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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2640-2643
Copyright © 2000 by The Endocrine Society

Special Articles

Lack of Association of Nonautoimmune Hyperfunctioning Thyroid Disorders and a Germline Polymorphism of Codon 727 of the Human Thyrotropin Receptor in a European Caucasian Population1

T. Mühlberg, K. Herrmann, W. Joba, M. Kirchberger, H.-J. Heberling and A. E. Heufelder

Division of Endocrinology and Metabolism, Department of Internal Medicine, Philipps University (W.J., A.E.H.), D-35033 Marburg; and Städtisches Klinikum Leipzig-West (T.M., K.H., M.K., H.-J.H.), D-04177 Leipzig, Germany

Address all correspondence and requests for reprints to: Armin E. Heufelder, M.D., Division of Endocrinology and Metabolism, Zentrum für Innere Medizin, Philipps University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: heufeld{at}mailer.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Constitutively activating mutations of the human TSH receptor (hTSHR) gene have been implicated as a major cause of hyperfunctioning nonautoimmune thyroid disease. However, significant geographic differences in the prevalence of these mutations have been observed. Recently, a high frequency of a germline polymorphism at codon 727 of the cytoplasmic tail of the hTSHR has been demonstrated in patients with toxic multinodular goiter. In the present study we assessed whether the codon 727 polymorphism is associated with hyperfunctioning thyroid adenomas. PCR followed by restriction enzyme digestion were used to genotype a total of 128 European Caucasian patients with toxic nonautoimmune thyroid disease (83 with toxic adenoma, 31 with toxic multinodular goiter, and 14 with disseminated autonomy) and to compare their codon 727 polymorphism frequencies with those of 99 healthy controls and 108 patients with Graves’ disease. All individuals were drawn from an identical ethnic background. Sequencing of PCR products was used to confirm the mutation analysis. We found no significant differences in codon 727 polymorphism frequencies between patients with autonomously functioning thyroid disorders (13.3%) and the healthy control group (16.2%; P = 0.57). Moreover, the subtypes of toxic nonautoimmune thyroid disease (toxic adenoma, 13.2%; multinodular goiter, 9.6%; disseminated autonomy, 21.4%) were not related to significant differences in codon 727 polymorphism frequencies compared with the healthy control group (P = 0.67, P = 0.40, and P = 0.70, respectively). Additionally, there were no significant differences between patients with Graves’ disease (21.3%) and healthy controls (P = 0.38). In conclusion, our data do not support an association between the codon 727 polymorphism of the hTSHR and toxic thyroid adenomas or toxic multinodular goiter in our study population. Thus, the codon 727 polymorphism of the hTSHR does not appear to be involved in the evolution of autoimmune or nonautoimmune hyperthyroidism in the European Caucasian population.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THYROID HYPERFUNCTIONING adenomas represent benign tumors of the thyroid gland characterized by TSH-independent autonomous growth, hypersecretion of thyroid hormones, and TSH suppression. These adenomas constitute a frequent cause of clinical hyperthyroidism. In endemic goiter areas, autonomously functioning thyroid disorders occur as solitary toxic nodule, toxic multinodular goiter, or disseminated autonomy. The pathogenesis of these benign thyroid abnormalities is still unknown. Constitutively activating mutations of the human TSH receptor (hTSHR) gene have been implicated as major causes in thyroid hyperfunctioning adenomas (1, 2). TSH-dependent signaling is primarily transduced via the TSHR-adenylate cyclase-cAMP pathway. Cytoplasmic concentrations of cAMP act to control the expression of thyroid-specific genes, the level of functional activity of the gland, and growth (3). Recent studies have suggested that numerous mutations in the hTSHR gene are capable of inducing constitutive, TSH-independent activation of the cAMP regulatory cascade (4). The majority of these mutations have been mapped to exon 10 of the TSHR gene. A majority of these mutations have been located in the third cytoplasmic loop and the sixth transmembrane segment of the hTSHR (1, 2, 3, 4). Recently, Morris et al. reported a remarkably high frequency (33.3%) of a germline polymorphism in the cytoplasmic tail of the hTSHR in patients with toxic multinodular goiter (5). This polymorphism results in an amino acid substitution of aspartate to glutamate at codon 727. Functional analysis of the codon 727 mutation revealed a significantly greater cAMP response of the polymorphic hTSHR to TSH stimulation compared to that of wild-type receptor, whereas basal cAMP production rates were similar (5). Intriguingly, significant geographic differences in the prevalence of hTSHR mutations have been observed, suggesting that additional mechanisms may contribute to the pathogenesis of thyroid hyperfunctioning adenomas (6). To examine further the role of the codon 727 polymorphism of the hTSHR in the evolution of hyperfunctioning thyroid lesions, we assessed the frequency of this polymorphism in a large sample of European patients with nonautoimmune hyperthyroidism compared to that in patients with Graves’ disease (GD) and healthy controls.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study participants

Investigations adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from the participants after the nature and possible consequences of the study had been explained to them. The research program was approved by the institutional human experimentation committees.

A total of 128 white German patients (31 men and 97 women) with scintigraphically proven thyroid autonomy, all native residents of Saxony, were genotyped for the codon 727 hTSHR polymorphism and compared with 99 healthy control individuals (31 men and 68 women) and 108 patients with GD (17 males and 91 females), all of whom were natives of the same geographic region. Thyroid hyperfunctioning nodules and GD were diagnosed by standard criteria (clinical examination, thyroid function tests, ultrasonography, TSHR and thyroid peroxidase antibody titers, and quantitative technetium scintigraphy). Patients with thyroid autonomy were divided into 3 subpopulations: those with solitary toxic adenoma (n = 83), those with toxic multinodular goiter (n = 31), and those with disseminated autonomy (n = 14). All healthy control participants had documented normal thyroid function and no evidence of thyroid autoimmune or nonautoimmune disease, as determined by a careful personal and family history, clinical examination, thyroid function, and thyroid autoantibody testing.

Polymorphism typing

Genomic DNA was extracted from ethylenediamine tetraacetate-anticoagulated peripheral blood. The polymorphic region within codon 727 of the hTSHR gene was amplified by PCR. Oligonucleotide primers used for analysis of mutant hTSHR gene expression were 5'-AACGCCAGGCTCAGGCATAC-3' and 5'-AAGTTCCCCTACCATTGTGA-3'. These primers should generate a product that is 232 bp in length. Amplifications were performed using 5 µL of each DNA template, 5 µL 10 x PCR reaction buffer, 1 µL 10 mmol/L deoxy-NTP, 0.6 µL of each oligonucleotide primer (50 µmol/L), and 2.5 U Taq DNA polymerase in a final volume of 50 µL. Amplifications were performed in an automated thermocycler (Thermodux, Wertheim, Germany) with 5 min of denaturation at 94 C, followed by 35 cycles of 45 s at 94 C, 1 min at 58 C, and 1 min at 72 C and a final 5-min extension at 72 C. Subsequently, restriction enzyme digestion was performed to distinguish mutant from wild-type hTSHR fragments. To this purpose, PCR transcripts were precipitated and resuspended in Aqua bidest. The restriction enzyme NlaIII (2.5 U) was used for hydrolysis in a final volume of 36 µL of the appropriate buffer. After incubation at 37 C for 1 h, digested fragments were resolved on 3% NuSieve agarose gels containing 10 mg/ml ethidium bromide, visualized by UV light, and compared with a 100-bp molecular size control ladder. The mutation at position 727 of the hTSHR gene alters the restriction site for NlaIII, producing two DNA fragments from the amplicon rather than three generated with wild-type DNA. Thus, the wild-type strand produces fragments of 129, 82, and 21 bp, whereas the codon 727 mutant DNA produces fragments of 129 and 103 bp, respectively.

Sequencing

To confirm wild-type sequence or point mutation, respectively, PCR products of several homozygous and heterozygous individuals were resolved on an ethidium bromide-stained agarose gel. DNA fragments were excised, isolated, and ligated overnight at 16 C into the DNA-plasmid pCR (Invitrogen, Groningen, The Netherlands). Using the Easyject Plus Electroporation System (Eurogentec, Seraing, Belgium), transfection of competent Escherichia coli XL1-blue cells was performed. Plasmid DNA of three picked clones was purified with QIAGEN-tip 20 (QIAGEN, Hilden, Germany) and sequenced with a Sequenase kit (version 2.0, U.S. Biochemical Corp., Cleveland, OH) using [{alpha}-35S]deoxy-ATP for labeling. The reaction mixes were then run on denaturing 6% (wt/vol) polyacrylamide-7 mol/L urea gels and exposed to x-ray films.

Statistical analysis

Allelic frequencies (number of copies of a specific allele divided by total number of alleles in the group) were calculated for the study groups. Statistical significance was determined using Fisher’s exact test. P < 0.05 indicated a statistically significant difference. Data were analyzed using InStat 3.00 for Win 3.1 (1997) software (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The hTSHR codon 727 polymorphism was present in 56 of 335 (16.7%) unrelated individuals by restriction enzyme digestion screening (Fig. 1Go). Polymorphic alleles, containing an aspartate to glutamate substitution of codon 727, were confirmed by sequencing of cloned PCR transcripts. There was no significant difference in codon 727 polymorphism frequencies between patients with toxic thyroid adenomas (13.3%) and the healthy control group (16.3%; P = 0.57; Table 1Go). In addition, the polymorphism was not associated with GD (21.3%; P = 0.37; Table 1Go). Within the gender-stratified subgroups, 6 of 31 males from the healthy control group carried the polymorphism, whereas only 1 of 31 males with toxic thyroid adenoma and 2 of 17 males in the GD group carried this polymorphism. Because of the small number of affected males within these groups, no statistical significance could be attributed to these observations. Moreover, comparison of codon 727 polymorphism in females failed to reveal a statistically significant difference between the patient groups (toxic thyroid adenoma and GD) and the control group (P = 0.57 and P = 0.38). Furthermore, patient subgroups with toxic nonautoimmune thyroid disease, including toxic adenoma (13.2%), toxic multinodular goiter (9.6%), and disseminated autonomy (21.4%), were not related to significant differences in codon 727 polymorphism frequencies compared with the healthy control group (P = 0.67, P = 0.56, and P = 0.70, respectively; Table 2Go). Of the 56 individuals found to carry the polymorphism, 47 were heterozygous, and 9 were homozygous (4 with toxic thyroid adenoma, 4 with GD, and 1 from the control group) for the polymorphic allele.



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  		Figure 1. A representative example of NlaIII restriction enzyme digestion of hTSHR-specific PCR fragments amplified from genomic DNA. The wild-type strand produces fragments of 129, 82, and 21 bp, whereas the codon 727 mutant DNA produces fragments of 129 and 103 bp. Lane 1, One hundred-base pair DNA ladder; lanes 2, 3, 6, 7, and 9–11, DNA from individuals homozygous for the wild-type allele; lanes 4 and 8, DNA from individuals heterozygous for the hTSHR codon 727 mutation; lane 5, DNA from an individual homozygous for the hTSHR codon 727 mutation; lane 12, negative control with Aqua bidest.

 

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  		Table 1. Frequencies of hTSHR codon 727 mutation in patients with thyroid hyperfunctioning adenomas, patients with GD, and healthy controls

 

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  		Table 2. Frequencies of hTSHR codon 727 mutation among the subgroups of patients with nonautoimmune hyperfunctioning thyroid disorders

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In endemic goiter areas, the three main types of hyperfunctioning nonautoimmune thyroid disease include solitary toxic nodules, toxic multinodular goiter, and disseminated autonomy. Constitutively activating mutations of the hTSHR and the gene encoding the {alpha}-subunit of the stimulatory G protein (Gs{alpha}) have been implicated as major causes of thyroid hyperfunctioning adenomas (4, 7, 8, 9). Recently, Morris et al. reported a surprisingly high frequency of a germline polymorphism of codon 727 of hTSHR in patients with toxic multinodular goiter, suggesting that it may be a predisposing factor in toxic multinodular goiter (5). Functional analysis of the codon 727 polymorphism of the hTSHR revealed a significantly greater cAMP response of the mutant hTSHR to TSH stimulation than wild-type receptor, although basal cAMP production levels were similar (5). However, these data have been contradicted by a recent study (10) in which the researchers were unable to detect any significant difference in activation of the cAMP cascade between wild-type TSH receptor and the variant D727E TSH receptor. Moreover, in contrast to the data reported by Gabriel et al. (5), we failed to detect a significant difference in codon 727 polymorphism between patients with toxic multinodular goiter and the healthy control group. Similarly, none of the other patient groups, including patients with solitary toxic adenoma, disseminated autonomy, or GD, were related to a significant difference in their codon 727 polymorphism frequencies compared to the healthy control group. Several studies have recently detected somatic activating mutations of the hTSHR gene in single hyperfunctioning nodules contained in multinodular goiter (11, 12, 13, 14). It is still debated whether an activating mutation of the hTSHR gene or the Gs{alpha} gene is by itself sufficient to promote the evolution of toxic adenomas and goiter nodules. Similar clinical features in patients with thyroid hyperfunctioning adenomas with and without hTSHR/Gs{alpha} receptor mutations have been reported by Arturi et al. (15), suggesting that additional mechanisms may contribute to the pathogenesis of toxic adenomas. Moreover, the prevalence of somatic hTSHR mutations (ranging from 0–82%) and that of Gs{alpha} mutations (4–38%) is still controversial (6, 16, 17). Differences in methodology, recruitment, geographical origin of patients, and environmental factors, such as iodine intake, may explain some of these discrepancies. Although mutational activation of the adenylate cyclase cascade has been experimentally shown to cause increased proliferation and function of thyroid cells in vivo and in vitro (18, 19), Derwahl et al. failed to establish any correlation between basal or TSH-stimulated adenylate cyclase activity and the levels of Gs{alpha} and Gi{alpha} expressed by toxic adenomas with or without activating mutations and in nodular and extranodular tissues of toxic multinodular goiter with proven germline mutation in the hTSHR gene (20). These data suggest that hTSHR and Gs{alpha} gene mutations alone may not be sufficient to cause hyperfunctioning nonautoimmune thyroid disease. Low iodine intake in our European Caucasian study population may be largely responsible for the pathological thyroid conditions studied, thus explaining some of the discrepancies of TSHR genotyping compared to the results of Gabriel et al. (5), who studied individuals developing thyroid abnormalities despite sufficient iodine supply. In conclusion, our current data do not support an association between the codon 727 polymorphism of the hTSHR gene and hyperfunctioning thyroid abnormalities in a European population. Thus, the codon 727 hTSHR gene polymorphism does not appear to be involved in the evolution of toxic nonautoimmune thyroid disease.


   Acknowledgments
 
We are grateful to F. Herrmann (Leipzig, Germany) for supplying blood samples.


   Footnotes
 
1 This study represents part of K.H.’s medical thesis at Leipzig Medical School, University of Leipzig, Germany. Back

Received December 2, 1999.

Revised March 21, 2000.

Accepted March 29, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Porcellini A, Fenzi G, Avvedimento EV. 1997 Mutations of thyrotropin receptor gene. J Mol Med. 75:567–575.[CrossRef][Medline]
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  3. Vassart G, Dumont JE. 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev. 13:596–611.[CrossRef][Medline]
  4. van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G. 1995 Somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab. 80:2577–2585.[CrossRef][Medline]
  5. Gabriel EM, Bergert ER, Grant CS, van Heerden JA, Thompson GB, Morris JC. 1999 Germline polymorphism of codon 727 of human thyroid stimulating hormone receptor induces increased cyclic AMP response to TSH stimulation. J Clin Endocrinol Metab84 :3328–3335.
  6. Takeshita A, Nagayama Y, Yokoyama N, et al. 1995 Rarity of oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in Japan. J Clin Endocrinol Metab. 80:2607–2611.
  7. Führer D, Holzapfel HP, Wonerow P, Scherbaum WA, Paschke R. 1997 Somatic mutations in the thyrotropin receptor gene and not in the Gs{alpha} protein gene in 31 toxic thyroid nodules. J Clin Endocrinol Metab. 82:3885–3891.[Abstract/Free Full Text]
  8. Duprez L, Parma J, Costagliola S, Hermans J, van Sande J, Dumont J, Vassart G. 1997 Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain. FEBS Lett. 409:469–474.[CrossRef][Medline]
  9. Murakami M, Kamiya Y, Yanagita Y, et al. 1998 Primary culture of cells from hyperfunctioning thyroid adenoma with an activating mutation of Gs{alpha}. Mol Cell Endocrinol. 138:137–142.[CrossRef][Medline]
  10. Nogueira CR, Kopp P, Arseven OK, Santos CLS, Jameson JL, Medeiros-Neto G. 1999 Thyrotropin receptor mutatons in hyperfunctioning thyroid adenoms from Brazil. Thyroid 9:1063–1068.
  11. Tonacchera M, Vitti P, Agretti P, et al. 1998 Activating thyrotropin receptor mutations in histologically heterogeneous hyperfunctioning nodules of multinodular goiter. Thyroid. 8:559–64.[Medline]
  12. Tonacchera M, Chiovato L, Pinchera A, et al. 1998 Hyperfunctioning thyroid nodules in toxic multinodular goiter share activating thyrotropin receptor mutations with solitary toxic adenoma. J Clin Endocrinol Metab. 83:492–98.[Abstract/Free Full Text]
  13. Holzapfel HP, Führer D, Wonerow P, Weinland G, Scherbaum WA, Paschke R. 1997 Identification of constitutively activating somatic thyrotropin receptor mutations in a subset of toxic multinodular goiter. J Clin Endocrinol Metab. 82:4229–33.[Abstract/Free Full Text]
  14. Duprez L, Hermans J, van Sande J, Dumont J, Vassart G, Parma J. 1997 Two autonomous nodules of a patient with multinodular goiter harbor different activating mutations of the thyrotropin receptor gene. J Clin Endocrinol Metab. 82:306–308.[Free Full Text]
  15. Arturi F, Capula C, Chiefari E, Filetti S, Russo D. 1998 Thyroid hyperfunctioning adenomas with and without Gsp/TSH receptor mutations show similar clinical features. Exp Clin Endocrinol Diabetes. 106:234–236.[Medline]
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  17. Pinducciu C, Borgonovo G, Arezzo A, Torre GC, Giordano G, Cordera R. 1998 Toxic thyroid adenoma: absence of DNA mutations of the TSH receptor and Gs{alpha}. Eur J Endocrinol. 138:37–40.[Abstract]
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  19. Muca C, Vallar L. 1994 Expression of mutationally activated Gs{alpha} stimulates growth and differentiation of thyroid FRTL5 cells. Oncogene. 9:3647–53.[Medline]
  20. Derwahl M, Hamacher C, Russo D, et al. 1996 Constitutive activation of the Gs{alpha} protein-adenylate cyclase pathway may not be sufficient to generate toxic thyroid adenomas. J Clin Endocrinol Metab. 81:1898–1904.[Abstract]



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