Generalized thyroid hormone resistance (GTHR) is a familial syndrome characterized by elevated levels of free thyroid hormones, resistance to thyroid hormone action, and inappropriately normal or elevated levels of thyroid-stimulating hormone. Kindreds with GTHR have variables clinical pathology presumably related to differences in regulation by thyroid hormones. Linkage studies in several kindreds with GTHR suggest that abnormalities in the C-erbA-beta thyroid hormone receptor gene are responsible for the syndrome. It has been hypothesized that the mutant receptors may interact with the wild-type receptor and inhibit its function, thus behaving as dominant negative mutations. This project will elucidate the molecular defects in C-erbA-beta in four different kindreds, A, D, S, and W with thyroid hormone resistance. These kindreds have variant phenotypes with affected members from some of these kindreds exhibiting short stature, mental retardation,and hyperactivity. C-erbA-beta cDNAs will be obtained by reverse-transcription-polymerase chain reaction amplifications from fibroblast total RNA and sequenced. Changes from the published wild-type sequence that alter amino acid structure will be verified in genomic sequence, and tested as mutations by measuring the frequency of the sequence alterations in random alleles using allelic specific hybridization. Mutations will be tested for linkage to GTHR, and by constructing mutant receptors and measuring their T3-binding- affinity in vitro. Also, Scatchard plots of mixtures of the wild-type beta-receptor and mutant receptors will be generated to see if a dominant negative effect can be detected in vitro. Finally, transformed fibroblast lines will be established from these kindreds and the relative steady-state levels of C-erbA-beta 1, and C-erbA-alpha1, and C-erbA alpha2, measured in primary and transformed fibroblasts of affected and unaffected kindred members. Correlation of the c-erbA-beta mutations with the tissue-specific clinical pathology in these four kindreds will help elucidate the tissue- specific roles of the beta-receptor. This project will also provide genetic tests for GTHR for neonates within the kindreds which may have therapeutic value in the future. Isolation and characterization of these putative, mutant beta-receptors will provide new insights into the complex mechanisms of thyroid hormone action in man.
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