The thyroid hormone receptor β (THRβ), a member of the nuclear receptor (NR) superfamily, governs thyroid hormone (TH or T3) signalling crucial for a wide range of developmental and physiological processes in the body, including metabolism, growth, and neuronal differentiation (Anyetei-Anum et al., 2018; Bassett et al., 2003; Rehman et al., 2023). The canonical structure of NRs is largely conserved, comprising four domains: a highly variable N-terminal domain (NTD), a conserved DNA binding domain (DBD), a hinge region/domain, and a C-terminal ligand binding domain (LBD). THR, typically forms heterodimers with retinoid X receptors (RXR), although it is also reported to form homodimers (Bassett et al., 2003; Evans and Mangelsdorf, 2014). THR can bind to the thyroid hormone response elements (TREs) in both unliganded and liganded state, with different transcriptional outcomes. In its unliganded state, THR associates with corepressors, maintaining a basal level of transcription. Upon binding to its ligand, T3, the receptor undergoes a conformational change that results in the displacement of corepressors and recruitment of coactivators, initiating transcription (Moeller and Führer, 2013). The THR-coactivator complex then binds to TRE located within the promoter regions of target genes, thereby modulating T3-mediated transcriptional activity. THRβ is widely expressed in multiple tissues, including the kidney, liver, hypothalamus, brain, and pituitary gland (Zhu et al., 2016). It plays several critical roles, including acting as a tumor suppressor in various cancers and regulating both the metabolism and feedback mechanism of the ‘hypothalamic-pituitary-thyroid’ (HPT) axis (Ke et al., 2021; Sun et al., 2020).

The non-synonymous genetic variations, which result in the substitution of an amino acid residue in the protein sequence, are classified as missense mutations (Stefl et al., 2013). These mutations can have profound effects on protein structure and function. Numerous studies have revealed that genetic variations in NRs, including THR, can disrupt the normal receptor function (Liu et al., 2018a; Rehman et al., 2023). Specifically, non-synonymous variations in the protein-coding region of THRβ can alter the amino acid sequence, leading to mutated or dysfunctional receptors that may associate with various pathological conditions and affect patient's susceptibility to illness and responses to diverse drugs (Liu et al., 2018a; Stefl et al., 2013).

One significant clinical manifestation of THRB genetic variations is ‘Resistance to Thyroid Hormone β’ (RTHβ), which can arise as either an autosomal recessive or dominant genetic disorder. Patients with RTHβ exhibit diminished responsiveness of target cells to thyroid hormone and experience developmental abnormalities, psychology disturbances, goiter, hair loss, sinus tachycardia, and recurrent nose, ear, and throat infections (Sun et al., 2020). The clinical markers of RTHβ include elevated levels of free triiodothyronine in serum and inadequate secretion of thyroid stimulating hormone (TSH). Recent findings have also established a strong correlation between THRβ mutations and cancer (Fang et al., 2022). However, the precise underlying mechanisms by which THRβ mutations contribute to cancer progression remain unclear. Although many clinical studies have explored the correlation between THRB variation and TH level, a comprehensive and comparative analysis of THRβ variants at the cellular level is lacking.

To comprehend the functional implications of naturally occurring THRβ non-synonymous variants, we initiated our study with in silico analysis to predict the effects of critical single nucleotide polymorphisms (SNPs) on THRβ function, followed by a series of cellular assays designed to validate our findings. Additionally, we characterized these naturally occurring disease-inflicting THRβ SNP variants by employing multiple cellular test parameters, including subcellular localization, interaction with T3 hormone, transcriptional response, interaction with RXR, and mitotic chromatin association. Our findings indicate that genetic variations in the coding region of the THRB gene adversely affect the structural and functional behaviour of the receptor at multiple levels, contributing to disease states. This consolidated analysis of THRβ variants aims to enhance our understanding of the mechanisms underlying disease development at both the cellular and molecular levels, paving the way for potential therapeutic interventions and personalized medicine approaches.