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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1488
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1760-1767
Copyright © 2005 by The Endocrine Society

A De Novo Mutation in an Already Mutant Nucleotide of the Thyroid Hormone Receptor ß Gene Perpetuates Resistance to Thyroid Hormone

Joaquin Lado-Abeal1, Alexandra M. Dumitrescu1, Xiao-Hui Liao, Ronald N. Cohen, Joachim Pohlenz, Roy E. Weiss, Marie-Christine Lebrethon, Alain Verloes and Samuel Refetoff

Departments of Medicine (J.L.-A., X.-H.L., R.N.C., J.P., R.E.W., S.R.), Pediatrics (S.R.), and Human Genetics (A.M.D.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Clinical Genetic Unit (A.V.), Hôpital Robert Debré, 75019 Paris, France; and Pediatric Endocrinology (M.-C.L.), Hôpital de la Citadelle, 4000 Liège, Belgium

Address all correspondence and requests for reprints to: Samuel Refetoff, University of Chicago, MC 3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Resistance to thyroid hormone (RTH) is a syndrome of reduced sensitivity to thyroid hormone, most commonly caused by mutations in the thyroid hormone receptor (TR) ß gene. Mutations are mostly located in the ligand-binding domain of the TRß, decreasing T3 binding to the mutant TRß molecule, which in turn interferes with the function of the wild-type (WT) TR. A total of 122 different TRß gene mutations have been identified so far, with 46 occurring in more than one family. We now report a family with two novel TRß mutations occurring in the same nucleotide. The proposita had two children from each of her two marriages. One daughter and one son from each marriage had severe RTH with free T4 and T3 levels 3- to 4-fold the mean normal values and unsuppressed TSH, mental retardation, and deafness. The proposita had a missense mutation (GTG to GGG) in codon 458 of the TRß gene, resulting in the replacement of the normal valine with glycine (V458G). Although this mutation was transmitted to her affected son, the mutated codon in her affected daughter was GAG, encoding glutamic acid (V458E). Haplotype analysis showed that this de novo mutation occurred on the already mutant allele of the proposita. Cotransfection of each of these mutant TRßs with the wild-type TRß showed a potent dominant negative effect. Large amounts of T3 were required to dissociate homodimers of the mutant TRß bound to DNA. In addition, and in contrast to other mutant TRßs with severe T3-binding defects, homodimer release failed to recruit the steroid receptor coactivator. No defects in heterodimerization with retinoid X receptor-{alpha} or association with a nuclear receptor corepressor, were identified. These in vitro data are in agreement with the in vivo phenotype of severe RTH. Unique and previously unreported in human inherited diseases is the occurrence of a de novo mutation at an already mutant nucleotide. Because the occurrence by chance is extremely unlikely, it is postulated that the presence of three guanines in the sequence created by the mutant nucleotide of the proposita results in a mutagenic site prone to de novo mutation.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE SYNDROME OF resistance to thyroid hormone (RTH) is characterized by reduced tissue sensitivity to thyroid hormone (1, 2). Patients with RTH are identified by the elevation of serum free T4 and T3 concentrations with persistence of normal or slightly elevated serum TSH levels. The clinical phenotype presenting stigmata of both hypo- and hyperthyroidism suggests a variable degree of RTH in different tissues. The most common clinical findings are goiter, tachycardia, attention deficit disorder, learning disability, and delayed bone age. Less common findings are reduced intelligence quotient, short stature, and hearing loss (1, 2, 3).

We have studied 142 families that, with the exception of 21 families, presented mutations in the thyroid hormone receptor (TR) ß gene, located on chromosome 3. In the majority of subjects, these mutations are dominantly inherited (1) and occur in the T3-binding domain and adjacent hinge domain of the TRß gene, reducing the bioactivity of the mutant TRß molecule (4, 5) and, more rarely, altering its association with cofactors (6). TRs form homodimers or heterodimers with the retinoid X receptors (RXRs) and bind to specific DNA sequences termed thyroid hormone response elements (TREs). In the absence of T3, TR homodimers and heterodimers are associated with corepressors that repress or silence the transcription of genes positively regulated by thyroid hormone. Binding of T3 to TRs releases the corepressors and recruits nuclear coactivators, which stimulate gene transcription (7). Mutant TRßs interfere with the functions of the wild-type (WT) TRs, a phenomenon termed dominant negative effect (8). This involves the occupation of a TRE by a mutant TR that has one of the following properties alone or in combination: no binding of T3 (9); reduced affinity for the ligand (9); tighter affinity for the corepressors (6, 10, 11); and reduced ability to recruit coactivators necessary to enhance gene transcription (12, 13). For the dominant negative effect to occur, TR has to bind to TRE, which explains why no mutations have been identified in the DNA-binding domain.

We now report on a family with a severe clinical and biochemical phenotype of RTH caused by two novel TRß mutations that occurred in the same codon and nucleotide. Of interest, and not previously reported in human inherited diseases, is the fact that a de novo mutation occurred at an already mutant nucleotide. Both TRß mutants had severely impaired transcriptional activity and exhibited strong dominant negative effect in vitro. Both mutant TRs were able to form homodimers and heterodimers with RXR{alpha} but had no obvious increase in affinity to the nuclear receptor corepressor (NCoR).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The proposita is a 41-yr-old female of Wallon origin with a goiter since childhood. At 3 yr of age, she was treated with Lugol solution with no change in goiter size. By the age of 9 yr, due to increase in serum thyroid hormone levels, an antithyroid drug was given for 8 months, which resulted in an increase in the thyroid gland size. At the age of 16 yr, she underwent subtotal thyroidectomy. On physical examination, at age 41 yr, the patient had hearing loss, mental retardation, and a large goiter. Signs suggestive of thyrotoxicosis included tachycardia and body mass index of 16 kg/m2, despite increased appetite and retraction of the eyelids. Menstrual cycles were normal. With 350 µg of levothyroxine daily, serum total T4 was 21.2 µg/dl (normal range, 5–12 µg/dl), total T3 was 420 ng/dl (normal range, 90–180 ng/dl), and TSH was 8.5 mU/liter (normal range, 0.4–3.6 mU/liter). Thyroid-stimulating antibodies, as well as thyroglobulin (TG) and thyroid peroxidase antibodies, were negative. Thyroidal uptake of radioiodide was increased to 77% (normal range, 10–35%). Serum levels of PRL, LH, and FSH were in the normal range. Intravenous administration of 100 µg TRH increased the serum TSH concentration from a baseline of 8.5 mU/liter to 56 mU/liter at 30 min. Administration of 75 µg of L-T3 daily for 10 d reduced the basal TSH to 3.3 mU/liter and only slightly reduced the response to TRH to 43 mU/liter at 30 min. Magnetic resonance imaging showed no abnormalities of the hypothalamus or pituitary.

The proposita (II-2) conceived two girls from one husband and two boys from another unrelated husband (Fig. 1Go). An 18-yr-old daughter (III-2) from the first marriage had hearing loss, mental retardation, and epilepsy. An 11-yr-old boy (III-3) from the second marriage had hearing loss, mental retardation (intelligence quotient, 52), attention deficit disorder with hyperactivity, and myopathy. The proposita’s mother and maternal grandmother, both deceased, had goiters, but no additional clinical data are available. The proposita had a twin sister who died at birth and four asymptomatic brothers, three of whom agreed to be tested. Informed consents were obtained for this study, and the study was approved by the Institutional Review Board of the University of Chicago.



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  		FIG. 1. Pedigree and results of thyroid function tests for the Mb family at the time of study. Abnormal values are in bold. The number at the left of each pedigree symbol represents age in years, and the number to the right is the identification number for the generation. Shaded symbols indicate subjects with TRß mutation, and the arrow indicates the proposita. FT4D, FT4 measured by dialysis; FT4I, FT4 index. TT4: conversion to SI (nM) = 12.84; TT3 and total rT3 (TrT3): conversion to SI (nM) = 0.0154; free T4 (FT4) I: conversion to SI = 12.84; FT4D: conversion to SI (pM) = 12.84.

 
Measurement of hormones in serum

Total T4 (TT4), total T3 (TT3), and TSH were measured by chemiluminescence using Elecsys 2010 technology (Roche Diagnostic Corp., Indianapolis, IN). Total reverse rT3 and TG were measured by RIAs. Free T4 was measured by dialysis (Quest/Nichols, San Juan Capistrano, CA) and was also estimated from the T4 and the resin T4 uptake ratio as the free T4 index. Antithyroid antibodies were measured by an agglutination method.

Genetic analysis

Genomic DNA was extracted from peripheral blood mononuclear cells of the proposita and members of her family. DNA was also isolated from cultured skin fibroblasts of the proposita. Exon 10 of the TRß1 gene was amplified by PCR with a forward oligonucleotide primer (5'-AGTCTGCAGAGGCCTGGAATTGGACAAAGC-3') and a reverse primer (5'-TCCCTCCCAAATAATCCCTCCCAACACAAAG-3') at an annealing temperature of 55 C. PCR products were confirmed by direct sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems, Foster City, CA).

Intragenic polymorphic markers used for haplotype analysis were obtained from the GenBank (http://www.ncbi.nlm.nih.gov/) and Marshfield databases (http://research.marshfieldclinic.org/genetics/) as listed in Table 1Go. Oligonucleotide primer sequences and the expected PCR product sizes are also given. The UT6471 marker was amplified by a two-step PCR. After the first PCR amplification, the product was used as a template for a second PCR reaction with nested primers. PCR conditions were as follows: initial denaturation at 94 C for 4 min followed by 35 cycles at 94 C for 30 sec, annealing at 55 C for 30 sec and 72 C for 20 sec, and terminal extension at 72 C for 5 min.


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  		TABLE 1. Genetic markers

 
Spreadex Gel kits (Elchrom Scientific AG, Cham, Switzerland) were used to analyze the AFM336zb5 marker (Spreadex Gel kit 70–250) and GGAA22h08 marker (Spreadex Gel kit 100–350) in a Mini Gel Cassette of 8 cm in length. Electrophoresis was run at a constant 200 V for 1 h (AFM336zb5) or 2 h (GGAA22h08). The products of PCR amplification containing the UT6471 marker were sequenced to determine the number of AAGG and AAAG repeats. The amplified DNA segments containing the polyT markers were cloned into a pGem-T Easy Vector System (Promega Corp., Madison, WI), and the inserts of individual clones were sequenced to determine the number of Ts.

Construction of plasmids

Expression vectors, containing the mutant TRß 458G and 458E, were constructed as follows. A 140-bp fragment of the corresponding mutant TRßs, amplified from DNA of the proposita (Fig. 1Go, II-2) and her affected daughter (Fig. 1Go, III-2), were cloned into pGem-T Easy Vector and transformed in Escherichia coli JM109 (Promega). Clones were verified by sequencing. Fragments of 114 bp, carrying the 458G or 458E TRß gene mutations, were excised from the pGem-T Easy Vectors with XmaI and EcoRI restriction enzymes and subcloned into the corresponding sites of the WT TRß1 cDNA cloned in the expression vector pcDNAI/Amp-TRß1 (9).

A Palx3-Luc plasmid that expresses the firefly luciferase (Luc) gene, which has, at the 5' end, three copies of a palindromic TRE (AGGTCA-TGACCT), was used as reporter in the transient transfection assays (9).

Cell culture

HepG2 (human hepatoblastoma cell line), CV-1 (African green monkey kidney fibroblasts), and COS-7 cells (simian virus 40-transformed African green monkey kidney fibroblasts) were grown in DMEM (Invitrogen Corp., Grand Island, NY) containing 10% fetal bovine serum (FBS), 50 µg/ml gentamicin at 37 C in 100% humidity, and 10% CO2. Before transfection, cells were transferred to 12-well (HepG2 and COS-7) or six-well (CV-1) culture plates and grown in the same medium until they reached 70–80% confluence. On the day of transfection, the growth medium was removed, cells were washed twice with Hank’s buffered saline solution (Invitrogen), and 1 ml (12-well plates) or 2 ml (six-well plates) of the transfection solution containing DMEM, 10% thyroid hormone-stripped FBS, Lipofectamine (Invitrogen), and expression and reporter plasmids were added. After 5 h, cells were washed twice with Hank’s solution and incubated for 72 h in DMEM containing 10% thyroid hormone-stripped FBS and gentamicin. After incubation with this thyroid hormone-depleted medium for 24 h, cells were treated with two different T3 doses (10–7 M and 10–8 M) and incubated for 48 h. Untreated cells were cultured in parallel as controls. Cells were harvested, and luciferase activity was determined as previously described (9).

The reporter plasmid Palx3-Luc was transfected at a concentration of 1 µg/well, whereas pcDNAI/Amp-TRß1 was transfected at 5 ng/well (HepG2 cells), 30 ng/well (COS-7 cells), and 40 ng/well (CV-1 cells). These concentrations were optimal for near maximal activity of the WT TRß1 in the presence of T3. The total amount of transfected DNA was adjusted to 1.5 µg/well with empty carrier plasmid. Dominant negative effect was tested at a T3 concentration of 10–7 M by transfection of the expression plasmid containing the WT TRß1 with equal, double, and quadruple amounts of the plasmid containing the mutant TRß1s. Experiments were performed at least twice, with three to five replicates per experiment.

EMSA

WT and mutant TRß1 and RXR{alpha} proteins were synthesized by in vitro transcription/translation using the TNT Coupled Reticulocyte Lysate System (Promega). Proteins were synthesized in the presence of 35S-Met, and the product was separated by 10% SDS-PAGE. The interacting domains of the corepressor NCoR and the steroid receptor coactivator (SRC1) were placed downstream of glutathione-S-transferase (GST) in the vector PGEX4T1 (10). GST-NCoR, GST-SRC1, and GST were synthesized in E. coli, bound to Sepharose beads, and extensively washed; proteins were eluted off the beads and then used in EMSA. For EMSA, the in vitro-translated proteins were synthesized in the absence of 35S-Met and were mixed with 32P-labeled DR4 thyroid response element (AGGTCACAGGAGGTCA). Reactions were carried out in binding buffer containing 20 mM HEPES, 50 mM KCl, 20% glycerol, 1 mM dithiothreitol, 0.1 mg/ml polydeoxy (inositate-cytidilate), and salmon sperm DNA. After a 20-min incubation period at room temperature, the samples were separated on a 5% polyacrylamide gel at 220 V for 2 h and subjected to autoradiography.

Data analysis

Data are expressed as mean ± SEM of the absolute luciferase activity or percentage of change in luciferase activity. Statistical analyses were performed using ANOVA.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Pedigree and thyroid function tests are shown in Fig. 1Go. The proposita, II-2, and her two affected children, III-2 and III-3, have a thyroid phenotype suggestive of RTH, namely high levels of total and free iodothyronines with nonsuppressed TSH. TG concentration was also high in the affected subjects, except for the proposita who was on levothyroxine replacement because of previous thyroidectomy. Antibodies to TG and thyroid peroxidase were not detected. Thyroid function tests in other unaffected family members were within the normal range except for an isolated mild elevation of serum T3 concentration of the proposita’s first husband (II-1), a mild elevation of TSH of her unaffected son (III-4), and increased FT4I of one of her brothers (II-7). As shown in Fig. 2Go, the affected subjects had the highest degree of thyrotrophs T4 resistance index among the subjects with RTH studied in our laboratory harboring different TRß gene mutations, except for 438fs -> 442X (14). This index, which is considered to best define the severity of RTH at the level of the thyrotrophs (15), was 1059 and 1821 for the two untreated subjects (normal mean ± SD, 136 ± 73).



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  		FIG. 2. Correlation of the serum free T4 (FT4; expressed as percentage of the upper limit of normal) to the TSH (mU/liter) in normal subjects and patients with RTH. Affected members of Mb family have a very severe RTH phenotype as indicated by the high FT4 to TSH ratio of our series.

 
In the proposita (II-2), we identified a missense mutation in exon 10 of the TRß gene, codon 458 (GTG -> GGG), that results in the replacement of the normal valine with a glycine (V458G). Although this mutation was transmitted to her affected son (III-3), the affected daughter (III-2) had a different mutation. In fact, in III-2, the codon 458 was GAG, encoding for glutamic acid (V458E). The V458E mutation was identified in two separate DNA samples obtained from blood drawn on two occasions, and the V458G mutation was identified in a blood sample, as well as from genomic DNA isolated from cultured fibroblasts derived from a skin biopsy of the proposita. As shown in Fig. 3Go, both TRß gene alleles of the fathers and unaffected children were WT (Fig. 3Go).



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  		FIG. 3. Identification of TRß gene mutations in family Mb. The proposita (II-2) and her affected son (III-3) have a missense mutation at codon 458 (GTG -> GGG) of exon 10 of the TRß gene. Her affected daughter (III-2), however, has a different mutation at the same codon (GAG). Both TRß alleles of the fathers and unaffected children are WT. V, Valine; G, glycine; E, glutamic acid.

 
We used intragenic markers to determine which of the two maternal alleles had undergone de novo mutation in the affected daughter. The haplotype analysis showed that the already mutated maternal allele underwent a de novo mutation, resulting in the replacement of the mutant glycine with a mutant glutamic acid (Fig. 4Go). This analysis could not determine whether the mutation found in the proposita’s TRß occurred also de novo because no parental samples were available; the proposita’s mother was deceased and the father refused to participate in the study. We could identify only three of the four parental alleles in the progeny of generation II. Therefore, it is not possible to determine whether the haplotype of the mutant allele shared with three of her brothers is identical with her mutant allele.



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  		FIG. 4. Genotype analysis of all available family members. Haplotypes are indicated below each symbol of the pedigree, and identical alleles have the same shading. The markers on chromosome 3 are indicated to the left. The proposita (II-2) and her two affected children (III-2 and III-3) share the same allele. The two unaffected children (III-1 and III-4) inherited the allele not bearing the mutation in the TRß gene. This proves that the already mutated maternal allele underwent a de novo mutation, resulting in the replacement of the mutant glycine with a mutant glutamic acid in her daughter (III-2). Because samples from the proposita’s parents are not available and because only three distinct alleles have been identified in the proposita and her siblings, it is uncertain whether the mutation identified in the proposita was inherited or occurred de novo.

 
The T3-dependent transactivation of the Palx3-Luc reporter, observed in HepG2 and COS-7 cells transfected with WT TRß1, was abolished when cells were transfected with either 458G or 458E mutants (Fig. 5Go). In addition, both mutants showed a strong dominant negative effect in the three cell lines tested. This dominant negative effect was even more pronounced than that observed with the mutant TRß1 G345R (Fig. 6Go), which is considered to be among the most potent mutant TRß1 studied (9, 16).



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  		FIG. 5. Functional analysis of the mutant TRßs. HepG2 cells transfected with WT TRß1 plasmid showed a T3-dependent transactivation of the Palx3-Luc reporter. HepG2 cells transfected with TRß1 458G and 458E mutants did not show any transactivation activity. Values are presented as mean and SE (n = 5).

 


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  		FIG. 6. Dominant negative effect of the mutant TRßs. HepG2 cells were transfected with the mutant TRß1s expression vectors alone or together with the WT TRß1, and transactivation of the Palx3-Luc reporter was tested in the presence of 10–7 M T3. At all ratios of WT to mutant TRßs, the dominant negative effect was stronger with the 458G and 458E mutants than the 345R mutant.

 
Direct binding of T3 to the mutant TRß1 proteins could not be detected because the method is not sensitive at affinities lower than 1–2% that of WT. To show whether the mutant receptors preserved minimal ligand-binding property, we tested the ability of large amounts of T3 to dissociate TRß homodimers bound to DNA. Whereas 10 nM of T3 produced a clear dissociation of the WT TRß1 homodimer (data not shown), at least 100 nM T3 was required to detect dissociation of TRß1 458G and 458E bound to DNA as homodimers (Fig. 7AGo). The mutant TRß1 345R was even more resistant to T3-induced homodimer dissociation (Fig. 7AGo). Both mutants TRßs 458G and 458E retained the ability to form heterodimers with RXR{alpha} (Fig. 7BGo).



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  		FIG. 7. EMSAs. In vitro synthesized WT and mutant TRß1 proteins were tested for binding to a TRE-DR4 oligonucleotide sequence in the absence and presence of increasing concentrations of T3. A, Larger amounts of T3 were required to dissociate homodimers formed by the mutant TRß1 458G and 458E than the WT TRß1. However, TRß1 345R was even more resistant to T3-induced homodimer dissociation. B, The mutant TRßs retained the ability to form heterodimers with RXR{alpha}.

 
CV1 cells transfected with the WT TRß1 and the mutant TRß1s 458G, 458E, or 345R showed, in the absence of ligand, the expected inhibition of basal transcription of Palx3-Luc reporter gene relative to the empty pcDNA3 plasmid as control (Fig. 8Go).



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  		FIG. 8. Inhibition of basal transcription by the unliganded TRßs. WT and mutant TRß1s were transfected together with the Palx3-Luc reporter plasmid into CV1 cells. Nonsignificant differences were found between WT and mutant TRß1s (458G, 458E, and 345R). The empty plasmid pcDNA3 transfected with Palx3-Luc reporter gene was used as control. Each bar is the mean of six determinations.

 
As shown in Fig. 9Go, the binding affinity of the various mutant TRß1s to GST-NCoR fusion protein was not different for the three mutant TRß1s tested (458G, 458E, and 345R) compared with the WT TRß1.



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  		FIG. 9. Association of the corepressor NCoR with TRß1 as determined by EMSA. Binding of GST-NCoR to WT TRß1 and mutant TRß1s was not different.

 
Recruitment of SRC1 requires a ligand-dependent dissociation of the corepressor or homodimers. As shown in Fig. 10Go, formation of complexes between SRC1 and the WT and 345R TRß1 was proportional to the ability of T3 to dissociate the respective homodimers. In contrast, the TRß1s 458G and 458E failed to associate with SRC1 even though T3 dissociated their homodimers more readily than those formed with TRß1 345R.



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  		FIG. 10. EMSA with the coactivator SRC1. In contrast to the WT TRß1 and TRß1 345R, both mutant TRß1 458G and 458E failed to recruit SRC1, even though T3 was more effective in dissociating their homodimers than those formed with TRß1 345R.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We identified a family with RTH caused by novel missense mutations in codon 458 of the TRß gene. Located in the ligand-binding domain of TRß, the mutations were present in three of 10 family members studied. An interesting and unique finding is that affected family members had two distinct mutations in the same codon and allele. Whereas the proposita and one affected child harbored a TRß V458G (replacement of the normal valine for glycine), the affected daughter had a TRß V458E (replacement of the normal valine for glutamic acid). Haplotype analysis showed that the defective TRß present in the daughter (458E) was a de novo mutation. Even more unique was the finding that the mutant nucleotide, an adenine, replaced the guanine of the mutant maternal allele rather than the normal thymidine on the normal allele. Contrary to previous findings in mutant TRß genes (17, 18, 19), this de novo mutation did not occur in a CpG dinucleotide hot spot. However, it did occur as a transition in a guanidine triplet created by the mutant maternal codon 458. It is uncertain whether the location of this de novo mutation occurred by chance or as result of mutagenesis in a propitious environment created by mutant guanidine.

Transitions are more frequent than transversions in inherited nucleotide substitutions in humans (20). The relative single-base pair rate of substitution is higher for G to A transition (3.13) than for T to G transversion (0.41). Nearest-neighbor analysis of single-base pair substitutions has also shown that relative substitution rates are higher for GG to GA (1.76) than for GT to GG (0.30). Visual inspection of exon 10 revealed a quasipalindromic DNA sequence in the region between codons 449 and 459. Such sequence structures permit the formation of DNA hairpins, and where complementarity is imperfect, frame shift and base-substitution mutations have been described as a result of abnormal DNA repair (21, 22). Computerized analysis using Mfold version 3.1 (23) of a 200-bp single-strand DNA (as observed during double-strand DNA dissociation) with the mutant triplet located in the middle of the sequence showed a hairpin structure of 33 bp with imperfect DNA complementarity at codon 458 that is corrected in GAG mutation (Fig. 11Go). Whether noncomplementarity in this palindrome was responsible for the GTG to GGG and later for the GGG to GAG mutations remains unresolved. Thermodynamical analysis showed that, although single-strain DNA loop carrying GGG nucleotide has slightly lower free energy level compared with the WT, the loop carrying GAG has much lower free energy than both of them, indicating that GGG transition to GAG is thermodynamically favorable (Fig. 11Go). The high rate of G to A transitions and GG to GA substitutions, the hairpin structure around the mutation area, and the higher free energy level of the single-strand DNA chain containing the GGG mutation vs. GAG mutation suggest that GAG mutation may have not occurred by chance. Because we were not able to obtain DNA from the parents of the proposita, the information derived from haplotyping of the three bothers was insufficient to establish whether the proposita had also a de novo mutation.



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  		FIG. 11. Modeling of the DNA at the mutation site. A 33-bp hairpin structure containing the mutated nucleotide was obtained by computerized simulation (Mfold version 3.1) of a sequence containing 100 bp on each side of the mutant nucleotide. The calculated free energy levels (dG) showed decreasing values in the following order: GTG > GGG > GAG.

 
All affected family members exhibited a severe clinical and biochemical RTH phenotype. They presented with a goiter, hearing deficit, and mental retardation. Thyroid hormone levels were the highest of 376 subjects with RTH and harboring TRß gene mutations studied in our laboratory. The results of the in vitro experiments are in agreement with the clinical findings of a severe form of RTH. Both mutant TRßs, 458G and 458E, had severely impaired ligand binding and showed no T3-inducible increase in transcriptional activity using a reporter plasmid positively regulated by thyroid hormone. Both mutant TRßs had a strong dominant negative effect that was more pronounced than TRß 345R, a mutation with virtually no T3 binding, producing a severe RTH phenotype (4, 16). The more severe functional impairment of the two TRß mutants, 458G and 458E, is most likely due to their inability to recruit the coactivator, even at high T3 concentrations that effectively dissociated the mutant TRß1 homodimers. This is in contrast to the TRß1 345R, which, although more resistant to T3-induced homodimer dissociation, binds readily to SRC1. Indeed, the mutations at codon 458 are at the AF2 region, which is the site of coactivator binding. These findings showing both impairment of T3 binding and recruitment of coactivator are in agreement with the severe RTH phenotype. Seven natural mutations in the carboxyl terminus of TRß have been reported with, apparently, a not as severe phenotype as observed in affected members of the family described herein. These mutations are L454V (12), L454S (24), F455S (25), F455L (26), V458A (27, 28), F459C (29), and E460K (18, 30). When T3 binding to the mutant receptors was examined, it was normal or only modestly reduced 22–67% that of the WT TRß (12, 18, 24, 29), indicating that the phenotype was due principally to impaired interaction with the coactivator (12, 24).

The occurrence of a mutation in a mutant nucleotide is a unique finding that has not been described before. In our attempt to determine the molecular mechanisms responsible for the severe RTH phenotype in our patients, we show that TRßs 458G and 458E retained the ability to form heterodimers with RXR{alpha}. Furthermore, the unliganded TRßs 458G and 458E had a strong inhibitory effect on basal transcription of a reporter construct positively regulated by T3. Inability to recruit coactivator appears to be the principal molecular basis for the unusually severe manifestation of RTH.


   Acknowledgments
 
We are grateful to Dr. Neal H. Scherberg and his laboratory staff for performing the tests of thyroid function in sera and thank all members of the families for their willingness to participate in this study. We acknowledge the contribution of the late Dr. Jean Louis Vandalem from the University of Liege for being the first to identify the proposita and to provide samples from some of the family members. We are also grateful to Dr. Carlos Rey from the University of Santiago de Compostela School of Physics for his help with the thermodynamic analysis.


   Footnotes
 
This work was supported in part by Grants RR00055, RR18372, DK07011, DK58281, and DK15070 from the National Institutes of Health. A.M.D. is a Howard Hughes Medical Institute Predoctoral Fellow.

Present address for J.L.-A.: Department of Medicine, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain.

Present address for J.P.: Children’s Hospital of the Johannes Guttenberg University, D-55101 Mainz, Germany.

First Published Online December 14, 2004

1 J.L.-A. and A.M.D. contributed equally to this study. Back

Abbreviations: FBS, Fetal bovine serum; GST, glutathione-S-transferase; NCoR, nuclear receptor corepressor; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SRC1, steroid receptor coactivator 1; TG, thyroglobulin; TR, thyroid hormone receptor; TRE, thyroid hormone response element; TT3, total T3; TT4, total T4; WT, wild type.

Received July 27, 2004.

Accepted December 6, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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