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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0812
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 12 4766-4770
Copyright © 2007 by The Endocrine Society

Lack of Mutations in the Thyroid Hormone Receptor (TR) {alpha} and β Genes but Frequent Hypermethylation of the TRβ Gene in Differentiated Thyroid Tumors

Biju Joseph1, Meiju Ji1, Dingxie Liu, Peng Hou and Mingzhao Xing

Division of Endocrinology and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

Address all correspondence and requests for reprints to: Michael Mingzhao Xing, M.D., Ph.D., Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 813 Hunterian Street, Baltimore, Maryland 21287. E-mail: mxing1{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: It remains inconclusive whether mutations in thyroid hormone receptor (TR) genes naturally occur in thyroid cancer and whether these genes could be suppressors of this cancer.

Objectives: Our objectives were to examine further mutations of TR{alpha} and TRβ genes in thyroid cancer and also to examine their methylation as an epigenetic silencing mechanism in thyroid cancer.

Experimental Design: Instead of using a cDNA sequencing approach used in previous studies, we used genomic DNA to sequence directly the coding regions of the TR{alpha} and TRβ genes to search mutations in various differentiated thyroid tumors and used methylation-specific PCR to analyze promoter methylation of these genes. Allelic zygosity status at TRβ was also analyzed.

Results: We found no TR{alpha} gene mutation in 17 papillary thyroid cancers (PTCs) and 11 follicular thyroid cancers (FTCs), and no TRβ gene mutation in 16 PTCs and 12 FTCs. We also found no methylation of the TR{alpha} gene in 33 PTCs, 31 FTCs, 20 follicular thyroid adenomas (FTAs), and 10 thyroid tumor cell lines. In contrast, we found hypermethylation of the TRβ gene in 10 of 29 (34%) PTCs, 22 of 27 (81%) FTCs, five of 20 (25%) follicular thyroid adenomas, and three of 10 (30%) thyroid tumor cell lines, with the highest prevalence in FTC. We additionally examined loss of heterozygosity at TRβ and found it in three of nine (33%) PTCs and three of nine (33%) FTCs.

Conclusions: Mutation is not common in TR genes, whereas hypermethylation of the TRβ gene as an alternative gene silencing mechanism is highly prevalent in thyroid cancer, particularly FTC, consistent with a possible tumor suppressor role of this gene for FTC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR EPITHELIAL cell-derived thyroid cancer is the most common endocrine malignancy, and can be classified into differentiated thyroid cancer and undifferentiated thyroid cancer. The former consists of papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC), which collectively account for the vast majority of thyroid cancers (1). Follicular thyroid adenoma (FTA) is a benign tumor, which may be a precursor of FTC. Genetic abnormalities are the basis for the development of thyroid cancer and have been extensively explored in the last one to two decades. Several major oncogenic genetic alterations have been characterized in thyroid cancer, including, e.g. RET/PTC rearrangements, Ras mutations, and BRAF mutations in the MAPK signaling pathway (2, 3, 4), PIK3CA mutations and amplifications and phosphatase and tensin homolog mutations in the phosphatidylinositol 3-kinase/Akt signaling pathway, and PAX8/peroxisome proliferator-activated receptor {gamma} rearrangements (5, 6, 7, 8). Epigenetic alteration, such as aberrant methylation of tumor suppressor genes, is another widely described molecular mechanism involved in thyroid tumorigenesis (9).

A particularly interesting new area in thyroid cancer genetics that has drawn attention in recent years is thyroid hormone receptor (TR) genes, including the TR{alpha} and TRβ genes, as potential tumor suppressor genes (10, 11). TRs are nuclear transcription factors that mediate the biological activities of thyroid hormones through binding T3 and DNA with their corresponding binding domains. TR{alpha} and TRβ are derived from two separate genes, which, through alternative splicing, yield several isoforms, including {alpha}-1, {alpha}-2, β-1, β-2, and β-3. TR{alpha} and TRβ genes consist of 10 exons, and exons 3–10 are the main coding regions (12, 13). Mutation-mediated inactivation of the TRβ gene causes the thyroid hormone resistance syndrome in humans through a dominant-negative inhibitory mechanism (14). The TR mutations are generally found in the domains of the two genes (exons 7–9 for TR{alpha} and exons 8–10 for TRβ) that are responsible for thyroid hormone binding. Mutations, deletions, and decreased expression of TR genes were found in various human cancers, raising the possibility that TR genes may be tumor suppressors (10, 11).

In a knock-in transgenic mouse model, creation of a homozygous germline mutation in the hormone-binding domain of the TRβ gene that impaired thyroid hormone binding resulted in the development of aggressive FTC, suggesting that mutations of this gene could also play a role in human thyroid tumorigenesis (15). This notion was supported by a Polish study published in this journal that reported a high prevalence of both TR{alpha} and TRβ gene mutations in PTC (16). However, these TR gene mutations were not found in a similar Japanese study published in this journal (17), raising uncertainty on the frequency of naturally occurring mutations of TR genes and on the relevance of these mutations as a mechanism in human thyroid tumorigenesis.

The present study was undertaken to investigate further TR gene mutations in differentiated thyroid cancers. We also investigated aberrant methylation and loss of heterozygosity (LOH) of the TRβ gene that could function as an alternative mechanism in disrupting the function of the TR gene in thyroid cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor samples and DNA isolation

The study was conducted with institutional review board approval, and, where required, appropriate patient consent was obtained. Tumor samples were originally obtained and processed, and DNA was isolated as described previously (7, 18). Thyroid tumor cell lines were obtained and cultured as described previously (18). Both fresh and paraffin-embedded PTC, FTC, and FTA samples were used. All the FTC tumors used were the classical type. For DNA isolation, fresh tumor tissues or cell pellets of various thyroid cell lines were directly subjected to protein digestion with 1% sodium dodecyl sulfate and 0.5 mg/ml proteinase K at 48 C for 48 h. For paraffin-embedded samples, an 8-h treatment at room temperature with xylene was pursued first to remove paraffin. Several mid-interval aliquots of concentrated sodium dodecyl sulfate-proteinase K were added to facilitate the digestion. DNA was subsequently isolated by standard phenol-chloroform extraction and ethanol precipitation, and resuspended in Tris-EDTA buffer.

Mutational analysis of TR{alpha} and TRβ genes

Mutation analysis of the coding regions (exons 3–10) of TR{alpha} and TRβ genes was performed by direct genomic DNA sequencing on DNA samples isolated from fresh thyroid tumor tissues. The PCR primer sequences for genomic DNA amplification for the TR{alpha} gene were specifically designed in the present study (Table 1Go), and those for the TRβ gene were from a previous study (19). PCR amplification for TR{alpha} and TRβ genes was performed in 20 µl of a reaction mixture containing 2 mM MgCl2, 10 mM 2-mercaptoethanol, 16.6 mM ammonium sulfate, 67 mM Tris (pH 8.8), 200 µM each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 200 nM forward and reverse primers, 0.5 U Taq DNA polymerase (Denville Scientific Inc., Metuchen, NJ), and about 60 ng genomic DNA as the template. To enhance the specificity, step-down PCR was performed as follows: after a 5-min denaturing at 95 C, the PCR was run with each temperature for 45 sec at five touch-down steps, for two cycles each. The details are shown in Table 2Go.


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  		TABLE 1. PCR amplification primers for sequencing of the TR{alpha} gene and MSP primers for TR{alpha} and TRβ genes

 

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  		TABLE 2. PCR amplification conditions for TR genes

 
The integrity of PCR products was confirmed by electrophoresis on a 1.5% agarose gel. The PCR products were then subjected to direct sequencing PCR with Big Dye terminator V 3.0 cycle sequencing reagents (Applied Biosystems, Foster City, CA) with the following cycles: 95 C for 30 sec for one cycle; and 95 C for 15 sec, 50 C for 15 sec, and 60 C for 4 min for 25 cycles. DNA sequencing was obtained and analyzed on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) at the Johns Hopkins Synthesis & Sequencing Facility (Baltimore, MD).

Methylation analysis of TR{alpha} and TRβ genes

For gene methylation analysis, genomic DNA from either fresh or paraffin-embedded tissues was treated with bisulfite as described previously (18). Briefly, about 2 µg genomic DNA in 20 µl water containing 5 µg salmon DNA was denatured with 0.3 M NaOH at 50 C for 20 min, followed by addition of 500 µl freshly prepared sodium bisulfite-hydroquinone mixture, containing 2.5 M and 0.125 M, respectively, for 3 h at 70 C. The bisulfite-modified DNA was purified using a Promega Wizard DNA Clean-Up system (Promega Corp., Madison, WI) and dissolved in 20 µl water. Methylation-specific PCR (MSP) for TR{alpha} and TRβ methylation was performed on bisulfite-treated DNA in 20 µl reaction mixture containing about 50 ng bisulfite-treated DNA, 16.6 mM ammonium sulfate, 67 mM Tris (pH 8.8), 2 mM MgCl2, 10 mM 2-mercaptoethanol, 200 µM each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 200 nM forward and reverse primers, and 0.5 U Taq DNA polymerase (Denville Scientific Inc.). MSP primer sequences are shown in Table 1Go. The PCR cycling conditions used are presented in Table 2Go. The MSP products were resolved with 2% agarose gel electrophoresis and visualized with ethidium bromide staining.

LOH analysis of the TRβ gene

Thyroid tumors and matched normal tissues from the opposite lobe of the thyroid gland or peripheral white blood cells were used for LOH analysis for the TRβ gene. The polymorphic marker chosen for this LOH analysis was in locus TRβ in map position 3p24. The amplifying primers were 5'-FAM-GATCACAAGGATGCT AGAGT-3' (forward) and 5'-TCAAAGGAGTCAGGCTGTAG-3' (reverse), as described previously (20), and the resulting amplimer corresponded to a region in intron 4 of the TRβ gene. The forward primer was labeled with florescent dye FAM (ResGen; Invitrogen, Frederick, MD). Twenty nanograms of genomic DNA were amplified using these primers in 10 µl reaction mixture containing 16.6 mM ammonium sulfate, 67 mM Tris (pH 8.8), 10 mM 2-mercaptoethanol, 400 nM of each primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 0.25 U Taq DNA polymerase. The template DNA was denatured initially at 95 C for 5 min. A touch-down protocol as shown in Table 2Go was used, with annealing temperature starting at 66 C, down to 64 C, 62 C, 60 C, 58 C, and 56 C, and with denaturation at 95 C and extension at 72 C for 40 sec. Then 35 cycles of denaturation at 95 C for 40 sec, annealing at 55 C for 40 sec, and extension at 72 C for 40 sec were done. A final extension at 72 C for 10 min was used as the last step. The amplified products were genotyped by capillary electrophoresis on ABI 3700 semi automated DNA sequencer (Applied Biosystems) at the Genetic Resources Core Facility Fragment Analysis Facility of the Johns Hopkins School of Medicine (Baltimore, MD) and analyzed using GeneMapper v3.7 (Applied Biosystems). The alleles of paired tumor and normal tissue DNA were compared with assessed LOH at the TRβ locus. Samples were considered informative for analysis when two distinct alleles were detected in the matched normal tissue. Samples were excluded if one allele (homozygous) or other noninformative allelic patterns were shown in the normal tissues. Allelic loss was estimated by LOH index calculated using the formula (T2/T1)/(N2 /N1), where T2 and T1 represent the peaks of smaller and larger alleles from the tumor sample, and N2 and N1 are the peaks of smaller and larger alleles from the matched normal tissue. LOH was defined as the LOH index less than 0.6 or more than 1.67 (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of mutations in TR{alpha} and TRβ genes in thyroid cancers

Genomic DNA samples isolated from fresh thyroid cancer tissues were used for mutational analysis in TR{alpha} and TRβ genes. The coding and adjacent intronic regions were successfully sequenced for the TR{alpha} gene in 17 PTCs and 11 FTCs, and for the TRβ gene in 16 PTCs and 12 FTCs. No mutation was found in either of the two genes in any of these tumor samples (Table 3Go). Several additional cases, whose coding regions for the two TR genes were partially successfully sequenced, also did not show any mutation. Therefore, mutations in the TR{alpha} and TRβ genes are not common events in differentiated thyroid cancers.


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  		TABLE 3. Mutation and methylation status of TR{alpha} and TRβ genes in thyroid tumors [positive/total (%)]

 
Frequent hypermethylation of the TRβ gene in thyroid cancers

With no mutations in TR genes found that could disrupt the function of these genes, we next pursued epigenetic analysis on these genes. We specifically examined the status of methylation of these genes because this epigenetic alteration is a common gene-silencing event in thyroid cancers (9). No promoter methylation in the TR{alpha} gene was identified in any of the 33 PTC, 31 FTC, 20 FTA, and 10 thyroid tumor cell lines (Table 3Go), although in vitro methylated DNA as a positive control did show methylation on MSP analysis under these conditions (Fig. 1AGo). In contrast, we found promoter hypermethylation of the TRβ gene (Fig. 1BGo) in 10 of 29 (34%) PTCs, 22 of 27 (81%) FTCs, five of 20 (25%) FTAs, and three of 10 (30%) thyroid tumor cell lines (Table 3Go), with the highest prevalence found in FTC. No TRβ gene methylation was seen in the two cases of undifferentiated thyroid cancer examined. We observed both unmethylated and methylated forms in the analysis of some samples (Fig. 1BGo). This might be due to the presence of nonhomogeneous cell composition of tumor tissues with respect to gene methylation status. For example, the tumor tissues might be contaminated with nontumor tissues that carried wild-type gene alleles. Alternatively, a subpopulation of tumor cells might have methylation on neither allele of a gene or only on one allele (a heterozygous epigenetic alteration), whereas another subpopulation of tumor cells might have methylation on both alleles of the gene (a homozygous genetic alteration).


Figure 1
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  		FIG. 1. Representative presentation of methylation analysis of TR{alpha} (A) and TRβ (B) genes by MSP. Details are as described in the Materials and Methods. Arrows indicate the specific unmethylation (U) or methylation (M) bands. P, Positive control (in vitro methylated DNA).

 
LOH at TRβ in differentiated thyroid cancers

Given the frequent methylation of the TRβ gene suggesting that this gene may be a tumor suppressor, we next analyzed LOH at this gene, which is a common genetic event with tumor suppressor genes. Among the samples analyzed that resulted in informative data, we found LOH at the locus of TRβ in three of nine (33%) PTCs and three of nine (33%) FTCs. Figure 2Go shows positive and negative representative samples.


Figure 2
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  		FIG. 2. LOH analysis of the TRβ gene. A, A representative case of microsatellite amplified PCR products from a FTC tumor using the TRB marker showing retention of allelic heterozygosity (LOH index = 1.18). B, A positive case of LOH in a FTC tumor with partial allelic loss (LOH index = 1.809). C, A positive case of LOH in a PTC tumor with complete allelic loss. Arrows indicate the alleles that are matched in the normal (top) and tumor (bottom) tissues.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutations in TR genes, particularly the TRβ gene, are commonly found in the thyroid hormone resistance syndrome (14). These mutations occur mostly in the thyroid hormone binding sites of the TRs, resulting in the loss of the ability of TR to bind thyroid hormone. These TR gene mutations are also found in several human cancers, such as liver, breast, and kidney, suggesting that these mutations may play a role in the tumorigenesis of these cancers (10, 11). However, results on TR gene mutations in human thyroid cancer are conflicting. A Polish study by Puzianowska-Kuznicka et al. (16) was first to investigate TR gene mutations in thyroid cancer. Using the cDNA sequencing approach, the authors examined 16 PTC samples and found a frequent occurrence of TR gene mutations in PTC, with a prevalence of 94% for the TRβ gene and 64% for the TR{alpha} gene. A subsequent Japanese study by Takano et al. (17) that used a similar cDNA sequencing approach to analyze the TRβ gene, also on 16 PTC samples, did not find any mutation. Several factors were thought to potentially explain this discrepancy, including different specificities of the Taq DNA polymerase and PCR primers used, and different ethnic backgrounds of the patients in the two studies (17). Although the cDNA sequencing approach in searching gene mutations is less tedious than direct genomic DNA sequencing of the entire coding regions of a gene, the former is associated with a higher risk of generating artifacts from RT, particularly when low-quality Taq DNA polymerase is used. Therefore, it has remained inconclusive whether TR gene mutations naturally exist in thyroid cancer. Takano et al. (17) suggested that this controversy could be resolved by directly examining mutations in the genome, ideally in a different population, such as one from the United States. This prompted us to conduct the present study by performing direct genomic DNA sequencing of the coding regions of the TR genes from a cohort of thyroid tumor patients in the United States. We did not find any mutation in the TR{alpha} gene in PTC. We also found no mutation in the TRβ gene in PTC, consistent with the results of Takano et al. (17). We additionally analyzed a series of FTC and similarly did not find mutations in the two TR genes. Although the number of tumors analyzed in our study was comparable to that in the two previous studies (16, 17), it was relatively small. Therefore, the present study could have missed some rare mutations in these genes in thyroid cancers. Nevertheless, our results from direct genomic DNA sequencing provide convincing evidence that TR gene mutations are at least uncommon, if not absent, in differentiated thyroid cancers. In the course of this investigation, a study by Rocha et al. (22) was published recently on partial genomic sequencing of the coding regions of the TRβ gene in thyroid cancer, also showing no mutations in this gene, consistent with our findings.

We for the first time demonstrated hypermethylation of TRβ gene in thyroid cancers. This finding is consistent with a tumor suppressor role of this gene, particularly in FTC, which harbored this epigenetic alteration with the highest prevalence. Interestingly, the TRβ methylation occurred also in some cases of FTA, suggesting an early role of this epigenetic event in thyroid tumorigenesis, which is consistent with a model that this epigenetic alteration may be involved in the transformation of FTA to FTC. The lack of TR{alpha} gene mutation and methylation suggests that this gene is unlikely to be a major tumor suppressor gene in thyroid tissues. The prevalence of LOH at TRβ in FTC in the present study is somewhat lower than that from a previous study using the same LOH marker in FTC (20). However, the "FTC" group in this study consisted mostly of oxyphilic Hürthle cell thyroid cancer, which is a genetically different thyroid tumor than classical FTC used in the present study. The relatively small number of cases investigated in both studies could also contribute to the varying rates of LOH found in the two studies. Another previous study by Rodrigues-Serpa et al. (23) reported a high prevalence of LOH in FTC, but not LOH in PTC. This study used two markers, D3S1263 (3p25-p24.2) and D3S1286 (3p ter-p24.2), for LOH in the 3p24 region in PTC, which flanked a large region and, therefore, would detect only large deletions. In our study we used an intragenic marker within the TRβ gene for LOH analysis, which lies between the two marker regions (3p24) in the Rodrigues-Serpa et al. (23) study. To be precise, it was in intron 4 of the TRβ gene and may, therefore, more reliably reflect the integrity status of this gene. This could account for the difference in the rates of LOH found in the current study and that of Rodrigues-Serpa et al. (23).

Expression of the TRβ gene, but not the TR{alpha} gene, was previously decreased in thyroid cancers in comparison with normal thyroid tissues (24, 25). This result is consistent with the differential methylation patterns of the TR{alpha} and TRβ genes found in the present study. These and our current data are consistent with the notion that the TRβ gene is a tumor suppressor in thyroid tissue, whose inactivation through aberrant methylation may be a relevant event in the tumorigenesis of differentiated thyroid cancer, particularly FTC. This notion is consistent with the results of transgenic mouse studies in which introduced homozygous inactivating TRβ mutations caused FTC (15), although natural mutations of the TRβ gene do not occur commonly in thyroid cancer.


   Acknowledgments
 
We thank those authors in Refs. 7 and 18 who originally contributed tumor tissues and cell lines for those previous studies, which were partially included in the present study.


   Footnotes
 
This work was supported by a Thyroid, Head and Neck Cancer Foundation thyroid research grant and an American Cancer Society Research Scholar Grant RSG-05-199-01-CCE (to M.X.).

Disclosure Statement: The authors have no interest conflict to disclose.

First Published Online October 2, 2007

1 B.J. and M.J. contributed equally. Back

Abbreviations: FTA, Follicular thyroid adenoma; FTC, follicular thyroid cancer; LOH, loss of heterozygosity; MSP, methylation-specific PCR; PTC, papillary thyroid cancer; TR, thyroid hormone receptor.

Received April 11, 2007.

Accepted September 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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