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Clonal Origin of Toxic Thyroid Nodules with Constitutively Activating Thyrotropin Receptor Mutations¹

Department of Internal Medicine III, University of Leipzig, D-04103 Leipzig, Germany

Address all correspondence and requests for reprints to: R. Paschke, M.D., Departmentof Internal Medicine III, University of Leipzig, Ph. Rosenthal Strasse 27, D-04103 Leipzig, Germany.

	Abstract

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Constitutively activating TSH receptor mutations have recently been detected in toxic nodules. In vitro studies suggest that mutated receptor signaling constitutively elevates cAMP, which causes hyperfunction and proliferation of thyrocytes. Therefore, toxic nodules with constitutively activating somatic TSH receptor mutations should result from clonal expansion of a single mutated cell. To test this hypothesis, we studied the clonal origin of 27 toxic nodules. In 13 of 27 nodules, a somatic mutation in the TSH receptor was identified. A PCR-based clonality assay that analyzes X-chromosome inactivation was used. The assay amplifies a polymorphism located in the androgen receptor gene. Of 27 toxic nodules studied, 23 (85%) were informative for the polymorphism. In the group that contains a somatic mutation in the TSH receptor, 10 of 11 cases showed nonrandom X inactivation, indicating clonal expansion. In only one toxic nodule with a TSH receptor mutation was random X inactivation detected. In the group without detectable mutations in exons 9 and 10 of the TSH receptor and exons 7–10 of the G_s protein, only 6 of 12 toxic nodules show nonrandom X-chromosome inactivation. Therefore, the majority of toxic nodules with constitutively activating TSH receptor mutations are of clonal origin. This finding supports the hypothesis that toxic nodules arise from aberrant growth of a single cell. It is widely accepted that somatic mutations might initiate monoclonal growth. The TSH receptor mutations in these toxic nodules together with G_s mutations in others are the most likely candidates for the initiation of this thyroid tumor. The clonal origin of toxic nodules in the group without detected mutations in the TSH receptor or the G_s protein suggests somatic mutations in genes that are unknown to date.

	Introduction

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IT IS A widely accepted paradigm in tumor biology that the origin of neoplasia is a single mutated somatic cell (1) whereas extrinsic growth-promoting factors cause a hyperplastic lesion. In keeping with this concept, somatic constitutively activating mutations in the TSH receptor (TSHR) and the G_s protein (gsp) have been suggested as the molecular basis of toxic thyroid nodules (TTNs) (2, 3). Thus, it is expected that TTNs with a TSHR or gsp mutation result from clonal expansion of a single cell. A TSHR or gsp mutation that causes a stimulation of the cAMP cascade would explain thyroid hyperfunction caused by a scintigraphically delineated nodule with high technetium uptake.

Determination of X-chromosome inactivation has been used to study the clonality of a group of cells in vivo and in vitro. According to the Lyon hypothesis (4), functional inactivation of one of the two X-chromosomes occurs in all female somatic cells in early embryogenesis. Functional inactivation is accomplished by DNA methylation at CpG sites. The once established pattern of X-chromosome inactivation is passed on from a given cell to its progeny. Therefore, a polyclonal tissue presents a mosaic of DNA methylation due to random inactivation of the paternal or maternal X-chromosome. In contrast, a group of cells resulting from clonal expansion of a single progenitor shows an identical pattern of X-chromosome inactivation.

To test the hypothesis that TTNs with constitutively activating TSH receptor mutations result from clonal expansion of a single mutated cell, we investigated the clonal origin of consecutive TTNs that had been screened for mutations in the TSHR gene and the gsp gene (5). As mutation events for TSHR or gsp genes were not detectable in all TTNs (for review, see Ref.5), we also intended to answer the question of whether TTNs without TSHR or gsp mutations are of polyclonal origin. A polyclonal origin would suggest a role for extrinsic factors in the etiology of hyperfunctioning thyroid nodules.

To date, data concerning the clonality of thyroid lesions are only available for a very heterogeneous entity of thyroid nodules, mostly defined by histopathological, rather than functional or molecular, criteria (6, 7, 8). In these nodules both monoclonal and polyclonal origins have been demonstrated.

	Materials and Methods

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Thyroid tissue samples

Specimen of 27 macroscopically visible thyroid toxic nodules from uninodular goiters and surrounding normal tissue of female patients were obtained at surgery. All patients were hyperthyroid. Part of the surrounding tissue or the area of increased technetium uptake was prepared for histological analysis. The remaining tissue was frozen in liquid nitrogen and stored at -80 C. This study was approved by the local ethics committee. Informed consent was obtained from all patients before surgery.

X-chromosome inactivation

To determine random vs. nonrandom X-chromosome inactivation, methods have been established that detect X inactivation of maternal or paternal chromosomes.

In our study we use a PCR approach described by Allen et al. (9). Amplification of a variable number of tandem repeats in the first exon of the human androgen receptor (HUMARA) (10) is studied after digestion with the methylation-sensitive HpaII restriction enzyme. Undigested DNA heterozygous for the variable number of tandem repeats results in two distinct PCR fragments corresponding to maternal or paternal alleles. In the case of nonrandom X-chromosome inactivation, amplification of one allele is diminished because maternal or paternal alleles are methylated to a different degree. This difference is attributed to a clonal origin of the respective tissue.

Frozen thyroid tissue was mechanically disrupted, and DNA extraction was carried out using the QIAamp tissue kit (Qiagen, Chatsworth, CA), according to the manufacturer’s guidelines. One hundred nanograms of DNA from nodular and surrounding tissue were digested at 37 C overnight with 8 U of the restriction enzyme HpaII in the digestion buffer supplied with the enzyme (MBI Fermentas, Vilnius, Lithuania). In parallel, an aliquot of 100 ng DNA was incubated in digestion buffer without enzyme. The reactions were terminated, and the enzyme was heat inactivated at 95 C for 10 min. Two microliters of HpaII-digested or undigested DNA were used in a 20-µL PCR reaction to amplify 230 bp of the HUMARA (10). The PCR reaction was carried out with the PrimeZyme PCR kit (Biometra) and the following primers at a concentration of 200 nmol/L: forward primer, 5'-CTC TAC GAT GGG CTT GGG GAG AAC-3' (11); and reverse primer, 5'-TCC AGA ATC TGT TCC AGA GCG TGC-3' (9).

For detection of the resulting PCR products we used incorporation of radiolabeled [-³²P]deoxy-ATP (>3000 Ci/mmol; ICN, Amersham , Arlington Height, IL). during PCR (cases 5–62) or a fluorescence-labeled forward primer 6-carboxy-fluorescein labeled; PE Applied Biosystems, Foster City, CA) according to the method of Delabesse et al. (12) (for cases 67–70 and a random selection of cases 5–62). Radiolabeled PCR fragments were separated on 6% polyacrylamide-8 mol/L urea denaturing gels. Gels were dried and exposed to x-ray film (X-Omat AR, Eastman Kodak, Rochester, NY) or to a PhosphorImager screen. Signal intensities were analyzed using the program ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Fluorescence-labeled PCR products were examined on a ABI 310 Genetic Analyzer (PE Applied Biosystems). The GeneScan software supplied with the system allows automatic quantification of fluorescence-labeled PCR products.

Quantification of X-chromosome inactivation

After quantification of radio- or fluorescence-labeled PCR products, clonality was evaluated as described by Delabesse et al. (12). Briefly, signal intensities on PhosphorImager screens or the calculated peak area for both alleles were quantified. The ratio of the numbers for the two alleles was calculated, and the ratio for the undigested DNA was divided by the ratio of the HpaII-digested DNA. If necessary, the resulting number was inverted to give a value greater than 1. The resulting index gave a value close to 1 when the sample was polyclonal. Values below 2 were interpreted as polyclonal, and numbers above 2 as monoclonal.

	Results

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X-Chromosome inactivation

X-Chromosome inactivation was studied in 27 consecutive TTNs and surrounding tissue from female patients. In Fig. 1, autoradiographs of four representative cases are shown. Twenty-three (85%) cases were heterozygous for the (CAG)_n-polymorphism in exon 1 of the HUMARA (for example, see Fig. 1, cases 8, 59, and 62). Four cases were not informative for this gene locus (Fig. 1, case 7). Figure 2 presents two informative cases with an identified mutation in the TSHR gene. Results are summarized in Table 1. In the group of TTNs that contain a somatic mutation in the TSH receptor, 10 of 11 cases showed nonrandom X-chromosome inactivation characterized by a loss of amplification for 1 allele after PCR from HpaII-digested DNA (Fig. 1, cases 8 and 62; Fig. 2, cases 12 and 70). In only 1 toxic nodule with a TSH receptor mutation was random X inactivation detected (Fig. 1, case 59). In the group of TTNs without detectable mutations in exons 9 and 10 of the TSH receptor and exons 7–10 of the G_s protein, only 6 of 12 toxic nodules show nonrandom X-chromosome inactivation. In toxic nodules with a somatic TSH receptor mutation, nonrandom inactivation was also found in the surrounding tissue (Fig. 2, case 12).

View larger version (44K): [in this window] [in a new window]   Figure 1. Autoradiographs of 32P-labeled PCR products from the HUMARA gene locus as described in Materials and Methods. Amplified DNA from TTNs or surrounding tissue (ST) of four cases with (+) or without (-) HpaII digest is shown. Lanes 1–4, Case 59; lanes 6–9, case 62; lanes 11–14, case 7; lanes 16–19, case 8; lanes 5, 10, 15, and 20, DNA size standards 220 and 269 bp. Loss of PCR amplification for one allele after HpaII digest in the TTNs of case 8 and 62 suggests clonal expansion. For case 59, no loss of PCR amplification suggests polyclonal origin. Case 7 is homozygous for the polymorphism and, therefore, noninformative.

View larger version (48K): [in this window] [in a new window]   Figure 2. Separation of fluorescence-labeled PCR products from the HUMARA gene locus, as described in Material and Methods. Amplified DNA from TTNs or surrounding tissue of two cases with (+HpaII) or without (-HpaII) HpaII digest is shown. Upper four panels, Case 12; lower four panels, case 70. PCR products for the two alleles are indicated. Loss of PCR amplification for one allele in the TTNs of cases 12 and 70 as well as the surrounding tissue of case 12 suggests clonal expansion.

View larger version (13K): [in this window] [in a new window]   Figure 3. Clonality index for all thyroid tissue samples studied. ST, Surrounding tissue; TSHR, TSH receptor. The vertical line represents the cut-off to decide clonal vs. polyclonal origin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In general, the clonal origin of the majority (70%) of TTNs was apparent in our study. Moreover, hot nodules with a somatic mutation in the TSH receptor are predominantly monoclonal. These findings support the hypothesis that most toxic nodules arise from aberrant growth of a single cell. It is widely accepted that somatic mutations might initiate monoclonal growth. The TSH receptor mutations in these toxic nodules together with G_s mutations in others are the most likely candidates for the initiation of this thyroid tumor. In only one toxic nodule with a TSH receptor mutation was random X inactivation detected. A comparison of two independent methods for the study of X-chromosome inactivation reported conflicting results in up to 10% of all cases evaluated (9). Thus, the one polyclonal toxic nodule could merely be a technical or a methodological problem. Alternatively, either a minor clonal expansion of a cell containing a TSHR mutation in an otherwise polyclonal TTN or a TTN with two independent mutations could also be postulated (see discussion below).

In the group without detectable mutations in exons 9 and 10 of the TSH receptor and exons 7–10 of the G_s protein, the clonal origin of TTNs is evident in half of all cases. This suggests somatic mutations in genes that are unknown to date.

Ideally, amplification of DNA from monoclonal tissue after HpaII digest should result in one fragment from either the maternal or paternal allele. However, a TTN usually contains cells of polyclonal origin, such as fibroblasts and peripheral blood cells, that contribute to the DNA extracted from hyperfunctioning thyroid nodules. These cells form a polyclonal background, which sometimes makes a clear decision between mono- and polyclonal patterns difficult. We, therefore, quantified amplification of the two alleles and calculated an index to decide random vs. nonrandom inactivation. In line with Delabesse et al. (12), we interpret an index below 2 as polyclonal origin, but because of the polyclonal background we decided to regard an index above 2 instead of 10 as monoclonal origin, which is in contrast to the report by Delabesse et al. (12), who studied clonality of purified hematopoietic stem cells sorted by fluorescence activation without polyclonal background. This was confirmed by the average index obtained for thyroid tissue from 18 Graves’ disease patients (see Results). In an additional dilution experiment, an index above 2 has been calculated for a sample containing more than 60% polyclonal tissue.

Also in the surrounding tissue of two toxic nodules with a somatic TSH receptor mutation, nonrandom inactivation was also found. Although this finding might be the result of the methodological problem discussed above, the nonrandom X inactivation could alternatively indicate true monoclonality for two reasons. First, small areas of autonomous tissue are often detectable in thyroid tissue surrounding TTNs (13). If areas such as these are contained in the sample studied, nonrandom X inactivation is a possible outcome. The second explanation for the monoclonality of the surrounding tissue is more hypothetical because it involves two independent somatic mutations: a primary mutation that is not restricted to the TTN, and a secondary mutation (the TSHR mutation) that is restricted to the TTN. This hypothesis would imply that the first somatic mutation is not sufficient to cause the TTN.

In areas with iodine deficiency, there is a 3-fold higher incidence for thyroid autonomy compared to iodine-sufficient areas (14, 15). Iodine deficiency leads to impaired thyroid hormone synthesis. In this context, a constitutive activation of the cAMP cascade (i.e. TSHR or gsp mutation) could lead to functional compensation. Along this line, a first response to iodine deficiency might be the production of growth factors and, hence, hyperplasia, which could propagate the aberrant growth of a single cell containing a somatic mutation (16), thus explaining the increased prevalence of thyroid autonomy in areas with iodine deficiency. In addition, the model could explain the occurrence of latent autonomy and iodine-induced hyperthyroidism in euthyroid goiters. Functional compensation through constitutively activating TSHR or gsp mutations is likely to reduce production of growth factors, which in turn could reduce exogenous propagation. Recent support for this hypothesis comes from two mathematical models by Tomlinson et al. (17) and Tomlinson and Bodmer (18) showing that an advantageous mutation is more important for tumorigenesis than changes in mutation rate and that clonal expansion of an advantageous mutation reaches an equilibrium rather than remaining at an exponential growth rate.

	Footnotes

 
¹ This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG/Pa423/3–1) and IZKF Leipzig, BMB+F, Interdisciplinary center for clinical research at the University of Leipzig 9504, (01KS 9504, project B5-W).

Received July 2, 1997.

Revised September 9, 1997.

Accepted September 24, 1997.

	References

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