STR mutations on chromosome 15q cause thyrotropin resistance by activating a primate-specific enhancer of MIR7-2/MIR1179.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.

Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie Allan-Herndon-Dudley syndrome.

Patients with mutations in the thyroid hormone (TH) cell transporter monocarboxylate transporter 8 (MCT8) gene develop severe neuropsychomotor retardation known as Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain during both intrauterine and postnatal developmental stages, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated cerebral organoids (COs) using human induced pluripotent stem cells (iPSCs) from MCT8-deficient patients. MCT8-deficient COs exhibited (i) altered early neurodevelopment, resulting in smaller neural rosettes with thinner cortical units, (ii) impaired triiodothyronine (T3) transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (iii) reduced expression of genes involved in cerebral cortex development, and (iv) reduced T3 inducibility of TH-regulated genes. In contrast, the TH analogs 3,5-diiodothyropropionic acid and 3,3',5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient COs, constituting proof of concept that lack of T3 transport underlies the pathophysiology of AHDS and demonstrating the clinical potential for TH analogs to be used in treating patients with AHDS. MCT8-deficient COs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for patients with AHDS.

ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.

Perturbation of endoplasmic reticulum proteostasis triggers tissue injury in the thyroid gland.

Defects in endoplasmic reticulum (ER) proteostasis have been linked to diseases in multiple organ systems. Here we examined the impact of perturbation of ER proteostasis in mice bearing thyrocyte-specific knockout of either HRD1 (to disable ER-associated protein degradation [ERAD]) or ATG7 (to disable autophagy) in the absence or presence of heterozygous expression of misfolded mutant thyroglobulin (the most highly expressed thyroid gene product, synthesized in the ER). Misfolding-inducing thyroglobulin mutations are common in humans but are said to yield only autosomal-recessive disease - perhaps because misfolded thyroglobulin protein might undergo disposal by ERAD or ER macroautophagy. We find that as single defects, neither ERAD, nor autophagy, nor heterozygous thyroglobulin misfolding altered circulating thyroxine levels, and neither defective ERAD nor defective autophagy caused any gross morphological change in an otherwise WT thyroid gland. However, heterozygous expression of misfolded thyroglobulin itself triggered significant ER stress and individual thyrocyte death while maintaining integrity of the surrounding thyroid epithelium. In this context, deficiency of ERAD (but not autophagy) resulted in patchy whole-follicle death with follicular collapse and degeneration, accompanied by infiltration of bone marrow-derived macrophages. Perturbation of thyrocyte ER proteostasis is thus a risk factor for both cell death and follicular demise.

Effect of the Fetal THRB Genotype on the Placenta.

CONTEXT: Pregnant women with mutations in the thyroid hormone receptor beta (THRB) gene expose their fetuses to high thyroid hormone (TH) levels shown to be detrimental to a normal fetus (NlFe) but not to an affected fetus (AfFe). However, no information is available about differences in placental TH regulators. OBJECTIVE: To investigate whether there are differences in placentas associated with a NlFe compared with an AfFe, we had the unique opportunity to study placentas from 2 pregnancies of the same woman with THRB mutation G307D. One placenta supported a NlFe while the other an AfFe. METHODS: Sections of placentas were collected and frozen at -80 °C after term delivery of a NlFe and an AfFe. Two placentas from healthy women of similar gestational age were also obtained. The fetal origin of the placental tissues was established by gDNA quantitation of genes on the X and Y chromosomes and THRB gene. Expression and enzymatic activity of deiodinases 2 and 3 were measured. Expression of following genes was also quantitated: MCT10, MCT8, LAT1, LAT2, THRB, THRA. RESULTS: The placenta carrying the AfFe exhibited a significant reduction of deiodinase 2 and 3 activities as well as the expression of the TH transporters MCT10, LAT1 and LAT2, and THRA. CONCLUSION: We present the first study of the effect of the fetal THRB genotype on the placenta. Though limited by virtue of the rarity of THRB mutations and sample availability, we show that the fetal THRB genotype influences the levels of TH regulators in the placenta.

Iodotyrosines Are Biomarkers for Preclinical Stages of Iodine-Deficient Hypothyroidism in Dehal1-Knockout Mice.

Background: Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and diiodotyrosines (MIT, DIT) to sustain TH synthesis when iodine supplies are scarce, but its role in the dynamics of storage and conservation of iodine is unknown. Methods: Dehal1-knockout (Dehal1KO) mice were generated by gene trapping. The timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-beta-galactosidase protein produced in fetuses and adult mice. Adult Dehal1KO and wild-type (Wt) animals were fed normal and iodine-deficient diets for 1 month, and plasma, urine, and tissues were isolated for analyses. TH status was monitored, including thyroxine, triiodothyronine, MIT, DIT, and urinary iodine concentration (UIC) using a novel liquid chromatography with tandem mass spectrometry method and the Sandell-Kolthoff (S-K) technique throughout the experimental period. Results: Dehal1 is highly expressed in the thyroid and is also present in the kidneys, liver, and, unexpectedly, the choroid plexus. In vivo transcription of Dehal1 was induced by iodine deficiency only in the thyroid tissue. Under normal iodine intake, Dehal1KO mice were euthyroid, but they showed negative iodine balance due to a continuous loss of iodotyrosines in the urine. Counterintuitively, the UIC of Dehal1KO mice is twofold higher than that of Wt mice, indicating that S-K measures both inorganic and organic iodine. Under iodine restriction, Dehal1KO mice rapidly develop profound hypothyroidism, while Wt mice remain euthyroid, suggesting reduced retention of iodine in the thyroids of Dehal1KO mice. Urinary and plasma iodotyrosines were continually elevated throughout the life cycles of Dehal1KO mice, including the neonatal period, when pups were still euthyroid. Conclusions: Plasma and urine iodotyrosine elevation occurs in Dehal1-deficient mice throughout life. Therefore, measurement of iodotyrosines predicts an eventual iodine shortage and development of hypothyroidism in the preclinical phase. The prompt establishment of hypothyroidism upon the start of iodine restriction suggests that Dehal1KO mice have low iodine reserves in their thyroid glands, pointing to defective capacity for iodine storage.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Congenital Hypothyroidism in Two Sudanese Families Harboring a Novel Iodotyrosine Deiodinase Mutation (IYD R279C).

Background: Congenital hypothyroidism due to defects in iodotyrosine deiodinase has variable phenotypes and can present as hypothyroid or with normal thyroid testing. Methods: Whole exome sequencing was performed in individuals from two families originating from different regions of Sudan. Mass spectrometry of urine and serum iodotyrosines was performed on subjects from both families. Results: A novel iodotyrosine deiodinase (IYD) mutation (c.835C>T; R279C) was identified in individuals from two Sudanese families inherited as autosomal recessive. The mutation was identified by multiple in silica analyses to likely be detrimental. Serum and urine monoiodotyrosine (MIT) and diiodotyrosine (DIT) were markedly elevated in the homozygous subjects. Conclusion: Measurement of serum and urine DIT and MIT was more sensitive than that of urine iodine or serum thyroid function tests to determine the effect of the IYD mutation.

Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism.

The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.

Foxe1 Deletion in the Adult Mouse Is Associated With Increased Thyroidal Mast Cells and Hypothyroidism.

CONTEXT: Foxe1 is a key thyroid developmental transcription factor. Germline deletion results in athyreosis and congenital hypothyroidism. Some data suggest an ongoing role for maintaining thyroid differentiation. OBJECTIVE: We created a mouse model to directly examine the role of Foxe1 in the adult thyroid. METHODS: A model of tamoxifen-inducible Cre-mediated ubiquitous deletion of Foxe1 was generated in mice of C57BL/6J background (Foxe1flox/flox/Cre-TAM). Tamoxifen or vehicle was administered to Foxe1flox/flox/Cre mice aged 6-8 weeks. Blood was collected at 4, 12, and 20 weeks, and tissues after 12 or 20 weeks for molecular and histological analyses. Plasma total thyroxine (T4), triiodothyronine, and thyrotropin (TSH) were measured. Transcriptomics was performed using microarray or RNA-seq and validated by reverse transcription quantitative polymerase chain reaction. RESULTS: Foxe1 was decreased by approximately 80% in Foxe1flox/flox/Cre-TAM mice and confirmed by immunohistochemistry. Foxe1 deletion was associated with abnormal follicular architecture and smaller follicle size at 12 and 20 weeks. Plasma TSH was elevated in Foxe1flox/flox/Cre-TAM mice as early as 4 weeks and T4 was lower in pooled samples from 12 and 20 weeks. Foxe1 deletion was also associated with an increase in thyroidal mast cells. Transcriptomic analyses found decreased Tpo and Tg and upregulated mast cell markers Mcpt4 and Ctsg in Foxe1flox/flox/Cre-TAM mice. CONCLUSION: Foxe1 deletion in adult mice was associated with disruption in thyroid follicular architecture accompanied by biochemical hypothyroidism, confirming its role in maintenance of thyroid differentiation. An unanticipated finding was an increase in thyroidal mast cells. These data suggest a possible explanation for previous human genetic studies associating alleles in/near FOXE1 with hypothyroidism and/or autoimmune thyroiditis.

Maintaining the thyroid gland in mutant thyroglobulin-induced hypothyroidism requires thyroid cell proliferation that must continue in adulthood.

Congenital hypothyroidism with biallelic thyroglobulin (Tg protein, encoded by the TG gene) mutation is an endoplasmic reticulum (ER) storage disease. Many patients (and animal models) grow an enlarged thyroid (goiter), yet some do not. In adulthood, hypothyroid TGcog/cog mice (bearing a Tg-L2263P mutation) exhibit a large goiter, whereas adult WIC rats bearing the TGrdw/rdw mutation (Tg-G2298R) exhibit a hypoplastic thyroid. Homozygous TG mutation has been linked to thyroid cell death, and cytotoxicity of the Tg-G2298R protein was previously thought to explain the lack of goiter in WIC-TGrdw/rdw rats. However, recent studies revealed that TGcog/cog mice also exhibit widespread ER stress-mediated thyrocyte death, yet under continuous feedback stimulation, thyroid cells proliferate in excess of their demise. Here, to examine the relative proteotoxicity of the Tg-G2298R protein, we have used CRISPR-CRISPR-associated protein 9 technology to generate homozygous TGrdw/rdw knock-in mice in a strain background identical to that of TGcog/cog mice. TGrdw/rdw mice exhibit similar phenotypes of defective Tg protein folding, thyroid histological abnormalities, hypothyroidism, and growth retardation. TGrdw/rdw mice do not show evidence of greater ER stress response or stress-mediated cell death than TGcog/cog mice, and both mouse models exhibit sustained thyrocyte proliferation, with comparable goiter growth. In contrast, in WIC-TGrdw/rdw rats, as a function of aging, the thyrocyte proliferation rate declines precipitously. We conclude that the mutant Tg-G2298R protein is not intrinsically more proteotoxic than Tg-L2263P; rather, aging-dependent difference in maintenance of cell proliferation is the limiting factor, which accounts for the absence of goiter in adult WIC-TGrdw/rdw rats.

A Novel Pathogenic Variant in PAX8 Leads to Familial Congenital Hypothyroidism.

We report a 10-month-old girl with familial congenital hypothyroidism harboring a novel heterozygous pathogenic variant in the paired DNA-binding domain of PAX8 (NM_003466:c.110T>C:p.Leu37Pro). Genotype-phenotype correlation revealed complete penetrance of this PAX8 defect in this family, in which the affected father and half-brother carry the same mutation. This deleterious variant has not been reported in any of the available databases [MAFgnomAD = 0, dbSNP (-)], and the amino acid leucine at position 37 is highly conserved across species. Establishing the molecular diagnosis expands our knowledge on the cause of thyroid dysgenesis and provides a guide for counseling and early treatment.

AAV9-MCT8 Delivery at Juvenile Stage Ameliorates Neurological and Behavioral Deficits in a Mouse Model of MCT8-Deficiency.

Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8-/y) male mice increased triiodothyronine (T3) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. Methods: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8-/y/Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/ - double knockout (dKO) mouse model of AHDS, which, unlike Mct8-/y mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120-P140 after which tissues were collected and analyzed. Results: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120-P140, which was accompanied by a near normalization of T3 content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T3/reverse T3 (rT3) ratio and in liver deiodinase 1 expression. Conclusion: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Thyroidal Transcriptomic Profiles of Pathoadaptive Responses to Congenital Hypothyroidism in XB130 Knockout Mice.

Congenital hypothyroidism is a genetic condition in which the thyroid gland fails to produce sufficient thyroid hormone (TH), resulting in metabolic dysfunction and growth retardation. Xb130-/- mice exhibit perturbations of thyrocyte cytoskeleton and polarity, and develop postnatal transient growth retardation due to congenital hypothyroidism, leading ultimately to multinodular goiter. To determine the underlying mechanisms, we performed transcriptomic analyses on thyroid glands of mice at three age points: week 2 (W2, before visible growth retardation), W4 (at the nadir of growth); and W12 (immediately before full growth recovery). Using gene set enrichment analysis, we compared a defined set of thyroidal genes between Xb130+/+ and Xb130-/- mice to identify differentially enriched gene clusters. At the earliest postnatal stage (W2), the thyroid glands of Xb130-/- mice exhibited significantly downregulated gene clusters related to cellular metabolism, which continued to W4. Additionally, mutant thyroids at W4 and W12 showed upregulated gene clusters related to extracellular matrix, angiogenesis, and cell proliferation. At W12, despite nearly normal levels of serum TH and TSH and body size, a significantly large number of gene clusters related to inflammatory response were upregulated. Early postnatal TH deficiency may suppress cellular metabolism within the thyroid gland itself. Upregulation of genes related to extracellular matrix and angiogenesis may promote subsequent thyroid growth. Chronic inflammatory responses may contribute to the pathogenesis of multinodular goiter in later life. Some of the pathoadaptive responses of Xb130-/- mice may overlap with those from other mutations causing congenital hypothyroidism.

TSHB R75G is a founder variant and prevalent cause of low or undetectable TSH in Indian Jews.

Objective: Bi-allelic loss-of-function mutations in TSHB, encoding the beta subunit of thyroid-stimulating hormone (TSH), cause congenital hypothyroidism. Homozygosity for the TSHB p.R75G variant, previously described in South Asian individuals, does not alter TSH function but abrogates its detection by some immune detection-based platforms, leading to erroneous diagnosis of hyperthyroidism. We set out to identify and determine the carrier rate of the p.R75G variant among clinically euthyroid Bene Israel Indian Jews, to examine the possible founder origin of this variant worldwide, and to determine the phenotypic effects of its heterozygosity. Design: Molecular genetic studies of Bene Israel Jews and comparative studies with South Asian cohort. Methods: TSHB p.R75G variant tested by Sanger sequencing and restriction fragment length polymorphism (RFLP). Haplotype analysis in the vicinity of the TSHB gene performed using SNP arrays. Results: Clinically euthyroid individuals with low or undetectable TSH levels from three apparently unrelated Israeli Jewish families of Bene Israel ethnicity, originating from the Mumbai region of India, were found heterozygous or homozygous for the p.R75G TSHB variant. Extremely high carrier rate of p.R75G TSHB in Bene Israel Indian Jews (~4%) was observed. A haplotype block of 239.7 kB in the vicinity of TSHB shared by Bene Israel and individuals of South Asian origin was detected. Conclusions: Our findings highlight the high prevalence of the R75G TSHB variant in euthyroid Bene Israel Indian Jews, demonstrate that heterozygosity of this variant can cause erroneous detection of subnormal TSH levels, and show that R75G TSHB is an ancient founder variant, delineating shared ancestry of its carriers.

XB130 Plays an Essential Role in Folliculogenesis Through Mediating Interactions Between Microfilament and Microtubule Systems in Thyrocytes.

Background: XB130 (actin filament-associated protein 1-like 2, AFAP1L2) is a thyroid-abundant adaptor/scaffold protein. Xb130-/- mice exhibit transient growth retardation postnatally due to congenital hypothyroidism with diminished thyroglobulin iodination and release at both embryonic and early postnatal stages due to disorganized thyroid apical membrane structure and function. We hypothesized that XB130 is crucial for polarity and folliculogenesis by mediating proper cytoskeletal structure and function in thyrocytes. Methods: Primary thyrocytes isolated from thyroid glands of Xb130-/- mice and their wild-type littermates at postnatal week 2 were cultured in 10% Matrigel for different time periods. Folliculogenesis was studied with immunofluorescence staining, followed by confocal microscopy. Cells were also transfected to express human XB130 fused Green Fluorescent Protein (XB130-GFP) or Green Fluorescent Protein (GFP) only before morphological analysis. Cytoskeletal structures from embryo and postnatal thyroid glands were also studied. Results: In three-dimensional cultures of thyrocytes, XB130, aligned with actin filaments, participated in defining the site of apical membrane formation and coalescence to form a thyroid follicle lumen. Xb130-/- thyrocytes displayed delayed folliculogenesis, reduced recruitment of a microtubule (MT)-associated proteins, and disorganized acetylated tubulin under the apical membrane, resulting in delayed folliculogenesis with reduced efficiency in formation of the thyroid follicle lumen. Conclusions: XB130 critically regulates thyrocyte polarization by functioning as a link between the actin filament cortex and MT network at the apical membrane of thyrocytes. Defects of adaptor scaffold proteins may affect cellular polarity and cytoskeletal structure and function and result in disorders of epithelial function, such as congenital hypothyroidism.

Triiodothyroacetic Acid Cross-Reacts With Measurement of Triiodothyronine (T3) on Various Immunoassay Platforms.

OBJECTIVES: Thyroid hormone analog 3,5,3'-triiodothyroacetic acid (TRIAC) is effective in reducing the hypermetabolism in monocarboxylate transporter 8 (MCT8)-deficient individuals. Because of the structural similarity between TRIAC and 3,3',5'-triiodothyronine (T3), we sought to investigate the degree of cross-reactivity of TRIAC with various commercially available total and free T3 assays. METHODS: Varying concentrations (50-1,000 ng/dL) of TRIAC (Sigma Aldrich) were added to pooled serum and assayed for total T3 (TT3) and free T3 (FT3) on the following platforms: e602 (Roche Diagnostics), Architect (Abbott Diagnostics), Centaur (Siemens Healthcare Diagnostics), IMMULITE (Siemens Healthcare Diagnostics), DxI (Beckman Coulter), and Vitros (Ortho Clinical Diagnostics). TT3 competition assay with TRIAC was performed by adding increasing amounts of T3 to pooled serum samples that contained a constant concentration of TRIAC (250 ng/dL). RESULTS: Significant overestimation of TT3 and FT3 assays were observed across all platforms corresponding to increasing concentrations of TRIAC. The TRIAC effect at 250 ng/dL showed a constant interference of approximately 190 ng/dL TT3. CONCLUSIONS: All commercial TT3 and FT3 assays tested in this work cross-react significantly with TRIAC. Therefore, patients undergoing TRIAC therapy should have T3 hormone response monitored using alternative nonimmunoassay-based methods to avoid misinterpretation of thyroid function profiles.

Pathogenesis of Multinodular Goiter in Elderly XB130-Deficient Mice: Alteration of Thyroperoxidase Affinity with Iodide and Hydrogen Peroxide.

Background: Multinodular goiter (MNG) is the most common disorder of the thyroid gland. Aging and genetic mutations that impair thyroid hormone (TH) production have been implicated in the development of MNG. XB130 is an adaptor/scaffold protein predominantly expressed in the thyroid gland. XB130 deficiency leads to transient postnatal growth retardation in mice due to congenital hypothyroidism. We studied the formation of MNG and possible mechanisms in elderly mice. Methods: Thyroid glands of male and female Xb130-knockout (Xb130-/-), heterozygous (Xb130+/-), and wild-type (Xb130+/+) mice at the ages of 12-20 months were harvested for visual examination, histopathological, and immunohistological analyses. Blood and thyroid samples were collected after feeding elderly mice with a low iodine diet for 125I uptake and perchlorate discharge assay. The activity of thyroperoxidase (Tpo) was examined by spectrophotometric evaluation of iodide oxidation. Results: While moderate MNG was seen in Xb130+/+ and Xb130+/- mice, severe MNG, characterized by multiple nodules intermixed with dilated colloid-rich macrofollicles, was found only in Xb130-/- mice at 18 months. Thyrocyte cytoskeletal structure and cell adhesion molecules were disorganized, and TH production was significantly reduced. Reduced iodide organification was seen in elderly Xb130+/+ mice and further enhanced in Xb130-/- mice. In Xb130+/+ mice, Tpo shows high affinity with hydrogen peroxide (H2O2) throughout aging, but reduced affinity with iodide in an age-dependent manner. By contrast, in elderly Xb130-/- mice, the affinity of Tpo for iodide remained high, but the affinity of Tpo for H2O2 was reduced. Conclusions: The pathophysiological features in the thyroid glands of aged Xb130-/- mice closely resemble the features of MNG in humans. Moderate MNG in elderly mice was dramatically aggravated by XB130 deficiency. Reduced affinity of Tpo for H2O2 may contribute to MNG development via oxidative stress. This could be specific to XB130 deficiency but also could be a common mechanism in MNG. Its clinical relevance should be further investigated.