Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.

Congenital Hypothyroidism

Congenital hypothyroidism (CH) is a disorder of thyroid hormone deficiency which develops secondary to incomplete thyroid development or inadequate thyroid hormone production. State-mandated newborn screening throughout the United States has increased the detection rate of CH, allowing for early intervention. Although the overall mortality rate of CH is low, delayed or omitted treatment can lead to devastating neurocognitive outcomes. As such, CH is regarded as the leading cause of preventable intellectual disability in children. Early identification, facilitated by astute neonatal nursing and medical care, is contingent upon an active working knowledge of the disease process and awareness of the limitations of the newborn screen.

ZFP36L2 Role in Thyroid Functionality

Thyroid hormone levels are usually genetically determined. Thyrocytes produce a unique set of enzymes that are dedicated to thyroid hormone synthesis. While thyroid transcriptional regulation is well-characterized, post-transcriptional mechanisms have been less investigated. Here, we describe the involvement of ZFP36L2, a protein that stimulates degradation of target mRNAs, in thyroid development and function, by in vivo and in vitro gene targeting in thyrocytes. Thyroid-specific Zfp36l2-/- females were hypothyroid, with reduced levels of circulating free Thyroxine (cfT4) and Triiodothyronine (cfT3). Their hypothyroidism was due to dyshormonogenesis, already evident one week after weaning, while thyroid development appeared normal. We observed decreases in several thyroid-specific transcripts and proteins, such as Nis and its transcriptional regulators (Pax8 and Nkx2.1), and increased apoptosis in Zfp36l2-/- thyroids. Nis, Pax8, and Nkx2.1 mRNAs were also reduced in Zfp36l2 knock-out thyrocytes in vitro (L2KO), in which we confirmed the increased apoptosis. Finally, in L2KO cells, we showed an altered response to TSH stimulation regarding both thyroid-specific gene expression and cell proliferation and survival. This result was supported by increases in P21/WAF1 and p-P38MAPK levels. Mechanistically, we confirmed Notch1 as a target of ZFP36L2 in the thyroid since its levels were increased in both in vitro and in vivo models. In both models, the levels of Id4 mRNA, a potential inhibitor of Pax8 activity, were increased. Overall, the data indicate that the regulation of mRNA stability by ZFP36L2 is a mechanism that controls the function and survival of thyrocytes.

Upregulation of GBP1 in thyroid primordium is required for developmental thyroid morphogenesis

Purpose Congenital hypothyroidism (CH) is a common congenital endocrine disorder in humans. CH-related diseases such as athyreosis, thyroid ectopy, and hypoplasia are primarily caused by dysgenic thyroid development. However, the underlying molecular mechanisms remain unknown. Methods To identify novel CH candidate genes, 192 CH patients were enrolled, and target sequencing of 21 known CH-related genes was performed. The remaining 98 CH patients carrying no known genes were subjected to exome sequencing (ES). The functions of the identified variants were confirmed using thyroid epithelial cells in vitro and in zebrafish model organisms in vivo. Results Four pathogenic GBP1 variations from three patients were identified. In zebrafish embryos, gbp1 knockdown caused defective thyroid primordium morphogenesis and hypothyroidism. The thyroid cells were stuck together and failed to dissociate from each other to form individual follicles in gbp1-deficient embryos. Furthermore, defects were restored with wild-type human GBP1 (hGBP1) messenger RNA (mRNA) except for mutated hGBP1 (p.H150Y, p.L187P) overexpression. GBP1 promoted β-catenin translocation into the cytosol and suppressed the formation of cellular adhesion complexes. Suppression of cell–cell adhesion restored the thyroid primordium growth defect observed in gbp1-deficient zebrafish embryos. Conclusion This study provides further understanding regarding thyroid development and shows that defective cellular remodeling could cause congenital hypothyroidism.

Perchlorate and Nitrate Treatments Disrupt the Endoderm and Thyroid Development Through Epigenetic Mechanisms

Nitrate and perchlorate competitively inhibit iodide uptake in thyrocytes and disrupt thyroid function in rodents and humans. Our previous data indicated that the intrauterine exposure to perchlorate or nitrate induced thyroid dysfunction in the offspring rats during adult life. Therefore, this study aimed to investigate the effects of these endocrine disruptors during the embryonic period on the endoderm and thyroid development. Additionally, it was also investigated the role of epigenetic modifications in the programming of gene expression of the evaluated tissues. For this purpose, CD1 pregnant female mice received filtered water (control) or filtered water supplemented with sodium perchlorate (0.3 or 1 ppm) or sodium nitrate (20 or 50 ppm). At gestational day 16.5 (GD16.5), the embryonic thyroid lobes were collected and processed for molecular analysis. Besides the in vivo model, the effect of these thyroid disruptors was also evaluated during the differentiation of mouse embryonic stem cells (mESc) into endoderm and thyroid cells. Endoderm cells differentiation was achieved through the treatment of mESc with several growth factors. During the entire protocol the cells were exposed or not to sodium perchlorate or sodium nitrate (10-5 or 10-7 M). The effects of perchlorate and nitrate were also evaluated during the differentiation of mESC into thyroid cells. For this purpose, mESC-derived endoderm cells were transiently transfected with Pax8/Nkx2.1 expressing vectors. During the endoderm-to-thyrocytes differentiation protocol, the cells were also exposed or not to perchlorate or nitrate (10-5 or 10-7 M). The results demonstrated that both thyroid disruptors reduced the mRNA and protein expression of several endoderm markers (Foxa1, Gata4, Sox17) in the mESc-derived endoderm cells. Moreover, perchlorate or nitrate treatment also reduced the expression of thyroid transcription factors (Pax8, Nkx2.1, Foxe1) and thyroid differentiation markers (Slc5a5, Tpo, Tshr, Tg) both in the embryonic thyroid lobes and in the mESc-derived thyrocytes. Epigenetic mechanisms related to transcription repression seem to be involved in the gene expression downregulation both in vivo and in vitro, since perchlorate and nitrate increased the mRNA expression of Dnmt1, Dnmt3, Hdac and reduced the expression of Hat. Additionally, the methylation of histone H3 was increased, and the acetylation status of this histone was decreased in perchlorate- or nitrate-exposed thyroid lobes and mESc-derived endoderm/thyroid cells. In conclusion, our data strongly suggest that the programming of thyroid dysfunction induced by intrauterine exposure to perchlorate or nitrate involve the disruption of the endoderm and thyroid development during embryonic life through epigenetic mechanisms.

Unraveling the Complex Interplay Between Transcription Factors and Signaling Molecules in Thyroid Differentiation and Function, From Embryos to Adults

Thyroid differentiation of progenitor cells occurs during embryonic development and in the adult thyroid gland, and the molecular bases of these complex and finely regulated processes are becoming ever more clear. In this Review, we describe the most recent advances in the study of transcription factors, signaling molecules and regulatory pathways controlling thyroid differentiation and development in the mammalian embryo. We also discuss the maintenance of the adult differentiated phenotype to ensure the biosynthesis of thyroid hormones. We will focus on endoderm-derived thyroid epithelial cells, which are responsible for the formation of the thyroid follicle, the functional unit of the thyroid gland. The use of animal models and pluripotent stem cells has greatly aided in providing clues to the complicated puzzle of thyroid development and function in adults. The so-called thyroid transcription factors – Nkx2-1, Foxe1, Pax8 and Hhex – were the first pieces of the puzzle identified in mice. Other transcription factors, either acting upstream of or directly with the thyroid transcription factors, were subsequently identified to, almost, complete the puzzle. Among them, the transcription factors Glis3, Sox9 and the cofactor of the Hippo pathway Taz, have emerged as important players in thyroid differentiation and development. The involvement of signaling molecules increases the complexity of the puzzle. In this context, the importance of Bmps, Fgfs and Shh signaling at the onset of development, and of TSH, IGF1 and TGFβ both at the end of terminal differentiation in embryos and in the adult thyroid, are well recognized. All of these aspects are covered herein. Thus, readers will be able to visualize the puzzle of thyroid differentiation with most – if not all – of the pieces in place.

Genetic Variability of the Paired Box Transcription Factor; PAX8 Gene: Guidance Towards Treatment Strategies in a Cohort of Congenital Hypothyroidism

The contribution of PAX8 genetic variants to congenital hypothyroidism (CH) is not well understood. We aimed to study the genetic variability of exons 3 and 5 of PAX8 gene among a cohort of children with congenital hypothyroidism in correspondence to their clinical aspect. Blood samples were collected from 117 children (63 girls and 54 boys) with CH and enrolled as cases (Group I). All cases underwent biochemical confirmation with low FT4 and high TSH levels and thyroid gland imaging, along with equal number of matched apparently healthy individuals who served as controls (Group II). Genomic materials for exons 3 and 5 of PAX8 gene were extracted, amplified by PCR, detected by electrophoresis, purified, and sequenced by the Sanger technique through the application of ABI 3730x1 DNA Sequencer. Out of 117 cases, eight different effective PAX8 mutations were detected in exon 3 (G23D, V35I, I34T, Q40P, p.R31C, p.R31H, p.R31A, and p.I47T) in 14 patients with their sonographic findings ranged from normal, hypoplastic to thyroid agenesis. Besides the reported mutations, one novel mutation; R31A was detected in 1 euotopic case. Exon 5 analysis revealed no detected mutations elsewhere. In contrast, all healthy control children showed no mutation and normal sonographic findings. Mutations in exon 3 of PAX8 gene, implies its important role in thyroid development and function, as a first estimate of PA8 mutation rate in Egyptian patients with CH having normal and dysgenetic gland. Using ultrasound is mandatory for diagnosis and guiding the treatment of children with CH.

Effects of photoperiod on thyroid gland development and function in growing chicks: a biochemical and morphometric study

Context Light treatment has a regulatory role in some growth-related functions, including thyroid development in chicks. Aims This study aimed to investigate the effects of different photoperiod treatments on thyroid organ weight and serum thyroid hormone concentrations of broilers by use of biochemical and histological methods. Methods After the hatching, 120 broiler chicks (Ross) were divided into two main groups according to sex. Both groups were then split into two sub-groups based on photoperiod treatment: 16 h (i.e. 16 h light:8 h dark) and 24 h (24 h light:0 h dark). Thyroid gland and blood samples of six animals from each group were obtained after slaughtering at 7-day intervals from Day 14 after hatching to Day 42. Serum concentrations of free triiodothyronine (FT3), free thyroxin (FT4) and thyroid-stimulating hormone (TSH) were determined by the chemiluminescence method for all groups. Thyroid weight, bodyweight and thyroid follicle diameter were also measured. Key results Thyroid weight:bodyweight ratio generally started to increase from Day 14 to Day 42, with no significant (P > 0.05) difference among the groups at the same age. For both male and female broiler chicks, morphometric measures increased as birds grew. Serum FT3 and TSH concentrations slightly decreased and serum FT4 concentrations increased in growing chicks of both sexes. Conclusions Extending the photoperiod from 16 to 24 h had no effects on thyroid gland development or functions in terms of both biochemical and morphometric parameters in broiler chicks. Implications Continuous light has minimal effects on thyroid functions of growing broiler chicks to Day 42.

Timed mesodermal FGF and BMP govern the multi-step thyroid specification

SummaryThe thyroid plays an essential role in homeostasis and development, but the extrinsic regulation of its embryonic development remains poorly understood. Recently, we have identified the FGF and BMP pathways to be crucial for thyroid specification and have confirmed the hypothesis that the cardiac mesoderm provides the FGF and BMP ligands to regulate this process. However, it is not clear how these ligands control thyroid specification. To study the molecular mechanisms underlying early thyroid development, we combined a pharmacological approach in zebrafish embryos with genetic models, to modulate the activity of the FGF and BMP pathways at different embryonic stages. We first characterized the expression of the transcription factors pax2a and nkx2.4b - the two main early thyroid markers - in the anterior foregut endoderm and observed that pax2a was expressed from 18 hours post fertilization (hpf) and marked a large endodermal cell population while nkx2.4b was expressed from 24 hpf and marked only a subset of the pax2a-positive endodermal cells. Interestingly, the activity profiles of FGF and BMP coincided with the detection of pax2a and nkx2.4b expression, respectively. Brief modulations of the FGF and/or BMP pathways support the hypothesis that the FGF pathway regulates the expression of pax2a and the BMP pathway regulates the expression of nkx2.4b. Furthermore, inhibition of the BMP pathway during early segmentation has dramatic effects on thyroid specification, probably via the FGF pathway. Together with our previous observations, we propose here, an updated model of early thyroid development in which the foregut endoderm receives several synchronized waves of FGF and BMP signals from the cardiac mesoderm, which result in sequential activation of pax2a and nkx2.4b gene expression and subsequent thyroid specification.