Biochemical characterization of four novel mutations in the thyroid hormone receptor beta gene in patients with resistance to thyroid hormone.

In resistance to thyroid hormone (RTH), decreased tissue responsiveness to thyroid hormones is usually caused by mutations in the thyroid hormone receptor beta (THRB) gene. Subsequently, in serum the level of thyroid stimulating hormone (TSH) is not suppressed despite increased concentrations of thyroxine (T4) and triiodothyronine (T3). In our laboratory, DNA sequences of exon 7 to 10 in the THRB gene have been analysed in individuals with biochemical signs of RTH. Four novel point mutations were identified (I250T, A279E, T327A and L440P) and their effects on T3 binding activity characterized. The mutations were introduced into a vector carrying the wild-type THRB cDNA by in vitro mutagenesis. T3-binding activity was measured by a filter-binding assay procedure in receptors generated from the vector by in vitro transcription and translation. Specific binding was calculated as total activity subtracted by non-specific activity. The association constants (Ka) of the wildtype (WT) and mutant receptors were determined by Scatchard analysis. No specific T3-binding was observed for the receptor with the A279E mutation. The binding affinity was reduced by 74% in the T327A mutant and by about 50% in the I250T and L440P mutants compared to the WT receptor (Ka = 4.2 x 10(10) L/mol). The reduction of T3-binding affinity caused by the four mutations in our study is comparable to the effects of THRB gene mutations found in other patients with RTH and supports the assumption that the signs of RTH observed in our patients are caused by the mutations.