The Musashi-1-type 2 deiodinase pathway regulates astrocyte proliferation.

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2 mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

Combined deletion of Mct8 and Dio2 impairs SVZ neurogliogenesis and olfactory function in adult mice.

Within the adult mouse subventricular zone (SVZ), neural stem cells (NSCs) produce neuroblasts and oligodendrocyte precursor cells (OPCs). T3, the active thyroid hormone, influences renewal and commitment of SVZ progenitors. However, how regulators of T3 availability affect these processes is less understood. Using Mct8/Dio2 knockout mice, we investigated the role of MCT8, a TH transporter, and DIO2, the T3-generating enzyme, in regulating adult SVZ-neurogliogenesis. Single-cell RNA-Seq revealed Mct8 expression in various SVZ cell types in WT mice, while Dio2 was enriched in neurons, astrocytes, and quiescent NSCs. The absence of both regulators in the knockout model dysregulated gene expression, increased the neuroblast/OPC ratio and hindered OPC differentiation. Immunostainings demonstrated compromised neuroblast migration reducing their supply to the olfactory bulbs, impairing interneuron differentiation and odor discrimination. These findings underscore the pivotal roles of MCT8 and DIO2 in neuro- and oligodendrogenesis, offering targets for therapeutic avenues in neurodegenerative and demyelinating diseases.

Determination of HLA class II risk alleles and prediction of self/non-self-epitopes contributing Hashimoto's thyroiditis in a group of Iranian patients.

One of the probable hypotheses for the onset of autoimmunity is molecular mimicry. This study aimed to determine the HLA-II risk alleles for developing Hashimoto's thyroiditis (HT) in order to analyze the molecular homology between candidate pathogen-derived epitopes and potentially self-antigens (thyroid peroxidase, TPO) based on the presence of HLA risk alleles. HLA-DRB1/-DQB1 genotyping was performed in 100 HT patients and 330 ethnically matched healthy controls to determine the predisposing/protective alleles for HT disease. Then, in silico analysis was conducted to examine the sequence homology between epitopes derived from autoantigens and four potentially relevant pathogens and their binding capacities to HLA risk alleles based on peptide docking analysis. We identified HLA-DRB1*03:01, *04:02, *04:05, and *11:04 as predisposing alleles and DRB1*13:01 as a potentially predictive allele for HT disease. Also, DRB1*11:04 ~ DQB1*03:01 (Pc = 0.002; OR, 3.97) and DRB1*03:01 ~ DQB1*02:01 (Pc = 0.004; OR, 2.24) haplotypes conferred a predisposing role for HT. Based on logistic regression analysis, carrying risk alleles increased the risk of HT development 4.5 times in our population (P = 7.09E-10). Also, ROC curve analysis revealed a high predictive power of those risk alleles for discrimination of the susceptible from healthy individuals (AUC, 0.70; P = 6.6E-10). Analysis of peptide sequence homology between epitopes of TPO and epitopes derived from four candidate microorganisms revealed a homology between envelop glycoprotein D of herpes virus and sequence 151-199 of TPO with remarkable binding capacity to HLA-DRB1*03:01 allele. Our findings indicate the increased risk of developing HT in those individuals carrying HLA risk alleles which can also be related to herpes virus infection.

A DIO2 missense mutation and its impact on fetal response to PRRSV infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. RESULTS: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. CONCLUSIONS: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

Single-cell and bulk RNA sequencing reveal heterogeneity and diagnostic markers in papillary thyroid carcinoma lymph-node metastasis.

PURPOSE: Papillary thyroid carcinoma (PTC) is characterized by lymph-node metastasis (LNM), which affects recurrence and prognosis. This study analyzed PTC LNM by single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing (RNA-seq) to find diagnostic markers and therapeutic targets. METHODS: ScRNA-seq data were clustered and malignant cells were identified. Differentially expressed genes (DEGs) were identified in malignant cells of scRNA-seq and bulk RNA-seq, respectively. PTC LNM diagnostic model was constructed based on intersecting DEGs using glmnet package. Next, PTC samples from 66 patients were used to validate the two most significant genes in the diagnostic model, S100A2 and type 2 deiodinase (DIO2) by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC). Further, the inhibitory effect of DIO2 on PTC cells was verified by cell biology behavior, western blot, cell cycle analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, and xenograft tumors. RESULTS: Heterogeneity of PTC LNM was demonstrated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. A total of 19 differential genes were used to construct the diagnostic model. S100A2 and DIO2 differ significantly at the RNA (p < 0.01) and protein level in LNM patient tissues (p < 0.001). And differed in PTC tissues with different pathologic typing (p < 0.001). Further, EdU (p < 0.001) and cell biology behavior revealed that PTC cells overexpressed DIO2 had reduced proliferative capacity. Cell cycle proteins were reduced and cells are more likely to be stuck in G2/M phase (p < 0.001). CONCLUSIONS: This study explored the heterogeneity of PTC LNM using scRNA-seq. By combining with bulk RNA-seq data, diagnostic markers were explored and the model was established. Clinical diagnostic efficacy of S100A2 and DIO2 was validated and the treatment potential of DIO2 was discovered.

In vitro assessment of thyroid peroxidase inhibition by chemical exposure: comparison of cell models and detection methods.

Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

A regulatory element associated to NAFLD in the promoter of DIO1 controls LDL-C, HDL-C and triglycerides in hepatic cells.

BACKGROUND: Genome-wide association studies (GWAS) have identified genetic variants linked to fat metabolism and related traits, but rarely pinpoint causative variants. This limitation arises from GWAS not considering functional implications of noncoding variants that can affect transcription factor binding and potentially regulate gene expression. The aim of this study is to investigate a candidate noncoding functional variant within a genetic locus flagged by a GWAS SNP associated with non-alcoholic fatty liver disease (NAFLD), a condition characterized by liver fat accumulation in non-alcohol consumers. METHODS: CRISPR-Cas9 gene editing in HepG2 cells was used to modify the regulatory element containing the candidate functional variant linked to NAFLD. Global gene expression in mutant cells was assessed through RT-qPCR and targeted transcriptomics. A phenotypic assay measured lipid droplet accumulation in the CRISPR-Cas9 mutants. RESULTS: The candidate functional variant, rs2294510, closely linked to the NAFLD-associated GWAS SNP rs11206226, resided in a regulatory element within the DIO1 gene's promoter region. Altering this element resulted in changes in transcription factor binding sites and differential expression of candidate target genes like DIO1, TMEM59, DHCR24, and LDLRAD1, potentially influencing the NAFLD phenotype. Mutant HepG2 cells exhibited increased lipid accumulation, a hallmark of NAFLD, along with reduced LDL-C, HDL-C and elevated triglycerides. CONCLUSIONS: This comprehensive approach, that combines genome editing, transcriptomics, and phenotypic assays identified the DIO1 promoter region as a potential enhancer. Its activity could regulate multiple genes involved in the NAFLD phenotype or contribute to defining a polygenic risk score for enhanced risk assessment in NAFLD patients.

Two pathways regulate insulin-like growth factor genes in the brain and liver of the tropical damselfish Chrysiptera cyanea: A possible role for melatonin in the actions of growth and thyroid hormones.

External and internal factors are involved in controlling the growth of fishes. However, little is known about the mechanisms by which external factors trigger stimulus signals. This study explored the physiological roles of melatonin in the transcription of growth-related genes in the brain and liver of Chrysiptera cyanea, a tropical damselfish with long-day preference. In brain samples of this species collected at 4-h intervals, the transcript levels of arylalkylamine N-acetyltransferase2 (aanat2), the rate-limiting enzyme of melatonin synthesis, and growth hormone (gh) peaked at 20:00 and 00:00, respectively. Concomitantly, the transcript levels of insulin-like growth factors (igf1 and igf2) in the brain and liver were upregulated during the scotophase. Levels of iodothyronine deiodinases (dio2 and dio3), enzymes that convert thyroxine (T4) to triiodothyronine (T3) and reverse T3, respectively, increased in the brain (dio2 and dio3) and liver (dio2) during the photophase, whereas dio3 levels in the liver showed the opposite trend. Fish reared in melatonin-containing water exhibited significant increases in the transcription levels of gh and igf1 in the brain and igf1 in the liver, suggesting that growth in this fish is positively regulated by the GH/IGF pathway on a daily basis. Melatonin treatment also stimulated the transcript levels of dio2 and dio3 in the liver, but not in the brain. Fish consuming pellets containing T3, but not T4, showed significant increases in gh and igf1 in the brain and igf1 and igf2 in the liver, suggesting that the intercellular actions of the TH/IGF pathway have an impact on growth on a daily basis. In summary, IGF synthesis and action in the brain and liver undergo dual regulation by distinct hormone networks, which may also be affected by daily, seasonal, or nutritional factors.

Is There a Link between Thyroid Peroxidase Gene Promoter Polymorphisms and Autoimmune Thyroiditis in the Polish Population?

(1) Autoimmune thyroiditis (AIT) is the most common cause of primary hypothyroidism and one of the most frequent organ-specific autoimmune diseases. Its pathogenesis is polygenic and still requires further research. The aim of the study was to assess, for the first time in the Caucasian population, the role of selected TPO gene promoter polymorphisms (rs2071399 G/A, rs2071400C/T, rs2071402 A/G, and rs2071403 A/G) in the development of AIT. A total of 237 patients diagnosed with AIT and 130 healthy controls were genotyped for four TPO gene polymorphisms, and the results were statistically analyzed to check for the role of these polymorphisms. There were no significant differences in the genotype and allele frequencies of the studied TPO gene promoter polymorphisms between patients and controls (p > 0.05). The haplotype distribution (rs2071400-rs2071402-rs2071403) between the two studied groups was similar for the most common variants (CGA, CAG, TGG). Only a rare haplotype (CGG) occurred more frequently among patients compared to controls (p = 0.04). The studied TPO gene promoter polymorphisms did not show an association with susceptibility to AIT in the Caucasian Polish population, contrary to the results in Japanese patients.

Effect of DIO2 Gene Polymorphism on Thyroid Hormone Levels and Its Correlation with the Severity of Schizophrenia in a Pakistani Population.

Low levels of triiodothyronine (T3) in the brain lead to increased dopamine receptor sensitivity, potentially resulting in schizophrenia. Iodothyronine deiodinase 2 (DIO2) is the only enzyme which converts tetraiodothyronine (T4) to T3 in the brain. DIO2 polymorphism of rs225014 results in the expression of non-functioning DIO2. Therefore, this study aimed to investigate the association of rs255014 with schizophrenia and its impact on thyroid hormone levels. This study included 150 schizophrenia cases and 150 controls. DNA was extracted from blood and subjected to PCR and amplicon sequencing. Serum thyroid profiles were determined using chemiluminescent magnetic microparticle immunoassay. Statistical analyses involved independent sample t-tests, Chi-square, and Pearson's correlation tests. The results revealed a higher frequency of the reference genotype (TT) in controls compared to cases (p < 0.05). However, rs225014 did not influence serum thyroid levels or the severity of schizophrenia (p > 0.05). Interestingly, control subjects exhibited significantly higher T3 levels (p < 0.001) than cases. Regardless of the genotype (TT or CC), the control group had higher mean T3 levels than the corresponding case group (p < 0.05). In conclusion, rs225014 is associated with schizophrenia and has no effect on serum thyroid hormone levels.

LncRNAs in the Dlk1-Dio3 Domain Are Essential for Mid-Embryonic Heart Development.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

Frequency of Mutations in the TPO Gene in Patients with Congenital Hypothyroidism Due to Dyshormonogenesis in Chile.

Background and Objectives: Congenital thyroid dyshormonogenesis is caused by alterations in the synthesis of thyroid hormones in a newborn. Additionally, 10 to 20% of these cases are hereditary, caused by defects in proteins involved in hormonal synthesis. One of the most common causes is mutations in the thyroid peroxidase (TPO) enzyme gene, an autosomal recessive disease. We aimed to detect mutations of the TPO gene in 12 Chilean patients with congenital hypothyroidism due to dyshormonogenesis (CHD) and to characterize these patients clinically and molecularly. Materials and Methods: Twelve patients under 20 years of age with CHD, controlled at San Juan de Dios Hospital in Santiago, Chile, were selected according to the inclusion criteria: elevated neonatal TSH, persistent hypothyroidism, and thyroid normotopic by imaging study. Those with deafness, Down syndrome, and central or transient congenital hypothyroidism were excluded. Blood samples were taken for DNA extraction, and the 17 exons and exon-intron junctions of the TPO gene were amplified by PCR. The PCR products were sequenced by Sanger. Results: Two possibly pathogenic mutations of the TPO gene were detected: c.2242G>A (p.Val748Met) and c.1103C>T (p.Pro368Leu). These mutations were detected in 2 of 12 patients (16.6%): 1 was compound heterozygous c.1103C>T/c.2242G>A, and the other was heterozygous for c.2242G>A. In the diagnostic confirmation test, both patients presented diffuse hyper-uptake goiter on thyroid scintigraphy and high TSH in venous blood (>190 uIU/mL). Conclusions: The frequency of patients with possibly pathogenic mutations in TPO with CHD was 16.6%. Its study would allow for genetic counseling to be offered to the families of affected patients.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis.

Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.

In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.

A Computational Approach: The Functional Effects of Thyroid Peroxidase Variants in Thyroid Cancer and Genetic Disorders.

PURPOSE: Thyroid peroxidase (TPO) is essential for the synthesis of thyroid hormones. However, specific mutations render TPO antigenic and prone to autoimmune attacks leading to thyroid cancer, TPO deficiency, and congenital hypothyroidism (CH). Despite technological advancement, most experimental procedures cannot quickly identify the genetic causes of CH nor detect thyroid cancer in the early stages. METHODS: We performed saturated computational mutagenesis to calculate the folding energy changes (∆∆G) caused by missense mutations and analyzed the mutations involved in post-translational modifications (PTMs). RESULTS: Our results showed that the functional important missense mutations occurred in the heme peroxidase domain. Through computational saturation mutagenesis, we identified the TPO mutations in G393 and G348 affecting protein stability and PTMs. Our folding energy calculations revealed that seven of nine somatic thyroid cancer mutations destabilized TPO. CONCLUSION: These findings highlight the impact of these specific mutations on TPO stability, linking them to thyroid cancer and other genetic thyroid-related disorders. Our results show that computational mutagenesis of proteins provides a quick insight into rare mutations causing Mendelian disorders and cancers in humans.

Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues.

Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroid hormones, exhibit a phase shift in locomotor activity, suggesting altered circadian rhythm. To better understand the physiological and molecular basis of this phenotype, we used Dio3+/+ and Dio3-/- mice of both sexes at different zeitgeber times (ZTs) and analyzed corticosterone and thyroxine (T4) levels, hypothalamic, hepatic, and adipose tissue expression of clock genes, as well as genes involved in the thyroid hormone action or physiology of liver and adipose tissues. Wild type mice exhibited sexually dimorphic circadian patterns of genes controlling thyroid hormone action, including Dio3. Dio3-/- mice exhibited altered hypothalamic expression of several clock genes at ZT12, but did not disrupt the overall circadian profile. Expression of clock genes in peripheral tissues was not disrupted by Dio3 deficiency. However, Dio3 loss in liver and adipose tissues disrupted circadian profiles of genes that determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes in serum T4 and corticosterone as a result of DIO3 deficiency. The circadian alterations manifested sexual dimorphism. Most notable, the time curve of serum corticosterone was flattened in Dio3-/- females. We conclude that Dio3 exhibits circadian variations, influencing the circadian rhythmicity of thyroid hormone action and physiology in liver and adipose tissues in a sex-specific manner. Circadian disruptions in tissue physiology may then contribute to the metabolic phenotypes of DIO3-deficient mice.

Evaluating the link between DIO3-FA27 promoter methylation, biochemical indices, and heart failure progression.

BACKGROUND: Heart failure (HF) is a disease that poses a serious threat to individual health, and DNA methylation is an important mechanism in epigenetics, and its role in the occurrence and development of the disease has attracted more and more attention. The aim of this study was to evaluate the link between iodothyronine deiodinase 3 promoter region fragment FA27 (DIO3-FA27) methylation levels, biochemical indices, and HF. RESULTS: The methylation levels of DIO3-FA27_CpG_11.12 and DIO3-FA27_CpG_23.24 significantly differed in HF patients with different degrees. Multivariate logistic regression analysis indicated that the relative HF risk in the third and fourth quartiles of activated partial thromboplastin time and fibrin degradation products. The results of the restricted cubic spline model showed that the methylation levels of DIO3-FA 27_CpG_11.12 and DIO3-FA 27_CpG_23.24 were associated with coagulation indicators, liver function, renal function, and blood routine. CONCLUSIONS: Based on the differential analysis of CpG methylation levels based on DIO3-FA27, it was found that biochemical indicators combined with DIO3-FA27 promoter DNA methylation levels could increase the risk of worsening the severity classification of HF patients, which provided a solid foundation and new insights for the study of epigenetic regulation mechanisms in patients with HF.

Dissociating Mechanisms That Underlie Seasonal and Developmental Programs for the Neuroendocrine Control of Physiology in Birds.

Long-term programmed rheostatic changes in physiology are essential for animal fitness. Hypothalamic nuclei and the pituitary gland govern key developmental and seasonal transitions in reproduction. The aim of this study was to identify the molecular substrates that are common and unique to developmental and seasonal timing. Adult and juvenile quail were collected from reproductively mature and immature states, and key molecular targets were examined in the mediobasal hypothalamus (MBH) and pituitary gland. qRT-PCR assays established deiodinase type 2 (DIO2) and type 3 (DIO3) expression in adults changed with photoperiod manipulations. However, DIO2 and DIO3 remain constitutively expressed in juveniles. Pituitary gland transcriptome analyses established that 340 transcripts were differentially expressed across seasonal photoperiod programs and 1,189 transcripts displayed age-dependent variation in expression. Prolactin (PRL) and follicle-stimulating hormone subunit beta (FSHß) are molecular markers of seasonal programs and are significantly upregulated in long photoperiod conditions. Growth hormone expression was significantly upregulated in juvenile quail, regardless of photoperiodic condition. These findings indicate that a level of cell autonomy in the pituitary gland governs seasonal and developmental programs in physiology. Overall, this paper yields novel insights into the molecular mechanisms that govern developmental programs and adult brain plasticity.

Genetic Background Strongly Influences the Impact of Carrying the Thr92Ala-DIO2 Polymorphism in the Male Mouse.

About half of the world population carries at least one allele of the Ala92-DIO2, which slows down the activity of the type 2 deiodinase (D2), the enzyme that activates T4 to T3. Carrying the Ala92-DIO2 allele has been associated with increased body mass index and insulin resistance, but this has not been reproduced in all populations. To test if the genetic background affects the impact of this polymorphism, here we studied the genetically distant C57Bl/6J (B6) and FVB/N (FVB) mice carrying the Ala92-Dio2 allele as compared to control mice carrying the Thr92-Dio2 allele. Whereas B6-Ala92-Dio2 and B6-Thr92-Dio2 mice-fed chow or high-fat diet-behaved metabolically similar in studies using indirect calorimetry, glucose- and insulin tolerance tests, and measuring white adipose tissue (WAT) weight and liver steatosis, major differences were observed between FVB-Ala92-Dio2 and FVB-Thr92-Dio2 mice: carrying the Ala92-Dio2 allele (on a chow diet) resulted in hypercholesterolemia, smaller WAT pads, hepatomegaly, steatosis, and transcriptome changes in the interscapular brown adipose tissue (iBAT) typical of ER stress and apoptosis. Acclimatization at thermoneutrality (30 °C) eliminated most of the metabolic phenotype, indicating that impaired adaptive (BAT) thermogenesis can be involved. In conclusion, the metabolic impact of carrying the Ala92-Dio2 allele depends greatly on the genetic background of the mouse, varying from no phenotype in B6 mice to a major phenotype in FVB mice. These results will help the planning of future clinical trials studying the Thr92Ala-DIO2 polymorphism and may explain why some clinical studies performed in different populations across the globe have obtained inconsistent results.

The Perioperative Period of Heart Transplantation Is Affected by Thyroid Hormone Status.

Background: Orthotopic heart transplantation (HTx) is a long-term surgical therapeutic approach for patients with end-stage heart failure. The objective of the present study was to uncover associations between altered thyroid hormone (TH) status and adverse outcomes after HTx. Methods: In this prospective, single-center cohort study, 283 patients underwent HTx between 2013 and 2020 at the Heart and Vascular Center of Semmelweis University in Hungary. We measured serum free triiodothyronine (fT3), free thyroxine (fT4), and thyrotropin (TSH) pre- and postoperatively. TaqMan qPCR was used to measure type 2 deiodinase and type 3 deiodinase mRNA (Dio2 and Dio3, respectively) levels from the diseased heart bioptates. To assess the local TH action of the heart, mRNA levels of Hcn2 and Myh7 were measured in a subgroup of patients receiving extracorporeal membrane oxygenation (ECMO) postoperatively. Groups were compared using nonparametric tests. Cox regression analysis and logistic regression test were used to investigate the outcomes. The connection between serum TH parameters and cardiac gene expressions was assessed using linear regression. Results: Serum TSH (p = 0.009), fT3 (p < 0.001), and fT4 (p < 0.001) levels were lower after HTx than preoperatively. Levothyroxine (LT4) administered to donors was associated with better survival after 30 days (p = 0.049). LT4 replacement given to recipients after HTx was associated with better survival after 30 days (p = 0.018), 1 year (p = 0.002), and 2 years (p = 0.001). Dio3 mRNA level was significantly increased in patients who were treated with ECMO (p = 0.026), left ventricular assist device (LVAD) (p = 0.008), and biventricular assist device (BiVAD) (p = 0.013) preoperatively, and ECMO (p = 0.042) postoperatively, compared with those who did not require any type of mechanical circulatory support (MCS). We found no significant difference in the expression of the Hcn2 and Myh7 marker genes between patients on postoperative ECMO and those without MCS, and neither did they correlate with serum hormone levels (p = 0.519 and p = 0.056, respectively). Conclusions: We conclude that TH status plays an important role in HTx patients, and monitoring of TH status in the perioperative period may contribute to improved treatment outcomes. Our findings require independent confirmation in a randomized controlled clinical trial.