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. 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, 0.2–3); free T3, 9.2 pmol/L [normal range, 2.2–6.8; 5.98 pg/mL (normal range, 1.4–4.4)]; and free T4, 27.6 pmol/L[ normal range, 9–23; 21.42 pg/mL (normal range, 7–18)]. 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. 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. 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. 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 Gsα gene, respectively. The prevalence of TSH receptor and Gsα 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. 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 1267–1630): forward primer, 5′-aatacgactcactatagggttcgttagtctgctggctc-3′(a); reverse primer, 5′-caggaaacagctatgacccaaccatgatggcacatg-3′(b); and fragment B including the left nodule mutation (position 1567–1960): 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 manufacturer’s 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). 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). After the demonstration that solitary toxic adenomas are mainly caused by activating mutations in the TSH receptor and to a lesser extent by Gsα 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 Gsα 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 Gsα (15:2) may explain the higher proportion of TSH receptor over Gsα mutations observed in solitary toxic adenomas. An open question is whether the occurrence of TSH receptor or Gsα 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 Gsα, 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 (11–13). 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 Gsα) 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 Plummer’s 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 Gsα mutations in populations of severe endemic goiter areas that show a burst of thyrotoxicosis after iodine supplementation (3).
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Duprez et al. (1997) studied this question.
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