Mechanisms of thyrotropin receptor-mediated phenotype variability deciphered by gene mutations and M453T-knockin model.

The clinical spectrum of thyrotropin receptor-mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T-knockin model revealed that the phenotype was dependent on the mutation's signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

Screening for Mutations in Isolated Central Hypothyroidism Reveals a Novel Mutation in Insulin Receptor Substrate 4.

Background: Central hypothyroidism (CeH) is a rare condition affecting approximately 1:16 000- 100 000 individuals. Congenital forms can harm normal development if not detected and treated promptly. Clinical and biochemical diagnosis, especially of isolated CeH, can be challenging. Cases are not usually detected in neonatal screening, which, in most countries, is focused on detection of the more prevalent primary hypothyroidism. Until now, five genetic causes for isolated CeH have been identified. Here we aimed to identify the genetic cause in two brothers with impaired growth diagnosed with CeH at the age of 5 years. We further evaluated the candidate gene variants in a large genetic database. Methods: Clinical and biochemical characterization together with targeted next-generation sequencing (NGS) was used to identify the genetic cause in a family of two brothers presenting with CeH. Screening of insulin receptor substrate 4 (IRS4) variants was carried out in the FinnGen database. Results: A novel monoallelic frameshift mutation c.1712_1713insT, p.Gly572Trp fs*32 in the X-linked IRS4 gene was identified by NGS analysis in both affected males and confirmed using Sanger sequencing. Their mother was an unaffected carrier. In addition to the declined growth at presentation, central hypothyroidism and blunted TRH test, no other phenotypic alterations were found. Diagnostic tests included head MRI, thyroid imaging, bone age, and laboratory tests for thyroid autoantibodies, glucose, insulin and glycosylated hemoglobin levels. Examination of the IRS4 locus in FinnGen (R5) database revealed the strongest associations to a rare Finnish haplotype associated with thyroid disorders (p = 1.3e-7) and hypothyroidism (p = 8.3e-7). Conclusions: Here, we identified a novel frameshift mutation in an X-linked IRS4 gene in two brothers with isolated CeH. Furthermore, we demonstrate an association of IRS4 gene locus to a general thyroid disease risk in the FinnGen database. Our findings confirm the role of IRS4 in isolated central hypothyroidism.

Hyperthyroidism and Papillary Thyroid Carcinoma in Thyrotropin Receptor D633H Mutant Mice.

BACKGROUND: Constitutively active thyrotropin receptor (TSHR) mutations are the most common etiology of non-autoimmune hyperthyroidism (NAH). Thus far, the functionality of these mutations has been tested in vitro, but the in vivo models are lacking. METHODS: To understand the pathophysiology of NAH, the patient-derived constitutively active TSHR D633H mutation was introduced into the murine Tshr by homologous recombination. RESULTS: In this model, both subclinical and overt hyperthyroidism was observed, depending on the age, sex, and genotype. Homozygous mice presented hyperthyroidism at two months of age, while heterozygous animals showed only suppressed thyrotropin. Interestingly, at six months of age, thyroid hormone concentrations in all mutant mice were analogous to wild-type mice, and they showed colloid goiter with flattened thyrocytes. Strikingly, at one year of age, nearly all homozygous mice presented large papillary thyroid carcinomas. Mechanistically, this papillary thyroid carcinoma phenotype was associated with an overactive thyroid and strongly increased stainings of proliferation-, pERK-, and NKX2-1 markers, but no mutations in the "hot-spot" areas of common oncogenes (Braf, Nras, and Kras) were found. CONCLUSIONS: This is the first study to reveal the dynamic age-, sex-, and genotype-dependent development of NAH. Furthermore, the study shows that a constitutively active TSHR can trigger a malignant transformation of thyrocytes.