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 (TS HR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese pat ients because no TSHR activating mutation has been found by previous incomp lete studies. Design: A typical AFTN developed in a 69-year-old Japanese woman was studie d. Methods: The entire exon 10 of the TSHR cDNA was sequenced. Functional stud ies were done by site-directed mutagenesis and transfection of a mutant con struct into COS-7 cells. Results: We identified a novel heterozygous TSHR gene mutation, Leu512-->Ar g (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the a djacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TS HR expression vector exhibited a 3.3-fold increase in basal cAMP level comp ared with that of cells transfected with wild-type TSHR DNA, confirming tha t 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 m utagenesis study encompassing residues 505-513 revealed that mutations invo lving 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 dev eloped in Japanese patients remains to be elucidated. In addition, function al studies suggested that amino acid residues in the third transmembrane he lix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with a nother helix.