Mechanisms of thyrotropin receptor–mediated phenotype variability deciphered by gene mutations and M453T-knockin model
Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli Linnossuo, Johanna Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, FinnGen FinnGen, Mary Pat Reeve, Jukka Kero
Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli Linnossuo, Johanna Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, FinnGen FinnGen, Mary Pat Reeve, Jukka Kero
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Research Article Endocrinology Genetics

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

Abstract

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.

Authors

Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli Linnossuo, Johanna Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, FinnGen FinnGen, Mary Pat Reeve, Jukka Kero

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Figure 3

Functional characterization of the TSHR mutations in vitro.

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Functional characterization of the TSHR mutations in vitro. (A) Accumula...
(A) Accumulation of cAMP in basal condition (constitutive activity) and after stimulation with high dose (10 mU/mL) of bovine TSH between TSHR WT, I640V, and S237N mutants using transiently transfected COS-7 cells. TSHR cell surface expression was assessed in live cells using flow cytometry (FC). (B) Representative FC profiles of COS-7 cells transfected with WT TSHR (red), S237N (blue), or I640V (orange) variants or negative control (green). (C) A graph with the relative median fluorescence intensity (MFI) of COS-7 cells expressing the TSHR WT or I640V and S237N mutants (n = 4). (D and E) Effect of different amino acid substitutions at TSHR position 237 (p.S237A, p.S237D, and p.S237V) on basal TSH-stimulated (10 mU/mL) cAMP compared with WT and previously described TSHR CAMs M453T and D633H mutants. (F) Production of cAMP in a response to different doses of human chorionic gonadotropin (hCG) using cells expressing TSHR WT, I640V, or S237N variants. Box-and-whisker plots show 95th–5th percentile, with median (line) and mean (+) symbols. The cAMP concentration were tested with 2-way ANOVA, whereas relative cell membrane TSHR expression was tested with 1-way ANOVA. Both tests were corrected for multiple hypothesis. The hCG-response assay was tested using univariate ANOVA. Family-wise error rate was controlled using Dunnett’s method, considering WT TSHR as the reference group in both conditions. Data are representative of 2 or 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.