The In Vitro Functional Impairment of Thyroid Hormone Receptor Alpha 1 Isoform Mutants Is Mainly Dictated by Reduced Ligand Sensitivity.

Background: Thyroid hormone (TH) acts on TH receptors (TRs) and regulates gene transcription by binding of TRs to TH response elements (TREs) in target gene promoters. The transcriptional activity of TRs is modulated by interactions with TR-coregulatory proteins. Mutations in TRα cause resistance to thyroid hormone alpha (RTHα). In this study, we analyzed if, beyond reduced triiodothyronine (T3) affinity, altered interactions with cofactors or different TREs could account for the differential impaired transcriptional activity of different mutants. Methods: We evaluated four mutants derived from patients (D211G, M256T, A263S, and R384H) and three artificial mutants at equivalent positions in patients with RTHß (T223A, L287V, and P398H). The in vitro transcriptional activity was evaluated on TRE-luciferase reporters (DR4, IR0, and ER6). The affinity for T3 and interaction with coregulatory proteins (nuclear receptor corepressor 1 [NCoR1] and steroid receptor coactivator 1 [SRC1]) were also determined. Results: We found that the affinity for T3 was significantly reduced for all mutants, except for TRα1-T223A. The reduction in the T3 sensitivity of the transcriptional activity on three TREs, the dissociation of the corepressor NCoR1, and the association of the coactivator SRC1 recruitment for each mutant correlated with the reduced affinity for T3. We did not observe mutation-specific alterations in interactions with cofactors or TREs. Conclusions: In summary, the degree of impaired transcriptional activity of mutants is mainly determined by their reduced affinity for T3.

Resistance to Thyroid Hormone due to Heterozygous Mutations in Thyroid Hormone Receptor Alpha.

BACKGROUND: Thyroid hormone (TH) acts via nuclear thyroid hormone receptors (TRs). TR isoforms (TRα1, TRα2, TRß1, TRß2) are encoded by distinct genes (THRA and THRB) and show differing tissue distributions. Patients with mutations in THRB, exhibiting resistance within the hypothalamic-pituitary-thyroid axis with elevated TH and nonsuppressed thyroid-stimulating hormone (TSH) levels, were first described decades ago. In 2012, the first patients with mutations in THRA were identified. Scope of this review: This review describes the clinical and biochemical characteristics of patients with resistance to thyroid hormone alpha (RTHα) due to heterozygous mutations in THRA. The genetic basis and molecular pathogenesis of the disorder together with effects of levothyroxine treatment are discussed. CONCLUSIONS: The severity of the clinical phenotype of RTHα patients seems to be associated with the location and type of mutation in THRA. The most frequent abnormalities observed include anemia, constipation, and growth and developmental delay. In addition, serum (F)T3 levels can be high-normal to high, (F)T4 and rT3 levels normal to low, while TSH is normal or mildly raised. Despite heterogeneous consequences of mutations in THRA, RTHα should be suspected in subjects with even mild clinical features of hypothyroidism together with high/high-normal (F)T3, low/low-normal (F)T4, and normal TSH.

Role of the Bile Acid Transporter SLC10A1 in Liver Targeting of the Lipid-Lowering Thyroid Hormone Analog Eprotirome.

The thyroid hormone (TH) analog eprotirome (KB2115) was developed to lower cholesterol through selective activation of the TH receptor (TR) ß1 in the liver. Interestingly, eprotirome shows low uptake in nonhepatic tissues, explaining its lipid-lowering action without adverse extrahepatic thyromimetic effects. Clinical trials have shown marked decreases in serum cholesterol levels. We explored the transport of eprotirome across the plasma membrane by members of three TH transporter families: monocarboxylate transporters MCT8 and MCT10; Na-independent organic anion transporters 1A2, 1B1, 1B3, 1C1, 2A1, and 2B1; and Na-dependent organic anion transporters SLC10A1 to SLC10A7. Cellular transport was studied in transfected COS1 cells using [14C]eprotirome and [125I]TH analogs. Of the 15 transporters tested initially, the liver-specific bile acid transporter SLC10A1 showed the highest eprotirome uptake (greater than a sevenfold induction after 60 minutes) as well as TRß1-mediated transcriptional activity. Uptake of eprotirome by SLC10A1 was Na+ dependent and saturable with a Michaelis constant of 8 µM. Eprotirome transport was inhibited by known substrates for SLC10A1 (e.g., cholate and taurocholate), and by TH analogs such as triiodothyropropionic acid and triiodothyroacetic acid. However, no significant SLC10A1-mediated transport was observed of these [125I]TH analogs. We also studied the plasma disappearance and biliary excretion of [14C]eprotirome injected in control and Slc10a1 knockout mice. Although eprotirome is also transported by mouse Slc10a1, the pharmacokinetics of eprotirome were not affected by Slc10a1 deficiency. In conclusion, we have demonstrated that the liver-specific bile acid transporter SLC10A1 effectively transports eprotirome. However, Slc10a1 does not appear to be critical for the liver targeting of this TH analog in mice. Therefore, the importance of SLC10A1 for liver uptake of eprotirome in humans remains to be elucidated.

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis.

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis. Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients. Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points. Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs. Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.

Increased circulating interleukin-8 in patients with resistance to thyroid hormone receptor α.

Innate immune cells have recently been identified as novel thyroid hormone (TH) target cells in which intracellular TH levels appear to play an important functional role. The possible involvement of TH receptor alpha (TRα), which is the predominant TR in these cells, has not been studied to date. Studies in TRα0/0 mice suggest a role for this receptor in innate immune function. The aim of this study was to determine whether TRα affects the human innate immune response. We assessed circulating interleukin-8 concentrations in a cohort of 8 patients with resistance to TH due to a mutation of TRα (RTHα) and compared these results to healthy controls. In addition, we measured neutrophil and macrophage function in one of these RTHα patients (mutation D211G). Circulating interleukin-8 levels were elevated in 7 out of 8 RTHα patients compared to controls. These patients harbor different mutations, suggesting that this is a general feature of the syndrome of RTHα. Neutrophil spontaneous apoptosis, bacterial killing, NAPDH oxidase activity and chemotaxis were unaltered in cells derived from the RTHαD211G patient. RTHα macrophage phagocytosis and cytokine induction after LPS treatment were similar to results from control cells. The D211G mutation did not result in clinically relevant impairment of neutrophil or pro-inflammatory macrophage function. As elevated circulating IL-8 is also observed in hyperthyroidism, this observation could be due to the high-normal to high levels of circulating T3 found in patients with RTHα.

Effects of thyroid hormone transporters MCT8 and MCT10 on nuclear activity of T3.

Transport of thyroid hormone (TH) across the plasma membrane is necessary for the genomic action of T3 mediated by its nuclear T3 receptor. MCT8 and MCT10 have been identified as important TH transporters. Mutations in MCT8 result in severe psychomotor retardation. In addition to TH transport into the cell, MCT8 and MCT10 also facilitate TH efflux from cells. Therefore, the aim of this study was to examine if MCT8 and MCT10 increase the availability of T3 for its nuclear receptor rather than generate a rapid equilibrium between cellular and serum T3. T3 action was investigated in JEG3 cells co-transfected with TRß1 and a T3 response element-driven luciferase construct, and T3 metabolism was analyzed in cells transfected with type 3 deiodinase (D3). In addition, cells were transfected with MCT8 or MCT10 and/or the cytoplasmic T3-binding protein mu-crystallin (CRYM). Luciferase signal was markedly stimulated by incubating cells for 24 h with 1 nM T3, but this response was not augmented by MCT8 or MCT10 expression. Limiting the time of T3 exposure to 1-6 h and co-transfection with CRYM allowed for a modest increase in luciferase response to T3. In contrast, T3 metabolism by D3 was potently stimulated by MCT8 or MCT10 expression, but it was not affected by expression of CRYM. These results suggest that MCT8 and MCT10 by virtue of their bidirectional T3 transport have less effect on steady-state nuclear T3 levels than on T3 levels at the cell periphery where D3 is located. CRYM alters the dynamics of cellular TH transport but its exact function in the cellular distribution of TH remains to be determined.

Diverse Genotypes and Phenotypes of Three Novel Thyroid Hormone Receptor-α Mutations.

CONTEXT: Recently several patients with resistance to thyroid hormone (RTH)-α due to T3 receptor-α (TRα) mutations were identified. The phenotype of these patients consists of varying degrees of growth impairment, delayed bone, mental and motor development, constipation, macrocephaly, and near-normal thyroid function tests. OBJECTIVE: The objective of the study was to describe the clinical phenotype of three new families with RTHα and thereby gain more detailed knowledge on this novel syndrome. DESIGN, SETTING, AND PARTICIPANTS: RTHα was suspected in three index patients from different families. Detailed clinical and biochemical assessment and imaging and genetic analyses were performed in the patients and their relatives. In addition, functional consequences of TRα mutations were investigated in vitro. RESULTS: We studied 22 individuals from three families and identified 10 patients with heterozygous TRα mutations: C380fs387X, R384H, and A263S, respectively. The frame-shift mutation completely inactivated TRα, whereas the missense mutations produced milder defects. These mutations were associated with decreasing severity of the clinical phenotype: the patient in family 1 showed severe defects in growth, mental, and motor development, whereas the seven patients in family 3 had only mild clinical features. The most frequent abnormalities were anemia, constipation, and a delay in at least one of the developmental milestones. Serum free T3 ranged from high-normal to high and serum free T4 and rT3 from normal to low. TSH levels were normal in all patients. CONCLUSIONS: This large case series underlines the variation in the clinical phenotype of RTHα patients. RTHα should be suspected in subjects when even mild clinical and laboratory features of hypothyroidism are present along with high/high-normal free T3, low/normal free T4, and normal TSH.

Resistance to Thyroid Hormone Alpha in an 18-Month-Old Girl: Clinical, Therapeutic, and Molecular Characteristics.

BACKGROUND: Recently, the first patients with resistance to thyroid hormone alpha (RTHα) due to inactivating mutations in the thyroid hormone receptor alpha (TRα) were identified. These patients are characterized by growth retardation, variable motor and cognitive defects, macrocephaly, and abnormal thyroid function tests. The objective was to characterize a young girl (18 months old) with a mutation in both TRα1 and TRα2, and to study the effects of early levothyroxine (LT4) treatment. METHODS: The patient was assessed clinically and biochemically before and during 12 months of LT4 treatment. In addition, the consequences of the mutation for TRα1/2 receptor function were studied in vitro. RESULTS: At 18 months of age, the patient presented with axial hypotonia, delayed motor development, severe growth retardation, and abnormally elevated triiodothyronine (T3)/thyroxine (T4) ratios. RTHα was suspected, and concomitantly a c.632A>G/p.D211G missense mutation was identified, affecting both the TRα1 and TRα2 proteins. This mutation was also found in the girl's father. LT4 treatment was started, resulting in a marked improvement of her hypotonia, motor skills, and growth. Functionally, the missense mutation led to decreased transcriptional activity of TRα1, which could be overcome by higher T3 levels in vitro. The mutant TRα1 showed a moderate dominant negative activity on wild type (WT) TRα1. In contrast, WT TRα2 and mutant TRα2 had negligible transcriptional activity and showed no dominant-negative effect over TRα1. CONCLUSIONS: This report describes the phenotype of a young RTHα patient with a mild TRα mutation before and during early LT4 treatment. Treatment had beneficial effects on her muscle tone, motor development, and growth.