Resistance to thyroid hormone caused by a mutation in thyroid hormone receptor (TR)α1 and TRα2: clinical, biochemical, and genetic analyses of three related patients

Abstract

Background: The thyroid hormone receptor α gene (THRA) transcript is alternatively spliced to generate either thyroid hormone receptor (TR)α1 or a non-hormone-binding variant protein, TRα2, the function of which is unknown. Here, we describe the first patients identified with a mutation in THRA that affects both TRα1 and TRα2, and compare them with patients who have resistance to thyroid hormone owing to a mutation affecting only TRα1, to delineate the relative roles of TRα1 and TRα2.

Methods: We did clinical, biochemical, and genetic analyses of an index case and her two sons. We assessed physical and radiological features, thyroid function, physiological and biochemical markers of thyroid hormone action, and THRA sequence.

Findings: The patients presented in childhood with growth failure, developmental delay, and constipation, which improved after treatment with thyroxine, despite normal concentrations of circulating thyroid hormones. They had similar clinical (macrocephaly, broad faces, skin tags, motor dyspraxia, slow speech), biochemical (subnormal ratio of free thyroxine:free tri-iodothyronine [T3], low concentration of total reverse T3, high concentration of creatine kinase, mild anaemia), and radiological (thickened calvarium) features to patients with TRα1-mediated resistance to thyroid hormone, although our patients had a heterozygous mis-sense mutation (Ala263Val) in both TRα1 and TRα2 proteins. The Ala263Val mutant TRα1 inhibited the transcriptional function of normal receptor in a dominant-negative fashion. By contrast, function of Ala263Val mutant TRα2 matched its normal counterpart. In vitro, high concentrations of T3 restored transcriptional activity of Ala263Val mutant TRα1, and reversed the dominant-negative inhibition of its normal counterpart. High concentrations of T3 restored expression of thyroid hormone-responsive target genes in patient-derived blood cells.

Interpretation: TRα1 seems to be the principal functional product of the THRA gene. Thyroxine treatment alleviates hormone resistance in patients with mutations affecting this gene, possibly ameliorating the phenotype. These findings will help the diagnosis and treatment of other patients with resistance to thyroid hormone resulting from mutations in THRA.

Funding: Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Marie Curie Actions, Foundation for Development of Internal Medicine in Europe.

Figure 1. Phenotypic Features of the Patients.

Photographs of the patients (Panel A) illustrate broad facies, flattened nasal bridge (P1 left, P3 right) full lips (P2 middle, P3 right) and long philtrum. Multiple skin tags are evident (P2 and P3). Head circumferences (Panel B) for height and gender (adapted with permission from reference 27) are markedly increased. Skull radiograph of P1 (Panel C) shows thickened calvarium. FT4:FT3 ratios, compared with gender-matched healthy subjects of similar age (females 35-64yrs, males 20-40yrs), are subnormal (Panel D).

Figure 2. Functional Properties and Molecular Modelling of A263V TRα1

JEG-3 cells were transfected with empty, WT or A263V TRα1 expression vectors together with a thyroid hormone responsive reporter gene, assaying T3-dependent activation (Panel A); the inset shows an electrophoretic mobility shift assay with comparable interaction of unliganded or hormone bound – WT/RXR and A263V mutant TRα/RXR heterodimers with a direct repeat thyroid response element from the malic enzyme gene. Dominant negative inhibition was tested (Panel B) in cells cotransfected with reporter gene and equal combinations of expression vectors. Panel C. Quantitative RT-PCR (internal control: 36B4, acidic ribosomal phosphoprotein) showing expression of KLF9 in peripheral blood mononuclear cells from the patients (A263V) or control subjects with increasing T3 concentrations. For Panels B and C * p<0·05; **p<0·01; ***p<0·001. Crystallographic modelling of the TRα1 ligand binding domain (LBD) bound to T3 (blue) (Panel D), highlighting the normal amino acid (alanine 263, green), with substitution of the larger valine residue (red) predicting steric hindrance to T3 binding. Panel E. Schematic representation showing the similar domain structure of TRα1, variant α2 and TRβ1 proteins. Conservation of alanine at position 263 in THRA from different species or THRB, suggests its functional importance. The A263V mutation is common to both TRα1 and α2, whereas THRA mutations described previously (E403X, F397fs406X, A382PfsX7) are unique to TRα1. Three clusters of TRβ mutations (I, amino acids 426-460; II, 309-353; III, 234-282) are associated with RTHβ and a homologous TRβ mutation (A317V) localises to one of these.

Figure 3. Functional Properties of A263V TRα2

Panel A shows 293 cells transfected with either GFP or GFP-tagged WTα2 or A263V mutant α2 expression vectors with visualisation of nuclei (blue), plasma membrane (red), GFP-fusion (green) by immunofluorescence and a composite merged image. Transcriptional function of WTα2 and A263V mutant α2 proteins was tested in JEG-3 cells cotransfected with reporter gene and increasing amounts (5 to 250ng) of empty, WT or A263V α2 expression vectors in the absence of ligand (Panel B), or a fixed amount of empty, TRα1, WT α2 and A263V mutant α2 expression vectors with increasing T3 concentrations (Panel C), or a fixed amount of WT TRα1 and increasing ratio (1:1 to 1:50) of α2 expression vectors (Panel D).