regions have fewer (11).
The role of hTSHR mutations in TMNG remains unknown. Two previous reports from a single laboratory have examined this question (12, 13). These investigators found somatic hTSHR transmembrane domain mutations in patients who demonstrated a solitary functioning nodule or adenoma in the setting of a multinodular goiter. However, most patients with TMNG express multiple hyperfunctioning thyroid nodules bilaterally, thereby demonstrating diffuse autonomy of the gland. Thus, the relevance of those previous findings to the majority of patients with TMNG is unclear.
In this study we have examined, by DNA sequence analysis, the entire transmembrane domain of hTSHR for mutations in a series of patients with TMNG. The results suggest that a polymorphism of hTSHR within the carboxyl-terminal tail is associated with the disease and may therefore play a role in its pathogenesis.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Surgical specimens and blood samples were obtained from 31
consecutive patients undergoing total or partial thyroidectomy for
toxic nodular thyroid disease. All patients demonstrated suppression of
TSH concentration, as measured by third generation assay, and elevation
of free T4 levels when measured in the clinical laboratory.
The diagnosis of AFTN (n = 7) was based upon palpation findings as
well as the presence of a solitary hyperfunctioning nodule with
suppression of surrounding thyroid tissue and the contralateral lobe
when visualized by thyroid scintillation scanning. All patients with
TMNG (n = 24) demonstrated multinodular goiters by palpation, and
thyroid scanning demonstrated bilateral, heterogeneous, and nodular
uptake of the tracer (Fig. 1
). All
patients were tested for thyroid antibodies, including anti-TSHR
antibodies. None had evidence of thyroid autoimmunity. In addition, all
surgical thyroid specimens were histologically examined for the
presence of infiltration of inflammatory cells.
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Nodular and nonnodular regions of the surgical samples were carefully dissected under visual inspection and examined separately. DNA was extracted using the FastDNA extraction kit (BIO 101, Inc., Vista, CA), checked for purity, quantified by spectrophotometry, and stored at -21 C until used. Ten milliliters of peripheral blood were also obtained, allowed to clot, and centrifuged to separate the plasma and cell pellet. Then genomic DNA was extracted as described above.
PCR
Unless otherwise noted, PCR was performed in a 100-µL reaction
volume with variable annealing temperature dependent upon the primers
used. All of the primers used in this study were synthesized by the
Mayo Molecular Biology Core Facility (Rochester, MN). The reaction
buffer contained 1 x PCR buffer [10 mmol/L Tris-HCl (pH 8.3),
500 mmol/L KCL, 1.5 mmol/L MgCl2, and 0.001% (wt/vol)
gelatin; GeneAmp, Perkin Elmer Corp., Foster City, CA],
10 IU DNA polymerase (AmpliTaq Gold, Perkin Elmer Corp.),
and 1.25 mmol/L each of deoxy (d)-ATP, dCTP, dGTP, and dTTP. To amplify
the transmembrane domain and the cytoplasmic tail of the human TSHR
(1154 bp) contained in exon 10 (14), specific primers (5'-primer TAC
CCC CAA GTC CGA TGA GT and 3'-primer GGG ATT GGA ATG CAT ATT CAA G)
were designed, and genomic DNA was used as a template (PCR-I). The
product of PCR-I was purified and used as a template to generate four
overlapping short PCR segments (PCR-II), designated S1, S2, S3, and S4.
Each of the four sets of oligonucleotide primers used to generate
PCR-II was tailed with -21M13-forward (TGT AAA ACG ACG GCC AGT) or
M13-reverse (CAG GAA ACA GCT ATG ACG) primer sequence at the 5'- and
3'-ends, respectively. PCR reactions were conducted at optimum
conditions using a GeneAmp 2400 thermal cycler and amplification
reagents (Perkin Elmer Corp.). All PCR amplicons were
analyzed by 2% agarose gel electrophoresis, demonstrated a single band
of the expected molecular size, and were purified using the QIAquick
PCR Purification Kit (QIAGEN, Chatsworth, CA). The
oligonucleotide primers used in the study are listed in Table 2.
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The PCR product of the four overlapping segments of TMD+CT-hTSHR
(designated S1, S2, S3, and S4) were sequenced in both forward and
reverse directions of the sense and antisense strands, respectively.
Direct automated fluorescent DNA sequencing was performed using a
sequencing kit with premixed -21M13-forward and M13-reverse universal
dye primers (Perkin Elmer Corp.) (15, 16, 17) and an ABI PRISM
377 DNA Sequencer (Perkin Elmer Corp.). The DNA sequencing
data were analyzed by a computer using FACTURA 1.2/6 and Sequence
Navigator 1.0.1 (PE Applied Biosystems, Foster City, CA)
for the presence of heterozygote base changes. All sequences were also
checked and verified by visual inspection. Furthermore, sequences that
showed D727E polymorphism in S4 were confirmed by DNA fingerprinting
(restriction digestion) that specifically identifies D727E heterozygote
base changes. The DNA fingerprinting method is described below, and
Fig. 2 shows representative data.
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The S4 amplicon (the PCR product of the fourth segment of the transmembrane and cytoplasmic tail of the hTSHR) was digested with NlaIII (New England Biolabs, Inc., Beverly, MA) for 1 h at 37 C. The NlaIII restriction enzyme has two restriction sites on S4 of the wild-type DNA strand. Restriction digestion of S4 of the wild-type DNA strand with the NlaIII restriction enzyme yields three DNA fragments of 252, 124, and 21 bp. A polymorphism (single base change) of codon D727E eliminates the second NlaIII restriction site on the S4 of the polymorphic DNA strand. Therefore, restriction digestion with NlaIII produces two DNA fragments of 273 and 124 bp. The digested fragments were analyzed by electrophoresis on 3-mm thick, 3.5% high resolution agarose gel (Metaphor, FMC BioProducts, Rockland, ME) run at 6.7 V/cm for 4 h.
Site-directed mutagenesis
Generation of variants of hTSHR DNA was performed by PCR-based
site-directed mutagenesis of the segment between nucleotides 1702 and
2478 of the wild-type pSVL-hTSHR construct (provided by Dr. Gilbert
Vassart, Brussels, Belgium). Using the site-directed mutagenesis
technique as described previously by other laboratories (20, 21, 22), the
mutant variant inserts were generated (PCR-III). Then, the variants of
hTSHR were reconstructed by restriction digestion of the wild-type
pSVL- hTSHR and the various inserts (products of PCR-III) with two
restriction enzymes, Bsu36 and BamHI, followed by
gel purification and ligation of inserts to pSVL-hTSHR with T4 DNA
ligase (Life Technologies, Inc., Grand Island, NY). The
two sites of ligations and the flanking regions were checked for
accuracy of sequence by sense and antisense sequencing. Later, HD5
Escherichia coli (Life Technologies, Inc.) was
transformed with the wild-type and polymorphic variant of pSVL-hTSHR
constructs and large quantities of pSVL-hTSHR DNA were generated by
standard recombinant DNA techniques. As a positive control, a
previously described mutation (A623V) of hTSHR known to induce
constitutive activity was generated by the same techniques and included
in the study (23, 24, 25, 26).
Transfection and functional assays
Transient expression of the wild-type and variant hTSHR was
accomplished using the lipofection technique in 3-cm culture dishes
with Lipofectamine (Life Technologies, Inc.) according to
the manufacturer’s recommendations. Total protein was measured by the
Bio-Rad protein assay (based on the method of Bradford; Bio-Rad Laboratories, Inc., Richmond, CA) as a control for cell numbers.
All assays were performed 48 h after transfection.
[125I]TSH binding to whole cells was performed in
sodium-free, isotonic buffer as previously described (27, 28). Binding
curves generated from the [125I]TSH binding assay were
analyzed mathematically for the total amount of receptor expressed, the
receptor affinity (Ka), and the measurement error
() using an algorithm published previously (29). Simultaneously with
the [125I]TSH binding assay, cAMP accumulation in a
separate set of wells was measured after 2 h of incubation
in buffer containing 0.5 mmol/L 3-isobutyl-1-methylxanthine and
variable concentrations of TSH (27, 30, 31). Each data point for both
the [125I]TSH binding assay and cAMP measurements were
determined in triplicate wells.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patient characteristics are summarized in Table 1
. Twenty-four
patients with TMNG and 7 patients with AFTN participated in this study.
As expected, the patients with TMNG were older as a group than the
patients with AFTN. Forty-nine patients with GD and 52 normal
individuals were also included in the study as controls for patients
with TMNG.
For bidirectional automated dye primer sequencing, the four PCR
segments of the transmembrane domain and cytoplasmic tail were
amplified with excellent fidelity using the primers listed in Table 2.
Bidirectional automated dye primer sequencing of the four PCR segments
of the transmembrane domain and cytoplasmic tail of hTSHR DNA from
nodules of seven cases of AFTN demonstrated only a single silent
polymorphism involving codon 459 in which G
A at position 1477 the
GCG was substituted with GCA, both of which
encode for alanine. Thus, no alterations that resulted in a change in
amino acid sequence were identified in this group.
However, sequencing of exon 10 of the 24 patients with TMNG identified
several sequence alterations, which are summarized in Table 3. Eight patients (33.3%;
P = 0.019 vs. normal subjects) were
heterozygous for CG transition at position 2281 within codon 727,
resulting in substitution of glutamic acid for aspartic acid (D727E)
within the carboxyl-terminal tail of the receptor (Fig. 2). Three other
alterations that resulted in amino acid substitution were also found,
although at lower frequency: A703G in 3 patients (12.5%) and I606M and
Q720E each in a single patient. One patient possessed multiple sequence
alterations, including I606M, A703G, Q720E, and D727E, whereas in all
other patients the variations were singular (Table 3). Amino acids
affected by these base changes are depicted in Fig. 3. No sequence differences were seen
using nodular or nonnodular DNA and DNA extracted from peripheral blood
from the affected patients (data not shown), suggesting that the
changes were genomic rather than somatic in nature.
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). Examination of peripheral blood
genomic DNA from the affected individuals confirmed the sequencing
results in all cases. The sequence variants were confirmed to be
genomic in nature and heterozygous in all patients. These results also
exclude the possibility of contamination of the thyroid DNA samples or
PCR artifacts as the etiology of the findings.
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). Five of
52 normal individuals were heterozygous for the D727E polymorphism, and
8 of 49 patients with GD were similarly positive (P =
0.1 vs. normal subjects). All positive cases in these series
were also sequenced, and the results confirmed the DNA fingerprinting
findings. These data indicate that the D727E polymorphism is
significantly associated with TMNG, suggesting that its presence is
important in the pathogenesis of the disease. Functional characterization of D727E
To characterize the functional activity of the variant hTSHR,
wild-type hTSHR complementary DNA from the pSVL-hTSHR construct (7) was
modified by site-directed mutagenesis. Transient transfection of the
wild-type and variant constructs into COS-7 cells was accomplished
using liposome-mediated transfection techniques. Expression of hTSHR
proteins on the cell surface was monitored by binding of
[125I]TSH, as demonstrated in Fig. 5. The binding activity of D727E was
indistinguishable from that of wild-type hTSHR, indicating that this
receptor is incorporated into the membrane and interacts with ligand in
a fashion similar to that of the native receptor (Table 4).
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(solid star). Basal cAMP levels were not different from
wild-type levels in cells transfected with either D727E. However, the
D727E variant showed an exaggerated response to TSH stimulation
compared to the wild type (Fig. 6, striped star). Although
the overall accumulation of cAMP produced by transfected cells varied
slightly (5–10%) from experiment to experiment, the relative values
between variants were consistent, and the observation was highly
reproducible, as seen in five different experiments performed on
different dates and with multiple DNA preparations.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Polymorphisms of receptors or enzymes have been implicated in variability in intracellular signal transduction, cellular metabolism, and the pathogenesis of several diseases. Examples include insulin receptor polymorphisms in type II diabetes (35), N-acetyltransferase polymorphisms (NAT1 and NAT2) in colorectal cancers (36), angiotensin I/II-converting enzyme polymorphism in renal and cardiac diseases (37, 38), and polymorphisms of the aldehyde dehydrogenase (ALDH2) allele or the TaqI A1 allele in alcoholism (39). Polymorphism of the leptin receptor gene was reported to be associated with obesity in humans (40). Polymorphisms may also influence the immune response to an immunogenic agent. An example of this is a polymorphism of the transporter associated with antigen processing (TAP2) gene that is associated with class II major histocompatibility complex and influences an antibody response to measles virus vaccine (41) or the large molecular weight proteasome and transporter associated with antigen processing polymorphism in autoimmune disease, such as rheumatoid arthritis (42). Among the extensively studied gene polymorphisms are enzymatic differences in drug metabolism and disposition (43, 44, 45, 46, 47). However, this is the first report describing the association of a germline polymorphism of hTSHR with nodular thyroid disease.
Multinodular goiter is a diffuse disease of the thyroid that begins as simple or colloid goiter early in life (3). As the thyroid continues to grow, it becomes nodular through irregular growth patterns, hemorrhage, degeneration, and deposition of fibrous material throughout the gland. In most instances, the nature of the nodules is not that of a true neoplasm or adenoma, in that they do not demonstrate full fibrous encapsulation, the histology within the nodular areas is not homogeneous and clearly distinguishable from that outside the nodules, and the perinodular tissue does not demonstrate compression from the nodule itself (48). TMNG occurs in a subset of patients affected by multinodular goiter who develop autonomous function of the gland and become thyrotoxic. The hyperthyroidism of TMNG occurs in older patients than does GD, with peak incidence occurring in the sixth and seventh decades of life, and a positive family history of multinodular goiter or TMNG is common (2), suggesting a genetic component in its pathogenesis. Given these characteristics, any genetic alteration that contributes to the pathogenesis of TMNG in the most common presentation of the disease 1) must be expressed throughout the thyroid diffusely and is therefore likely to be genomic rather than somatic, and 2) must manifest its effect on growth and thyroid function over many decades and is therefore likely to be subtle in magnitude.
During the execution of our experiments, a report of activating point mutations occurring in thyrotoxic patients with multinodular rather than uninodular goiters was published, in which the mutations found were similar or identical to those previously reported in AFTN (13). However, with one exception, the patients involved in that report demonstrated the presence of a single functioning nodule in the setting of a multinodular goiter, and the function of the remaining nodules and perinodular tissue was suppressed. The patients examined in our experiments clearly demonstrated diffuse thyroid disease and autonomy rather than a solitary functioning neoplasm; thus, the patients included in our study better represent the majority of patients affected by TMNG. This difference in patient population probably explains the discrepancy between our data and those of the previous report, in that we did not find constitutively activating mutations in any of our TMNG patients because none of them had functioning thyroid adenomata, but, instead, demonstrated diffuse multinodular disease with widespread autonomy.
Clearly, the presence of the heterozygous state for the D727E variant hTSHR is not sufficient for the development of TMNG, because approximately 10% of normal individuals possess this polymorphism. However, its presence may predispose to abnormal thyroid growth and function as part of a multi-hit process, in which other genetic or environmental factors participate in the pathogenesis. These other factors are unknown at the present time, but may involve other components of the intracellular signaling mechanism, transcription factors, elements influencing growth factor secretion or response, or environmental processes, such as iodine exposure. Much additional investigation is needed to examine these questions. Also, the frequency of the polymorphisms we encountered in euthyroid goiter and their potential role in that disorder remain unexplored.
Despite sequencing of the entire transmembrane domain encoded in exon 10 of hTSHR we failed to find mutations in any of the seven AFTN patients we examined. Although the number is low, the finding suggests that the frequency of mutation of hTSHR in our patients with AFTN is very low compared to that in the European AFTN cases studied (25, 26). However, our findings are not unlike those of Takeshita et al. (11) from a study performed in Japanese patients in which only 1 of 34 AFTN was found to possess a hTSHR mutation. Others have suggested that low iodine intake may increase the frequency of hTSHR mutations because of chronic low level stimulation to the gland, although no direct evidence currently supports this hypothesis.
In conclusion, our findings indicate that a polymorphism of the hTSHR involving the carboxyl-terminal intracellular tail (D727E) is significantly associated with TMNG (P = 0.019). Altered intracellular signaling of the variant receptor after stimulation by TSH may contribute to the pathogenesis of the disease. However, this study does not elucidate the mechanism of how the exaggerated cAMP associated with D727E polymorphism contributes to the pathogenesis of TMNG. Therefore, our hypothesis is that the D727E polymorphism induces increased localized sensitivity to TSH stimulation accompanied with enhanced growth in the nodules. Further more, as D727E is a germline polymorphism, the mechanism is likely to be mutifactorial, perhaps involving linkage disequilibrium with another gene in the region. As Bignell et al. recently described linkage between a locus on chromosome 14 in the region of the hTSHR and benign goiters (49), the D727E polymorphism may need to be accompanied by other, as yet undefined, genetic and/or environmental factors to predispose patients to the development of multinodular goiters and thyroid autonomy when expressed over decades of life.
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Acknowledgments |
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Footnotes |
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2 Recipient of NIH Research Training Fellowship Grant DK-07352.
Received January 8, 1999.
Revised May 4, 1999.
Accepted May 26, 1999.
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References |
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protein gene in 31 toxic thyroid
nodules. J Clin Endocrinol Metab. 82:3885–3891.-subunit. J Biol Chem. 268:21613–21617.-subunit peptides on
thyrotropin interaction with its receptor. Endocrinology. 123:456–462.-subunit peptides on
thyroid-stimulating immunoglobulin activity. J Clin Endocrinol
Metab. 67:707–712.This article has been cited by other articles:
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  O. J. Brand, J. C. Barrett, M. J. Simmonds, P. R. Newby, C. J. McCabe, C. K. Bruce, B. Kysela, J. D. Carr-Smith, T. Brix, P. J. Hunt, et al. Association of the thyroid stimulating hormone receptor gene (TSHR) with Graves' disease Hum. Mol. Genet., May 1, 2009; 18(9): 1704 - 1713. [Abstract] [Full Text] [PDF]
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 F Palos-Paz, O Perez-Guerra, J Cameselle-Teijeiro, C Rueda-Chimeno, F Barreiro-Morandeira, J Lado-Abeal, the Galician Group for the Study of Toxic Multinod, D Araujo Vilar, R Argueso, O Barca, et al. Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain Eur. J. Endocrinol., November 1, 2008; 159(5): 623 - 631. [Abstract] [Full Text] [PDF]
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R. P Peeters, W. M van der Deure, and T. J Visser Genetic variation in thyroid hormone pathway genes; polymorphisms in the TSH receptor and the iodothyronine deiodinases. Eur. J. Endocrinol., November 1, 2006; 155(5): 655 - 662. [Abstract] [Full Text] [PDF]
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 H. Hiratani, D. W. Bowden, S. Ikegami, S. Shirasawa, A. Shimizu, Y. Iwatani, and T. Akamizu Multiple SNPs in Intron 7 of Thyrotropin Receptor Are Associated with Graves' Disease J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2898 - 2903. [Abstract] [Full Text] [PDF]
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R. P. Peeters, H. van Toor, W. Klootwijk, Y. B. de Rijke, G. G. J. M. Kuiper, A. G. Uitterlinden, and T. J. Visser Polymorphisms in Thyroid Hormone Pathway Genes Are Associated with Plasma TSH and Iodothyronine Levels in Healthy Subjects J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2880 - 2888. [Abstract] [Full Text] [PDF]
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K. Krohn and R. Paschke Progress in Understanding the Etiology of Thyroid Autonomy J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3336 - 3345. [Full Text] [PDF]
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M. Tonacchera and A. Pinchera Thyrotropin Receptor Polymorphisms and Thyroid Diseases J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2637 - 2639. [Full Text]
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M. Tonacchera, P. Agretti, L. Chiovato, V. Rosellini, G. Ceccarini, A. Perri, P. Viacava, A. G. Naccarato, P. Miccoli, A. Pinchera, et al. Activating Thyrotropin Receptor Mutations Are Present in Nonadenomatous Hyperfunctioning Nodules of Toxic or Autonomous Multinodular Goiter J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2270 - 2274. [Abstract] [Full Text]
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