Download 16) Clifton-Bligh RJ, de Zegher F, Wagner RL, Collingwood TN

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Resistance to Thyroid Hormone - emerging definition of a disorder of thyroid
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hormone action
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Carla Moran and Krishna Chatterjee
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University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC, Institute of
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Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom
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Abbreviated title: RTH.
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Key terms: Resistance to Thyroid Hormone, dominant negative inhibition; THRA mutation
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Disclosure statement: The authors have nothing to disclose
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Word Count: text (1651); figures 1
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Address for correspondence and reprint requests:
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V Krishna K Chatterjee, University of Cambridge, Metabolic Research Laboratories, Wellcome Trust-
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MRC Institute of Metabolic Science, Level 4, Box 289, Addenbrooke’s Hospital, Cambridge, CB2
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0QQ. Tel: (44)-1223-336842; Fax: (44)-1223-330598; Email: [email protected]
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Funding: Our research is supported by funding from the Wellcome Trust (095564/Z/11/Z to KC) and
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National Institute for Health Research Cambridge Biomedical Research Centre (CM, KC).
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Thyroid hormones (TH) act via nuclear receptors, encoded by separate genes (THRA, THRB), with
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differing tissue distributions (TR1: central nervous system, myocardium, skeletal muscle,
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gastrointestinal tract; TR1: liver, kidney; TR2: hypothalamus, pituitary, cochlea, retina).
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Alternative splicing of THRA generates a variant, non TH-binding, protein (TR2), with a divergent
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carboxyterminal domain, whose function is not understood (1).
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Resistance to thyroid hormone  (RTH), usually due to heterozygous mutations in THRB, is
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characterised by raised T4 and T3 levels, non-suppressed TSH and a variable phenotype
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encompassing both hyperthyroid (e.g. failure to thrive, raised metabolic rate, tachycardia, low bone
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density, anxiety) and hypothyroid (e.g. dyslipidaemia) features (2). Consonant with its dominant
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mode of inheritance, dominant negative inhibition of wild type receptor function by mutant TR is
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central to the pathogenesis of this disorder (3). Although TR and  proteins exhibit marked
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aminoacid sequence similarity, such that introduction of a  receptor mutation causing RTH into the
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murine TR gene is associated with an abnormal phenotype (e.g. growth retardation, locomotor
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abnormalities) (4), the homologous human disorder (Resistance to Thyroid Hormone , RTH),
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eluded discovery for over a decade probably because it was not associated with a readily-recognisable
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diagnostic signature.
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RTH, due to a highly deleterious mutation (E403X), truncating the carboxyterminal region of TR1,
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was first identified in a six yr old girl with hypothyroid features (e.g. skeletal dysplasia with growth
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retardation, neurodevelopmental delay, constipation) in selected target tissues, but associated with
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near-normal (borderline low or normal T4, borderline high or normal T3, normal TSH) thyroid
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function tests (5). Subsequently, further childhood plus adult male and female cases, harbouring
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frameshift/premature stop mutations also involving the carboxyterminus of TR1 were found, with
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recognition of additional, potentially pathognomonic, features (macrocephaly, anaemia, dysmorphic,
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facies) (6,7). Indeed, recognition of a distinct, shared, phenotype encompassing macrocephaly and
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dysmorphic features in a single clinic, led to the identification of a further five cases with similar,
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carboxyterminal TR1 mutations (8). Where functionally characterised, these TR1 mutants
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exhibited negligible hormone binding and functional activity, with failure to dissociate from
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transcriptional corepressor and recruit coactivator proteins – properties that are highly analogous to
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those of dominant negative TRmutants in RTH (3). From these reports, a biochemical signature for
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RTH also seemed to emerge; although TH levels could be in the normal range, a computed T4/T3
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ratio or reverse T3 levels (where ascertained) were subnormal (5,8,9). Following this, a missense
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THRA mutation (A263V), located in a receptor region common to both TR1 and TR2 proteins, was
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identified in three members of a family with similar clinical (growth and developmental retardation,
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constipation, macrocephaly) features. The A263V TR1 mutant showed partial loss-of-function,
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being transcriptionally impaired at low TH concentrations in vitro, with reversal of this at higher T3
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levels. It was tempting to speculate that such partial preservation of mutant TR1 function accounted
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for a milder phenotype in this family. However, the possibility that thyroxine treatment of these cases
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since early childhood had also contributed to amelioration of the phenotype, could not be discounted.
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Concordant with no measurable gain- or loss-of-function of A263V mutant TR2 compared with its
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normal counterpart, no added phenotypes attributable to mutated TR2, could be discerned in these
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cases (10). In contrast, some unusual added clinical features (micrognathia, clavicular agenesis,
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metacarpal fusion and syndactyly of digits, hyperparathyroidism, chronic diarrhoea) were reported in
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an adult female harbouring a different mutation (N359Y), common to both TR1 and TR2 proteins
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(11).
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In this context, the report by Demir and colleagues in the current issue of JCEM (12), describing three
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different THRA mutations (C380fs387X, R384H, A263S) in 10 RTH patients from 3 different
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families, together with associated clinical features, clarifies several aspects of this disorder. The
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authors were able to discern a gradation in phenotype associated with different genotypes: a child,
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harbouring a highly deleterious mutation (C380fs387X) involving the carboxyterminus of TR1, is
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growth retarded with cognitive and motor deficits that result in severe handicap; moderate cognitive
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deficit, motor abnormalities and growth retardation in infancy but less evident in adulthood, were
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recorded in a child and mother with a partial loss-of-function mutation (R384H) in TR1; in a large
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family, harbouring A263S mutant TR1 with slightly impaired function, mild growth retardation and
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slight developmental delay in childhood culminated in average/low-average adult IQ, with
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constipation being a relatively conserved clinical feature.
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The A263S mutation in THRA reported by Demir et al, as well as another RTH-associated mutation
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(D211G) reported recently by the same group, also involve the TR2 protein (12,13). No added
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phenotypic characteristics were observed in nine affected individuals harbouring one or other of these
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mutated residues. Accordingly, it is conceivable that the unusual skeletal and other abnormalities
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unique to the patient with the N359Y THRA mutation, are attributable to a defect in another pathway;
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although whole exome sequencing was undertaken in this case, a pathogenic abnormality (for
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example in a non-coding gene region) has not been excluded (11). Alternatively, as depicted when
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RTH mutations reported hitherto are mapped on the crystal structure of TR1, the Asparagine to
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Tyrosine change at codon 359 (N359Y), involves a residue that is in an unusual location in the
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receptor protein compared to the position of other RTH-associated mutations (Figure 2); therefore,
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the possibility of the N359Y substitution inducing a specific functional alteration in the receptor that
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is linked to unique, additional phenotypes, cannot be discounted.
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With the identification of a more functionally diverse repertoire of TR1 mutations, a specific
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biochemical signature for RTH seems less clearcut. Specifically, in some cases, particularly
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associated with mild TR mutations (e.g. A263S, D211G), normal reverse T3 levels have been
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documented (12,13). The T4/T3 or T3/rT3 ratio remains abnormal in most cases, overlapping
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minimally with values in unaffected family members (12), but this abnormal thyroid function test
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pattern is a recognised feature of other disorders (e.g. MCT8 deficiency) (14); likewise, although mild
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anaemia is almost universal in RTH, this parameter is unhelpfully nonspecific. Studies in a
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transgenic model of RTH raised the possibility of circulating trace element (selenium) levels having
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diagnostic utility (15), but did not translate readily into the human context (7). In RTH, even subtle
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TR mutants exhibit impaired negative feedback regulation of hypothalamo-pituitary target genes
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(TRH, TSH) (16), reflected in pathognomonic, raised, TH levels. It has been suggested that a
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combination of raised type 1 deiodinase and reduced type 3 deiodinase enzyme activities accounts for
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thyroid biochemical abnormalities in RTH17); whether another TH metabolite that reflects such
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altered deiodinase activity more consistently or a biomarker associated with another universally
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dysregulated pathway in this disorder, can be identified, remains to be seen.
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With the estimated prevalence of RTH being ~1 in 40,000, with over 160 different TR mutations
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recorded hitherto, it is likely that RTH is more common, but underdiagnosed. Both maternal and
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paternal transmission of TR mutations has now been recorded in several families (6, 8, 10, 12, 13),
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suggesting that, unlike in a murine transgenic model (18), fertility in either gender is not unduly
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compromised. However, screening for THRA defects based on current, documented, phenotypes may
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artificially enrich for identification of particular TR mutations (e.g. involving the carboxyterminal
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receptor region). Supporting this notion, whole exome sequencing (WES), identified a TR variant
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(R384C), known to be pathogenic in transgenic mice (4), associated with autism spectrum disorder –
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an unexpected phenotype (19). Indeed, interrogation of exome databases (e.g. ExAC;
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http://exac.broadinstitute.org/gene/ENSG00000126351) reveals many rare, nonsyonymous THRA
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variants including several in TR1 predicted to be damaging, based either on homology to known
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RTH mutations or structural modelling. However, in the absence of definitive diagnostic criteria
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(clinical, biochemical) for RTH, it may be necessary to develop functional assays to assess the
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putative pathogenicity of receptor variants, as has been done for PPARG variants identified in WES of
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type 2 diabetes cohorts (20). TR mutations causing RTH cluster within three regions of its hormone
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binding domain and, as illustrated in Figure 1, many RTH-associated TR mutations have TR
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counterparts, involving the same aminoacids, which localise to these clusters. As the repertoire of
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RTH-associated TR mutations grows, it will be interesting to see whether they also localise to
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particular receptor regions. One functional constraint on RTH-associated TR mutations is
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preservation of the ability to exert dominant negative activity, such that receptor regions mediating
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functions (DNA binding, interaction with corepressor and RXR) that are required for such activity are
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devoid of naturally-occuring mutations (3); if dominant negative inhibition is also central to the
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pathogenesis of RTH, this property may also constrain localisation of RTHmutations within TR.
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Seven out of fourteen TR mutations recorded hitherto have occurred de novo, with mutations
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involving some residues (e.g. Alanine 263, Arginine 384, Glutamic Acid 403) occurring more than
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once in unrelated families; as has been shown in RTH (21), it is possible that nucleotide substitutions
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involving mutation-prone, CpG dinucleotides will occur more frequently, resulting in some aminoacid
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changes being overrepresented.
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Although not identified via neonatal screening (in a program with both T4 and TSH measurement),
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thyroxine therapy from age 18 months has been beneficial for growth, motor development and
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constipation in a childhood case with a partial loss-of-function mutation (D211G) (13). Despite the
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first recorded patient harbouring a more deleterious E403X TR mutation (5), we have observed
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similar benefit following five years of thyroxine therapy (Moran, Chatterjee and Dattani unpublished
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observations). Accordingly, despite the relative paucity of data, a trial of thyroxine therapy in RTH
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cases harbouring both mild and severe loss-of-function TR mutations may be indicated. However,
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careful surveillance to not only assess therapeutic benefit but also monitor for unwanted tissue toxicity
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(e.g in liver, which expresses mainly TR), is warranted. In this context, as in RTH, reliable
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biomarkers of tissue responses to TH are needed. In the future, it also conceivable that genetic
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stratification may guide therapy of RTH patients, with thyroxine or selective thyromimetics being
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used in patients harbouring TR mutations with residual functional activity, with alternative
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approaches (e.g. blocking formation or activity of receptor-transcriptional corepressor complexes) in
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cases with severe, hormone-refractory, TR defects.
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ACKNOWLEDGEMENTS
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We thank Dr Louise Fairall, Department of Molecular and Cell Biology, University of Leicester, UK
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for the crystallographic modelling shown in Figure 2.
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FIGURE LEGENDS
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Figure 1.
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Schematic alignment of TR1, TR2 and TR showing functional regions (N-terminal, DNA
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binding domain DBD, hormone binding domains); the divergent carboxyterminus of TRis cross-
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hatched. The location of recorded RTH mutations, involving either TR1 alone or both TR1 and
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2 proteins, is superimposed. Three regions of TR1 (aminoacids 234 to 282, 309 to 353 and 426 to
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460), within which RTH mutations cluster are depicted. Within these clusters, published or our
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unpublished (D265A) RTH mutations, involving aminoacids homologous to residues mutated in
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RTH, are shown.
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Figure 2: The crystal structure of the hormone binding domain of human TR1 bound to T3 (PDB
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2H77, magenta) is shown. The location of aminoacids which are mutated (missense or premature stop
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or frameshift/premature stop) in RTH is superimposed (red balls). The carboxyterminal region
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(distal helix 11 to helix 12) within which RTH mutations occur, is highlighted in yellow.
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