Prevalence of activating thyrotropin receptor and gsa gene mutations in paediatric thyroid toxic adenomas: a multicentric italian study

Toxic thyroid adenomas or functioning nodules are rare in children and adolescents, representing less than 3% of all causes of hyperthyroidism.[1] Somatic gain-of-function mutations of the TSHr gene (accession number NM_00369) have been identified as a cause of toxic thyroid adenomas and functioning nodules of toxic multinodular goitre in the adult.[2] In patients with toxic thyroid adenomas coming from an area of iodine deficiency, activating somatic mutations in the TSHr gene have been detected in up to 80% and in the Gs\u3b1 gene up to 25%, suggesting that these genetic anomalies may play a role in the pathogenesis of functioning nodules in adults. In contrast, the incidence of gain-of-function somatic mutations of the TSHr or Gs\u3b1 genes in rare toxic thyroid adenoma in the paediatric population is poorly understood.[3] In this study, we searched for somatic mutations of TSHr and Gs\u3b1 genes in a group of nine children affected by toxic thyroid adenomas or functioning nodules that had undergone surgery. Nine children from five Italian paediatric Endocrinology Units who underwent surgery for toxic thyroid adenoma or functioning nodules were included in this study. At diagnosis, six patients were clinically thyrotoxic and three had normal FT4 and FT3 and low TSH serum levels. Thyroid glands were studied by clinical examination, thyroid ultrasound, scintiscan imaging using iodine-123 (123I) or technetium-99 m (99mTc) and histology. Thyroid autoimmunity was excluded by the absence of circulating antithyroid antibodies. Genomic DNA was extracted from formalin-fixed paraffin-embedded thyroid sections, and direct sequencing of exons 9 and 10 of the TSHr gene and exons 8 and 9 of the Gs\u3b1 gene was performed. The TSHr gene mutant harbouring the single-nucleotide missense substitution was generated by site-directed mutagenesis; COS-7 cells were transiently transfected using the DEAE-Dextran technique and used for cAMP production assay, 125I-bTSH binding and microchip flow cytometry analysis. Two heterozygous TSHr gene point mutations were identified in genomic DNA from nodular tissues with a prevalence of two of 9 (about 22%). No alteration was identified in normal tissues or blood cells. A residue change at position 568 of the TSHr (I568F) due to the heterozygous nucleotide substitution ATC/TTC (Fig. 1a) was revealed in one nodule. In genomic DNA from another nodule, a residue change at position 453 of the TSHr (M453T) due to the heterozygous nucleotide substitution ATG/ACG (Fig. 1a) was identified. Sequence of exons 8 and 9 of the Gs\u3b1 gene was normal in each patient. The I568F mutant was functionally characterized in this study, while the functional characteristics of M453T mutation were well known.[4] The I568F mutant was characterized by increased basal constitutive activity in terms of cAMP accumulation when compared with the wtTSHr. Moreover, the mutant showed a very low response to stimulation with increasing doses of bTSH, suggesting that this mutation caused sufficient receptor activation that TSH stimulation had little further effect (Fig. 1b). The I568F mutant showed TSH binding capacity (Bmax values) similar to that of the wtTSHr, suggesting a not modified expression at the cell surface (Fig. 1c). Results obtained by microchip flow cytometry analysis confirmed that I568F mutant showed a cell surface expression similar to that of the wtTSHr (Fig. 1d). image Figure 1. Genetic and functional characteristics of the TSHr gene mutations. (a) Automated fluorescence-based sequencing chromatograms showing I568F and M453T mutants. (b) cAMP response to bTSH stimulation in COS-7 cells transfected with wtTSHr and I568F mutation. (c) 125I-bTSH binding characteristics of wtTSHr and I568F mutation. (d) Expression analysis of I568F mutation obtained by microchip flow citometry using the BA8 monoclonal antibody. Juvenile thyrotoxicosis is a rare disorder with an incidence of 0\ub78 per 100\ub7000 person-years between 0 and 15 years in Caucasians. Graves' disease accounts for more than 96% of cases, while toxic adenomas or autonomously functioning thyroid nodules account for less than 3%.[1] Activating mutations of the TSHr gene have been shown to be a cause of toxic adenomas or functioning thyroid nodules in adults with a prevalence up to 80% in patients coming from areas of iodine deficiency.[2] Thyroid toxic adenomas or hyperfunctioning thyroid nodules in children and adolescents are uncommon, and few studies have been published on the incidence of TSHr and Gs\u3b1 mutations.[3] In addition, these nodules in children and adolescents may have a significantly different biological potential with a more rapid progression towards toxicity and a higher incidence of thyroid carcinoma, although all nodules we studied were follicular adenomas and no evidence of cancer was identified at histological examination. In this study, we analysed the sequence of TSHr and Gs\u3b1 genes in nine children subjected to surgery for thyroid toxic adenomas or functioning nodules coming from different clinical Italian centres living in areas of moderate iodine deficiency. In two children, I568F and M453T activating TSHr mutations were identified in genomic DNA from nodules. Frequency of TSHr mutations in this group of paediatric toxic thyroid adenomas or functioning nodules was about 22%, lower with respect to the prevalence of TSHr mutations reported in thyroid toxic adenomas in adults. The naturally occurring mutation I568F was described for the first time in a child with autonomous thyroid nodules,[3] but the functional study was not performed. However, activating mutations at the same codon (I568T and I568V) have been reported in functioning thyroid adenomas and familial and sporadic nonautoimmune hyperthyroidism. The I568T mutation caused very strong activation of the cAMP-regulatory cascade and stimulated constitutively the inositol phosphate-diacylglycerol cascade. I568V mutation conferred constitutive activation of the cAMP pathway, but not of the inositol phosphate cascade.[5] Our results showed that I568F mutant was expressed similarly to wtTSHr but had a basal constitutive activity in terms of cAMP production higher with respect to the wtTSHr. Inositole phosphate (IP) accumulation of I568F mutant was not studied but from previous studies using site-directed mutagenesis[5] basal and ligand-stimulated IP levels were comparable with that of the wtTSHr. It is not surprising in our case that the replacement of the nonpolar aminoacid isoleucine at position 568 with the nonpolar phenylalaninehad little affect IPs pathway. The isoleucine residue at position 568, located within the ECL2 of TSHr, has important functional properties affecting the signalling activity of the receptor.[5] In conclusion, activating somatic mutations of TSHr and Gs\u3b1 genes in thyroid toxic adenomas may be identified in childhood but are rarer than in adult population. In this small series of toxic adenomas or functioning nodules, no cancer was detected. Acknowledgements Special thanks go to our late master, Professor Aldo Pinchera, for his precious teaching. This work was supported by grants from Ministero dell'Universit\ue0 e della Ricerca Scientifica (MURST) and Ministero della Sanit\ue0, Ricerca Finalizzata. We are grateful to MURST and Ministero della Sanit\ue0 for funding. Disclosure All authors have nothing to declare