Genetic and functional studies on the thyroid hormone axis

Abstract: This thesis has two main parts. The first is based on the first three papers with a focus on understanding the etiology and pathogenesis of thyroid malfunction and its effects on different organs. The second is about gaining an in-depth understanding of the role of the TSHr in adipose tissue and, for this purpose, a tissue-specific knockout mouse model of TSHr in adipocytes was generated and used as an experimental tool. In Paper I, a family meeting clinical and biological criteria for autosomal dominant non-autoimmune hyperthyroidism was enrolled; we investigated the pathogenesis of severe neuromuscular symptoms in the index patient. The TSHr gene was investigated by direct sequencing. Sequence analysis revealed a heterozygous missense mutation, glycine 431 for serine in the first transmembrane segment. Functional properties of the mutant TSHr revealed constitutive activity when investigated during transient expression in COS-7 cells. There was no indication of autoimmune disorder. All symptoms disappeared upon treatment. The data imply that neuromuscular symptoms can be caused by excessive thyroid levels rather than by autoimmunity In Paper II, we addressed the questions of whether TSHr antibody-negative Graves disease is associated with somatic mutations in the TSHr or Gsalpha genes and whether histopathologically defined thyroid lesions are associated with such mutations. We investigated 43 patients undergoing thyroid surgery. The patients were diagnosed with sporadically occurring TSAbs-negative Graves disease (n = 11), TSAbs-positive Graves disease (n = 4), hyperfunctioning follicular adenoma (n = 9), nonfunctioning follicular adenoma (n = 5), and toxic (n = 9) or non-toxic multinodular goiter (n =5). The diagnoses were based on the clinical, biochemical, radiological, and histopathological criteria. Antibody detection assays were performed using porcine TRAb. In the thyroid tissue DNA, all exons of the TSHr gene were sequenced from the 15 cases of Graves disease; in the remaining 28 cases, exons 9 and 10 of the TSHr gene were sequenced. Exons 8 and 9 of the Gsalpha gene were sequenced in all 43 cases. No mutations, but three germ-line polymorphisms, were found in patients withTSHr antibody-negative Graves disease. Two heterozygous somatic TSHr mutations were found in two hyperfunctioning adenomas and in two toxic multinodular goiters. The lack of TSHr and Gsalpha mutations in TSHr antibody-negative Graves disease patients indicates that such mutations are neither primary nor secondary events in this disease. In Paper III, we studied the effect of hypothyroidism and thyroxin therapy on renal function in children with long-term follow-up. The glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were studied in 31 patients with symptomatic acquired hypothyroidism. Children with hypothyroidism were examined before starting thyroxin therapy and 1 week and 1, 3, and 6 months after starting thyroxin therapy. Thirteen patients were reinvestigated at 6, 12, 18, 36, and 60 months after initiating thyroxin therapy. In children investigated before or within 1 week after starting thyroxin therapy, GFR and ERPF were < 2 SD in 58% and 45%, respectively. 31% and 6% of the children studied 1 to 6 months after thyroxin therapy had a GFR and ERPF < 2 SD. At the last investigation, 1 to 5 years after the start of treatment, the GFR was still significantly lower in children with hypothyroidism than in controls. The data imply that acquired hypothyroidism during childhood may have a longterm impact on renal function. In Paper IV, we have selectively removed the TSHr gene in adipocytes by using the Cre-loxP recombination system. Mice lacking TSHr in adipocytes were apparently normal. In epididymal adipocytes, TSH-induced sensitivity was ten times lower in the targeted animals. However, TSH-induced maximum response, catecholamine-induced lipolysis, and insulin-induced inhibition of lipolysis were unaltered. Adipocyte sizes were increased in the targeted animals. Thus, our results indicate that TSHr has a physiological role in adipocyte growth and development.