The actual frequency of constitutively activating thyrotropin receptor or Gsalpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7-9 of the Gsalpha protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser-->Leu) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or Gsalpha mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsalpha play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or Gsalpha genes as the molecular cause of these hot nodules.

Neonatal hyperthyroidism in the absence of maternal autoimmune thyroid disease and without thyroid-stimulating antibodies in the child is rare. We here describe a boy with severe intrauterine hyperthyroidism and advanced bone age in the absence of thyroid-stimulating autoantibodies. After long term antithyroid treatment and relapse of hyperthyroidism, a near-total thyroid resection was performed. The necessity to progressively decrease postoperative thyroid hormone replacement indicates thyroid tissue regrowth in the small thyroid remnant. Analysis of the genomic DNA of the child's peripheral leukocytes showed a G to A base exchange that led to a heterozygous Ser to Asn conversion at position 505 in the third transmembrane region of the TSH receptor (TSHR). The absence of the Ser505 Asn mutation in all other family members identifies the child's TSHR mutation as a sporadic germline mutation. Transient expression of the mutated TSH receptor in COS-7 cells showed a constitutively activated cAMP cascade. We thus identified a new constitutively activating germline mutation. Neonates with persistent nonautoimmune hyperthyroidism should be investigated for TSHR germline mutations. Because of frequent relapses, patients with sporadic congenital nonautoimmune hyperthyroidism should be treated with early subtotal to near-total thyroid resection. Moreover, post-operative radioiodine treatment should be considered.

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L L-Arrestins mediate agonist dependent desensitization of G protein-coupled receptors. Somatic TSH receptor mutations were identified in the majority of hot thyroid nodules. When transiently overexpressed in COS 7 cells these mutations resulted in constitutive activation of the cAMP pathway. However, the in vivo mechanisms and the in vivo desensitization of these TSH receptor mutations are unknown. Moreover, constitutively activated L L-adrenergic receptors are known to be constitutively desensitized. Therefore, we investigated the expression of L Larrestins in toxic thyroid nodules (TTNs) with and without somatic TSH receptor mutation and in cold thyroid

The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.