DIVERSITY AND PREVALENCE OF SOMATIC MUTATIONS IN THE THYROTROPIN RECEPTOR AND G(S)ALPHA GENES AS A CAUSE OF TOXIC THYROID ADENOMAS

A total of 33 different autonomous hot nodules from 31 patients, origi nating mainly from Belgium, were investigated for the presence of soma tic mutations in the TSH receptor and G(s) alpha genes. This constitut es an extension of our previous study, including the first 11 nodules of the series. The complete coding sequence of the TSH receptor gene a nd the segments of G(s) alpha known to harbor mutations impairing guan osinetriphosphotase activity were studied by direct sequencing of geno mic DNA extracted from the nodules. DNA from the juxtanodular tissue o r peripheral white blood cells was analyzed in all patients to confirm that the mutations identified were somatic. Twenty-seven mutations (8 2%) were found in the TSH receptor gene, affecting a total of 12 diffe rent residues or locations. All these mutations but 2 (see below) have been identified previously as activating mutations. Only 2 mutations were found in G(s) alpha (6%). In 4 nodules, no mutation was detected. Five residues (Ser281, Ile486, Ile568, Phe631, and Asp633) were found mutated in 3 or 4 different nodules, making them hot spots for activa ting mutations. Phe631 and Asp633 belong to a cluster of 5 consecutive residues (629-633) in the N-terminal half of transmembrane segment VI , which harbor together 44% of the mutations identified in this cohort . Two novel mutations were identified: a point mutation causing substi tution of Phe for Leu at position 629 (L629F); and a deletion of 12 ba ses removing residues 658-661 at the C-terminal portion of exoloop 3 ( del658-661). When tested by transfection in COS-7 cells, both mutant r eceptors display increase in constitutive stimulation of basal cAMP ac cumulation. Although it is still capable of binding TSH, the del658-66 1 mutant has completely lost the ability to respond to the stimulation by the hormone. Our results demonstrate that, in a cohort of patients from a moderately iodine deficient area, somatic mutations increasing the constitutive activity of the TSH receptor are the major cause of autonomous hot nodules.