Resistance to thyrotropin

Abstract

Resistance to thyrotropin (RTSH) is broadly defined as reduced sensitivity of thyroid follicle cells to stimulation by biologically active TSH due to genetic defects. Affected individuals have elevated serum TSH in the absence of goiter, with the severity ranging from nongoitrous isolated hyperthyrotropinemia to severe congenital hypothyroidism with thyroid hypoplasia. Conceptually, defects leading to RTSH impair both aspects of TSH-mediated action, namely thyroid hormone synthesis and gland growth. These include inactivating mutations in the genes encoding the TSH receptor and the PAX8 transcription factor. A common third cause has been genetically mapped to a locus on chromosome 15, but the underlying pathophysiology has not yet been elucidated. This review provides a succinct overview of currently defined causes of nonsyndromic RTSH, their differential diagnoses (autoimmune; partial iodine organification defects; syndromic forms of RTSH) and implications for the clinical approach to patients with RTSH.

1. Introduction

Thyroid-stimulating hormone (thyrotropin; TSH) is secreted by the specialized cells (thyrotrophs) residing in the anterior pituitary and acts on follicular thyroid cells via binding to its cognate receptor (TSHR) to stimulate hormone production and secretion as well as differentiation and growth of the thyroid gland. It is thereby integral part of the pituitary-thyroid feedback control of thyroid function. Resistance to TSH (RTSH) is broadly defined as reduced sensitivity of thyroid follicle cells to stimulation by biologically active TSH due to genetic defects. This definition would exclude autoimmunity with TSHR-blocking antibodies mimicking the RTSH phenotype. Affected individuals have elevated serum TSH levels with normal or low levels of thyroid hormones (triiodothyronine, T₃ and thyroxine, T₄) in the presence of a eutopic, hypoplastic or normal-sized thyroid glands. They are frequently identified at birth through TSH-based neonatal screening for congenital hypothyroidism (CH).

Conceptually, defects leading to RTSH impair both aspects of TSH-mediated action: thyroid hormone synthesis and thyroid gland growth, and can be envisioned to be caused by either 1) inactivating mutations in the TSHR gene, 2) reduced quantity of TSHR secondary to defects in factors controlling TSHR expression, 3) postreceptor defects in signal transduction, e.g. defect in G proteins, and 4) defects in transcriptional master regulators required for both normal differentiated function and growth of thyroid cells.

2. RTSH due to loss-of-function mutations in TSHR

2.1. TSHR physiology

The TSHR is a G-protein coupled receptor expressed at the basolateral surface of thyroid follicle cells. It consists of a classical seven transmembrane domain (TMD) connected via a linker region (hinge region) to a large extracellular domain (ECD) principally composed of a sequence of several leucine-rich repeat regions (LRR) (Fig. 1). The latter assemble into a horseshow-like structure with the beta-strands of the LRRs forming a concave surface for ligand binding. The TMD consists of alpha helical transmembrane spanning segments connected by extracellular loops in contact with the liganded ECD, and intracellular loops involved in G-protein coupling.

Fig. 1.

Topology model of TSHR with location of confirmed or putative inactivating mutations identified in subjects with RTSH. Missense mutations are indicated in orange, nonsense mutations in red, insertions/deletions in the coding sequence in yellow, and intronic mutations in blue. Residues forming the beta strands of the leucine-rich repeats within the extracellular ligand-binding domain are marked by green arrows. SP, signal peptide sequence (removed in mature protein).

Activation by TSH binding generates a complex structural rearrangement transmitted to the intracellular G-protein binding surface formed by TMD and intracellular loops (reviewed in [1]). TSHR can signal through both Gs and Gq G-proteins. Thus, in terms of second messenger, binding of TSH activates both the cAMP pathway (via Gsα) as wells as the phosphoinositol/calcium (IP/Ca2+; via Gq) signaling cascades. While the former is linked to iodide uptake, thyroid hormone secretion, and gland growth and differentiation, the IP/Ca2+ pathway is rate-limiting for hormone synthesis by stimulating iodide organification.

Another feature relevant for TSHR physiology and the manifestation of TSHR defects is the propensity of TSHR to form dimers and/or oligomers at the surface of thyroid cells. This phenomenon provides an explanation for the interference observed with some mutant receptors when coexpressed with the wild type and should be relevant for the observed dominant transmission of some heterozygous TSHR defects [2].

2.2. Inactivating mutations of TSHR

First described in 1995 [3], at least 68 distinct TSHR loss-of-function (LOF) mutations have now been reported in patients with RTSH phenotype (Fig. 1). Except for rare deletions [4–6], the described mutations have been either point or small indel mutations in the coding sequence causing amino acid replacement (missense) or truncation (nonsense or frameshift) of the predicted protein [7–54]. TSHR LOF mutations are found throughout the receptor structure, in contrast to the gain-of-function mutations causing hyperthyroidism, which are located primarily in the TMD of the receptor. Decreased action of TSH results in reduced T₄ and T₃ synthesis and secretion, with compensatory increase in TSH secretion. The absence of goiter despite high serum level of biologically active TSH is compatible with the dominant role of TSHR-induced cAMP signaling on the growth of the thyroid gland. Although the majority of TSHR LOF mutations impair overall receptor expression level and/or ligand binding, some mutations have differential effects on the coupling of either Gs or Gq proteins. In a small number of patients, Gq-dominant mutations have been linked to an RTSH phenotype with paradoxically increased thyroidal iodine uptake, a feature associated with impaired iodine organification (“nonclasscial RTSH”) [16, 55]. Since TSHR LOF mutations have to date been rarely evaluated for both Gs and Gq coupling, it remains an open question whether there are clear clinical correlates to mutations with differential effects on dual G protein coupling.

The magnitude of functional impairment of TSHR correlates to some degree with the severity of the RTSH phenotype: complete loss of TSHR function due to biallelic complete LOF mutations produces severe CH [7]. In these cases, severe hypoplasia with absent radiotracer uptake can be mistaken for athyreosis, but serum thyroglobulin (TG) is always detectable (“apparent athyreosis”). Biallelic defects (compound heterozygous or homozygous) with residual receptor function allow for either partial compensation (mild hypothyroidism) or full compensation (isolated hyperthyrotropinemia, approximately one third of cases) by high serum TSH. The inheritance of RTSH due to TSHR defects is typically considered recessive, since monoallelic TSHR defects are not regularly detected in neonatal screening using TSH cut-off value >20 uU/ml [10, 52]. Heterozygous TSHR mutations do, however, play a more prominent role in the pathogenesis of isolated non-autoimmune hyperthyrotropinemia (NAHT) diagnosed after the neonatal period. For instance, in the largest cohort of pediatric NAHT patients studied so far [10], about 12% of the patients carried potentially pathogenic heterozygous mutations (compared to a estimated frequency of <1% of heterozygous mutation carriers in the general population) [33, 52].

The mutational spectrum of TSHR mutations differs among different populations, in part due to the frequency of population-specific founder mutations. It is thus not surprising that the reported overall prevalence of TSHR mutations in patients with non-autoimmune hyperthyrotropinema varies widely between studies of different populations. In various East-Asian cohorts with nonsyndromic congenital hyperthyrotropinemia (hypothyroidism), between 4.2% and 9.4% harbored mono- or biallelic TSHR mutations. About 75% of which were of the R450H variant that is found at a prevalence of about 0.5% in the corresponding general populations [33, 34, 46, 49]. TSHR LOF mutations are the most common cause of non-goitrous CH in consanguineous families [28] and specific founder mutations have been found in over half of patients with subclinical hypothyroidism in a consanguineous Arab-Muslim population [31]. Genetic analysis of the TSHR gene should therefore especially be considered if there is parental consanguinity or a family history suggestive of autosomal recessive inheritance of the RTSH phenotype.

3. RTSH due to loss-of-function mutations in PAX8

3.1. PAX physiology

PAX8 is a member of the paired box domain containing transcription factors that plays an essential role in the morphogenesis of the thyroid gland, the maintenance of a thyroid-differentiated phenotype [56], and the survival of differentiated thyroid follicle cells [57, 58]. PAX8, together with the homeobox protein NKX2-1, is the earliest marker of thyroid cell specification in the median thyroid anlage of both human and mice. The essential role of PAX8 for thyroid development was first shown in Pax8 knockout mice, in which the thyroid is hypoplastic with residual tissue only containing C cells derived from the lateral thyroid anlage [59]. In synergy with NKX2-1, PAX8 expression promotes the differentiation of functional thyroid tissue from embryonic stem cells and with the aid of TSH regulates expression of terminal differentiation markers, including thyroglobulin (TG), thyroid peroxidase (TPO), and the sodium-iodide symporter (SLC5A5; NIS) producing a fully functional thyroid gland synthesizing T₄ [60].

3.2. PAX8 mutations in RTSH

Although initially associated with thyroid dysgenesis [61], PAX8 mutations are not a relevant cause of sporadic thyroid ectopy or genuine agenesis [62–64] but found in a minority of cases (e.g. 1/28 German, 1/16 Chinese) within the normotopic hypoplasia subgroup [65–68]. More generally, heterozygous PAX8 LOF mutations have to be considered as another cause of RTSH that is clinically and by thyroid function tests indistinguishable from that caused by TSHR mutations. The clinical severity can thus range from subclinical hypothyroidism with normal-sized gland to overt hypothyroidism with severe thyroid gland hypoplasia. The most common mechanism involves mutations in the paired box domain disrupting binding to target sites, thereby leading to reduced expression of target genes (Fig. 2). The presence of RTSH-associated PAX8 promoter variants [69–71], the observation of a frameshift mutation with demonstrated protein instability [72], and the autoregulation of PAX8 by binding to its own promoter [73] are also consistent with a haploinsufficiency mechanism. A noteworthy mutational hotspot is the CpG dinucleotide at codon 31, for which frequent mutational events (R31H and R31C) have been reported [61, 65, 67, 74–77]. For some of the reported mutations, the primary defect is the impaired synergism with other thyroid transcription factors (NKX2-1) or insufficient recruitment of coactivators (p300) without altering DNA binding [78–80].

Fig. 2.

PAX8 gene mutations identified in patients with RTSH phenotype. The PAX8 structure comprises an N-terminal paired box (prd) DNA-binding domain and C-terminal region crucial for transactivation activity. Colored boxes indicate the relative positions of prd domain, conserved octapeptide sequence, (partial) homeodomain-homolog region, and of regions containing repressor or activator activity [108]. The expanded view of the prd domain reveals two subdomains (PAI and RED), each defined by trihelical helix-turn-helix motifs with independent DNA-binding activities. Missense mutations within the PAI subdomain interfering with the DNA-binding induced-fit of the helix-turn-helix motif are the most common mutational events in PAX8-associated RTSH.

Inheritance of PAX8 linked RTSH follows an autosomal dominant segregation pattern [81], but often shows highly variable expressivity within affected members of the same family [82]. Thus, there is no clear correlation between the activity of mutant PAX8 proteins in vitro and the severity of RTSH in patients. In addition, incomplete penetrance [83], parental mosaicism [84], and late-onset of RTSH phenotype due to insufficient postnatal thyroid growth [64, 77, 85, 86] have been shown to potentially mask the inherited nature of the condition.

PAX8 is also expressed during mammalian kidney development and, at least in mice, plays a redundant role with PAX2 in formation of the initial pronephros [87]. Thus, kidney organogenesis in Pax8 mutant mice is generally normal [59]. Yet, several human carriers of PAX8 gene mutation were reported to have associated kidney and urogenital abnormalities [28, 77, 85, 88]. It is tempting to speculate that the PAX8 mutations may have contributed to these non-thyroidal developmental defects.

4. RTSH linked to a defect on the long arm of chromosome 15

Mutations in TSHR or PAX8 have only been found in a relatively small proportion of screened patients with RTSH phenotype suggesting that additional etiologies remain to be discovered. With expected locus heterogeneity in RTSH, one approach is to focus on large RTSH kindreds with sufficient statistical power for genome-wide linkage scans. Among six multigenerational families, in which non-syndromic RTSH segregated in autosomal dominant fashion with high penetrance, yet variable expressivity, only one harbored a mutation in the PAX8 candidate gene [78, 89]. In the remaining five families, the defect was mapped to a single, 2.9 Megabase interval on chromosome 15q25.3–26.1 (combined LOD score of 14.6) [90] (Fig. 3). Since there were no genealogical links or evidence for shared ancestral haplotypes, genetic defects in this locus are expected to be a rather prevalent event in RTSH. While none of the protein-coding genes in this interval appeared to be a plausible candidate gene, recent genome-wide association studies have found a significant association between common single nucleotide polymorphisms in the center of the linked region containing a micro-RNA cluster and TSH serum level in the general population [91, 92]. Thus, elucidating the precise genetic cause for this form of RTSH may shed light on a novel thyroid-specific expressed modulator of TSH-responsiveness.

Fig. 3.

Autosomal-dominant RTSH linked to a locus on the long arm of chromosome 15. A) Multipoint genetic linkage analysis of chromosome 15 in five extended families with dominant inherited RTSH. The analysis shown includes data from additional family members not available in the original publication [90]. B) Example of fine mapping of the critical recombinants in family 25 using short tandem repeat markers. The RTSH-associated haplotype can be narrowed to a 2.9 megabase interval containing sixteen positional candidate genes. Note that common single nucleotide polymorphisms within the central micro RNA cluster (mir7-2, mir1179) have recently been found to be significantly associated with serum TSH level in the general population [91, 92]. Adapted from ref. [90] with permission.

5. RTSH as part of complex syndromes

Abnormal thyroid function consistent with RTSH is also found as a feature of complex syndromes that obligatorily involve other organs. In these patients, the non-thyroidal abnormalities dominate the clinical presentation and the underlying genetic defects should not be considered candidate genes for patients with isolated RTSH phenotype.

5.1. RTSH caused by mutations in GNAS1 (Albright hereditary osteodystrophy)

Heterozygous germline mutations in the gene encoding the alpha subunit of G stimulatory protein (Gsα, GNAS1) cause hypocalcemia and hyperphosphatemia due to impaired signaling transduction from the parathormone receptor (pseudohypoparathyroidism, PHP Ia) [93]. Haploinsufficiency for GNAS1 also explains the resistance to other hormones, specifically gonadotropins and TSH. Clinically this syndrome is referred to as Albright hereditary osteodystrophy characterized by typical physical features (short stature, short neck, round face, obesity, brachymetacarpy, subcutaneous ossification) and mental retardation.

5.2. RTSH caused by mutations in NKX2-1

NKX2-1 (also known as thyroid transcription factor 1, TITF1) is a homeobox transcription factor critical for the development of thyroid gland, basal ganglia and lung parenchyma. It is involved in maintaining the expression of thyroid-specific genes (TPO, TG, TSHR) in apparent synergism with PAX8. Haploinsufficiency for NKX2-1, due to either chromosomal deletions encompassing the gene locus [94] or deleterious gene mutations ([95], [96], and recently reviewed in ref. [97]), produces a “brain-thyroid-lung” syndrome. The severity of the individual components of the syndrome is very variable, and includes: 1) RTSH (70% of patients), 2) “benign hereditary chorea” (90% of patients) manifesting as neonatal hypotonia preceding the development of juvenile choreoathetosis and ataxia, 3) respiratory distress (55% of patients) due to lung hypoplasia causing significantly increased mortality. Inheritance of the defect is autosomal dominant with variable penetrance, however, most of the reported mutations have apparently arisen de novo. The RTSH phenotype, if present, is in the majority of cases compensated (i.e., isolated hyperthyrotropinema) [98].

6. Organification defects presenting with hallmarks of RTSH

Defects in thyroid hormonogenesis due to impairment of the enzymatic machinery in iodine organification are classically associated with thyroid gland enlargement. However, in partial defects of iodine organification, goiter is frequently absent despite elevated serum TSH [99–102]. These patients thus present with the hallmarks of mild RTSH (elevated TSH, low or normal T₄, normal-sized gland). The common genetic defects in these patients are in DUOX2 and DUOXA2, which encode the heterodimeric dual oxidase enzyme complex that is rate limiting in the iodine organification [103]. For instance, only one out of twelve Korean CH patients with DUOX2 or DUOXA2 mutations was noted to have thyroid gland enlargement [101]. In contrast to genuine RTSH, whose postnatal course is either stable (TSHR, Chr15-associated) or tends to be progressive (PAX8) due to insufficient thyroid growth, partial defects in the DUOX2 system are often self-limiting only manifesting during the newborn period (transient CH) [100].

In this context the recent report on SLC26A4 (Pendrin) mutations in two patients with apparent RTSH and thyroid gland hypoplasia is noteworthy [104]. Biallelic SLC26A4 mutations are a cause of sensorineural hearing loss with bilateral enlargement of the vestibular aqueduct in combination with goiter and/or CH (Pendred syndrome). In the follicular thyroid cells, SLC26A4 is localized to the apical membrane and mediates the iodine efflux into the follicular lumen where organification takes place. It is believed that reduced thyroid gland size in these patients is a consequence of severe iodide deficiency within the follicular lumen concomitant with upregulation of the H₂O₂-generating enzymes leading to oxidative stress and secondary epithelial atrophy [105].

7. Recommendations for treatment and genetic screening

Individuals with uncompensated RTSH should be treated with levothyroxine (L-T₄), like any other patient with primary hypothyroidism. Since these subjects have normal responsiveness to thyroid hormone, the goal is to normalize their serum TSH concentration. Immediate initiation of replacement therapy with L-T₄ is crucial in all infants diagnosed with CH by neonatal screening, if the elevated blood TSH is confirmed on a serum sample on day 3 or 6 of life and is accompanied by low T₄.

In individuals with compensated RTSH (euthyroid hyperthyrotropinema), longitudinal studies of individuals with TSHR LOF mutation or with RTSH linked to Chr15 indicate that the elevated TSH concentrations stimulate an adequate production of thyroid hormones and L-T₄ therapy should thus be dispensable [10, 42, 89, 106, 107]. In fact, compared to a patient cohort receiving L-T₄ supplementation, untreated patients with compensated RTSH had no obvious signs of growth or neurological abnormalities [10]. There was also no evidence for tissue hypothyroidism or the development of pituitary hyperplasia as a consequence of chronic thyrotroph hyperstimulation [10].

In patients with TSHR LOF mutations and those in whom the defect has been linked to the chromosome 15q locus, the RTSH phenotype appears to be stable over time. In contrast, mutations in PAX8 have been repeatedly reported to manifest RTSH that progresses during postnatal growth of the thyroid gland indicating that the defect in growth and/or survival of follicular thyroid cells cannot be permanently compensated [64, 77, 85, 86]. Genetic analysis may therefore provide a diagnostic tool to guide therapy and follow-up of RTSH patients.

PRACTICE POINTS.

• RTSH should be considered in the differential diagnosis of all patients with non-autoimmune, nongoitrous hyperthyrotropinemia with or without low serum iodothyronines or clinical stigmata of hypothyroidism

• Genetic analysis has the potential to provide a definitive diagnosis with relevance for prognosis, follow-up and genetic counseling.

• Standard L-T₄ replacement aiming to normalize serum TSH level is required in all hypothyroid patients, but the need of therapy is questionable in individuals with fully compensated RTSH and isolated hyperthyrotropinemia.

RESEARCH AGENDA.

• Elucidating the precise genetic cause for RTSH linked to the Chr15q locus may shed light on a novel modulator of TSH-responsiveness in health and disease.

• The role of Gq signaling in TSHR LOF mutations and its potential relevance for distinct clinical RTSH subtypes requires more systematic investigation.