TSH receptor extracellular region mutations in thyroid functioning nodules: further evidence for the functional role of this region in the receptor activation

IntroductionA causative role for activating mutations of the TSHreceptor (TSHR) has been demonstrated in the majority ofthyroid hyperfunctioning nodules, as well as for thefamilial and sporadic congenital nonautoimmune hyper-thyroidism [1, 2] and also the rare hyperfunctioning car-cinomas [3, 4]. More than 30 different amino acidsubstitutions have been described so far, and their char-acterization, together with in vitro mutagenesis studieshave been exploited for a structure–function analysis of thereceptor, leading to a structural model in which the areasinvolved in the binding to TSH or signal transduction havebeen depicted [5]. The emerging picture suggests theexistence of a molecular constraint which limits the con-stitutive activity of the receptor, removed by the interactionof TSH with the extracellular (EX) portion, but with animportant role played also by the transmembrane regions inthe dimerization process and the functional activation ofthe receptor [5, 6]. Herein we describe the discovery of twounusual heterozygous mutations occurring in two of seventhyroid functioning nodules detected as single nodules or ina multinodular goiter.Materials and methodsSpecimen of nodular tissues were collected and immedi-ately frozen after surgical removal from seven patients withsingle or multinodular hyperfunctioning goiter. Non-nod-ular normal tissue from the contralateral lobe was alsocollected. After RNA extraction and cDNA synthesis, PCRamplification of the exons 9 and 10 of the TSHR gene wasperformed as previously described [3, 4] and the amplifiedproducts sequenced with a BigDye Terminator version 3.1Cycle Sequencing kit in an automated 3130xl analyzer(both from Applied Biosystems, Foster City, CA, USA).The study protocol was approved by the local ethicscommittee.ResultsSequencing of TSH receptor gene in a small cohort ofseven patients with thyroid hyperfunctioning nodulesrevealed the presence, in two cases, of unusual somaticheterozygous mutations. The first was a deletion of D403