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

Abstract

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 sitedirected mutagenesis and transfection of a mutant construct into COS-7 cells. Results: We identi®ed 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, con®rming 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 ®rst 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 a-helix, possibly making interhelical bonds with another helix.

Autonomously Functioning Thyroid Nodule Associated with Thyrotoxic Periodic Paralysis

“…In contrast, it has been reported to be very low in Japan [23]. However, Kosugi et al analyzed the entire exon 10 of the TSHR gene and found the first activating mutation in the TSHR gene in a Japanese patient with AFTN [17]. Vanvooren et al consistently identified four mutations in the TSHR gene in 10 Japanese AFTN patients and thus suggested that activating mutations in the TSHR gene may not be rare in the Japanese population [18].…”

“…Whereas we did not examine other exons of TSHR and Gsα genes, our results suggest that oncogenic mutations of these genes might not play a major role in the development of AFTN in Japan. Further studies on a larger number of AFTN patients are clearly required to determine the actual prevalence of gain-of-function mutations in the TSHR and Gsα genes in the Japanese population since previous studies have analyzed these mutations in only a small number of AFTN cases [17,18].…”

“…The polymerase chain reaction (PCR) was performed in a 25 µl reaction volume containing 100-200 ng of genomic DNA, 10 pmol of each upstream and downstream primer, 1.25 units of Taq polymerase (Takara, Otsu, Japan), 0.2 mM each dNTP, and 2 mM MgCl 2 . All the reactions were started by an initial denaturation at 95°C for 5 min (except for exon 9 of TSHR at 93°C for 2 min 30 sec and exon 10 of TSHR at 94°C for 2 min), followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec (except for exon 9 of TSHR at 61°C for 30 sec) and extension at 72°C for 1 min [17,18,22]. Aliquots of PCR products were run in 1% agarose gel electrophoresis and visualized with ethidium bromide to verify their quality.…”

“…TSH binds to its receptor (TSHR) and activates the α-subunit of the stimulatory G protein (Gsα), leading to adenylate cyclase (AC) activation and subsequently cyclic AMP (cAMP) production. In autonomously functioning thyroid nodule (AFTN), which is characterized by autonomous, TSH-independent hypersecretion of thyroid hormones, gain-of-function mutations have been identified in TSHR and Gsα genes [17][18][19][20]. These mutations result in the constitutive activation of the AC-cAMP pathway, leading to cellular hyperfunction and proliferation.…”

An interactive web-tool for molecular analyses links naturally occurring mutation data with three-dimensional structures of the rhodopsin-like glycoprotein hormone receptors

“…The second example focuses on three tightly-packed triad-like interacting hydrophobic residues, which constitute a hydrophobic patch for stabilization of the basal TSHR conformation. Several naturally occurring single CAMs are known at these positions ( Figure 2B) and might be related to hyperfunctioning thyroid nodules (Alberti et al, 2001;Esapa et al, 1999;Gozu et al, 2006;Holzapfel et al, 1997;Kosugi et al, 2000;Trulzsch et al, 2001). Each of these CAMs causes disruption of the interacting triad, leading to constitutive TSHR activation.…”