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.

Table S3: List of TSHR primers used for Sanger-sequencing.
Target site in TSHR Forward primer Reverse primer Ta Ta, annealing temperature of the primer pairs.
Table S4: List of primers used for quantitative RT-PCR.

Clinical characteristics of familial and sporadic NAH and cases with toxic thyroid nodules
Family A was nonconsanguineous kindred of Polish origin, with overt NAH across three generations (Figure 1).S3 and Table S1).Antibody tests for TSHR, thyroglobulin, and thyroid peroxidase were also negative, and thyroid ultrasound imaging was normal for both subjects in family C.
In the family D, the index case (#293) and his brother developed overt symptomatic NAH at the age of 13 months and 3 years, respectively, while the patients' mother and the sibling were asymptomatic.At the time of recruitment, his father had low serum TSH, but normal fT4 concentration with no hyperthyroid symptoms (Figure 1).

Patients with sporadic nonautoimmune congenital hyperthyroidism and toxic thyroid nodules
Patient #121 was born at 37 + 6 weeks of gestation as a third child to euthyroid parents with no history of thyroid disease (Supplemental Table S1).There were no complications or symptoms of hyperthyroidism during pregnancy or delivery, and no TSHR-stimulating antibodies were detected in the serum of the child or the mother.The umbilical cord blood TSH concentration, measured at birth during routine hypothyroidism screening, was below the detection limit.Diagnosis of hyperthyroidism was confirmed at the first day of life with very high serum fT4 concentration (73.3 pmol/L, reference: 10 -36 pmol/l) and TSH below the detection limit (<0.03 mU/L) (Figure 2A).He had normal linear growth (Figure 2B), but head circumference was initially -2 SD in Finnish children but increased to + 2 SD (Supplemental

Figure S3
).A total thyroidectomy was performed at the age of 6.6 years due to the permanent requirement for antithyroid medication and high variation in thyroid function test, as illustrated in Figure 2A.Thyroidectomy led to severe complications including transient bilateral palsy of the laryngeal nerve, permanent hypocalcemia and significant weight gain (Figure 2C).While thyroid ultrasound showed a normal size and location, the iodine uptake test revealed a relatively high accumulation of radioactivity in both lobes (Figure 2D).

Histological analysis of the thyroid showed normal follicles of variable size, several follicles
with vacuoles, and homogenous colloid staining (Figure 2E).
We  3A) and multiple nodules and hemigoiter in the other patient (#230).In both cases, the thyroid 99m Tc pertechnetate scan revealed high radioactive tracer uptake specifically in the nodules (Figure 3B).The patients underwent hemithyroidectomy without complications, and hyperthyroidism was resolved.
Histological analysis revealed that the nodules consisted of follicles of variable sizes, with only a small amount of colloid, a thick thyrocyte epithelial layer (Figure 3C), and diffuse hyperplastic thyrocytes in multiple nodules (Figure 3D).

Genetic screening of TSHR mutations in patients with CH or resistance to TSH
We previously utilized a thyroid gene panel to screen for mutations in individuals with congenital hypothyroidism (CH) (15) and found twins with a heterozygous variant in TSHR (Arg519Cys) (#67 and 68, Supplemental Table S2).Here, we performed segregation analysis of the family members and found that the father (#137) was a euthyroid carrier and lacked an additional rare thyroglobulin variant (TG, c.6952G>A, p.Asp2318Asn), that the twins and euthyroid mother possessed (#139, Table S2).Furthermore, in a new cohort of 30 patients with CH or resistance to TSH (RTSH), we identified a known pathogenic homozygous TSHR mutation (p.Pro162Ala) (16) in a child with RTSH diagnosed before 7 years of age (#143, Supplemental Tables S1 and S2).The patient´s parents were heterozygous carriers with slightly elevated serum TSH concentrations (#144 and #145, Supplemental Table S1).

Extended structural and mechanistic insights into the I640V and S273N CAMs
To explore the mechanism leading to constitutive receptor activation by the newly identified natural variant p.I640V, some general and detailed aspects of receptor activation, previously (17) and recently (12)

*
Classification: ID = number of the participant, A = pathogenic mutation (based on segregation, literature, and in vitro experiments), B = benign variant, VUS = variant of unknown significance, gnomAD MAF = minor allele frequency in The Genome Aggregation Consortium database.

Figure S1 .
Figure S1.Thyroid function tests (TSH, T4v and T3v concentrations) and a dose of antithyroid medication during pregnancy of the patients A) #266 and B) #279 with TSHR CAM Ile640Val mutation.C) Graph of gestational age specific reference ranges for total human chorionic gonadotropin (hCG) levels during pregnancy modified fromKorevaar et al. (10)  showing a peak in the 9 th and 10 th week of gestation.

Figure S3 .
Figure S3.Additional growth-, head growth-and weight curves of the children with TSHR CAM mutations.Patient (#75) with TSHR Ile640Val mutation and subclinical hyperthyroidism showing normal A) weight gain, B) linear growth, and C) head growth.D) Head growth curve of the patient (#121) with TSHR L629F mutation during the first two years of his life.

Figure S4 .
Figure S4.Panel A basal and Panel B TSH-stimulated (10mU/ml, lower panel) cAMP secretion of WT and TSHR mutants detected in FinnGen database.Two previously described constitutively active TSHR mutations Y601N and L629F were used as positive controls.Statistical comparisons were calculated using one-way ANOVA with Bonferroni's multiple comparisons test.**p< 0.01; ***p< 0.001.

Figure S5 .
Figure S5.An association of the TSHR variants to disease phenotypes in FinnGen database.Panel A variant rs1023586 (new lead SNP for rs179252 Thyrotoxicosis, TSHR intron 1), and Panel B rs12897126 (TSHR hypothyroidism lead) with the associated risk to hypothyroidism.

Figure S6 .
Figure S6.Generation and genotyping of the TSHR M453T knock-in mice using CRISPR/Cas9 and homology-directed repair technique.A) Genomic location and designed guide RNA (gRNA) construct.B) Middle panel shows, PCR primer locations, expected sizes and location of NlaIII restriction site altered by the TSHR c.1358 T>C mutation.Below is an example of agarose gel electrophoresis with different WT, HET, and HOM genotypes after digestion of the PCR product with NlaIII restriction enzyme.

Figure S8 .
Figure S8.Structural and molecular insights into TSHR variants identified and investigated in this study.Recently determined TSHR WT structures in different activity state conformations were used to highlight the impact of the described TSHR variants on TSHR structure-function relationships.A) The inactive state TSHR WT structure (PDB ID 7t9m (12)) with TSHR variants identified in the FinnGen database and in patients with CH or resistance to TSH.Underlined are newly identified variants.B) The active WT TSHR state structure (PDB ID 7t9i (13)) is highlighted with the CAMs identified and shown in this study.TSH and the Gprotein molecules are presented as surfaces with different colors.The receptor is visualized by a backbone cartoon.The hormone binding induced push-movement of the extracellular LRRD/hinge-TSH complex is indicated, as well as the pull-mechanism via the TSH-hinge interaction.In C) and D) both complexes are in superimposition for comparison at specific structural regions important to explain molecular backgrounds of receptor activation and the associated potential models of constitutive receptor activation.TMH -transmembrane helix, EL -extracellular loop, IL -intracellular loop.

Table S5 :
List of antibodiesPrimary antibodies FACS• Rabbit monoclonal HA-tag IgG (C29F4); Cell Signaling Technology (37245) The patient had no hyperthyroid symptoms, and of linear growth together with advanced bone age and a decrease in body weight at the time of diagnosis, typical for pediatric hyperthyroidism.There was no history of osteoporosis or fractures among the family members.Moreover, all other biochemical tests were normal in hyperthyroid cases (Figure1and Supplemental TableS1).Families B and C were Finnish nonconsanguineous kindreds.Every child had normal umbilical TSH values at neonatal screening for congenital hypothyroidism (CH)(Supplemental TableS1).All affected individuals were initially diagnosed with subclinical hyperthyroidism and had suppressed serum TSH, but normal fT4 and free T3 (fT3) levels.hypertensionat the age of 24 years.At diagnosis of subclinical hyperthyroidism, her TSH concentration was below detection limit (0.06 mU/L, reference: 0.5 -3.6 mU/L), but fT4 (17 pmol/L, reference: 9.0 -19 pmol/l) and fT3 (5.5 pmol/L, reference: 2.6 -6 pmol/L) concentrations were in range (Figure1).
levels 0.06 and <0.03 mU/L, reference: 0.51 -4.3 mU/L; fT4 23.6 and 31.5 pmol/L, reference: 7.7 -12.6 pmol/L) at the age of 12 and 10 years, respectively.Thyroid ultrasound revealed a single nodule in the right lobe (#229, Figure studied two children (#229 and 230) with typical hyperthyroid symptoms, no stimulating TSHR antibodies and overt hyperthyroidism in thyroid function tests at diagnosis (serum TSH received, must be considered.Activation of the TSHR and other GPHRs and supported by a strong dislocation of the Met637 side chain, which is in the group of GPHRs at position 6.48 instead of the more class A GPCR common Trp/Phe6.48(toggleswitchtryptophan).The mutation p.S237N is specifically interesting as it is located at the extracellular leucine rich repeat (LRR) 9, whereby the entire LRR domain is constituted by 11 repeats (amino acids Cys24-Asn288, FigureS8C).Moreover, Ser237 is not in the hormone binding site and not in a direct contact to any other side chain in the LRRD or in the TMD.This is in stark contrast to the known extracellular CAMs in position Ser281 (48-50).Ser281 is close to the TMD and especially the EL1, which brings the Ser281 side chain in direct spatial relation to the tethered ligand-associated activation mechanism.This explains from a structural perspective why at this residue receptor activation (red arrows in FigureS8C, D indicating structural shifts from inactive to active state conformations) can be initiated by a mutation in contrast to position Ser237 without any intra-or intermolecular interaction partner.Of note, further side-chain variations p.S237A, p.S237D, p.S237V did not result in constitutive activation, and this TSHR Weinstein class A GPCR numbering system P6.50), which participates in the constitution of the TMH6 helix-kink that is pivotal for TMH6 outward movement during receptor activation.This TMH6 movement -a hallmark of class A GPCR activation in G-protein coupling -is further reflectedCAM cannot be hyperstimulated by an increased affinity of CG to TSHR (Figure3D), which altogether supports a specific activation mechanism for variant p.S237N.