Detection of thyroid-stimulating hormone receptor and G(s)alpha mutations:in 75 toxic thyroid nodules by denaturing gradient gel electrophoresis

The actual frequency of constitutively activating thyrotropin receptor or G (s)alpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been repor ted. We studied a series of 75 consecutive TTNs and performed mutation scre ening by the more sensitive method of denaturing gradient gel electrophores is (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimul ating hormone receptor (TSHR) and exons 7-9 of the G(s)alpha protein gene w as determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TS HR mutations were identified. Six TSI-IR mutations were detected only by DG GE, underlining the importance of a sensitive screening method. Novel, cons titutively activating mutations were identified at positions 425 (Ser-->Leu ) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detecte d at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or G(s)alpha mutation n egative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutat ions appear to be a major cause of TTNs (57%), while mutations in G(s)alpha play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or G(s)alpha genes as the molecular cause of these hot nodules.