SOMATIC MUTATIONS IN THE THYROTROPIN RECEPTOR GENE AND NOT IN THE G(S)ALPHA PROTEIN GENE IN 31 TOXIC THYROID-NODULES

Studies on frequency and distribution pattern of TSH receptor (TSHR) a nd G(s) alpha protein (gsp) mutations in toxic thyroid nodules (TTNs) reported conflicting results, most likely also related to the differen t screening methods applied and the investigation of only part of exon 10 of the TSHR. Therefore, we screened a consecutive series of 31 TTN s for both TSHR and gsp mutations by direct sequencing of exon 9 and t he entire exon 10 of the TSHR gene and exons 7-10 of the gsp gene. Som atic TSHR mutations were identified in 15 of 31 TTNs. TSHR mutations w ere localized in the third intracellular loop (Asp(619)Gly and Ala(623 )Val), the sixth transmembrane segment (Phe(631)Leu and Thr(632)Ile, A sp(633)Glu) and the second extracellular loop (Ile(568)Thr). One mutat ion was found in the extracellular TSHR domain (Ser(281)Asn). Two new TSHR mutations were identified. One involves codon 656 in the third ex tracellular loop (Val(656)Phe). The other new mutation is a 27-bp dele tion in the third intracellular loop resulting in deletion of 9 amino acids at codons 613-621. Transient expression of the new TSHR mutation s in COS-7 cells demonstrated their constitutive activity. No mutation was found in exons 7-10 of the gsp gene. This finding was confirmed b y an allele-specific PCR for mutations in gsp codons 201 (Arg --> His, Cys) and 227 (Gln --> His, Arg). Our data indicate that constitutivel y activating TSHR mutations can be found in 48% of TTNs and thus curre ntly represent the most frequent molecular mechanism known in the etio pathogenesis of TTNs. Moreover, the absence of gsp mutations in our se ries argues for an only minor role of these mutations in TTNs. Constit utive activation of the TSHR by a deletion in a region that might be i nvolved in G protein coupling of the TSHR offers new insights into TSH R activation.