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Background: Previous Mendelian randomization (MR) studies showed an association between hypothyroidism and cataract and between high-normal free thyroxine (FT4) and late age-related macular degeneration (AMD), but not between FT4, thyroid stimulating hormone (TSH), or hyperthyroidism and diabetic retinopathy or cataract. These studies included a limited number of genetic variants for thyroid function and did not investigate autoimmune thyroid disease (AITD) or glaucoma, include bidirectional and multivariable MR (MVMR), and examine sex differences or potential mediation effects of diabetes. We aimed to address this knowledge gap. Methods: We examined the causality and directionality of the associations of AITD, and FT4 and TSH within the reference range with common age-related eye diseases (diabetic retinopathy, cataract, early and late AMD, and primary open-angle glaucoma). We conducted a bidirectional two-sample MR study utilizing publicly available genome-wide association study (GWAS) summary statistics from international consortia (ThyroidOmics, International AMD Genetics Consortium, deCODE, UK Biobank, FinnGen, and DIAGRAM). Bidirectional MR tested directionality, whereas MVMR estimated independent causal effects. Furthermore, we investigated type 1 diabetes (T1D) and type 2 diabetes (T2D) as potential mediators. Results: Genetic predisposition to AITD was associated with increased risk of diabetic retinopathy ( p = 3 × 10 -4 ), cataract ( p = 3 × 10 -3 ), and T1D ( p = 1 × 10 -3 ), but less likely T2D ( p = 0.01). MVMR showed attenuated estimates for diabetic retinopathy and cataract when adjusting for T1D, but not T2D. We found pairwise bidirectional associations between AITD, T1D, and diabetic retinopathy. Genetic predisposition to both T1D and T2D increased the risk of diabetic retinopathy and cataract ( p < 4 × 10 -4 ). Moreover, genetically predicted higher FT4 within the reference range was associated with an increased risk of late AMD ( p = 0.01), particularly in women ( p = 7 × 10 -3 ). However, we neither found any association between FT4 and early AMD nor between TSH and early and late AMD. No other associations were observed. Conclusions: Genetic predisposition to AITD is associated with risk of diabetic retinopathy and cataract, mostly mediated through increased T1D risk. Reciprocal associations between AITD, diabetic retinopathy, and T1D imply a shared autoimmune origin. The role of FT4 in AMD and potential sex discrepancies needs further investigation.

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n  = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n  = 8). We used unimmunized BALB/c mice as a control group ( n  = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.

Background: Thyroid dysfunction has been observed among some patients with coronavirus disease (COVID-19). It is unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (or its severity) leads to the development of thyroid dysfunction, or vice versa. In this study, we examined the bi-directional causal relationship between host genetic liability to three COVID-19 phenotypes (including SARS-CoV-2 infection, hospitalized and severe COVID-19) and three thyroid dysfunction traits (including hyperthyroidism, hypothyroidism, and autoimmune thyroid disease [AITD]) and three continuous traits of thyroid hormones (including thyrotropin [TSH] and free thyroxine [fT4] within reference range, and TSH in full range). Methods: Summary statistics from the largest available meta-analyses of human genome-wide association studies were retrieved for the following variables: SARS-CoV-2 infection ( n  = 1,348,701), COVID-19 hospitalization ( n  = 1,557,411), severe COVID-19 ( n  = 1,059,456), hyperthyroidism ( n  = 51,823), hypothyroidism ( n  = 53,423), AITD ( n  = 755,406), TSH within reference range ( n  = 54,288), fT4 within reference range ( n  = 49,269), and TSH in full range ( n  = 119,715). Using a two-sample Mendelian randomization (MR) approach, the inverse-variance weighted (IVW) method was adopted as the main MR analysis. Weighted median, contamination mixture, MR-Egger, and MR pleiotropy residual sum and outlier (MR-PRESSO) methods were applied as sensitivity analyses. Results: Host genetic susceptibility to SARS-CoV-2 infection was causally associated with hypothyroidism in the main IVW analysis (per doubling in prevalence of SARS-CoV-2 infection, odds ratio [OR] = 1.335; 95% confidence interval [CI]: 1.167-1.526; p  = 2.4 × 10 -5 , surpassing the Bonferroni multiple-testing threshold). Similar causal estimates were observed in the sensitivity analyses (weighted median: OR = 1.296; CI: 1.066-1.575; p  = 9 × 10 -3 ; contamination mixture: OR = 1.356; CI: 1.095-1.818; p  = 0.013; MR-Egger: OR = 1.712; CI: 1.202-2.439; p  = 2.92 × 10 -3 , and MR-PRESSO: OR = 1.335; CI: 1.156-1.542; p  = 5.73 × 10 -4 ). Host genetic liability to hospitalized or severe COVID-19 was not associated with thyroid dysfunction or thyroid hormone levels. In the reverse direction, there was no evidence to suggest that genetic predisposition to thyroid dysfunction or genetically determined thyroid hormone levels altered the risk of the COVID-19 outcomes. Conclusions: This bi-directional MR study supports that host response to SARS-CoV-2 viral infection plays a role in the causal association with increased risk of hypothyroidism. Long-term follow-up studies are needed to confirm the expected increased hypothyroidism risk.

Background: Untreated hypothyroidism is associated with acquired von Willebrand syndrome, and hyperthyroidism is associated with increased thrombosis risk. However, the causal effects of thyroid function on hemostasis, coagulation, and fibrinolysis are unknown. Methods: In a two-sample Mendelian randomization (MR) study with genome-wide association variants, we assessed causality of genetically predicted hypothyroidism ( N  = 134,641), normal-range thyrotropin (TSH; N  = 54,288) and free thyroxine (fT4) ( N  = 49,269), hyperthyroidism ( N  = 51,823), and thyroid peroxidase antibody positivity ( N  = 25,821) on coagulation (activated partial thromboplastin time, von Willebrand factor [VWF], factor VIII [FVIII], prothrombin time, factor VII, fibrinogen) and fibrinolysis (D-dimer, tissue plasminogen activator [TPA], plasminogen activator inhibitor-1) from the CHARGE Hemostasis Consortium ( N  = 2583-120,246). Inverse-variance-weighted random effects were the main MR analysis followed by sensitivity analyses. Two-sided p  < 0.05 was nominally significant, and p  < 0.0011[ = 0.05/(5 exposures × 9 outcomes)] was Bonferroni significant for the main MR analysis. Results: Genetically increased TSH was associated with decreased VWF [β(SE) = -0.020(0.006), p  = 0.001] and with decreased fibrinogen [β(SE) = -0.008(0.002), p  = 0.001]. Genetically increased fT4 was associated with increased VWF [β(SE) = 0.028(0.011), p  = 0.012]. Genetically predicted hyperthyroidism was associated with increased VWF [β(SE) = 0.012(0.004), p  = 0.006] and increased FVIII [β(SE) = 0.013(0.005), p  = 0.007]. Genetically predicted hypothyroidism and hyperthyroidism were associated with decreased TPA [β(SE) = -0.009(0.024), p  = 0.024] and increased TPA [β(SE) = 0.022(0.008), p  = 0.008], respectively. MR sensitivity analyses showed similar direction but lower precision. Other coagulation and fibrinolytic factors were inconclusive. Conclusions: In the largest genetic studies currently available, genetically increased TSH and fT4 may be associated with decreased and increased synthesis of VWF, respectively. Since Bonferroni correction may be too conservative given the correlation between the analyzed traits, we cannot reject nominal associations of thyroid traits with coagulation or fibrinolytic factors.

Background: Thyroxine (T4) to triiodothyronine (T3) deiodination in the hypothalamus/pituitary is mediated by deiodinase type-2 (D2) activity. Dio2 (-/-) mice show central resistance to exogenous T4. Patients with resistance to exogenous thyroxine (RETH) have not been described. The aim of this study was to identify hypothyroid patients with thyrotropin (TSH) unresponsiveness to levothyroxine (LT4) and to characterize the clinical, hormonal, and genetic features of human RETH. Methods: We investigated hypothyroid patients with elevated TSH under LT4 treatment at doses leading to clinical and/or biochemical hyperthyroidism. TSH and free T4 (fT4) were determined by chemiluminescence, and total T4, T3, and reverse T3 (rT3) by radioimmunoassay. TSH/fT4 ratio at inclusion and T3/T4, rT3/T4, and T3/rT3 ratios at follow-up were compared with those from patients with resistance to thyroid hormone (RTH) due to thyroid hormone receptor-β ( THRB ) mutations. DIO2, including the Ala92-D2 polymorphism, selenocysteine binding protein 2 ( SECISBP2 ), and THRB were fully sequenced. Results: Eighteen hypothyroid patients (nine of each sex, 3-59 years) treated with LT4 showed elevated TSH (15.5 ± 4.7 mU/L; reference range [RR]: 0.4-4.5), fT4 (20.8 ± 2.4 pM; RR: 9-20.6), and TSH/fT4 ratio (0.74 ± 0.25; RR: 0.03-0.13). Despite increasing LT4 doses from 1.7 ± 1.0 to 2.4 ± 1.7 μg/kg/day, TSH remained elevated (6.9 ± 2.7 mU/L). Due to hyperthyroid symptoms, LT4 doses were reduced, and TSH increased again to 7.9 ± 3.2 mU/L. In the euthyroid/hyperthyrotropinemic state, T3/T4 and T3/rT3 ratios were decreased (9.2 ± 2.4, RR: 11.3-15.3 and 2.5 ± 1.4, RR: 7.5-8.5, respectively) whereas rT3/T4 was increased (0.6 ± 0.2; RR: 0.43-0.49), suggesting reduced T4 to T3 and increased T4 to rT3 conversion. These ratios were serum T4-independent and were not observed in RTH patients. Genetic testing was normal. The Ala92-D2 polymorphism was present in 7 of 18 patients, but the allele dose did not correlate with RETH. Conclusions: Human RETH is characterized by iatrogenic thyrotoxicosis and elevated TSH/fT4 ratio. In the euthyroid/hyperthyrotropinemic state, it is confirmed by decreased T3/T4 and T3/rT3 ratios, and elevated rT3/T4 ratio. This phenotype may guide clinicians to consider combined T4+T3 therapy in a targeted fashion. The absence of germline DIO2 mutations suggests that aberrant post-translational D2 modifications in pituitary/hypothalamus or defects in other genes regulating the T4 to T3 conversion pathway could be involved in RETH.

Background: Novel molecular tests (MTs), such as ThyroSeq, may improve the management of thyroid nodules with indeterminate cytologic diagnoses; however, the impact of these tests on cost and outcome of management is unknown. Here, we evaluated the impact of ThyroSeq testing on the cytopathologic diagnosis, management, and cost of care in patients with thyroid nodules. Methods: In a retrospective study, using actual patient cohorts, the outcome and cost of management of patients with thyroid nodules seen before the introduction of ThyroSeq v2 at our institution (standard of care [StC] cohort) were compared with those seen after the introduction of this test (MT cohort). Results: A total of 773 consecutive patients entered the study (393 StC, 380 MT). The incidence of cytologically benign nodules decreased from 71.0% (StC) to 53.2% (MT) and those of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) increased from 8.9% (StC) to 21.3% (MT) and from 3.1% (StC) to 6.3% (MT), respectively. The overall rate of surgery did not change significantly (23.4% in StC vs. 23.2% in MT). Among patients who underwent surgery, the rate of overtreatment (surgeries performed on histologic benign nodules without clinical indication: compressive symptoms, hyperthyroidism resistant to medication, and nodule size >4 cm) slightly decreased from 18.8% (StC) to 16.7% (MT). The rate of malignancy decreased from 45.5% (StC) to 37.9% (MT) in AUS/FLUS and increased from 40.0% to 53.8% in FN/SFN. However, the overall rate of malignancy remained equal (47.8% in StC vs. 47.7% in MT). The average cost of care per patient in the AUS/FLUS and FN/SFN categories increased from $6,566 (StC) to $8,444 (MT) and from $9,313 (StC) to $10,253 (MT), respectively. Similarly, the overall average cost of care of a patient who underwent thyroid fine-needle aspiration increased from $3,088 (StC) to $4,282 (MT). Finally, the average cost per thyroid cancer detected increased from $26,312 (StC) to $38,746 (MT). Conclusions: Introduction of ThyroSeq v2 resulted in a shift toward indeterminate cytology results. The institutional rate of surgery, overtreatment, and malignancy did not change significantly. Lack of decrease in the rate of surgery along with the additional cost of ThyroSeq v2 increased the overall cost of care of patients including those with indeterminate cytology results.

Nonautoimmune hyperthyroidism caused by activating mutations in the GNAS gene is a rare condition. In this study, we report a five-year-old girl diagnosed with nonautoimmune hyperthyroidism and tall stature harboring a somatic mosaic gain-of-function mutation in the GNAS gene (NM&#95;080425.3: c.2530C>T;p.Arg844Cys previously reported as NM&#95;000516.5:c.601C>T;p.Arg201Cys) and referred to thereafter as R201C, in three of four quadrants of the thyroid gland. Provision of a molecular diagnosis may avoid unnecessary complete ablation of the thyroid gland.

Background: The secretion of pituitary hormones oscillates throughout the 24-hour period, indicating that circadian clock-mediated mechanisms regulate this process in the gland. Additionally, pituitary hormone synthesis has been shown to be altered in hypo- and hyperthyroidism. Although thyroid hormones can modulate the other peripheral clocks, the interaction between thyroid hormone levels and circadian clock gene expression in the anterior pituitary has yet to be elucidated., Methods: Male Wistar rats were divided into three groups: control, hypothyroid, and hyperthyroid. Following the experimental procedures, animals were euthanized every three hours over the course of a 24-hour period. The anterior pituitary glands were excised and processed for mRNA expression analysis by quantitative reverse transcriptase polymerase chain reaction. One- and two-way analysis of variance as well as cosinor analysis were used to evaluate the time-of-day-dependent differential expression for each gene in each experimental group and their interactions., Results: Hyperthyroidism increased the mRNA expression of core clock genes and thyrotrophic embryonic factor (Tef), as well as the mesor and amplitude of brain and muscle Arnt-like protein-1 (Bmal1) and the mesor of nuclear receptor subfamily 1 (Nr1d1) group D member 1, when compared to euthyroid animals. Hypothyroidism disrupted the circadian expression pattern of Bmal1 and period circadian regulator 2 (Per2) and decreased the mesor of Nr1d1 and Tef. Furthermore, it was observed that the pituitary content of Dio2 mRNA was unaltered in hyperthyroidism but substantially elevated in hypothyroidism during the light phase. The upregulated expression was associated with an increased mesor and amplitude, along with an advanced acrophase. The gene expression of all the pituitary hormones was found to be altered in hypo- and hyperthyroidism. Moreover, prolactin (Prl) and luteinizing hormone beta subunit (Lhb) displayed circadian expression patterns in the control group, which were disrupted in both the hypo- and hyperthyroid states., Conclusion: Taken together, the data demonstrate that hypo- and hyperthyroidism alter circadian clock gene expression in the anterior pituitary. This suggests that triiodothyronine plays an important role in the regulation of pituitary gland homeostasis, which could ultimately influence the rhythmic synthesis and/or secretion of all the anterior pituitary hormones.

Background: Constitutively active thyrotropin receptor (TSHR) mutations are the most common etiology of non-autoimmune hyperthyroidism (NAH). Thus far, the functionality of these mutations has been tested in vitro, but the in vivo models are lacking., Methods: To understand the pathophysiology of NAH, the patient-derived constitutively active TSHR D633H mutation was introduced into the murine Tshr by homologous recombination., Results: In this model, both subclinical and overt hyperthyroidism was observed, depending on the age, sex, and genotype. Homozygous mice presented hyperthyroidism at two months of age, while heterozygous animals showed only suppressed thyrotropin. Interestingly, at six months of age, thyroid hormone concentrations in all mutant mice were analogous to wild-type mice, and they showed colloid goiter with flattened thyrocytes. Strikingly, at one year of age, nearly all homozygous mice presented large papillary thyroid carcinomas. Mechanistically, this papillary thyroid carcinoma phenotype was associated with an overactive thyroid and strongly increased stainings of proliferation-, pERK-, and NKX2-1 markers, but no mutations in the "hot-spot" areas of common oncogenes (Braf, Nras, and Kras) were found., Conclusions: This is the first study to reveal the dynamic age-, sex-, and genotype-dependent development of NAH. Furthermore, the study shows that a constitutively active TSHR can trigger a malignant transformation of thyrocytes.

Background: Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal., Methods: Adult male mice were treated with daily injections of T3 (20 μg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics., Results: Although TH receptor (TRα and TRβ) mRNAs decreased slightly after chronic T3 treatment, only TRβ protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRβ protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal., Conclusions: Decreased hepatic TRβ protein expression occurred after chronic T3 exposure in adult male wild-type and Mct8-knockout mice. Gene array pathway and metabolomics analyses showed abnormalities in hepatic lipogenic gene expression and acylcarnitine levels, respectively, after withdrawal, despite normalization of serum TSH and TH levels. These findings may help explain the variable clinical presentations of some patients during hyperthyroidism and recovery, since TRβ protein, target gene expression, and metabolic adaptive changes can occur in individual tissues without necessarily being reflected by circulating TH and TSH concentrations.

Background: Congenital hyperthyroidism can be a cause of failure to thrive, hyperactivity, developmental delay, and craniosynostosis during infancy. Most commonly, the condition occurs in the setting of maternal autoimmune thyroid disease. Rarely, congenital hyperthyroidism can also occur secondary to activating mutations within the thyrotropin (TSH) receptor., Patient Findings: A Hispanic male infant presented at age 6 months with severe thyrotoxicosis. At the time of presentation he was being evaluated for squamosal suture synostosis and he was noted to have significant developmental delays., Summary: The patient's thyrotoxicosis was initially treated with antithyroid medication, and he subsequently underwent craniosynostosis repair leading to neurodevelopmental improvement. DNA from the patient and his parents was submitted for mutational analysis of exons 9 and 10 of the TSH receptor. He was found to carry a monoallelic transition 1895C>T in exon 10 that resulted in the substitution of threonine at position 632 by isoleucine (T32I). This mutation resulted in constitutive activation of the TSH receptor. Neither parent carried this mutation indicating that the child has acquired a de novo germline mutation., Conclusions: We report the first case of squamosal suture craniosynostosis in a patient with non-autoimmune hyperthyroidism. Squamosal suture craniosynotosis is rare, often has a subtle presentation, and should be considered in all patients with this condition because prompt treatment of hyperthyroidism and craniosynotosis repair can lead to normalization of neurodevelopment.

Background: We studied the frequency of thyrotropin (TSH) receptor mutations in hyperthyroid patients with diffuse goiter and negative TSH receptor antibodies (TRAb), and the clinical pictures of the hyperthyroid patients in the presence and absence of mutations., Patients and Methods: From 2003 through 2012, 89 hyperthyroid patients with diffuse goiter and negative TRAb based on a second- or third-generation assay underwent sequence analysis of the TSH receptor gene from peripheral leukocytes. The outcome of hyperthyroidism in patients with a TSH receptor mutation and their affected family members was compared with that in patients without any mutation after a 1-10-year follow-up., Results: Germline mutations of the TSH receptor occurred in 4 of the 89 patients (4.5%), including 3 definitive constitutively activating mutations (L512Q, E575K, and D617Y). The main difference in the clinical outcome of hyperthyroidism was that no patients with a TSH receptor mutation achieved euthyroidism throughout the follow-up, while 23.5% of patients without any mutation entered remission. The progression from subclinical to overt hyperthyroidism was not significantly different between patients with or without a mutation. Meanwhile, 10.3% of TRAb-negative patients without any TSH receptor mutation developed TRAb-positive Graves' hyperthyroidism during the follow-up., Conclusions: The prevalence of nonautoimmune hyperthyroidism with TSH receptor mutations is lower than that of latent Graves' disease in TRAb-negative patients with hyperthyroidism. However, all affected patients with a TSH receptor mutation showed persistent hyperthyroidism regardless of subclinical or overt hyperthyroidism throughout the follow-up.

Background: Autonomously functioning, "hot", thyroid nodules are not common in children and adolescents. Such nodules are not considered alarming because they are assumed to be benign adenomas. Herein, we present a 15-year-old girl with a papillary thyroid carcinoma of 3.5 cm in diameter, which was functionally autonomous and scintigraphically hot., Patient Findings: The patient, initially referred to our Endocrine Unit because of a thyroid nodule, returned 6 months later for symptoms of hyperthyroidism. Hyperthyroidism was confirmed biochemically. Radioactive iodine ((131)I) thyroid scintigraphy was consistent with an autonomous thyroid nodule. As per guidelines, the patient underwent surgery and a pathological examination revealed papillary carcinoma, follicular variant. The excised nodule was examined for activating mutations of the thyrotropin receptor (TSHR), Gsα (GNAS1), H-RAS, N-RAS, K-RAS, and BRAF genes by direct sequencing. No mutations were found. Nevertheless, two combined nonfunctioning mutations were detected: a single-nucleotide polymorphism (SNP) of the TSHR gene, in exon 7, at codon 187 (AAT→AAC, both encoding asparagine), and a SNP within exon 8 of the Gsα gene at codon 185 (ATC→ATT, both encoding isoleucine). Both SNPs were also identified in the germline DNA of the patient. The same SNPs were sought in the parents and brother of our patient. Her father was heterozygous for the TSHR SNP, her mother heterozygous for the Gsα SNP, and her brother was wild type., Conclusions: This case demonstrates that the presence of hyperfunctioning thyroid nodule(s) does not rule out cancer and warrants careful evaluation, especially in childhood and adolescence to overlook malignancy.

Background: This 8-year follow-up study is aimed at determining the relapse and development of Graves' disease (GD) and the potential risk factors that could be associated with the development of thyroid dysfunction and autoantibodies in Chinese pedigrees., Methods: Fifty-four Chinese Han GD pedigrees (322 members) were recruited in 2000. Forty-five pedigrees (263 members) were followed up. Their clinical and laboratory characteristics and fasting urinary iodine were measured with the same method at the two time points., Results: We found that the mean age for onset of GD in offspring was much younger than that of their parents (p=0.013). At baseline, the prevalence of hyperthyroidism, hypothyroidism, and subclinical hypothyroidism in first-degree relatives were 5.5%, 1.6%, and 1.1%, respectively. Individuals with thyroid dysfunction were positive for thyroid autoantibodies. The prevalence of positive thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TgAb), or TSH receptor antibody (TRAb) in the first-degree relatives with euthyroidism in these pedigrees was 18.6%, 17.4%, or 56.9%, respectively. At follow-up, individuals with positive TPOAb were at risk of developing thyroid dysfunction, whereas patients with positive TRAb had increased risk for relapse even after drug treatment. The percentage of nonsmokers with positive TPOAb and TgAb was significantly higher than that of smokers (p<0.05), but the levels of serum TRAb were significantly higher in smokers at follow-up than baseline (p<0.01)., Conclusions: Genetic factors are crucial for the development of autoimmune thyroid disease (AITD), thyroid dysfunction, and the outcomes of Graves' patients following treatment with medicines. Although smoking was negatively associated with the presence of thyroid antibodies (TPOAb/TgAb), smoking may induce or aggravate GD.

Background: Germline thyrotropin receptor (TSHR) mutations are associated with sporadic congenital nonautoimmune hyperthyroidism and familial nonautoimmune hyperthyroidism. Somatic TSHR mutations are associated with toxic thyroid nodules (TTNs). The objective of the study was to define a relation of the clinical appearance and the in vitro activity (IVA) of the TSHR mutations described by several authors for these thyroid disorders., Methods: We analyzed the IVAs published as linear regression analysis (LRA) of the constitutive activity as a function of the TSHR expression and the basal cyclic adenosine monophosphate (cAMP) values to determine differences between exclusively somatic, exclusively familial, and shared sporadic and somatic TSHR-mutations. Further, we investigated correlations of the LRAs/basal cAMP values with clinical activity characteristics (CACs) of TTNs, such as largest diameter of the TTN and the age of the patient at thyroid surgery., Results: Shared sporadic and somatic mutations showed higher median LRA (14.5) and higher median basal cAMP values (fivefold) than exclusively familial mutations (6.1, p = 0.0002; 2.9-fold, p < 0.0001, respectively). Moreover, mutations shared between sporadic congenital nonautoimmune hyperthyroidism and toxic thyroid nodules (TTNs) showed higher median LRA/basal cAMP values (p < 0.0001) than exclusively somatic mutations in TTNs (5.1; 3.89-fold, respectively). Exclusively somatic mutations and exclusively familial mutations showed no significant difference in their median LRA values (p = 0.786) but a significant difference for basal cAMP values (p = 0.0006). The two examined CACs showed no correlation with the IVA characterized by LRA/basal cAMP values or with the presence or absence of a TSHR-mutation., Conclusions: This systematic analysis of published constitutively activating TSHR-mutations, their CACs, and their IVA provides evidence for higher IVA of shared sporadic and somatic TSHR mutations as compared with familial TSHR mutations. CACs of somatic TSHR mutations in TTNs did not have a clear association with the IVA as characterized by LRA or basal cAMP values.

Background: Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene., Methods: The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism., Results: A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway., Conclusion: Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial.

Background: Germline activating mutations of the thyrotropin receptor (TSHR) gene have been considered as the only known cause of sporadic nonautoimmune hyperthyroidism in the pediatric population. Here we describe the long-term follow-up and evaluation of a patient with sporadic nonautoimmune primary hyperthyroidism who was found to have a de novo germline activating mutation of the TSHR gene., Summary: The patient was an infant who presented at the age of 10 months in an unconscious state with exsiccation, wet skin, fever, and tachycardia. Nonautoimmune primary hyperthyroidism was diagnosed, and brain magnetic resonance imaging and computed tomography showed also Arnold-Chiari malformation type I. Continuous propylthiouracil treatment resulted in a prolonged clinical cure lasting for 10 years. At the age of 11 years and 5 months the patient underwent subtotal thyroidectomy because of symptoms of trachea compression caused by a progressive multinodular goiter. However, 2 months after surgery, hormonal evaluation indicated recurrent hyperthyroidism and the patient was treated with propylthiouracil during the next 4 years. At the age of 15 years the patient again developed symptoms of trachea compression. Radioiodine treatment resulted in a regression of the recurrent goiter and a permanent cure of hyperthyroidism without relapse during the last 3 years of his follow-up. Sequencing of exon 10 of the TSHR gene showed a de novo heterozygous germline I630L mutation, which has been previously described as activating mutation at somatic level in toxic thyroid nodules., Conclusions: The I630L mutation of the TSHR gene occurs not only at somatic level in toxic thyroid nodules, but also its presence in germline is associated with nonautoimmune primary hyperthyroidism. Our case report demonstrates that in this disorder a continuous growth of the thyroid occurs without any evidence of elevated TSH due to antithyroid drug overdosing. This may justify previous recommendations for early treatment of affected patients with removal of as much thyroid tissue as possible.

Background: Two previously unreported thyrotropin (TSH) receptor mutations, A623F and I635V, were identified in toxic follicular thyroid adenoma specimens from two patients with hyperthyroidism. Our aim was to characterize both novel mutants in terms of the following: cAMP basal constitutive activity, cAMP response to TSH, plasma membrane expression levels, and TSH binding properties., Methods: We performed DNA extraction for TSHR gene sequencing. COS-7 cells were transiently transfected with wild-type and mutated TSH receptor constructs for determination of basal cAMP constitutive activity and dose-response accumulation of cAMP using recombinant human TSH. Flow cytometry analysis was performed to evaluate plasma membrane expression. Binding studies using bovine TSH as a ligand were performed to compare the affinities of wild-type and mutated TSH receptors for TSH., Results: Both mutants, A623F and I635V, had higher cAMP basal constitutive activities than the wild-type TSH receptor. A623F but not I635V showed lower plasma membrane expression than the wild-type receptor. IC50, an indirect measurement of ligand-receptor affinity, was lower in A623F and higher in I635V than in the wild-type TSH receptor, although no statistically significant differences were observed. No differences were observed in EC50 and although the absolute values of maximal stimulation achieved with both mutants were higher than the wild type, the differences did not achieve statistical significance., Conclusions: A623F and I635V are two naturally occurring TSH receptor mutations that increase basal cAMP accumulation and consequently promote the development of toxic follicular thyroid adenoma. cAMP response to increasing TSH dose is retained by A623F and I635V mutated receptors and the maximal stimulation obtained is not different from that of the wild-type receptor. Substitution of alanine 623 by phenylalanine 623 at the third intracellular loop of the TSH receptor decreases its plasma membrane expression, indicating that alanine 623 is important in directing the TSH receptor to the cell surface or in down-regulating the constitutive receptor. By contrast, isoleucine 635, located in the sixth transmembrane domain, is important in regulating TSH receptor basal activity but does not modify its plasma membrane expression.

Background: Activating mutations of the thyroid stimulating hormone receptor gene (TSHR) are rare in the neonate and in the pediatric population. They are usually present in the germline, and are either inherited or occur de novo. Somatic mutations in TSHR are unusual in the pediatric population., Methods: We describe a nine-month-old infant with thyrotoxicosis who harbored an activating somatic mutation in TSHR that was not present in the germline., Results: As genomic DNA analysis failed to show a TSHR gene mutation, a radioiodide scan was performed to reveal a unilateral localization of uptake suppressing the remaining thyroid tissue. Genomic and complementary DNA analyses of the active thyroid tissue, removed surgically, identified a missense mutation (D633Y) located in the sixth transmembrane domain of the TSHR. The absence of this TSHR mutation in circulating mononuclear cells and in unaffected thyroid tissue confirmed the somatic nature of this genetic alteration., Conclusions: To the authors' knowledge, this is the youngest patient to receive definitive treatment for hyperthyroidism due to an activating mutation of TSHR.

Background: Up to date, three thyroid-stimulating hormone receptor (TSHR) germline variants have been reported for which no functional consequences have been detected by in vitro characterizations. However, familial nonautoimmune hyperthyroidism and hot nodules are clearly associated with constitutively activating TSHR germline mutations. We describe a family with a new TSHR germline mutation that is associated with euthyroidism in 13 family members and hyperthyroidism in 1 family member., Methods: Mutation analysis of the TSHR gene was performed by denaturing gradient gel electrophoresis. TSHR constructs were characterized by determination of cell surface expression, 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation, and constitutive cAMP activity., Results: A novel TSHR germline mutation (N372T) was found in a man who presented with thyrotoxicosis. The mutation was also detected in 13 family members, all of whom were euthyroid. Interestingly, an additional constitutively active somatic mutation (S281N) was identified on the second parental TSHR allele of the hyperthyroid index patient. Linear regression analysis showed a lack of constitutive activity for N372T. Moreover, coexpression studies of N372T with S281N did not reveal any evidence for a functional influence of N372T on the constitutively active mutation (CAM)., Conclusions: N372T is unlikely to cause altered thyroid function. This is consistent with the finding that only the index patient with the additional somatic mutation S281N was hyperthyroid.

We report here the 20-year follow-up study of a male subject diagnosed at 15 months of age as a sporadic case of pituitary resistance to thyroid hormone on the combination of clinical hyperthyroidism, elevated serum thyroid hormone (TH) levels and inappropriate thyrotropin (TSH). On D-thyroxine (D-T(4)) therapy from 30 months of age to 12.5 years, hyperactivity and hyperthyroid signs and symptoms as well as growth abnormalities improved, serum L-thyroxine (L-T(4)) enantiomer normalized, and basal and stimulated TSH decreased significantly without complete suppression. After 8 years off D-T(4), at 20 years of age, clinical status was normal despite persisting high TH levels and inappropriate TSH. Evolution of serum markers of TH action and echocardiography measurements followed up from 15 months to 20 years of age either in basal condition or on triiodothyronine (T(3)), as well as the sequential determination of bone mineral density suggest differences in the tissue responses to T(3): normal in bone with a high remodelling rate, heterogeneity for various hepatic markers, and decreased at heart level. No mutations were found in the coding sequence of TRbeta1, TRbeta2, TRalpha1, RXRgamma, SMRT, NCoR1, and NCoA1. In this patient the putative long-term effects of the persisting high bone resorption are unknown.

Objective: Hereditary (familial) nonautoimmune hyperthyroidism (FNAH) is caused by activating thyroid-stimulating hormone (thyrotropin) receptor (TSHR) germline mutations. We describe a family with recurrent thyrotoxicosis and goiter across three generations, including an 8-year-old girl., Main Outcome: Sequences of the TSHR gene in the index patient, her father, her paternal grandmother, and a paternal uncle demonstrated the presence of an identical germline TSHR mutation. The mutation was heterozygous and determined the substitution of valine for methionine (codon 463; ATG-->GTG) in the second transmembrane domain of the TSHR in all the affected patients, but in none of the unaffected family members., Conclusions: We compared the clinical presentation of FNAH in the family reported by us with the other cases harboring the same mutation reported in the literature. This analysis revealed high variability in the phenotypical expression of the disease. In the family reported by us, we also observed a clear anticipation of the onset of the disease across generations, and we discussed whether such a phenomenon can be the consequence of the increased iodine supplementation in the area where the family lives.

Constitutively-activating germline mutations of the thyrotropin receptor (TSHR) gene are very rare and are considered the cause of hereditary nonautoimmune hyperthyroidism. We describe four affected individuals from a Caucasian family: a mother and her three children, and an unaffected father. The mother and her first two children presented in a similar manner: lifelong histories of heat intolerance, hyperactivity, fast heart rate, reduced energy, increased appetite, and scrawny build. They all developed goiter in childhood and showed a suppressed TSH and elevated thyroxine (T(4)). The last child, a 12-year-old female, presented with no clinical symptoms or palpable neck mass, but with a suppressed TSH, elevated T(4) and thyromegaly detected by ultrasound. Mutation analysis of the TSHR gene in all family members revealed a novel heterozygous germline mutation resulting in the substitution of phenylalanine (TTC) by serine (TCC) at codon 631 in transmembrane helix 6 in the mother and all three children. Functional characterization of this germline mutation showed constitutive activation of the G(s)-mediated cyclic adenosine monophosphate (cAMP) pathway, which controls thyroid hormone production and thyroid growth. Molecular characterization of F631S demonstrates that this activating mutation plays a key role in the development of hereditary hyperthyroidism in this family although the timing of onset of clinical manifestations in the subjects may depend on other, as yet unidentified, factors.

BALB/c mice are susceptible and C57BL/6 mice are resistant to Graves' hyperthyroidism induced by immunization with adenovirus encoding the thyrotropin receptor (TSHR) A-subunit. Both strains develop comparable levels of TSHR antibodies, but potent TSH blocking antibody activity in C57BL/6 mice likely blocks development of hyperthyroidism. We used microarrays to compare gene expression in spleens of mice immunized with A-subunit adenovirus (TSHR-Ad) or control adenovirus (Con-Ad). To preclude the effects of variable thyroxine (T(4)) levels, mice were studied when euthyroid as follows: BALB/c mice immunized three times with TSHR-Ad or Con-Ad and C57BL/6 mice immunized three times with TSHR-Ad or Con-Ad. Among the 14,000 expressed probe sets, there were no statistically significant differences in gene expression in BALB/c mice immunized with TSHR-Ad versus Con-Ad. In contrast, expression of 57 transcripts (representing 40 genes) changed in response to TSHR-Ad in C57BL/6 mice. Diverse genes were identified, including proteins involved in immune responses, inflammation, and cell cycling as well as heat-shock proteins and proteases. Down-regulation of chitinase 3- and-4 gene expression likely reflects cytokines produced by T-helper 2 (Th2) type cells. Indeed, the immunoglobulin (IgG) subclass for TSHR antibodies reflects a deviation away from Th2 cytokines and toward Th1 in C57BL/6 mice. In conclusion, TSHR-Ad immunization altered gene expression profiles in C57BL/6, but not in BALB/c, mice. This response primarily involved reduced gene expression. In C57BL/6 mice, decreased expression of genes such as cathelicidin, calgranulins, and lipocalin following TSHR A-subunit adenovirus immunization suggests the importance of innate immunity in this response.

Objective: Hereditary nonautoimmune hyperthyroidism is caused by activating thyrotropin receptor (TSHR) germline mutations. We describe a family from Saxony, Germany, with this condition. Recurrent thyrotoxicosis and goiter were prevalent in three generations, affecting in addition to the 16-year-old index patient, her father and late paternal grandmother. Hyperthyroidism in the girl was remarkable for its poor response to methimazole (40-60 mg/d) treatment., Methods and Results: Molecular analysis of genomic DNA extracted from peripheral blood leucocytes showed a TSHR germline mutation in the girl and her father. This mutation results in a new amino acid exchange of valine for isoleucine in TSHR codon 568 (Ile568Val). Only the wild-type TSHR sequence was found in the girl's mother. On functional characterization in COS-7 cells, the novel Ile568Val TSHR mutation conferred constitutive activation of the cAMP pathway (2- to 3-fold increase of basal cyclic adenosine monophosphate [cAMP]), but not of the inositol phosphate cascade. As a consequence of the molecular findings, total thyroidectomy has been performed in the girl. She is now euthyroid on levothyroxine replacement therapy., Conclusion: This is the second Saxonian family with autosomal-dominant nonautoimmune hyperthyroidism, adding to a total of 13 families and 11 individuals with activating TSHR germline mutations worldwide. We suggest that the condition may indeed be more frequent than previously thought and that consequent assessment of a family history in children as well as adults with thyroid autonomy will contribute to ensure correct diagnosis and adequate treatment of patients with activating TSHR germline mutations.

Peroxiredoxin 5 (PRDX5) is a newly discovered thioredoxin peroxidase able to reduce peroxides that is implicated in antioxidant protective mechanisms. We report here its expression in the human thyroid gland. Twenty-seven human thyroid specimens were examined by immunohistochemistry. They included six normal thyroid tissues, five multinodular goiters, nine hot nodules, two Hürthle cell adenomas, and five thyroids from patients with Graves' disease. In the control tissue, PRDX5 expression was heterogeneous, being stronger in cubical functionally active follicular cells than in flat quiescent thyrocytes. It was diffuse in the cytoplasm, occasionally localized in inclusions that most likely corresponded to mitochondria. This feature was particularly marked in the Hürthle cell adenoma case. In multinodular goiters, hot nodules, and Graves' thyroids, the cytosolic labeling was enhanced compared to the control tissue and a signal was also detected in few nuclei. To determine whether the level of expression was different between multinodular goiters and hyperthyroid Graves' thyroids, PRDX5 immunoblotting was performed in these two respective tissues. We observed that PRDX5 expression was higher in the thyroid gland of patients with Graves' disease compared to multinodular goiters. In conclusion, our data show that PRDX5 is expressed in the thyroid gland where it could act as antioxidant. The level of expression is directly correlated with the functional status of epithelial cells, being higher in multinodular goiters, and even more pronounced in hyperthyroid tissues, such as Graves' disease.

Muscle weakness in patients with thyrotoxicosis during hypokalemic episodes (thyrotoxic periodic paralysis [TPP]) occurs sporadically and mostly in males. It is treated by infusion or oral supplementation with potassium and with resolution of the thyrotoxicosis state. The clinical features of TPP resemble familial hypokalemic periodic paralysis (hypoKPP), which has been linked to two mutations in the gene encoding the skeletal muscle calcium channel alpha-1 subunit (CACN1AS; Arg528His and Arg1239His) and to the sodium channel alpha-subunit (SCN4A; Arg672His). We screened for the mutations (CACN1AS by polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP]; SCN4A by single-strand conformation polymorphism analysis) described in hypoKPP in 20 unrelated patients with documented episodes of TPP (mean age, 40.0 +/- 12.3 years 19 males). Forty-eight patients with hyperthyroidism resulting from Graves' disease (48.5 +/- 12.3 years; 13 males), 1 patient with idiopathic hypoKPP (a 32-year-old male) and 32 healthy subjects (41.0 +/- 19.1 years; 16 males) were included. We found none of the TPP patients carry CACN1AS and SCN4A mutations. The hyperthyroid patients and control subjects were also negative for the mutations. The patient with idiopathic hypoKPP was genotyped to have the Arg528His mutation. These results suggest that despite close similarities between TPP and hypoKPP, a likely genetic basis for TPP does not involve the same gene mutations associated with hypoKPP.

There is abundant evidence for a genetic influence on the development of autoimmune thyroid diseases (AITD). One measure of the magnitude of genetic contribution to the development of a disease is the sibling risk ratio (lambda(s)). Recent accurate prevalence data for hypothyroidism and hyperthyroidism in the United States reported from the National Health and Nutrition Examination Survey III (NHANES III) study have now allowed us to compute the sibling recurrence risk for AITD. Patients were recruited from our endocrine clinic on the basis of having AITD. The inclusion of patients in this study was unambiguously single ascertainment. We studied 155 patients (131 with Graves' disease [GD] and 24 with Hashimoto's thyroiditis [HT]) who had reliable information on the presence or absence of AITD in siblings. Nine probands had siblings with GD and 13 probands had siblings with HT. Using the prevalence rates from NHANES III for clinical hyperthyroidism and hypothyroidism, the calculated lambda(s) was 16.9 for AITD, 11.6 for GD, and 28.0 for HT. These results confirm the significant contribution of genetic factors to the development of AITD.

The etiopathogenesis of feline thyrotoxicosis is unknown. The transmembrane part (gene codons 480-640) of the thyrotropin receptor (TSHR) gene of hyperthyroid cats has already been investigated for the presence of stimulating mutations. No mutations were found in this part of the TSHR gene. We have investigated the TSHR gene codons 66-530 for gene mutations in 10 hyperthyroid cats and in 1 euthyroid cat. This part of the TSHR gene encodes the transmembrane part as well as most of the extracellular part of the receptor. The G(s alpha) gene of these cats was also sequenced and subjected to mutational analysis. Although our study revealed a polymorphism in the TSHR gene, no association was found with tumor formation. In 4 of 10 cats with hyperthyroidism a G(s alpha) gene mutation was found. This work suggests that mutations in the extracellular or transmembrane part of the TSHR gene are not likely the cause of feline hyperthyroidism. Mutations in the G(s alpha) gene, however, may play a role in the etiopathogenesis of this disease.

Hereditary nonautoimmune hyperthyroidism is caused by activating germline mutations in the thyrotropin (TSH) receptor (TSHR) gene. We describe an extended Welsh kindred with toxic thyroid hyperplasia affecting 8 family members in three generations and a history consistent with thyrotoxicosis in a further three generations now deceased. A novel heterozygous germline mutation (ATG --> GTG; Met463Val) was identified in the second membrane spanning TSHR region in 6 relatives with thyrotoxicosis and goiter and absence of TSHR antibodies. Screening of 5 additional family members led to the identification of 2 siblings with the mutation, who were asymptomatic at the time, although subsequent thyroid function tests in these children showed suppressed serum TSH and increased serum free triiodothyronine (FT3) and free thyroxine (FT4) concentrations. Functional studies of the novel TSHR germline mutation demonstrated a constitutive activation of the cAMP pathway, which in the thyroid controls both thyroid hormone production and stimulation of thyroid growth. The molecular diagnosis in this family has clinical implications: genetic counseling is required, and primary thyroid ablation should be advocated as the preferred treatment in the patients with the constitutively activating TSHR germline mutation.

Hot nodules are rarely found to be carcinomas. We report a case of a nonmetastatic follicular carcinoma that presented as a hot nodule that was causing hyperthyroidism. A base substitution (ATC for TTC) was found in codon 486 of the TSH receptor gene and this resulted in the substitution of an isoleucine for a phenylalanine in the first extracellular loop of the receptor. This was absent in the deoxyribonucleic acid from the surrounding normal thyroid tissue indicating its somatic origin. This mutation, which was previously reported to activate both cyclic adenosine monophosphate and the inositol phosphate-diacylglycerol cascades, may have been responsible for the constitutive activation of the thyrotropin receptor and resulting hyperfunction of this follicular carcinoma.

The de novo occurrence of germline-activating thyrotropin receptor (TSHR) gene mutations has been reported as the cause of sporadic nonautoimmune neonatal hyperthyroidism in eight children. We report the case of an Italian infant girl who presented at birth with severe hyperthyroidism and goiter. Ultrasonografic examination of the infant's thyroid showed a diffuse goiter with a normal echogenic pattern. Serum antithyroglobulin, antithyroperoxidase, and antithyrotropin receptor antibodies were undetectable. Treatment with propylthiouracyl, propranolol, and saturated potassium iodide solution started at 44 days of life with the resolution of thyrotoxic symptoms. Once euthyroidism was achieved, the dose of propylthiouracyl was tapered, but hyperthyroidism recurred. Auxological parameters showed an acceleration of linear growth and bone age. DNA was extracted from peripheral white blood cells of the patient, the sister, and the two parents. All of exon 10 of the TSHR gene was amplified by polymerase chain reaction (PCR) and subjected to direct sequencing. In the thyrotoxic infant girl, a substitution of cytosine to thymine was detected, changing isoleucine 568 into a threonine (1568T), located in the second extracellular loop. The normal sequence could also be detected, indicating heterozygosis of the mutated allele. This mutation was previously described as a somatic mutation in a patient with toxic thyroid adenoma. The sister and the parents of the propositus, all euthyroid, showed the wild-type TSHR gene. In conclusion, we describe a case of a de novo germinal mutation of the TSHR causing severe congenital hyperthyroidism.

Resistance to thyroid hormone (RTH) is a rare syndrome characterized by reduced sensitivity to thyroid hormone due to thyroid hormone receptor-beta (TRbeta) gene mutations or deletion. RTH has been classified on the basis of clinical features into generalized (GRTH) and pituitary (PRTH) resistance. There is, however, overlap of clinical and biochemical findings in patients with the two forms of resistance, and similar TRbeta gene mutations have been identified in both. The 2 subtypes of RTH, therefore, are considered to be different manifestations of a single genetic entity. We report a mutation of the TRbeta gene, an arginine to leucine substitution at codon 338 (R338L), in 2 unrelated RTH kindreds of northern Italian ancestry. The same mutation was already reported in a single unrelated kindred affected by PRTH. Five individuals, 3 in the first and 2 in the second family, were clinically evaluated and followed for 3-11 years. During the long-term follow-up, the patients manifested symptoms and signs of hyperthyroidism including palpitations, fine tremors, heat intolerance, increased sweating, increased deep tendon reflexes, moist and warm skin, cardiac rhythm abnormalities, reduced body weight, and reduced bone mineral density. The clinical features of these kindreds are consistent with a predominant PRTH phenotype.

Neonatal hyperthyroidism has mostly been described in the context of maternal Graves' disease. It has been estimated that about 0.2% of pregnant women have Graves' disease; however only 1% of the children born to these women are described as having hyperthyroidism. In most of the cases, the disease is due to maternal antibodies transferred from the mother into the fetal compartment, which stimulate the fetal thyroid by binding to the thyrotropin (TSH) receptor. In this form of neonatal hyperthyroidism, thyrotoxicosis disappears with the clearance of the maternal antibodies and usually signs disappear during the first 4 months of life. Rare forms of persistent, nonimmune neonatal hyperthyroidism are explained by molecular abnormalities of the TSH receptor. Prematurity is frequent, as well as hypotrophia. Tachycardia, goiter, hyperexcitability, poor weight gain, hepatomegaly and/or splenomegaly, stare and/or eyelid retraction are among the most frequent neonatal thyrotoxicosis clinical signs. Diagnosis is based on the determination of the blood level of thyroxine (T4), triiodothyronine (T3), and TSH. Even if these levels are normal in the cord blood, tests should be repeated 3 to 10 days later to detect possible delayed appearance of the disorder. These parameters should be interpreted according to the age of the neonate. To confirm the immune nature of this hyperthyroidism, thyroid-stimulating immunoglobulins (TSI) should be determined. The TSI determination is crucial in identifying nonimmune causes of neonatal hyperthyroidism: in this neonatal hyperthyroidism, TSI are not detected, either by radioreceptor assay and/or by functional assay, and molecular studies are needed to identify the mutation. Mutation of the TSH receptor leading to its constitutive activation and to neonatal hyperthyroidism have been described. Germline mutations are found in hereditary hyperthyroidism; de novo germline mutations can cause sporadic congenital hyperthyroidism.