A novel activating mutation in the thyrotropin receptor gene in an autonomously functioning thyroid nodule developed by a Japanese patient.

OBJECTIVE A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. DESIGN A typical AFTN developed in a 69-year-old Japanese woman was studied. METHODS The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. RESULTS We identified a novel heterozygous TSHR gene mutation, Leu512-->Arg (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, confirming that the mutation was the direct cause of the AFTN. TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505-513 revealed that mutations involving residues other than these three did not show constitutive activation. CONCLUSION This is the first TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with another helix.