Endocrine Abstracts

Study of the prevalence and mechanisms of action of TSH receptor and Gs protein alfa-subunit mutations, in toxic multinodular goiter and toxic adenoma from Galicia (Spain)

F Palos¹, O Perez¹, V Alvarez-Iglesias², J Cameselle², F Barreiro², D Araujo¹, R Argueso², M Botana², JM Cabezas², L Dominguez¹, T Martinez², J Nuño², JC Rueda² & J Lado-Abeal¹

¹UETeM. Departament of Medicine. University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain; ²Grupo Gallego para el Estudio del Adenoma Toxico y Bocio Multinodular Toxico, Galicia, Spain.

Toxic adenomas (TA) and toxic multinodular goiters (TMNG) are frequent causes of hyperthyroidism in Galicia, an endemic goiter area. In some European countries, 40–80% of toxic goiters are caused by TSH receptor (TSHr) and Gs alfa-subunit (Gsa) mutations that activate cAMP pathway.

Aims: To study 1) the prevalence of TSHr and Gsa mutations in TMNG and TA from Galicia, 2) the clonality of sequenced samples, 3) the constitutive activity of the identified mutations.

Material and methods: 1) Mutations search. 62 thyroid samples were obtained at surgery: TA (31), TA within MNG (20) and TMNG (11). TSHr exons 10 to 1 and Gsa exons 8 and 9 were PCR amplified followed by sequencing using dRhodamine. Identified mutations were confirmed by enzymatic restriction analysis or TA-cloning plus sequencing. 2) Clonality was performed by HUMARA assay, performing fragment analysis with 6FAM-labeled primer. 3) Constitutive activity. Mutants D633Y, T632I, F631L, L629F, Δ619, D619G, I568T were cloned in pSVL-TSHr; COS-7 cells were transfected with mutants plus a CRE-LUC reporter and incubated with and without hrTSH.

Results: 1) Mutations were found in 32 samples: 19 TA (65.5%), 13 TA within MNG (56.5%), and 0 TMNG; 28 mutations (87.5%) were found in TSHr exon 10, one in exon 9 and none in exons 1 to 8; only 3 mutations were in Gsa. TSHr D727E was found in 9%, P52T in 7.5% and 3 silent mutations were at aa 459. 2) 68% of samples were polyclonal, and 3) mutants had 2.5–6 times higher CRE-Luc activity than TSHr WT.

Conclusions: 1) In Galicia, 53% of toxic goiters have TSHr and Gsa mutations, 2) 68% of our samples were polyclonal, probably due to contamination by non-tumoral tissue, 3) tested mutants shown higher constitutive activity than TSHr-WT, suggesting that they are the cause the hyperthyroidism.

Financed by FIS PI030401, XUNTAPGIDIT04PXIC20801PN

Endocrine Abstracts (2006) 11 P809