The broad, long term objectives of this research proposal are to gain knowledge on the mechanisms of thyroid hormone action and the structural requirements of transport proteins for interaction with the hormone. The proposed approach to achieve these goals is to study naturally occurring mutations in man, namely, generalized resistance to thyroid hormone (GIRTH) and inherited defects of thyroxine-binding globulin (TBG). Recently developed methods for the clinical diagnosis of GIRTH will be adapted for use in an outpatient setting and validated by their application to non-resistant subjects. Epidemiological studies will include determination of the prevalence of GIRTH by the screening of newborns and children with the attention deficit/hyperactive disorder, a condition suspected of having high incidence of GIRTH. These data will provide a tissue diagnosis of GIRTH, cultures of fibronectin and collagen synthesis and their mRNA accumulation. Genetic linkage studies by restriction fragment polymorphism using the thyroid hormone receptor probes, c-erb-a alpha and beta will help localize the defect of GIRTH and provide an explanation for its heterogenous phenotype. Data form linkage analysis will help identify affected subjects suitable for the amplification of a specific thyroid hormone receptor gene in order to characterize the defect at the molecular level by gene sequencing. The remaining 7 of 11 TBG mutants so far identified will be characterized by gene sequencing. Their frequency will be determined in subjects with TBG defects which have been detected by standard clinical laboratory means. The properties of mutant TBG molecules, studies by either direct physiochemical methods or after their expression by gene transfection into heterologous cells, will serve to deduce the molecular requirements of normal TBG for its biological function. Studies on the regulation of TBG gene expression will exploit information derived from the structures of mutant TBG genes which exhibit abnormal expression of a normal TBG molecule, and data obtained from the expression of chimeric gene constructs in transfected liver cells. The effect of estrogen on the post-translational modification of the TBG molecule will be studied by analysis of the oligosaccharide moieties of TBG expressed in transfected rat liver cells in the presence or absence of estrogens. The deletion of each potential site of N-glycosylation by site-directed mutagenesis would provide information on the location of the oligosaccharide chains on the TBG peptide backbone and their roles in the proper folding of the molecule.
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