Genetic and Functional Studies of Patients with Thyroid Dyshormonogenesis and Defects in the TSH Receptor (TSHR)

Abstract

Genetic defects in the TSH receptor (TSHR) can cause poor thyroid differentiation (thyroid dysgenesis) and/or thyroid malfunction (thyroid dyshormonogenesis). The phenotype spectrum is wide: from severe congenital hypothyroidism to mild hyperthyrotropinemia. Over 250 TSHR variants have been published, many uncharacterized in vitro. We aimed to genetically characterize patients with thyroid dyshormonogenesis with TSHR defects and to study in vitro the effect of the genetic variants to establish the genotype-phenotype relationship. Pediatric patients with thyroid dyshormonogenesis (160 patients, Catalan CH neonatal screening program, confirmation TSH range: 18.4-100 mIU/L), were analyzed by a high-throughput gene panel. In vitro studies measuring the TSH-dependent cAMP-response-element activation were performed. Five patients with mild or severe thyroid dyshormonogenesis presented six TSHR variants, two unpublished. Each variant showed a different in vitro functional profile that was totally or partially deleterious. Depending on the genotype, some of the variants showed partial deficiency in both genotypes, whereas others presented a different effect. In conclusion, the percentage of patients with thyroid dyshormonogenesis and candidate variants in TSHR is 3.13%. Our in vitro studies contributed to the confirmation of the pathogenicity of the variants and highlighted the importance of studying the effect of the patient's genotype for a correct diagnostic confirmation.

Keywords: TSH receptor; TSHR; congenital hypothyroidism; functional studies; genetic variants; thyroid dyshormonogenesis; thyroid gland.

Figure 1

Genetic variants reported in this study in the peptide structure of the TSH receptor (TSHR, NP_000360.2). Five TSHR variants were located in the extracellular loops (LRR1-LRR10) and one in the membrane (TMD4). Colored squares identify the localization of each detected variant. SP: signal peptide; LRR: Leucine Reach Repeats (10 loops); C: C peptide; TMD: transmembrane domain; CM: Cytoplasmic Motifs; L: loop; ICL: intracellular. Underlined: novel variants; *: non-reported in vitro studies. Data from [6]; TSH Receptor Mutation Database (accessed on 2 August 2023, https://www.tsh-receptor-mutation-database.org/); UniProt (AC P16473; accessed on 2 August 2023, https://www.uniprot.org/uniprotkb/P16473/entry).

Figure 2

Multiple sequence alignment of the detected TSHR variants with eight species (Clustal Omega, EMBL-EBI). All affected amino acids except Asn256 are well conserved within species. Abbreviation (species/subspecies; name; reference sequence): HS (human; Homo sapiens; NP_000360.2), GG (gorilla; Gorilla gorilla; XP_055217156.1), PT (common chimpanzee; Pan troglodytes; XP_009426511.2), MF (crab-eating macaque; Macaca fascicularis; XP_015309546.1), BA (minke whale; Balaenoptera acutorostrata; XP_007188765.2), RN (brown rat; Rattus norvegicus; NP_037020.2), MM (house mouse; Mus musculus, NP_035778.3), XT (Western clawed frog; Xenopus tropicalis; XP_017951898.2), and GJ (Japanese gecko; Gekko japonicus; XP_015282512.1). Numbers indicate the amino acid position in the peptide sequence.

Figure 3

Results of in vitro functional studies of TSHR variants in our cohort. The TSH-dependent activation of Gsα-coupled signal transduction was studied in the detected variants in the homozygosis state. HEK293 cells were transiently transfected with a pGL4.29 (CRE) reporter and a TSHR-wild-type (WT) or a TSHR-mutant plasmid. The results of cells treated with different bovine TSH concentrations (0, 0.1, 1, 5, 10, 15 and 20 IU/L) are shown. Luciferase activity was measured with ONE-Glo™ Luciferase Assay System (Promega). Experiments were performed in duplicate. Results of the WT expression vector are shown in black and the ones from the mutant expression vectors in different colors. Data are shown in comparison with the empty vector values (FOV). The graphic shows impaired functional profiles in all the variants: completely deleterious [c.767dupA/p.(Asn256LysfsTer6)], two partially deleterious [c.484G/p.(Pro162Ala) and c.770T/p.(Thr257Ile)], and one mildly deleterious [c.202T/p.(Pro68Ser)]. Statistical significance was measured with a Student’s t-test (p-value < 0.05). Statistically significant differences with respect to WT are shown with *.

Figure 4

In vitro functional studies of our variants: studying the patients’ genotype in vitro. TSH-dependent activation of Gsα-coupled signal transduction was studied in all detected variants in homozygosis, heterozygosis, or cis-heterozygosis depending on the patient’s genotype. HEK293 cells were transiently transfected with a pGL4.29 (CRE) reporter and a TSHR-wild-type (WT), and/or a TSHR-mutant plasmid. The results of cells treated with different bovine TSH concentrations (0, 0.1, 1, 5, 10, 15, and 20 IU/L) are shown. Luciferase activity was measured with ONE-Glo™ Luciferase Assay System (Promega). Experiments were performed in duplicate. Results of the WT expression vector are shown in black, the ones of heterozygous mutant expression vectors in grey, and of homozygous mutant expression vectors in red. Data are shown in comparison with the empty vector values (FOV). Heterozygous variant c.202T/p.(Pro68Ser) in patient CH-77 showed mild impaired function. The homozygous c.484G/p.(Pro162Ala) variant in patient CH-72 showed partially impaired function. The two variants in the cis-heterozygosity of patient CH-71 showed partially impaired TSHR function, in spite of the presence of the completely deleterious c.767dupA/p.(Asn256LysfsTer6) variant. Statistical significance was measured with a one-way ANOVA test or a Kruskal–Wallis test depending on whether variances were equal or unequal, respectively (p-value < 0.05). Statistically significant differences: * variants versus WT are shown; & heterozygous versus homozygous variant.