Central Hypothyroidism and Novel Clinical Phenotypes in Hemizygous Truncation of TBL1X

Abstract

Transducin β-like 1 X-linked (TBL1X) gene encodes a subunit of the nuclear corepressor-silencing mediator for retinoid and thyroid hormone receptor complex (NCoR-SMRT) involved in repression of thyroid hormone action in the pituitary and hypothalamus. TBL1X defects were recently associated with central hypothyroidism and hearing loss. The current study aims to describe the clinical and genetic characterization of a male diagnosed with central hypothyroidism through thyroid hormone profiling, TRH test, brain MRI, audiometry, and psychological evaluation. Next-generation sequencing of known genes involved in thyroid disorders was implemented. The 6-year-old boy was diagnosed with central hypothyroidism [free T4: 10.42 pmol/L (normal: 12 to 22 pmol/L); TSH: 1.57 mIU/L (normal: 0.7 to 5.7 mIU/L)], with a mildly reduced TSH response to TRH. He was further diagnosed with attention-deficit/hyperactivity disorder (ADHD) at 7 years, alternating episodes of encopresis and constipation, and frequent headaches. MRI showed a normal pituitary but detected a Chiari malformation type I (CMI). At 10 years, audiometry identified poor hearing threshold at high frequencies. Sequencing revealed a nonsense hemizygous mutation in TBL1X [c.1015C>T; p.(Arg339Ter)] largely truncating its WD-40 repeat domain involved in nuclear protein-protein interactions. In conclusion, to our knowledge, we identified the first severely truncating TBL1X mutation in a patient with central hypothyroidism, hypoacusia, and novel clinical features like ADHD, gastrointestinal dysmotility, and CMI. Given the relevance of TBL1X and NCoR-SMRT for the regulation of transcriptional programs at different tissues (pituitary, cochlea, brain, fossa posterior, and cerebellum), severe mutations in TBL1X may lead to a distinct syndrome with a phenotypic spectrum wider than previously reported.

Central congenital hypothyroidism (CCH) is an underdiagnosed disorder characterized by deficient production of thyroid hormones due to reduced TSH synthesis, secretion, or bioactivity, which fails to properly stimulate an otherwise normal thyroid gland [1, 2]. To date, defects in only four genes have been identified in patients with CCH: TSHB (encoding the B-subunit of the TSH glycoprotein hormone), TRHR (the specific 7-transmembrane domain receptor for hypothalamic TRH), IGSF1 (a protein regulating the expression of TRHR in pituitary thyrotropes), and the recently identified TBL1X (transducin β-like 1 X-linked), a subunit of the nuclear corepressor-silencing mediator for retinoid and thyroid hormone receptor complex (NCoR-SMRT) complex modulating thyroid hormone action in thyrotropes [3].

TBL1X is located at the Xp22.3-p22.2 chromosome and encodes a protein highly expressed in pituitary and present in the hypothalamus, representing an essential subunit of the NCoR-SMRT involved in T3-regulated gene expression [3–5].

A recent publication reported the identification of missense and splice-site point mutations in TBL1X in six unrelated families, variably presenting congenital T4 deficiency or normal T4 levels at birth [3]. Defects in this gene have also been associated with hearing loss [3, 6]. TBLX1 was linked to autism in genomewide association studies; however, neurodevelopmental defects have not been yet reported in patients with TBL1X defects [3]. The pathogenic mechanism(s) underlying this X-linked disorder remains unknown.

Here, we present, to our knowledge, the first nonsense and de novo mutation in the TBL1X gene in a patient with CCH, hearing loss, attention-deficit and hyperactivity disorder (ADHD), encopresis, and CMI.

1. Materials and Methods

The coding regions of the candidate genes for CCH (TSHB, TRHR, and IGSF1) were amplified by PCR using appropriate primers flanking each exon. PCR products were purified and directly sequenced on an automated DNA sequencer (3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). A targeted panel of next-generation sequencing, including the coding region and flanking intronic boundaries of 252 genes involved in (human and experimental) thyroid disorders, was investigated using the NextSeq 500 platform (Illumina, San Diego, CA) as reported [7]. Genes included known or plausible candidates for hypothalamic/pituitary (TSHB, TRHR, TRH, IGSF1, TBL1X, NCOR1, RXRG, RXRA, THRB, THRA, TBL1XR1, GPS2, HDAC3, NCOA1, NCOA2, NCOA3, NCOA4, GATA2, NR4A1, PITX2, PITX1, PCSK1, CGA, LHX3, BMP4, SOX2, CREB1, MED1, MED12, DIO2, SLC16A2) or primary nonautoimmune hypothyroidism (TPO, TG, TSHR, DUOX2, DUOX1, DUOXA1, DUOXA2, NKX2-I, PAX8, SLC26A4, NKX2-5, RUNX2, TBX5, SLC5A5, FOXE1). The complete list of genes is available upon request. Genetic variants were filtered on population frequency and pathogenic prediction criteria using five publicly available databases (dbSNP, 1000 Genomes Project, The Exome Aggregation Consortium, Human Gene Mutation Database, and ClinVar). The presence of the mutation identified in the index case was investigated in the patient’s parents and sister by Sanger sequencing.

The patient was clinically (hormonally, imaging) and genetically characterized. Serum TSH, free T4 (FT4), total T3, prolactin, and cortisol were determined with chemiluminescent immunoanalysis in the Centaur XP (Siemens, Healthineers, Erlangen, Germany). IGF1, IGFBP3, and ACTH were measured with chemiluminescent immunoanalysis in the Immulite 2000 (Siemens, Healthineers, Erlangen, Germany). TRH stimulation test was performed as previously reported [8]. Thyroid volume was calculated from three-dimensional measurements of each lobule on ultrasounds according to the formula, volume (mL) = 0.479 × depth × width × length (cm), and compared with standards of the local population [9]. Informed consent for genetic studies was obtained from parents of the patient, according to protocols from the ethical committee of our institution.

2. Results

A. Clinical Case

The patient is a 10-year-old male of Spanish descent, born to nonconsanguineous parents in a twin pregnancy derived from in vitro fertilization of own parental sperm and oocytes (Fig. 1A). His father has type 1 diabetes mellitus and a familial history of type 2 diabetes mellitus but no antecedents of hypothyroidism. His mother and twin sister showed an unremarkable phenotype (Fig. 1A).

Figure 1.

Clinical and biochemical features of the patient with a TBL1X defect. (A) Pedigree of members of the family showing patient born of twin pregnancy from nonconsanguineous parents. Thyroid hormone profile and glycosylated hemoglobin were used to evaluate hypothyroidism and diabetes, respectively, in all members of the family. Italics represent values above normal ranges. (B) Thyrotropin response of the index patient (6 y of age without levothyroxine treatment) at the TRH test during 180 min. TSH and prolactin (Prl) values are represented in the graph through time during the 3 h of the test. Fold increase of TSH was calculated by dividing the TSH value at each time (0, 15, 30, 60, 120, or 180 min) by the TSH level at the beginning of the test (0 min). FT4 increase 3 h after TRH administration was calculated as reported [8], as an indirect measure of TSH bioactivity, and was normal. Underlining represents values below normal ranges. (C) Thyroid ultrasound at 6 y and 9 mo of age, representing three-dimensional measures of each lobule, showed mild hypoplasia of the gland. (D) Brain MRI in the index case at 6.5 y of age showing normal pituitary morphology and size (white asterisk) but a downward herniation of cerebellar tonsils protruding through the foramen magnum (CMI), which compresses the spinal cord (white arrows). (E) Hearing loss per frequency in the right and left ear of the index patient is represented in left and right panels, respectively. %FT4 Incr, percentage of FT4 increase; N.R., normal range; wt, wild-type.

The patient was not detected by the TSH-based Neonatal Screening Program for Congenital Hypothyroidism and had normal growth, weight gain, and neurologic development in infancy. At 5 years of age, he showed refractory episodes of constipation with encopresis requiring osmotic laxatives. At 6 years, he was referred to Pediatric Endocrinology for evaluation of abnormal thyroid function tests, including low FT4 (10.16 pmol/L; normal, 12 to 22 pmol/L) but normal TSH (1.06 mIU/L; normal, 0.7 to 5.7 mIU/L), consistent with central hypothyroidism (Table 1). TT3 was normal (1.46 ng/mL; normal, 0.8 to 2 ng/mL) (Table 1). Antithyroglobulin antibodies (<15 IU/mL; normal, <60 IU/mL) and antithyroid peroxidase antibodies (<28 IU/mL; normal, <60 IU/mL) were negative, ruling out autoimmune thyroid disease. A 180-minute TRH test was performed to investigate the origin of his hypothyroidism, showing reduced TSH stimulation (low TSH secretion capacity) and full recovery of the basal TSH level at the end of the test (Fig. 1B), suggesting a pituitary defect. His TSH-FT4 relation was studied in the model of Dietrich et al. [10]. Although initial values fell inside the green area representing the normal dynamic relationship between the two parameters, later determinations showed ratios falling outside such area, suggesting an evolving thyrotropic failure (data not shown). He did not have clear symptoms of hypothyroidism but performed poorly at school, requiring the help of extra teaching. Levothyroxine replacement was started at a low dose of 1.1 µg/kg/d, which normalized his FT4 levels (15.70 pmol/L) while notably reducing TSH (0.37 mIU/L) (Table 1), both characteristics of central hypothyroidism. Neck ultrasounds showed mild hypoplasia of the thyroid gland with 1.84 mL volume (p50: 3.05 mL) [9] (Fig. 1C). No defect in other pituitary axes was identified, with basal cortisol, ACTH, IGF1, IGFBP3, FSH, LH, and testosterone within normal ranges (Table 1). Brain MRI showed normal pituitary size and shape, but a CMI was identified, consisting of the aberrant displacement of the cerebellar tonsils into the foramen magnum (Fig. 1D). At 7 years of age, the patient was formally diagnosed with ADHD. Pharmacological treatment of ADHD was indicated, but he did not receive therapy due to parental decision. At 10 years of age, a pure-tone audiometry was performed, which identified poor hearing thresholds in the air conduction at high frequencies compared with the age-specific reference interval [11] (Fig. 1E). The patient showed adequate growth during his infancy and childhood, with weight and height around p50 (Spanish growth curves, 2010) (data not shown). In the last clinical visit at 10 years of age, his height [141.4 cm (p57), +0.19 SD], weight [35.2 kg (p45), −0.15 SD], and body mass index 17.61 (p40), −0.27 SD) were normal, as was his gonadal development, with bilateral testicular volumes of 3 mL in agreement with pubertal Tanner I stage [12].

Table 1.

Long-Term Follow-up of Thyroid and Other Pituitary Axes Profiles of the Patient With TBL1X Defect

Characteristic	Age, y
	5.5	6.25	6.3 a	6.75	7	7.75	8.3	8.8	9.3	10
TSH, mIU/L (0.7–5.7)	1.69	1.06	1.57	0.37	0.53	0.66	1.17	0.66	1.05	0.73
FT4, pmol/L (12–22)	11.58	10.16	10.42	15.70	14.70	14.54	12.48	12.09	11.60	13.5
TT3, ng/mL (0.8–2)	—	—	1.46	1.16	—	—	—	—	—	1.08
L-T4 dose, μg/kg/d	—	—	—	1.1	1.25	1.18	1.18	1.3	1.35	1.9
ACTH, pg/mL (5–46)	—	—	13	—	—	—	—	—	28	—
Cortisol, µg/dL (5–25)	—	—	9.9	—	—	—	—	—	21.3	—
IGF1, ng/mL	—	—	179	—	—	—	—	—	210	—
IGFBP3, μg/mL	—	—	4.00	—	—	—	—	—	4.06	—
FSH, mIU/mL (1.5–12.4)	—	—	1.45	—	—	—	—	—	3.73	—
LH, mIU/mL (1.7–8.6)	—	—	<0.1	—	—	—	—	—	0.11	—
Testosterone, ng/mL (1.8–15.9)	—	—	0.02	—	—	—	—	—	<0.1	—

Hormone parameters are represented in chronological order along the patient’s life. Italics represent hormone values below normal ranges. Values in parentheses represent reference ranges.

Abbreviations: L-T4, levothyroxine; TT3, total T3; —, not determined.

^aAge at clinical diagnosis of the patient, when the TRH test was performed.

B. Identification of the TBL1X Mutation

Molecular analysis for mutations by Sanger sequencing of three candidate genes associated with central hypothyroidism (TRHR, TSHB, and IGSF1) did not reveal any defects in the patient. Targeted next-generation sequencing of a panel of genes including candidates for central and primary hypothyroidism identified a rare nonsense hemizygous mutation in exon 11 of the TBL1X gene (c.1015C>T), changing arginine 339 into a stop codon [p.(Arg339Ter)] (transcript accession number NM_005647.3). The mutation was confirmed by Sanger sequencing and investigated in the family. The p.(Arg339Ter) was a de novo mutation because it was not identified in parents or the (dizygotic twin) sister of the patient (Fig. 1A and Fig. 2A), whose paternity was confirmed by a panel of 20 single tandem repeat microsatellite markers studied in the four members of the family (data not shown). This variant was not reported in databases for genomic variants.

Figure 2.

Genetics findings: de novo nonsense mutation in TBL1X, location in the protein levels, and proposed transcriptional implications involved in the syndrome. (A) Representative chromatograms showing wild-type and the hemizygous c.1015C>T TBL1X mutation in the patient. (B) Location of the nonsense p.(Arg339Ter) mutation at the third WD-repeat motif of TBL1X (red dot) and other TBL1X mutations previously identified (black dots). (C–F) Schematic models of TBL1X action within the NCoR-SMRT protein complex, including interactions with TBL1X receptor 1 (TBL1XR1), G protein pathway suppressor 2 (GPS2), and histone deacetylase 3 (HDAC3), regulating different T3-responsive genes. (C) TSH β subunit (TSHB) promoter is negatively regulated by thyroid hormone and retinoid receptors (TRβ-RXR) signaling through negative thyroid hormone–responsive elements (nTREs). In the absence of T3, unoccupied TRs allow basal transcription and secretion of TSH through interaction with the NCoR-SMRT complex, including TBL1X, whose defect may explain central hypothyroidism. (D) TR-T3 regulates the expression of genes involved in ear development, like pendrin (SLC26A4), prestin (SLC26A5), or potassium voltage-gated channel subfamily Q member 4 (KCNQ4) [13, 14]. Therefore, it is likely that auditory defects in TBLX1 deficiency could be mediated by dysregulation of TR-T3–dependent transcription. (E) NCoR-SMRT corepressor complexes regulate transcription of genes involved in synaptic homeostasis, like brain-derived neurotrophic factor (BDNF), cyclin D1 (CCND1), and achaete-scute homolog 2 (ASCL2) of brain neurons, associated with neurodevelopmental disorders like ADHD and ASDs [15]. NCoR-SMRT also regulates the expression of genes of the ENS. Notably, another component of the complex, highly expressed in ENS, is the methyl-CpG binding protein 2 (MECP2). This protein is involved in Rett syndrome (a distinct neurodevelopmental disorder), which prominently shows severe GI dysmotility [16]. (F) Finally, NCoR-SMRT regulates TR-dependent transcription in cerebellar growth and differentiation [17], and notably, TBL1XR1 plays a role in the expression of Wnt signaling proteins like dickkopf Wnt signaling pathway inhibitor 1 (DKK1) and LDL receptor-related protein 4 (LRP4) implicated in posterior fossa development of the skull, with TBL1XR1 defects being associated with CMI [18].

TBL1X protein structure contains an N-terminal (LisH), a putative F box domain for protein dimerization, and a C-terminal WD40 domain with eight WD repeats for interaction with other nuclear proteins [4] (Fig. 2B). The p.(Arg339Ter) is predicted to have deleterious effects on the function of TBL1X given its N-terminal location from the highly conserved WD40 repeat domain, which would become largely truncated. The mRNA containing c.1015C>T was not translated and characterized in vitro. Most mutant mRNAs containing nonsense mutations are unstable and degraded through nonsense-mediated decay. Nevertheless, if such mRNA were stable, it would encode a protein lacking six of the eight WD repeat domains by which TBL1X interacts with other nuclear factors toward the activation/repression of target gene transcription (Fig. 2B).

3. Discussion

TBL1X is an essential subunit of the NCoR-SMRT corepressor complexes that regulate transcription of many different genes [19]. TBL1X binds to its receptor protein TBL1XR1 (another component of NCoR-SMRT) and also interacts with other partners within corepressor complexes (Fig. 2C–2F). The function of such complexes is exerted through remodeling of nuclear chromatin by histone-deacetylase 3 and interaction with classical DNA-binding nuclear receptors at the promoter of target genes. Importantly, NCoR-SMRT regulates T3-dependent gene expression through the thyroid hormone receptors (TRs) and retinoid-dependent expression through retinoic acid receptors (RAR, RXR) [4]. Furthermore, TBL1X regulates β catenin–mediated Wnt-responsive genes, controlling cell proliferation and cell fate [20, 21], and is also involved in synaptogenesis and synaptic activity and homeostasis [15] (Fig. 2E–2F).

Given the involvement of TBL1X in multiple intracellular signaling pathways [15, 19–21], it may seem surprising that those mutations so far known in this protein lead to limited phenotypes of deafness and central hypothyroidism.

Many patients with CCH are detected at peripubertal ages in countries with TSH-based neonatal screening programs [1]. First patients reported with TBL1X mutations were detected through a T4-based neonatal screening with variable degrees of hypothyroxinemia [3].

Our patient was diagnosed with CCH at the age of 6 years by an abnormal thyroid hormone profile in the course of an etiological study of encopresis and constipation while not presenting typical symptoms of hypothyroidism. The TRH test showed poor TSH surge but normal secretion dynamics [8]. His TSH curve is in contrast with those found in previous patients with TBLX1 mutations (reported normal) probably because point mutations in the Dutch study plausibly led to milder effects on the protein function [3]. Our patient presented with mild thyroid hypoplasia, similar to patients previously described with TBL1X and IGSF1 defects [3, 22]. This reduction in thyroid volume is attributable to weak trophic stimulation of the gland by low TSH levels [22].

TSHB promoter is negatively regulated by T3 through interaction with the nuclear thyroid hormone receptor B isoform (TRB). However, in the absence of the T3 ligand, unoccupied TR allows the basal transcription and secretion of TSH through interaction with the NCoR-SMRT complex, including TBL1X (Fig. 2C) [3]. Therefore, it is not surprising that patients with TBL1X mutations show central hypothyroidism due to inadequate basal TSH secretion and stimulation of the thyroid gland (Fig. 2C). TBL1X is also expressed in the parvocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting coexpression with TRH [3]. Therefore, it could be speculated that the mechanism of TBL1X-dependent hypothyroidism may involve not only defective (pituitary) TSH synthesis but also abnormal (hypothalamic) TRH signaling.

Sensorineural hearing loss was specifically searched for during the course of this study, after identification of the TBL1X mutation in the patient. Hypoacusia was bilateral and mild, being restricted to high tones and so far not involving clinically relevant disability. Follow-up of the patient’s audition capacity is warranted because deafness related to TBL1X deletions seems to progress with age [6]. It is interesting to note that mutations in the TBL1X receptor (TBL1XR1) cause Pierpont syndrome, a constellation of features also including deafness [23].

Thyroid hormone is essential in development and differentiation of the inner ear, participating in its maturation from early fetal stages [13, 14, 24]. In this organ, thyroid hormone action is mediated by TRB, where T3 binding induces the exchange of transcriptional corepressors and coactivators, encompassing TBL1X (Fig. 2D). Thyroid hormone receptor β (Thrb)–deficient mice are deaf and have multiple morphological defects in the cochlea, a phenotype similar to that observed in humans with genetic defects in TBL1X [25–27]. Interestingly, defects in T3-TRB target genes like SLC26A4 (pendrin), SLC26A5 (prestin), or KCNQ4 also present phenotypes of sensorineural deafness [28–30]. Therefore, it is likely that auditory defects related to TBL1X (and TBLX1R1) deficiency could be mediated by abnormal interactions within the NCoR-SMRT complex and dysregulation of TRB-dependent expression in the inner ear [3, 6] (Fig. 2D).

ADHD and learning difficulties at school were revealed at 6 years of age in the course of an extended clinical study. Following the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), these neurodevelopmental features fall in the scope of autism spectrum disorders (ASDs) [31].

ASDs have been consistently linked to defects in synaptogenesis and synapsis homeostasis [15]. The NCoR-SMRT corepressor complexes are known to regulate transcription of genes related to ASDs, like BDNF (brain-derived neurotrophic factor), or Wnt target genes like CCND1 (cyclin D1) or ASCL2 (achaete-scute family bHLH transcription factor 2) [15], all important for synaptic homeostasis (Fig. 2E). Interestingly, polymorphisms in TBL1X have been associated with ASDs in a Genome Wide Association Study (GWAS) study [32]; however, no neurodevelopmental defects have been reported so far in patients with TBLX1 defects [3]. It is tempting to speculate that such patients, harboring missense (point) mutations in the gene, may retain residual protein function, leading to absent or milder neurodevelopmental phenotypes. In our case, cognition and behavior of parents are normal, in full consistency with the de novo nature of the mutation in the patient. Furthermore, maternal hypothyroidism during pregnancy cannot be claimed here as a factor influencing this phenotype because, again, the mutation is not inherited and the patient’s twin sister has normal behavior and learning capacity.

Encopresis or fecal incontinence is a disabling disorder derived from alterations of motility in the gastrointestinal (GI) tract, especially the colonic part, which may alternate with constipation episodes. The pathogenesis is not well understood, but genetic factors are proposed [33]. Strikingly, encopresis is very prevalent in patients with neurodevelopmental disorders like ADHD or autism [34, 35], and inversely, a notable proportion of children with functional defecation disorders presents with ADHD [36]. This may suggest etiological connections between these entities because motility of the GI tract is performed by the smooth muscle cells governed by the enteric neuron system (ENS). The NCoR-SMRT complex system is equally important for synaptic development and plasticity of ENS neurons, as it is for brain neurons. MECP2, another integral component of the NCoR-SMRT, is highly expressed in ENS, where it regulates intestinal motility [16]. Mutations in MECP2 cause Rett syndrome, a neurodevelopmental disorder characterized by severe cognitive impairment, motor dyspraxia, and seizures but also GI dysmotility. Therefore, it is tempting to speculate that encopretic/constipation symptoms in our patient relate to dysregulation of ENS gene transcription involved in normal intestinal motility (Fig. 2E).

CMI is a rare congenital abnormality of the craniocerebral junction, leading to a variety of neurologic symptoms [37]. Our patient complained of exertional headaches, especially after the practice of sports.

CMI is suspected to have a genetic basis because familial clustering has been reported [38, 39]. Thyroid hormone signaling through at least three different TRs (TRβ1, TRα1, TRα2) is crucial in cerebellar growth and differentiation [17]. Recently, Wnt–β catenin signaling has been implicated in the development of CMI [38]. Notably, the catenin-based regulation of transcription is governed by TLB1X-TBL1XR1, which are negative regulators of β catenin [20, 21]. Because CMI has been reported in relation to a TBL1XR1 mutation [18], it is tempting to speculate that the TBL1X defect in our patient could also cause CMI by dysregulated transcription of Wnt–β catenin target genes involved in the development of the cerebellum or patterning of the cranial mesenchyme forming the posterior fossa of the skull [39] (Fig. 2F).

In summary, we have described, to our knowledge, the first severely truncated TBL1X mutant identified in a male with central hypothyroidism and hypoacusia and expanded the phenotypic spectrum of the syndrome to neurodevelopmental disorders, GI dysmotility, and CMI as novel features.

Glossary

Abbreviations:

ADHD

attention-deficit/hyperactivity disorder

ASD

autism spectrum disorder

CCH

central congenital hypothyroidism

CMI

Chiari malformation type I

ENS

enteric neuron system

FT4

free T4

GI

gastrointestinal

NCoR-SMRT

nuclear corepressor-silencing mediator for retinoid and thyroid hormone receptor complex

TR

thyroid hormone receptor

TRB

thyroid hormone receptor B isoform