Rapid molecular diagnosis of mutations associated with generalized thyroid hormone resistance by PCR-coupled automated direct sequencing of genomic DNA: detection of two novel mutations.

Abstract

Generalized thyroid hormone resistance (GTHR) is a syndrome characterized by tissue nonresponsiveness to thyroid hormones and by variable clinical phenotype manifestations. This syndrome has also been implicated as a predisposing factor in some cases of attention deficit-hyperactivity disorder (ADHD). GTHR results from single mutations in the gene encoding the thyroid hormone receptor. These mutations are clustered in two major sites surrounding the ligand-binding domain. Mutations in 10 previously described patients as well as in five new THR cases have been identified using PCR amplification of genomic DNA coupled with automated direct sequencing with commercially available "universal" fluorescent dye-labeled primers. This strategy allows for the accurate and automated base-calling of normal and mutated nucleotides at the same position in a heterozygote. The rapid molecular diagnostic protocol, from whole blood to DNA sequence data, takes approximately 15 hr, allowing for rapid, efficient, and unambiguous direct detection of the mutant alleles.