Corresponding Author:
Samuel Refetoff
University of Chicago, MC 3090
5841 S. Maryland Ave.
Chicago, IL 60637
Tel.: (773)-702-6939
FAX: (773)-702-6940
Email: refetoff@uchicago.edu

Supported by grants DK17050, DK20595 and RR00055 from the National Institutes of Health (S.R.) and Howard Hughes Medical Institute Predoctoral Fellowship (A.M.D.)


Resistance to thyroid hormone (RTH) is a syndrome of reduced end-organ responsiveness to thyroid hormone (TH) that manifests as persistent elevation of serum levels of T₄ and T₃ with non-suppressed TSH. Following its clinical identification in 1967 (1) various potential mechanisms including transport, metabolism and action have been explored to explain the etiology of the defect (2). In 1989, three years after cloning of the nuclear TH receptor (TR) (3, 4), the first two mutations in the TRβ gene were identified as the cause for RTH (5, 6). The finding of mutations in other subjects with RTH has established the link between the syndrome and defects of the TR, a transcription factor whose principal action at the level of the nucleus is modulated by TH (7, 8). Nevertheless, in a broader sense, reduced sensitivity to TH encompasses all defects that can interfere with the expression of the biological activity of a chemically intact hormone supplied in normal amounts. These could be due to defects in 1) TH entry into the cell, 2) its intracellular metabolism and distribution, 3) cytosolic (non genomic) effects, 4) translocation into the nucleus, 5) association with the receptor and 6) abnormalities in co-regulators or other post receptor effects required for the proper mediation of TH action (Fig.1).




Figure 1. Thyroid hormone action: from entrance into the cells to the nuclear and cytosolic action. For details see text.

Non-TR RTH
Important for the understanding of the mechanism of TH action is the occurrence of RTH in the absence of TRβ mutations as 15% of families with RTH do not harbor mutations in TR. The clinical manifestations and laboratory abnormalities in such subjects are not different from those with mutations in the TRβ gene (9). Several lines of evidence suggest that cofactors involved in the TR-mediated TH action are likely candidates in the etiology of RTH (10) and this type of mechanism has been labeled as nonTR-RTH (10, 11). In humans, combined resistance to glucocorticoids, mineralocorticoids and androgens have been reported in the absence of mutations in the respective receptors (12-14) and a lack of a putative cofactor has been suggested (14).
Failure to identify mutations in the TRα gene, have led to speculations that either defects are innocuous or are lethal. Unexpectedly, mice deficient in all forms of TRα are more sensitive to TH (15), and the brain of mice deficient in TRα1 is protected from the effects of hypothyroidism (16). In contrast, KI mice, heterozygous for a mutant TRα, have severe postnatal developmental and growth retardation, as well as reduced fertility, increase in body fat, insulin resistance and decreased cold-induced thermogenesis (17-19). Homozygotes do not survive, emphasizing the noxious effect of unliganded TRα1.
Recent investigations have identified and explored the roles of cell membrane TH transporters (20-22) and the pathways of intracellular metabolism that lead to either TH activation by conversion of the secreted prohormone T₄ to T₃, or its inactivation by conversion to rT₃. However, the physiological importance of these protein molecules did not become apparent until the very recent discovery of genetic defects that produce complex clinical phenotypes and characteristic abnormalities in thyroid function tests.


TH transporter defect
Defects in the X-linked monocarboxylate transporter (MCT) 8, a transmembrane TH transporter have been reported (23-26). Affected males present a syndrome characterized by abnormal thyroid tests, high T₃, low T₄ and rT₃, slightly elevated TSH and severe psychomotor developmental delay, no verbal communication, mental retardation, generalized dystonia combined with spasticity and poor coordination. As for most X-linked diseases, female carriers have only mild thyroid test abnormalities and no neurologic manifestations. Identification of neurologically asymptomatic females allows the provision of prenatal diagnosis and genetic counseling for this serious condition.


Defects in TH metabolism
Iodothyronine deiodinases (Ds) are selenoproteins that regulate intracellular TH (Fig.2). This unique class of proteins requires selenocysteine (Sec) for enzymatic activity. Sec is incorporated into the nascent protein chain through recoding of an in frame UGA stop codon. Several factors are required for Sec insertion, cis-acting sequences present in the mRNA of a selenoprotein (UGA codon and Sec insertion sequence, SECIS) and trans-acting factors [elongation factor eEF^Sec, tRNA^Sec and SECIS-binding protein (SBP2)].

D1 and D2 are the principal enzymes that convert T₄ to T₃ and rT₃ to 3,3’-diiodothyronine (T2), while D3 and to a lesser degree D1 convert T₄ to rT₃ and T₃ to T2 (27). D activities are modulated by the availability of substrate and environmental factors such as food intake and illness. While acquired changes in D activities are common, until recently inherited defects have not been identified in humans.
We identified two families with abnormal thyroid function tests (28) suggestive of abnormal TH metabolism. In a Bedouin Saudi family, two brothers and a sister had high serum T₄ (total and free), high rT₃, low T₃ and slightly increased TSH and transient growth retardation. The parents and other 4 siblings had normal tests. In an Irish family one child born from non-consanguineous parents had a similar phenotype. Linkage analysis and sequencing excluded abnormalities in all 3 DIO genes, yet in vivo TSH-suppression tests suggested a defect in T₄ to T₃ conversion in the affected children. In vitro tests of patients’ fibroblasts showed significantly reduced baseline and cAMP-stimulated D2 activity, despite a normal increase in DIO2 mRNA expression, suggesting a defect in generating or maintaining an active D2 enzyme. The selenocysteine present in the active center of D2 is required for proper enzymatic activity (27), and D2 is subject to ubiquitination (29, 30). Systematic linkage analysis with candidate genes excluded all known factors in these pathways except for SBP2.
At the SBP2 locus the affected Bedouin children shared homozygous haplotypes. A homozygous mutation R540Q was identified in these children, and the parents and the four unaffected siblings were heterozygous carriers of this mutation. The child of Irish origin harbored compound-heterozygous mutations in SBP2 not present in controls, a nonsense mutation K438X and an intronic mutation IVS8ds+29 G->A creating an alternative donor splice site and abnormal transcripts incorporating parts of intron 8.
All four affected children from both families had reduced levels of other selenoproteins. For example glutathione peroxidase (GPx) activity in serum and in fibroblasts was decreased, as were the levels of serum selenoprotein P (SePP) and total serum selenium. The global effect of SBP2 deficiency on the synthesis of selenoproteins has been documented and represents an interesting example of epistatic effect resulting in deficiency of selenoproteins. Although the reduction in GPx and SePP is not trivial, thyroid abnormalities resulting from decreased D2 activity and likely also D1 and D3, appear to dominate the clinical phenotype. Among the known selenoproteins, the UGA codon of DIO2 gene is most distant from the SECIS element and the half-life of the protein is less than 45 min. These factors and the hierarchy among selenoproteins might aggravate a deficit in Sec incorporation producing this specific thyroid phenotype.
It is believed that SBP2 is the major determinant of Sec incorporation as its in vitro addition increases selenoprotein synthesis by 20-fold, whereas its immunodepletion eliminates Sec incorporation (31). The relatively mild phenotype manifested in the patients described above is due to partial loss of SBP2 function. In the Saudi family, the missense mutation is likely to function as a hypomorphic allele. In the affected child of the Irish family the intronic mutation results in partial alternative splicing with preservation of about 25% normal transcripts. Insight into consequences of SBP2 gene defect is novel and represents the first report of mutations in a component of the machinery leading to the synthesis of selenoproteins, and the first instance of inherited deiodinase deficiency in humans.


Concluding overview
Two novel mechanisms of reduced sensitivity to TH (Fig.3, lines B and C respectively) have been uncovered. These forms or reduced sensitivity to TH also present different modes of inheritance due to gene location and to protein function. RTH due to TR mutations is inherited as an autosomal dominant trait with the exception of the autosomal recessive inheritance of a deleted TR in the index family. The dominant inheritance requires that the mutant TR interfere with the function of the normal TR (dominant negative effect). MCT8 defect has an X-linked inheritance, and SBP2 defect has autosomal recessive inheritance. For the rest of the families with non-TR RTH of unknown cause, the mode of inheritance is less clear though dominant is apparent in some. Other defects at putative steps in TH action are still to be identified and non-Mendelian modes of inheritance and defects with low penetrance should be considered.



Figure 3. Defects in thyroid hormone action. Shown in red are reported (straight lines A, B, C) and putative (dotted line, D, E, F) defects.


Summarizing the characteristics of the 3 forms of reduced sensitivity to TH (Table 1), it is apparent that the two novel forms have distinctive patterns of TH concentrations in serum compared to those characteristic of TR mutations. Subjects with MCT8 defect have a more complex phenotype in terms of thyroid and neurological manifestations.