Resistance to thyroid hormone due to defective thyroid receptor alpha

Thyroid hormones act via nuclear receptors (TRα1, TRβ1, TRβ2) with differing tissue distribution; the role of α2 protein, derived from the same gene locus as TRα1, is unclear. Resistance to thyroid hormone alpha (RTHα) is characterised by tissue-specific hypothyroidism associated with near-normal thyroid function tests. Clinical features include dysmorphic facies, skeletal dysplasia (macrocephaly, epiphyseal dysgenesis), growth retardation, constipation, dyspraxia and intellectual deficit. Biochemical abnormalities include low/low-normal T4 and high/high-normal T3 concentrations, a subnormal T4/T3 ratio, variably reduced reverse T3, raised muscle creatine kinase and mild anaemia. The disorder is mediated by heterozygous, loss-of-function, mutations involving either TRα1 alone or both TRα1 and α2, with no discernible phenotype attributable to defective α2. Whole exome sequencing and diagnostic biomarkers may enable greater ascertainment of RTHα, which is important as thyroxine therapy reverses some metabolic abnormalities and improves growth, constipation, dyspraxia and wellbeing. The genetic and phenotypic heterogeneity of RTHα and its optimal management remain to be elucidated.

Thyroid hormones act via nuclear receptors (TRa1, TRb1, TRb2) with differing tissue distribution; the role of a2 protein, derived from the same gene locus as TRa1, is unclear. Resistance to thyroid hormone alpha (RTHa) is characterised by tissue-specific hypothyroidism associated with near-normal thyroid function tests. Clinical features include dysmorphic facies, skeletal dysplasia (macrocephaly, epiphyseal dysgenesis), growth retardation, constipation, dyspraxia and intellectual deficit. Biochemical abnormalities include low/low-normal T4 and high/ high-normal T3 concentrations, a subnormal T4/T3 ratio, variably reduced reverse T3, raised muscle creatine kinase and mild anaemia. The disorder is mediated by heterozygous, loss-of-function, mutations involving either TRa1 alone or both TRa1 and a2, with no discernible phenotype attributable to defective a2. Whole exome sequencing and diagnostic biomarkers may enable greater ascertainment of RTHa, which is important as thyroxine therapy reverses some metabolic abnormalities and improves growth, constipation, dyspraxia and wellbeing.

Introduction
The diverse physiological actions of thyroid hormones (TH: thyroxine, T4; triiodothyronine, T3) include regulation of growth, control of metabolic rate, positive chronotropic and inotropic cardiac effects and development of the central nervous system (Table 1). TH synthesis is controlled by hypothalamic thyrotropin-releasing hormone (TRH) and pituitary thyroid stimulating hormone (TSH) and, in turn, T4 and T3 regulate TRH and TSH synthesis as part of a negative feedback loop. These physiological effects are mediated by thyroid hormone-dependent changes in expression of specific target genes in different tissues ( Table 1). The cellular entry of thyroid hormones , particularly in the central nervous system, is mediated by a membrane transporter [monocarboxylate transporter 8 (MCT8)] [1]. Intracellularly, deiodinase enzymes (DIOs) mediate hormone metabolism, with a high-affinity type 2 enzyme (DIO2) mediating T4 to T3 conversion in the central nervous system (CNS) including pituitary and hypothalamus, type I deiodinase (DIO1) generating T3 in peripheral tissues, and type 3 deiodinase (DIO3) mediating catabolism of thyroid hormones to inactive metabolites [2]. Thyroid hormones alter target gene expression via a receptor protein (TR), belonging to the steroid/nuclear receptor superfamily of ligand-inducible transcription factors. TR binds preferentially to regulatory DNA sequences (thyroid hormone response elements, TREs) in target gene promoters as a heterodimer with the retinoid X receptor (RXR), although the receptor can bind some TREs as a homodimer or monomer. In the absence of hormone, unliganded receptor homodimers/heterodimers recruit a protein complex containing corepressors (e.g. nuclear receptor corepressor [NCoR]; silencing mediator for retinoic acid and thyroid receptors [SMRT]) and histone deacetylase (HDAC) to repress basal gene transcription. Receptor occupancy by hormone (T3) results in dissociation of the corepressor complex and relief of repression together with recruitment of coactivator proteins which mediate transcriptional activation [3].
In humans, two highly homologous thyroid hormone receptors, TRa and TRb are encoded by genes (THRA, THRB) on chromosomes 17 and 3, respectively. Two different proteins are generated from the THRA locus by alternate splicing: TRa1 is an ubiquitously expressed receptor isoform, with particular abundance in the central nervous system, myocardium, gastrointestinal tract and skeletal muscle; a2 protein, which exhibits a divergent carboxy-terminal region such that it is unable to bind thyroid hormones ( Fig. 1), is expressed in a variety of tissues (e.g. brain and testis) and its biological function is poorly understood [4]. The REV-ERBa gene, located on the opposite strand of the THRA locus, is transcribed to generate a nuclear receptor which is involved in regulating circadian rhythm [5]. THRB generates two major receptor isoforms, TRb1 and TRb2, which differ in their amino-terminal regions; TRb1, which is widely expressed, is the predominant isoform in liver  [4]. Resistance to Thyroid Hormone beta (RTHb), a dominantly-inherited disorder due to THRB mutations, is readily recognized due to a characteristic biochemical signature of elevated circulating T4 and T3 with non-suppressed pituitary TSH levels reflecting central (hypothalamicepituitary) refractoriness to thyroid hormone action and is associated with variable resistance to hormone action in peripheral tissues [6]. The incidence of RTHb is~1 in 40,000, and several hundred heterozygous, b receptor mutations which localise to three hotspots or clusters within its ligand binding domain (LBD) have been identified in this disorder [7]. Consistent with its mode of inheritance, mutant b receptors in RTHb inhibit the function of their wild type receptor counterparts in a dominant negative manner; constitutive target gene repression due to failure of corepressor complex dissociation from mutant TRb represents a likely mechanism for such dominant negative inhibition [8].
Human TRb and TRa exhibit marked aminoacid sequence similarity, including (80%) in their hormone binding domains; accordingly, with~160 different receptor mutations known to be associated with RTHb, the identification of a homologous human disorder with defective TRa had been anticipated. Supporting this notion, murine transgenic models harbouring different, heterozygous, TRa mutations are viable and exhibit recognisable abnormalities, but with little perturbation of thyroid function [9e12]; such absence of an overt biochemical, thyroid, phenotype likely explains why the homologous human disorder had eluded discovery. However, human THRA mutations have now been identified in 14 cases from 10 different families, with hypothyroid features and thyroid hormone resistance in target tissues, but associated paradoxically with near-normal thyroid function tests [13e20]. Here, we review the clinical features, differential diagnosis, molecular genetics, pathogenesis and management of Resistance to Thyroid Hormone due to defective thyroid receptor alpha (RTHa).

Clinical features
At birth, some features (e.g. macroglossia, poor feeding, hoarse cry), recognized in hypothyroidism, have been noted [16,17]. Several patients were investigated in infancy for growth retardation which in some cases predominantly affected the lower segment [13,18]. Abnormal physical characteristics in the majority of cases include macrocephaly, broad facies, hypertelorism, a flattened nose, prominent tongue and thick lips [13e18]; indeed, five cases were identified following genetic investigation of a clinic patient cohort with these shared characteristics [18]. An excessive number of skin tags and moles have been noted, particularly in adults [13, 16,17].

Biochemical
The most consistent pattern of thyroid function tests comprises low or low-normal free T4, and high or high-normal free T3, resulting in an abnormally low T4/T3 ratio; reverse T3 levels were subnormal in severe cases [13e17] but can be normal [19,20]. A mild, usually normocytic anaemia [13,15e18] with normal haematinics (Iron, B12, folate) and haemolytic indices (reticulocyte count, circulating haptoglobin and lactate dehydrogenase) [16] and raised muscle creatine kinase levels [13,15e17] are a consistent abnormality. Raised total and LDL cholesterol levels have been documented [15,16], even in childhood cases.

Neurocognitive
In childhood, patients showed delayed milestones (motor, speech). Slow initiation of motor movement, together with fine and gross motor incoordination, manifesting as dyspraxia or a broadbased, ataxic gait and slow, dysarthric speech were a consistent feature. Their IQ was variably reduced, being markedly subnormal, with seizures in one case [16].

Gastrointestinal
Reduced frequency of bowel movements is a common finding, with severe constipation being a significant problem in several cases [13, 16,18].

Metabolic & endocrine
Resting energy expenditure (metabolic rate) was low in most patients [13,16,17,19]. Both male and female to offspring transmission of TRa defects has been recorded [14,17,18], suggesting that fertility in either gender is not unduly compromised. Table 2 summarises known clinical features of RTHa, together with clinical, biochemical and physiological investigations which can identify recognised abnormalities.

Differential diagnosis
RTHa could be suspected in childhood patients with dysmorphic features or retardation of growth and psychomotor development or adults with a history of such features. Whilst a low ratio of circulating T4/T3 levels is a consistent feature which could identify potential cases, this biochemical abnormality is also a feature of disorders (genetic or environmental) with dyshormonogenetic hypothyroidism or AllaneHerndoneDudley syndrome due to defects in the MCT8 gene. Table 3 shows clinical and biochemical features which could differentiate between these entities.

Molecular genetics
Affected individuals are heterozygous for THRA mutations which occurred de novo in six cases [13,18e20] or were familial [14,17,18]. Hitherto, two broad classes of receptor defect have been identified: either highly deleterious, frameshift/premature stop mutations; or less severe, missense, aminoacid changes (Fig. 1). None of the mutations affect the REV-ERBa gene, transcribed from the opposite strand of the THRA locus.
Most cases harbour mutations which selectively disrupt the carboxyterminal activation domain of TRa1 [13,14,17,18]. Consistent with this, where their functional properties have been elucidated, the mutant receptors fail to bind ligand and are devoid of transcriptional activity [13, 15,16]. Similar to TRb mutations in RTHb, TRa1 mutants inhibit the function of their wild type receptor counterparts in a  (Fig. 2). Expression of TH-responsive target genes in mutation-containing patient peripheral blood  [24]. Whole genome sequencing in human autism spectrum disorder has identified a patient with a de novo, missense, variant (R384C) in TRa1 [20]. This aminoacid change is almost certainly pathogenic, being functionally deleterious when studied in the context of murine TRa1 [9]. Interestingly, transgenic mice harboring this mutation exhibit locomotor (ataxia) and behavioural abnormalities (anxiety, depression) which can be alleviated by thyroid hormone treatment initiated even in adulthood [25,26].

Pathogenesis
Many clinical features in RTHa are typical of uncorrected hypothyroidism in childhood or adult life. Patent cranial sutures, delayed dentition, femoral epiphyseal dysgenesis (disordered, endochondral ossification) and wormian bones (disordered, intramembranous ossification) are recognized features of childhood thyroid hormone deficiency [27,28]; macrocephaly may reflect delayed fontanelle closure and hypothyroid facies includes a flattened nasal bridge; such skeletal dysplasia is associated with growth retardation (predominantly lower segmental) and delayed bone age in childhood or adult short stature. Similarly, diminished colonic motility resulting in slow-transit constipation with colonic dilatation (megacolon) or even ileus are reported in human hypothyroidism [29]. Skeletal abnormalities (growth retardation, delayed tooth eruption, patent cranial sutures, epiphyseal dysgenesis) and intestinal dysmotility in human RTHa are recapitulated in mutant TRa1 mutant mouse models [11,30].
Although borderline, the biochemical abnormalities found in RTHa cases (disproportionately raised/high-normal T3 and low/low-normal T4 levels, resulting in a markedly reduced T4/T3 ratio together with low rT3 levels in some cases) may reflect altered metabolism of thyroid hormones in these patients. One possibility is that, as has been documented in mice with a dominant negative TRa1 mutation (TRa1-PV) [10], increased hepatic DIO1 levels augment T4 to T3 conversion; alternatively, reduced tissue levels of DIO3, whose expression is TRa1 regulated [31], may contribute to these abnormalities with decreased inner-ring deiodination of T4 to rT3 and T3 to T2.
DIO3 is also expressed in skin and inhibition of the enzyme in this tissue enhances keratinocyte proliferation in mice [32]. Accordingly, it is tempting to speculate that cutaneous DIO3 deficiency in RTHa patients might, at least in part, mediate propensity to excess skin tags and moles.
Anaemia in RTHa patients correlates with documented abnormal erythropoiesis and reduced haematocrit in TRa null or mutant mice [33,34]. Normal haematinics in patients suggests defective proliferation or differentiation of erythroid progenitors, with the mechanism remaining to be elucidated.
Idiopathic epilepsy which was noted in one human case [16] correlates with heightened susceptibility to seizures following photic [11] or audiogenic [25] stimulation and aberrant development of GABAergic inhibitory interneurons [35] in mutant mice harbouring different TRa1 mutations.
Following thyroxine treatment in physiological dosage, tissues of RTHa patients exhibit variable responses: thus, TSH levels suppress readily, implying preserved sensitivity within the hypothalamicepituitaryethyroid axis; conversely, cardiac parameters, resting energy expenditure and muscle CK levels are less responsive [16,17]. Overall, these observations are consonant with thyroid hormone resistance in organs (e.g. myocardium, skeletal muscle, gastrointestinal tract) expressing predominantly TRa1, with preservation of TH sensitivity in TRb-expressing tissues (hypothalamus, pituitary, liver) (Fig. 3).

Treatment
Thyroxine therapy raises metabolic rate, serum IGF1 and SHBG and lowers elevated LDL cholesterol and muscle creatine kinase levels [13,15e17]; these changes may limit weight gain, especially in older patients. In the childhood case we first described [13], five years of thyroxine therapy has been clearly beneficial, improving overall height and subischial leg length, alleviating constipation (with associated restoration of contractile activity in colonic manometry) and improving wellbeing (Moran & Chatterjee, unpublished observations). Low-normal IGF1 levels prompted the addition of growth hormone to thyroxine therapy in another childhood case [15], but with little further improvement in growth. Treatment from early childhood in cases harbouring mutant TRa1 whose dysfunction is reversible at higher TH levels might have ameliorated their phenotype [17]. In adult life, these individuals report Fig. 3. Summary of the major tissue actions of thyroid hormone, together with the receptor subtypes mediating these effects. In RTHa, tissues expressing mainly TRa would be resistant to thyroid hormone action with TRb-expressing tissues being sensitive. that thyroxine therapy improves dyspraxia and enhances social interaction (Moran & Chatterjee, unpublished observations). In contrast, in most cases, anaemia persists following thyroxine therapy; and, relative to the rise in TH levels, changes in cardiac parameters (e.g. heart rate, indices of myocardial contractility) are blunted [16,17].
Following thyroxine treatment, TSH levels suppress readily with elevation of FT3 to supraphysiologic levels; serum SHBG may rise further from high-normal baseline levels [13] and biochemical markers of bone turnover became progressively elevated in one case [16]. These observations raise the possibility that chronic, excess TH exposure in thyroxine-treated RTHa patients might lead to unwanted toxicities in normal TRb-containing tissues. In this regard, future therapies which could be developed include TRa1-selective thyromimetics [36], to selectively activate either residual, normal TRa1 or partially defective, mutant TRa1 and overcome resistance in TRa-expressing tissues.
As described above, many THRA defects in RTHa abrogate hormone binding to receptor, such that dominant negative inhibition exerted by mutant TRa1 in vitro or in patient's cells studied ex vivo is irreversible, even following exposure to high T3 levels. Here, developing small molecules which either inhibit TR interaction with the corepressor complex or its histone deacetylase enzymatic activity, might represent a rational therapeutic approach. Supporting this notion, introduction of a mutation in NCoR that abrogates its interaction with TR [37] or administration of suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase [38], ameliorates phenotypic abnormalities (growth, bone development) in the murine TRa1-PV mutant model of RTHa.

Summary and conclusions
RTHa, a dominantly-inherited or sporadic disorder, due to heterozygous THRA mutations affecting TRa1 alone or in combination with variant a2 protein, is characterised by clinical, biochemical and physiological features of hypothyroidism in specific tissues, together with subtle abnormalities (low T4/T3 ratio, variably reduced rT3) of thyroid function. Preliminary experience suggests that thyroxine therapy is beneficial.
Given the estimated prevalence (~1 in 40,000) of RTHb, with over 160 different TRb mutations being recorded hitherto, it is highly likely that RTHa is more common but not fully ascertained, either because the disorder lacks a clearcut, diagnostic signature of biochemical abnormalities or is associated with unexpected phenotypes (e.g. autism spectrum disorder). In this context, it is interesting to note that interrogation of databases (e.g. ExAC, 60,000 Exomes) reveals at least 101 non synonymous variants in THRA (52 common to TRa1/a2; 3 TRa1-specific; 49 a2-specific); at least five variants are potentially damaging, with aminoacid changes in codons that are homologous to residues in TRb known to be mutated in association with RTHb (http://exac.broadinstitute.org/gene/ENSG00000126351).
The discovery of additional biomarkers in RTHa would be useful. Specifically, the discovery of a combination of abnormal metabolites and/or proteins which can constitute a specific diagnostic test, would enable more complete ascertainment of the disorder, with earlier commencement of TH treatment in cases being potentially more effective. Furthermore, during TH therapy, markers which better indicate correction of resistance in TRa-expressing tissues or toxicity in TRb-containing organs would be of utility.

Practice points
Growth retardation, macrocephaly, skeletal dysplasia and constipation are common clinical findings in TRa-mediated Resistance to thyroid hormone (RTHa). Biochemical abnormalities include low T4/T3 ratio, subnormal reverse T3, raised muscle creatine kinase and anaemia. Thyroxine therapy reverses hypothyroidism in hormone-resistant TRa target tissues and is of symptomatic benefit. However, careful monitoring for adverse sequelae of excessive TH exposure in hormone-sensitive TRb tissues, is warranted.

Disclosures
None of the authors have anything to disclose.