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To date only a fraction of the genetic footprint of thyroid function has been clarified. We report a genome-wide association study meta-analysis of thyroid function in up to 271,040 individuals of European ancestry, including reference range thyrotropin (TSH), free thyroxine (FT4), free and total triiodothyronine (T3), proxies for metabolism (T3/FT4 ratio) as well as dichotomized high and low TSH levels. We revealed 259 independent significant associations for TSH (61% novel), 85 for FT4 (67% novel), and 62 novel signals for the T3 related traits. The loci explained 14.1%, 6.0%, 9.5% and 1.1% of the total variation in TSH, FT4, total T3 and free T3 concentrations, respectively. Genetic correlations indicate that TSH associated loci reflect the thyroid function determined by free T3, whereas the FT4 associations represent the thyroid hormone metabolism. Polygenic risk score and Mendelian randomization analyses showed the effects of genetically determined variation in thyroid function on various clinical outcomes, including cardiovascular risk factors and diseases, autoimmune diseases, and cancer. In conclusion, our results improve the understanding of thyroid hormone physiology and highlight the pleiotropic effects of thyroid function on various diseases., Pattern Recognition and Bioinformatics

Contains fulltext : 304858.pdf (Publisher’s version ) (Open Access), To date only a fraction of the genetic footprint of thyroid function has been clarified. We report a genome-wide association study meta-analysis of thyroid function in up to 271,040 individuals of European ancestry, including reference range thyrotropin (TSH), free thyroxine (FT4), free and total triiodothyronine (T3), proxies for metabolism (T3/FT4 ratio) as well as dichotomized high and low TSH levels. We revealed 259 independent significant associations for TSH (61% novel), 85 for FT4 (67% novel), and 62 novel signals for the T3 related traits. The loci explained 14.1%, 6.0%, 9.5% and 1.1% of the total variation in TSH, FT4, total T3 and free T3 concentrations, respectively. Genetic correlations indicate that TSH associated loci reflect the thyroid function determined by free T3, whereas the FT4 associations represent the thyroid hormone metabolism. Polygenic risk score and Mendelian randomization analyses showed the effects of genetically determined variation in thyroid function on various clinical outcomes, including cardiovascular risk factors and diseases, autoimmune diseases, and cancer. In conclusion, our results improve the understanding of thyroid hormone physiology and highlight the pleiotropic effects of thyroid function on various diseases.

Item does not contain fulltext, OBJECTIVE: Thyroid hormone (TH) transport represents a critical first step in governing intracellular TH regulation. It is still unknown whether the full repertoire of TH transporters has been identified. Members of the solute carrier (SLC) 22 family have substrates in common with the known TH transporters of the organic anion-transporting peptide family. Therefore, we screened the SLC22 family for TH transporters. METHODS: Uptake of 1 nM of iodothyronines or sulfated iodothyronines in COS1 cells expressing SLC22 proteins was performed. RESULTS: We first tested 25 mouse (m) SLC22 proteins for TH uptake and found that the majority of the organic anion transporter (OAT) clade were capable of 3,3',5-triiodothyronine and/or thyroxine (T4) transport. Based on phylogenetic tree analysis of the mouse and human (h) SLC22 family, we selected eight hSLC22s that grouped with the newly identified mouse TH transporters. Of these, four tested positive for uptake of one or more substrates, particularly hSLC22A11 showed robust (3-fold over control) uptake of T4. Uptake of sulfated iodothyronines was strongly (up to 17-fold) induced by some SLC22s, most notably SLC22A8, hSLC22A9, mSLC22A27 and mSLC22A29. Finally, the zebrafish orthologues of SLC22A6/8 drOatx and drSlc22a6l also transported almost all (sulfated) iodothyronines tested. The OAT inhibitors lesinurad and probenecid inhibited most SLC22 proteins. CONCLUSIONS: Our results demonstrated that members of the OAT clade of the SLC22 family constitute a novel, evolutionary conserved group of transporters for (sulfated) iodothyronines. Future studies should reveal the relevance of these transporters in TH homeostasis and physiology.

Item does not contain fulltext, Background: A recent genome-wide association study identified the SLC17A4 locus associated with circulating free thyroxine (T4) concentrations. Human SLC17A4, being widely expressed in the gastrointestinal tract, was characterized as a novel triiodothyronine (T3) and T4 transporter. However, apart from the cellular uptake of T3 and T4, transporter characteristics are currently unknown. In this study, we delineated basic transporter characteristics of this novel thyroid hormone (TH) transporter. Methods: We performed a broad range of well-established TH transport studies in COS-1 cells transiently overexpressing SLC17A4. We studied cellular TH uptake in various incubation buffers, TH efflux, and the inhibitory effects of different TH metabolites and known inhibitors of other TH transporters on SLC17A4-mediated TH transport. Finally, we determined the effect of tunicamycin, a pharmacological inhibitor of N-linked glycosylation, and targeted mutations in Asn residues on SLC17A4 function. Results: SLC17A4 induced the cellular uptake of T3 and T4 by ∼4 times, and of reverse (r)T3 by 1.5 times over control cells. The uptake of T4 by SLC17A4 was Na(+) and Cl(-) independent, stimulated by low extracellular pH, and reduced by various iodothyronines and metabolites thereof, particularly those that contain at least three iodine moieties irrespective of the presence of modification at the alanine side chain. None of the classical TH transporter inhibitors studied attenuated SLC17A4-mediated TH transport. SLC17A4 also facilitates the efflux of T3 and T4, and to a lesser extent of 3,3'-diiodothyronine (T2). Immunoblot studies on lysates of transfected cells cultured in absence or presence of tunicamycin indicated that SLC17A4 is subject to N-linked glycosylation. Complementary mutational studies identified Asn66, Asn75, and Asn90, which are located in extracellular loop 1, as primary targets. Conclusions: Our studies show that SLC17A4 facilitates the transport of T3 and T4, and l

Contains fulltext : 251756.pdf (Publisher’s version ) (Open Access), OBJECTIVE: While most of the variation in thyroid function is determined by genetic factors, single nucleotide polymorphisms (SNPs) identified via genome-wide association analyses have only explained ~5% to 9% of this variance so far. Most SNPs were in or nearby genes with no known role in thyroid hormone (TH) regulation. Therefore, we performed a large-scale candidate gene study investigating the effect of common genetic variation in established TH regulating genes on serum thyrotropin [thyroid-stimulating hormone (TSH)] and thyroxine (FT4) concentrations. METHODS: SNPs in or within 10 kb of 96 TH regulating genes were included (30 031 TSH SNPs, and 29 962 FT4 SNPs). Associations were studied in 54 288 individuals from the ThyroidOmics Consortium. Linkage disequilibrium-based clumping was used to identify independently associated SNPs. SNP-based explained variances were calculated using SumHer software. RESULTS: We identified 23 novel TSH-associated SNPs in predominantly hypothalamic-pituitary-thyroid axis genes and 25 novel FT4-associated SNPs in mainly peripheral metabolism and transport genes. Genome-wide SNP variation explained ~21% (SD 1.7) of the total variation in both TSH and FT4 concentrations, whereas SNPs in the 96 TH regulating genes explained 1.9% to 2.6% (SD 0.4). CONCLUSION: Here we report the largest candidate gene analysis on thyroid function, resulting in a substantial increase in the number of genetic variants determining TSH and FT4 concentrations. Interestingly, these candidate gene SNPs explain only a minor part of the variation in TSH and FT4 concentrations, which substantiates the need for large genetic studies including common and rare variants to unravel novel, yet unknown, pathways in TH regulation.

Contains fulltext : 231654.pdf (Publisher’s version ) (Open Access)

CONTEXT: Genetic variants in SLC16A2, encoding the thyroid hormone transporter MCT8, can cause intellectual and motor disability and abnormal serum thyroid function tests, known as MCT8 deficiency. The C-terminal domain of MCT8 is poorly conserved, which complicates prediction of the deleteriousness of variants in this region. We studied the functional consequences of 5 novel variants within this domain and their relation to the clinical phenotypes. METHODS: We enrolled male subjects with intellectual disability in whom genetic variants were identified in exon 6 of SLC16A2. The impact of identified variants was evaluated in transiently transfected cell lines and patient-derived fibroblasts. RESULTS: Seven individuals from 5 families harbored potentially deleterious variants affecting the C-terminal domain of MCT8. Two boys with clinical features considered atypical for MCT8 deficiency had a missense variant [c.1724A>G;p.(His575Arg) or c.1796A>G;p.(Asn599Ser)] that did not affect MCT8 function in transfected cells or patient-derived fibroblasts, challenging a causal relationship. Two brothers with classical MCT8 deficiency had a truncating c.1695delT;p.(Val566*) variant that completely inactivated MCT8 in vitro. The 3 other boys had relatively less-severe clinical features and harbored frameshift variants that elongate the MCT8 protein [c.1805delT;p.(Leu602HisfsTer680) and c.del1826-1835;p.(Pro609GlnfsTer676)] and retained ~50% residual activity. Additional truncating variants within transmembrane domain 12 were fully inactivating, whereas those within the intracellular C-terminal tail were tolerated. CONCLUSIONS: Variants affecting the intracellular C-terminal tail of MCT8 are likely benign unless they cause frameshifts that elongate the MCT8 protein. These findings provide clinical guidance in the assessment of the pathogenicity of variants within the C-terminal domain of MCT8.

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in MCT8 deficiency, characterized by severe intellectual and motor disability. The MCT8 protein is predicted to have 12 transmembrane domains (TMDs) and is expressed as monomers, homodimers, and homo-oligomers. This study aimed to delineate the mechanism of

BACKGROUND: While thyroxine (T4), 3,3’,5-triiodothyronine (T3), and 3,3’,5’-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals. METHODS: Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards RESULTS: The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9–11.3 ng/dL), 3-monoiodothyronine (0.06 –0.41 ng/ dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3’-diiodothyronine (0.25–0.77 ng/dL), T3 (66.4–129.9 ng/dL), rT3 (15.0–64

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood–brain and blood–placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Contains fulltext : 215622.pdf (publisher's version ) (Closed access), CONTEXT: Thyroid hormone is important for normal brain development. The type 2 deiodinase (D2) controls thyroid hormone action in the brain by activating T4 to T3. The enzymatic activity of D2 depends on the incorporation of selenocysteine for which the selenocysteine-insertion sequence (SECIS) element located in the 3' untranslated region is indispensable. We hypothesized that mutations in the SECIS element could affect D2 function, resulting in a neurocognitive phenotype. OBJECTIVE: To identify mutations in the SECIS element of DIO2 in patients with intellectual disability and to test their functional consequences. DESIGN, SETTING, AND PATIENTS: The SECIS element of DIO2 was sequenced in 387 patients with unexplained intellectual disability using a predefined pattern of thyroid function tests. SECIS element read-through in wild-type or mutant D2 was quantified by a luciferase reporter system in transfected cells. Functional consequences were assessed by quantifying D2 activity in cell lysate or intact cell metabolism studies. RESULTS: Sequence analysis revealed 2 heterozygous mutations: c.5703C>T and c.5730A>T, which were also present in the unaffected family members. The functional evaluation showed that both mutations did not affect D2 enzyme activity in cell lysates or intact cells, although the 5730A>T mutation decreased SECIS element read-through by 75%. In the patient harboring the c.5730A>T variant, whole genome sequencing revealed a pathogenic deletion of the STXBP1 gene. CONCLUSIONS: We report on two families with mutations in the SECIS element of D2. Although functional analysis showed that nucleotide 5730 is important for normal SECIS element read-through, the two variants did not segregate with a distinct phenotype.

Item does not contain fulltext, Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter

Context: The two major forms of circulating thyroid hormones (THs) are T3 and T4. T3 is regarded as the biologically active hormone because it binds to TH receptors (TRs) with greater affinity than T4. However, it is currently unclear what structural mechanisms underlie this difference in affinity. Objective: Prompted by the identification of a novel M256T mutation in a resistance to TH (RTH)a patient, we investigated Met256 in TRa1 and the corresponding residue (Met310) in TRb1, residues previously predicted by crystallographic studies in discrimination of T3 vs T4. Methods: Clinical characterization of the RTHa patient and molecular studies (in silico protein modeling, radioligand binding, transactivation, and receptor–cofactor studies) were performed. Results: Structural modeling of the TRa1-M256T mutant showed that distortion of the hydrophobic niche to accommodate the outer ring of l

The gold standard for quantification of pain is a person's self-report. However, we need objective parameters for pain measurement when intensive care patients, for example, are not able to report pain themselves. An increase in pain is currently thought to coincide with an increase in stress hormones. This observational study investigated whether procedure-related pain is associated with an increase of plasma cortisol, adrenaline, and noradrenaline. In 59 patients receiving intensive care after cardiac surgery, cortisol, adrenaline, and noradrenaline plasma levels were measured immediately before and immediately after patients were turned for washing, either combined with the removal of chest tubes or not. Numeric rating scale scores were obtained before, during, and after the procedure. Unacceptably severe pain (numeric rating scale ≥ 4) was reported by seven (12%), 26 (44%), and nine (15%) patients, before, during and after the procedure, respectively. There was no statistically significant association between numeric rating scale scores and change in cortisol, adrenaline, and noradrenaline plasma levels during the procedure. Despite current convictions that pain coincides with an increase in stress hormones, procedural pain was not associated with a significant increase in plasma stress hormone levels in patients who had undergone cardiac surgery. Thus, plasma levels of cortisol, adrenaline, and noradrenaline seem unsuitable for further research on the measurement of procedural pain.

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and nonproteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as inWnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is cor

Context: Thyroid hormones (TH) are important for normal brain development and abnormal TH regulation in the brain results in neurocognitive impairments. The type 2 deiodinase (D2) is important for local TH control in the brain by generating the active hormone T3 from its precursor T4. Dysfunction of D2 likely results in a neurocognitive phenotype. No mutations in D2 have been reported yet. Objective: The objective of the study was to identify D2 mutations in patients with intellectual disability and to test their functional consequences. Design, Setting, and Patients: The patients were selected from the multicenter Thyroid Origin of Psychomotor Retardation study, which is a cohort of 946 subjects with unexplained intellectual disability. Based on characteristic serum TH values, the coding region of the DIO2 gene was sequenced in 387 patients. Functional consequences were assessed by in vitro D2 assays or intact cell metabolism studies using cells transfected with wild-type or mutant D2. Results: Sequence analysis revealed two heterozygousmutations: c.11TA (p.L4H) in three subjects and c.305CT (p.T102I) in one subject. Sequence analysis of family members revealed several carriers, but no segregation was observed with thyroid parameters or neurocognitive phenotype. Extensive tests with different in vitro D2 assays did not show differences between wild-type and mutant D2. Conclusion: This study describes the identification and functional consequences of novel genetic variation in TH activating enzyme D2. Family studies and functional tests suggest that these variants do not underlie the neurocognitiveimpairment. Altogether our data provide evidence of the existence of rare but apparently harmless genetic variants of D2.

The discovery of a (pro)renin receptor ((P)RR) in 2002 provided a long-sought explanation for tissue renin-angiotensin system (RAS) activity and a function for circulating prorenin, the inactive precursor of renin, in end-organ damage. Binding of renin and prorenin (referred to as (pro)renin) to the (P)RR increases angiotensin I formation and induces intracellular signalling, resulting in the production of profibrotic factors. However, the (pro)renin concentrations required for intracellular signalling in vitro are several orders of magnitude above (patho)physiological plasma levels. Moreover, the phenotype of prorenin-overexpressing animals could be completely attributed to angiotensin generation, possibly even without the need for a receptor. The efficacy of the only available putative (pro)renin receptor blocker handle region peptide remains doubtful, leading to inconclusive results. The fact that, in contrast to other RAS components, (P)RR knock-outs, even tissue-specific, are lethal, points to an important, (pro)renin-independent, function of the (P)RR. Indeed, recent research has highlighted ancillary functions of the (P)RR as an essential accessory protein of th

Prorenin binding to the prorenin receptor [(P)RR] results in nonproteolytic activation of prorenin but also directly (i.e., independent of angiotensin generation) activates signal transduction cascades that can lead to the upregulation of profibrotic factors. The (P)RR is an accessory protein of vacuolar-type H+-ATPase (V-ATPase) and is required for V-ATPase integrity. In addition, in collecting duct cells, prorenin-induced activation of Erk depends on V-ATPase activity. However, whether prorenin binding to the (P)RR directly regulates V-ATPase activity

During hypovolemia and hyperkalemia, the kidneys defend homeostasis by Na+ retention and K+ secretion, respectively. Aldosterone mediates both effects, but it is unclear how the same hormone can evoke such different responses. To address this, we mimicked hypovolemia and hyperkalemia in four groups of rats with a control diet, low-Na+ diet, high-K+ diet, or combined diet. The low-Na+ and combined diets increased plasma and kidney ANG II. The low-Na+ and high-K+ diets increased plasma aldosterone to a similar degree (3-fold), whereas the combined diet increased aldosterone to a greater extent (10-fold). Despite similar Na+ intake and higher aldosterone, the high-K+ and combined diets caused a greater natriuresis than the control and low-Na+ diets, respectively (P < 0.001 for both). This K+-induced natriuresis was accompanied by a decreased abundance but not phosphorylation of the Na+-Cl- cotransporter (NCC). In contrast, the epithelial Na+ channel (ENaC) increased in parallel with aldosterone, showing the highest expression with the combined diet. The high-K+ and combined diets also incre

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4-SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and los