Rarity of oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in Japan

Constitutively activating mutations have recently been identified in the thyrotropin receptor (TSHR) of hyperfunctioning thyroid adenomas and familial hyperthyroidism. In the present study, we evaluated the frequency of constitutively activating TSHR mutations in a large series of autonomously functioning thyroid nodules (AFTNs) in Japan. Forty-five AFTNs (38 solitary hyperfunctioning thyroid adenomas and 7 toxic multinodular goiters) were analyzed. Genomic DNA was extracted from paraffin-embedded tissue sections, from which DNA fragments encoding the mutational hot spots of the receptor (the third cytoplasmic loop and the sixth transmembrane segment) were amplified by polymerase chain reaction. In the single-stranded conformation polymorphism (SSCP) analysis, only one hyperfunctioning adenoma (no. 21) displayed a migration abnormality. In sequence analysis, an unusual mutation of alternate three-base deletions at nucleotides 1953-1957 (AAA GAT ACC to AAG TCC), resulting in one amino acid deletion (Asp at 619) and one conservative amino acid substitution (Thr to Ser at 620), was identified in tumor DNA but not in leukocyte DNA of no. 21. Further, the normal sequence in these regions was confirmed in 10 randomly selected samples with normal migrating patterns in SSCP analysis. The functional property of the mutant with delta 619 and T620S (designated TSHR delta 619) was then evaluated with in vitro mutagenesis and transfection studies. Unexpectedly, however, there were no significant differences in TSH binding affinity, and basal and TSH-stimulated levels of cAMP and inositol 1,4,5-triphosphate between the TSHR delta 619 and the wt-TSHR. In conclusion, the incidence of the constitutively activating TSHR mutations in AFTNs appears to be low in Japan. The oncogenic potential of a novel somatic mutant TSHR delta 619 identified in a hyperfunctioning adenoma in this study is at present uncertain because of its intact function.