Conflict of interest declaration: None

Correspondence to:
Roy E. Weiss, MD, PhD
Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism
The University of Chicago, MC 3090
5841 S. Maryland Ave
Chicago, IL 60637
TEL: 773-702-9266
FAX: 773-834-3966
email: rweiss@medicine.bsd.uchicago.edu

ABSTRACT
Resistance to thyroid hormone (RTH) is an inherited syndrome of reduced end-organ responsiveness to thyroid hormone (TH). It is characterized by elevated TH levels and nonsupressed serum TSH in the presence of a goiter. As the term implies, subjects with RTH have impaired responsiveness to TH manifested to variable degrees in different tissues. TH action is mediated by the TH receptors (TR) β and α. The etiology of RTH is usually due to a mutation in the TRβ gene. The mutant TRβ proteins have impaired TH binding and/or cause impaired activation of TH-responsive genes. However, 15% of subjects with a clinically identical RTH phenotype have no demonstrable mutations in the TRβ gene or in TRα gene, when examined. These subjects are classified as nonTR-RTH. The lack of TR gene mutation has been confirmed by sequencing both cDNA and gDNA and, in 4 families, TRβ mutations have additionally been excluded by linkage analysis. We have identified 39 affected individuals belonging to 29 kindreds with nonTR-RTH. This relatively large number of individuals has allowed us to appreciate subtle differences in the demographics of nonTR-RTH compared to RTH with TRβ mutations, including a female preponderance in the former (2.5:1). However, the key component to the phenotypes, namely TH and TSH levels, do not differ from RTH caused by TRβ gene mutations. Despite the discovery of nonTR-RTH 15 years ago, the molecular basis for this condition has remained elusive.

TH Action and Reduced Sensitivity to the Hormone
TH action requires more than 30 different cofactors which involve several distinct processes. The first step in TH action is for the hormone to enter the cell. This is achieved through active cell membrane transport. T4 and T3 transport is mediated by an active transport process through a family of TH transporters, including the monocarboxylate transporter 8 (MCT-8) (1). In the cell T4 is either activated by 5’ deiodination to form T3 or inactivated by 5-deiodination to form reverse T3. One mode of TH action is through rapid, non-genomic pathways, which are exerted at the level of the plasma membrane and cytoplasm (2). However, the principal, best-studied and characterized effect requires the translocation of the hormone into the nucleus where it interacts with TRs to activate or repress transcription of specific target genes. These genes contain nucleotide sequences at or near their promoter regions (TH response elements or TREs) recognized by TRs for binding. In the absence of TH, TRs homodimerize and associate with nuclear corepressors. These complexes have silencing effect on genes positively regulated by TH. T3 binding to TRs produces conformational changes, which trigger a chain of processes, including release of the corepressor, often heterodimerization of TR with the retinoid X receptor (RXR) and recruitment of coactivators and a large number of other proteins. In positively controlled genes by TH, this results in making the DNA more accessible for transcription (3). If any of the above molecules (transporters, TH activating enzymes, repressors, activators, etc.) were dysfunctional, a form of reduced TH sensitivity could ensue some sharing the phenotype of RTH. However, since some of the accessory molecules serve in more than one pathway, the phenotype resulting from a defect cannot be predicted.

Clinical Features of RTH and Course of the Disease
The cardinal features of RTH are: 1) elevated serum levels of free T4 and often free T3; 2) normal or slightly increased serum thyrotropin (TSH); and 3) absence of typical symptoms and metabolic consequences of TH excess (4, 5).
The precise incidence of RTH is not known as it is usually not detected by routine neonatal screening for hypothyroidism, using blood spot TSH determination. A limited screen for high T4 values found a prevalence of 1:40,000 life births (6).
Characteristic of the RTH syndrome is the paucity of specific clinical manifestations. When present, they are variable from one patient to another (4, 7) Presenting symptoms and signs are goiter, hyperactive behavior, learning disabilities, developmental delay and sinus tachycardia. The finding of elevated serum TH levels in association with nonsuppressed TSH usually leads to suspect the diagnosis.
The majority of subjects maintain a normal metabolic state at the expense of high TH levels. This compensation for the hyposensitivity to TH is variable not only among individuals but also in different tissues. As a consequence, clinical and laboratory evidence of TH deficiency and excess often coexist. For example, delayed growth and bone maturation and learning disabilities, suggestive of hypothyroidism, can be present along with hyperactivity and tachycardia, compatible with thyrotoxicosis. Common clinical features are given in Table 1. They occur with similar frequency in subject with TRβ gene mutations or without. Frank symptoms of hypothyroidism are more common in individuals who have received treatment to normalize their circulating TH levels.



Goiter is by far the most common finding, reported in 66-95% of cases. Enlargement is usually diffuse. Sinus tachycardia is also very common, which, together with goiter, often lead to the erroneous diagnosis of autoimmune thyrotoxicosis.
About one-half of subjects with RTH have some degree of learning disability with or without attention deficit hyperactivity disorder (4). One-quarter have intellectual quotients (IQ) less than 85 but frank mental retardation (IQ <60) was found only in 3% of cases. Deaf-mutism and color blindness occurred in all three affected members of a single family with TRß gene deletion (8).
The course of the disease is as variable as its presentation. Most subjects have normal growth and development, and lead a normal life at the expense of high TH levels and a small goiter.
Others present variable degrees of mental and growth retardation. Symptoms of hyperactivity tend to improve with age. Goiter usually recurs after surgery. As a consequence, some subjects have been submitted to several thyroidectomies or treatments with radioiodide (4).

RTH and TRβ Gene Mutations
In the majority of cases, RTH is caused by mutations in the TRβ gene, located on chromosome 3.
Mutations are found in the carboxyl terminus covering the ligand-binding domain and adjacent hinge domain of the TRβ protein (9-11). They are contained within three clusters rich in CG “hot spots”, separated by areas devoid of mutations (cold regions). The latter are located between codons 282 and 310, and with the exception of 383, codons 353 and 429. No mutation has been reported upstream of codon 234. As cold regions are not devoid of “hot spots”, the lack of mutations reflects the observation that mutations in the second cold region does not impair TR function and, therefore, is not expected to produce a phenotype (5)
TRβ gene defects have been identified in 473 families comprising more than 150 distinct mutations. The authors have found mutations in 148 families and a partial listing is available from http://www.receptors.org/cgi-bin/nrmd/nrmd.py. Though mostly missense, nucleotide deletion and insertions producing frameshifts have created nonsense proteins with two additional aminoacids or produced truncated receptors. In only one family complete TRβ gene deletion resulted in recessively inherited RTH. The mutant TRβ molecules have either reduced affinity for T3 (9, 10) or impaired interaction with one of the cofactors involved in the mediation of thyroid hormone action (10, 12-14).
As TR mutants are still able to bind to TREs on DNA and dimerize with normal TRs or the RXR partner, they interfere with the function of the normal TRs, explaining the dominant mode of inheritance. Therefore, it is not surprising that in the single family reported with a deletion of all coding sequences of the TRβ gene, only homozygotes manifest the phenotype of RTH (8).
No mutations in the TRα gene have been identified so far in humans. Based on observations in transgenic mice a putative TRα gene mutation should not cause typical thyroid function tests as seen in RTH.

nonTR-RTH: Definition and Demographics
In 1996, we reported a family in which RTH manifested in the absence of TRβ gene mutation and a TRβ gene transcripts of normal size and abundance (15). In addition abnormalities of TRβ were excluded in this family because of absence of phenotype cosegregation with the TRβ allele.
Nevertheless, fibroblasts were resistant to the in-vitro effect of TH. Recombinant wild-type (WT) TRβ interacted aberrantly with nuclear extracts of fibroblasts from affected individuals of the family but not from normal individuals or subjects with complete TRβ gene deletion and Far Western analysis revealed an additional 84 kD band. More families with nonTR-RTH were subsequently reported (16- 19).
We evaluated 39 affected subjects with nonTR-RTH and 139 unaffected first degree relatives from 29 different families. Comparison of the thyroid function test results of the 39 affected by nonTRRTH with the corresponding.473 subjects with TRβ gene mutations showed no differences (Table 2). While RTH caused by TRβ gene mutations has equal gender incidence, nonTR-RTH is more common in females (2.5:1). The possibility of an autoimmune component was excluded by the absence of higher frequency of thyroid autoantibodies. NonTR-RTH occurred mostly sporadically with only 6 families having more than one affected subject. Recessive inheritance and mosaicism need to be considered and when possible excluded.

Laboratory Diagnosis of nonTR-RTH
The laboratory diagnosis of nonTR-RTH is similar to that previously published for RTH. No single test is conclusive and diagnosis of RTH must rest on a combination of test and observations: 1) the absence of an elevated serum concentration of the alpha pituitary glycoprotein subunit; 2) stimulation of TSH following the administration of TSH-releasing hormone (TRH); 3) absence of elevated serum sex hormone-binding globulin concentration (SHBG), reflecting a euthyroid state; and 4) ability to suppress serum TSH with supraphysiological doses of L-T3.
The measurement of responses to the administration of incremental doses of TH is the best mean to assess the presence and magnitude of the hormonal resistance and obtain a clinical diagnosis of RTH. The rational for the use of L-T3 rather than L-T4 is its direct effect on tissues, independent of variations in T4 metabolism. The rapid onset of L-T3 action reduces the period of hormone administration and the shorter half-life of this hormone decreases the duration of symptoms that may arise in hormonally responsive subjects. It involves the administration of three incremental doses of L-T3, each for the duration of 3 days. Amounts range from just below to 3-times above replacement. Hospitalization for 11 days is required for the detailed study, which includes measurement of sleeping pulse, basal metabolic rate (BMR) and calorie balance for which food intake is controlled and urinary nitrogen excretion is measured (4). A TRH test is performed at baseline and at the time of the administration of the last L-T3 dose of each increment. Blood samples drawn over the period of 180 min are used to measure the TSH and prolactin responses as well as the nadir and peak of serum T3 achieved with each incremental dose. Measurements of TG and T4 assess the magnitude of thyroid gland suppression, while those of serum cholesterol, creatine kinase, ferritin, SHBG and osteocalcin (OC), the responses of peripheral tissues to the hormone. Whereas these tests can confirm or exclude RTH, they are unable to distinguish TRβ-RTH from nonTR-RTH (Figure 1).

Differential Diagnosis of nonTR-RTH
The combination of non-suppressed (normal or slightly elevated) serum TSH with increased concentrations of T₄, T₃ or both, is characteristic of the three syndromes of reduced sensitivity to TH.
However, the most difficult differential diagnosis to make is between RTH due to TRβ gene mutations and nonTR-RTH as appreciated from the overlapping phenotype and clinical characteristics. Gene sequencing of both cDNA and gDNA and ideally linkage data (when family size permits) can be very helpful to distinguish the two. In addition genetic analysis using several tissue as source of DNA can identify subject with mosaicism due to de-novo mutation.

1. MCT8 Mutation (Transport Defect)
Although the clinical presentation of TH cell transporter defects involving other cell-membrane transporters than MCT8, is unknown, the latter always presents in males accompanied by psychomotor abnormalities, including truncal hypotonia, limb spasticity, poor head control, dyskinetic movements and absent or garbled speech. However, presence of the characteristic thyroid test abnormalities is mandatory. Typical serum test abnormalities are high T3, low reverse T3 and often slightly reduced T4 concentrations.
The lowish serum T4 concentration and psychomotor abnormalities should enable the physician to distinguish MCT8 from RTH (20). Sequencing of the MCT8 gene in subjects with similar psychomotor manifestations but no characteristic thyroid test changes have yielded negative results (21).

2. SECISBP-2 Mutation (T4 to T3 Conversion Defect)
Elevated serum T4 can be observed in subjects with defects in the conversion of T4 to T3. Patients with defects in 5’ deiodination are unable to generate sufficient amount of T3 resulting in pituitary stimulation of TSH and increase in serum T4 concentration. To date the only gene mutation


Figure 1. A. Thyrotroph responses to TRH stimulation at baseline and after the administration of graded doses of L-T3. The hormone was given in three incremental doses, each for 3 days. Results are shown for patients with RTH in the presence (left) or absence (right) of a TRβ gene mutation, together with the unaffected mother of the patient with nonTR-RTH (center). B. Responses of peripheral tissues to the administration of L-T3 in the presence or absence of mutations in the TRβ gene. Note the stimulation of ferritin and sex hormone binding globulin (SHBG) and the suppression of cholesterol and creatine kinase (CK) in the normal subject. Responses in affected subjects, with or without a TRβ gene mutation, were blunted or paradoxical. [Modified from www.thyroidmanager.org, chapter 16c].

found to result in a iodothyronine deiodinase defect is selenocysteine incorporation sequence-binding protein 2 (SECISBP-2). The defect causes a selective, though generalized reduction in the synthesis of selenoproteins. These subjects are easily distinguished from RTH subjects due to the low T3 (22).
Growth retardation in childhood and azoospermia in adulthood are common.

3. Binding Defects (TBG Excess; FDH)
RTH is characterized by elevation of usually both free T4 and T3 levels with non suppressed TSH.
Subjects with familial dysalbuminemic hyperthyroxinemia caused by albumin gene mutations, or thyroxine binding globulin (TBG) excess present with elevated total T4 and T3, but the free hormone concentrations, when measured by equilibrium dialysis or ultrafiltration, are normal.

4. Mosaicism
Any subject expressing the RTH phenotype in whom no mutation can be demonstrated in a particular cell lineage may have mosaicism. If peripheral blood leukocytes (the most common source of DNA) are not found to harbor a TRβ gene mutation, DNA from skin fibroblasts, buccal epithelial cells, sperm (all easily accessible) or other available tissues should be analyzed (23). Such a patient was initially believed to have nonTR-RTH. In the list of subjects with nonTR-RTH (Table 2), the number of tissues examined are listed.

5. TSH Secreting Pituitary Tumor
Patients with TSH secreting tumors display thyroid function tests similar to those of subjects with nonTR-RTH and also have no detectable TRβ gene mutations. Pituitary microadenoma may be too small to be detected by imaging. More often a positive MRI may be associated with RTH. The finding of elevated serum α-subunit to TSH ratios and failure to respond to TSH releasing hormone (TRH) are useful tests to distinguish TSH secreting pituitary tumors from RTH, irrespective of the presence or absence of TRβ gene mutation. Furthermore, the presence of more than one family member with the same phenotype makes a TSH pituitary tumor unlikely. Rarely, somatic TRβ gene mutations can produce TSH secreting adenomas (24).
Treatment of nonTR-RTH
As treatment of RTH is not dictated by the presence and nature of the TRβ gene mutation, the therapeutic approach in nonTR-RTH is not different, being aimed at alleviating symptoms when present. Stigmata of TH deficiency are treated with L-T4 and symptoms are TH excess are treated with β adrenergic blockers. It is important not to treat asymptomatic, fully compensated, individuals with the sole purpose of correcting the laboratory test abnormalities. Prior ablative treatment, resulting from misdiagnosis, requires the judicious administration of TH, often in supraphysiological doses.