Severe congenital hyperthyroidism caused by a germ-line neo mutation in the extracellular portion of the thyrotropin receptor

Gain of function mutations in the TSH receptor (TSHR) have been identified as the molecular basis for congenital and acquired forms of autonomous thyroid function. Herein, we report the molecular characterization of a case of severe congenital hyperthyroidism with a history of hyperthyroidism in the paternal aunt and the paternal grandmother, who were both found to be heterozygous for a mutation (R528H) located in exon 10 of the TSHR gene. Functional expression of the mutant TSHR-R528H in COS-7 cells, however, did not result in constitutive activity of the TSHR. Subsequent analysis of exons 1-9 led to the detection of an additional heterozygous mutation (S281N) in the patient, but not in other family members. Interestingly, the latter mutation is located in the extracellular domain of the TSHR, and functional studies revealed a marked increase in basal cAMP levels when the mutant receptor was expressed in COS-7 cells. To address the question of whether both mutations were present on the same allele, a double mutant TSHR (S281N/R528H) was generated and characterized. These functional studies in conjunction with RT-PCR analysis of thyroid tissue obtained from subtotal thyroidectomy performed at the age of 6 yr revealed that the patient bears two distinct mutations on different alleles: the familial paternal R528H mutation to be regarded as a polymorphism and a de novo mutation (S281N) on the maternal allele accounting for the clinical picture. Thus, the main conclusions to be drawn from this case are 1) a search for mutations in cases of congenital nonautoimmune hyperthyroidism should not remain restricted to exon 10 of the TSHR gene, because germ-line gain of function mutations of the TSH receptor can be located outside of the transmembrane core of the receptor; and 2) this case illustrates the necessity for careful functional characterization of any novel mutation before a causal relationship to hyperthyroidism can be established.