Detection of thyroid-stimulating hormone receptor and Gs α mutations: in 75 toxic thyroid nodules by denaturing gradient gel electrophoresis

Abstract

The actual frequency of constitutively activating thyrotropin receptor or Gsalpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7-9 of the Gsalpha protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser-->Leu) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or Gsalpha mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsalpha 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 Gsalpha genes as the molecular cause of these hot nodules.

Autonomously Functioning Thyroid Nodule Associated with Thyrotoxic Periodic Paralysis

“…The frequency of these mutations varies among reports but amounts to 50-80% [19][20][21]. In contrast, it has been reported to be very low in Japan [23].…”

“…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]. In contrast to the TSHR gene, the incidence of the mutations in Gsα is reportedly low in Europe as well as in Japan [18][19][20]22]. In the present report, we tested whether our patient harbored the mutations in exons 9 and 10 of the TSHR gene and those in exons 8 and 9 of the Gsα gene, in which the known hot spots for mutations exist [19,28].…”

“…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.…”

“…The frequency with which these activating mutations in the TSHR gene are found in AFTN is variable, but reaches up to 80% [19]. In contrast, such mutations in the Gsα gene are reportedly less frequent [19,20]. AFTN is a frequent cause of hyperthyroidism in Europe but very rare in Japan [21], and the frequency of the gain-of-function mutations in the TSHR gene and/or Gsα gene is reportedly very low in Japan [22,23].…”

“…Here we report a case with AFTN who suffered from attacks of TPP. In addition, we analyzed mutations in exons 9 and 10 of the TSHR gene and exons 8 and 9 of Gsα gene, which are frequently detected in AFTN from European countries [18][19][20], as well as mutations in CACNA1S and KCNE3, which are identified as the cause of FHPP [5,6,8].…”