Immacolata Cristina Nettore

The molecular causes of thyroid dysgenesis: A systematic review

Background: Congenital hypothyroidism (CH) is a frequent disease occurring with an incidence of about 1/2500 newborns/year. In 80–85% of the cases CH is caused by alterations in thyroid morphogenesis, generally indicated by the term “thyroid dysgenesis” (TD). TD is generally a sporadic disease, but in about 5% of the cases a genetic origin has been demonstrated. In these cases, mutations in genes playing a role during thyroid morphogenesis (NKX2-1, PAX8, FOXE1, NKX2-5, TSHR) have been reported. Aim: This work reviews the main steps of thyroid morphogenesis and all the genetic alterations associated with TD and published in the literature.

Long period fiber grating nano-optrode for cancer biomarker detection

We report an innovative fiber optic nano-optrode based on Long Period Gratings (LPGs) working in reflection mode for the detection of human Thyroglobulin (TG), a protein marker of differentiated thyroid cancer. The reflection-type LPG (RT-LPG) biosensor, coated with a single layer of atactic polystyrene (aPS) onto which a specific, high affinity anti-Tg antibody was adsorbed, allowed the label-free detection of Tg in the needle washouts of fine-needle aspiration biopsies, at concentrations useful for pre- and post-operative assessment of the biomarker levels. Analyte recognition and capture were confirmed with a parallel on fiber ELISA-like assay using, in pilot tests, the biotinylated protein and HRP-labeled streptavidin for its detection. Dose-dependent experiments showed that the detection is linearly dependent on concentration within the range between 0 and 4 ng/mL, while antibody saturation occurs for higher protein levels. The system is characterized by a very high sensitivity and specificity allowing the ex-vivo detection of sub ng/ml concentrations of human Tg from needle washouts of fine-needle aspiration biopsies of thyroid nodule from different patients.

Identification and Functional Characterization of a Novel Mutation in the NKX2-1 Gene: Comparison with the Data in the Literature

BACKGROUND NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). METHODS The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. RESULTS The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. CONCLUSIONS We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome.

Sunshine vitamin and thyroid.

Vitamin D exerts its canonical roles on the musculoskeletal system and in the calcium/phosphorus homeostasis. In the last years, increasing evidences suggested several extra-skeletal actions of this hormone, indicating that vitamin D may produce effects in almost all the body tissues. These are mediated by the presence of vitamin D receptor (VDR) and thanks to the presence of the 1-α-hydroxylase, the protein that converts the 25-hydroxyvitamin (calcidiol) to the active form 1,25-dihydroxyvitamin (calcitriol). Several studies evaluated the possible role of vitamin D in the pathogenesis of thyroid diseases, and this review will focus on the available data of the literature evaluating the association between vitamin D and thyroid function, vitamin D and autoimmune thyroid diseases, including Hashimoto’s thyroiditis, Graves’ disease and post-partum thyroiditis, and vitamin D and thyroid cancer.

Selenium supplementation modulates apoptotic processes in thyroid follicular cells

Selenium (Se) is an essential micronutrient modulating several physiopathological processes in the human body. The aim of the study is to characterize the molecular effects determined by Se-supplementation in thyroid follicular cells, using as model the well-differentiated rat thyroid follicular cell line FRTL5. Experiments have been performed to evaluate the effects of Se on cell growth, mortality and proliferation and on modulation of pro- and antiapoptotic pathways. The results indicate that Se-supplementation improves FRTL5 growth rate. Furthermore, Se reduces the proportion of cell death and modulates both proapoptotic (p53 and Bim) and antiapoptotic (NF-kB and Bcl2) mRNA levels. In addition, incubation with high doses of Na-Se might prevent the ER-stress apoptosis induced by tunicamycin, as assessed by membrane integrity maintenance, reduction in caspase 3/7 activities, and reduction in Casp-3 and PARP cleavage. Taken together, these results provide molecular evidences indicating the role of Se supplementation on cell death and apoptosis modulation in thyroid follicular cells. These observations may be useful to understand the effects of this micronutrient on the physiopathology of the thyroid gland.

High-fat diet unveils an enhancer element at the Ped/Pea-15 gene responsible for epigenetic memory in skeletal muscle

BACKGROUND The impact of nutrition on the evolution towards type 2 diabetes has recently received increasing attention because of the effect on chromatin structure and gene expression. PURPOSE Evaluate the effect of high-fat diet on chromatin remodelling and expression of Ped/Pea-15, a gene commonly overexpressed in individuals at risk of type 2 diabetes. METHODS We used mouse and cell models to investigate Ped/Pea-15 transcriptional regulation by high-fat diet and glucose, respectively. Chromatin structure and histone modification marks were assessed by Micrococcal Nuclease Protection and Chromatin Immunoprecipitation assays. RESULTS Sixteen-week exposure of C57BL/6J mice to a high-fat diet impaired glucose tolerance and enhanced Ped/Pea-15 expression in their skeletal muscle tissue. This effect was associated with increased chromatin accessibility at specific regulatory sites at the Ped/Pea-15 gene. In particular, the region at -1900 to -1300 bp from Ped/Pea-15 transcription start site was revealed to feature enhancer activity as demonstrated by its function in the luciferase assay, increased p300 recruitment and H3K4me1 and H3K27Ac levels, all marks of functionally active enhancers. Returning mice to a standard chow diet was accompanied by rapid loss of acetylation of K27 on histone H3 and p300 recruitment at Ped/Pea-15. In contrast, the increased H3K4me1, which accompanied the high-fat diet exposure, remained stable. Incubation of muscle cells in culture medium supplemented with 25 mM glucose (HG) increased Ped/Pea-15 mRNA expression and H3K4me1 at the enhancer region. These effects became measurable upon 72 h of exposure to the HG medium and were not rescued upon returning the cells to the 5 mM glucose-containing medium. Interestingly, after 25 mM and sequential 5 mM glucose treatments, re-exposure of the same cells to HG medium further enhanced Ped/Pea-15 expression and increased H3K4me1 above the levels induced by the initial HG challenge already upon 24 h. CONCLUSION Transient exposure to HFD or HG unveiled the presence of an enhancer element at the Ped/Pea-15 gene. Epigenetic changes imposed at this region by diets, which impair glucose tolerance generate metabolic memory of the nutritional injury and leave Ped/Pea-15 induction in a poised state.

High-resolution melting analysis (HRM) for mutational screening of Dnajc17 gene in patients affected by thyroid dysgenesis.

BackgroundCongenital hypothyroidism is a frequent disease occurring with an incidence of about 1/1500 newborns/year. In about 75% of the cases, CH is caused by alterations in thyroid morphogenesis, defined “thyroid dysgenesis” (TD). TD is generally a sporadic disease but in about 5% of the cases a genetic origin has been demonstrated. Previous studies indicate that Dnajc17 as a candidate modifier gene for hypothyroidism, since it is expressed in the thyroid bud, interacts with NKX2.1 and PAX8 and it has been associated to the hypothyroid phenotype in mice carrying a single Nkx2.1 and Pax8 genes (double heterozygous knock-out).PurposeThe work evaluates the possible involvement of DNAJC17 in the pathogenesis of TD.MethodsHigh-resolution DNA melting analysis (HRM) and direct sequencing have been used to screen for mutations in the DNAJC17 coding sequence in 89 patients with TD.ResultsTwo mutations have been identified in the coding sequence of DNAJC17 gene, one in exon 5 (c.350A>C; rs79709714) and one in exon 9 (c.610G>C; rs117485355). The last one is a rare variant, while the rs79709714 is a polymorphism. Both are present in databases and the frequency of the alleles is not different between TD patients and controls.ConclusionsDNAJC17 mutations are not frequently present in patients with TD.

Nutritional and Environmental Factors in Thyroid Carcinogenesis

Several epidemiological studies suggest an increased incidence of thyroid carcinoma (TC) in recent years, especially for the papillary histotype (PTC), suggesting that specific carcinogens might promote molecular abnormalities that are typical of PTC. The increased incidence is probably attributed to more intensive and sensitive diagnostic procedures, even if recent data suggest that various toxic elements could explain the phenomenon. Ionizing radiation exposure represents the most accepted risk factor for differentiated thyroid cancer that includes both the follicular and papillary histotypes. In this review, we examined the other environmental carcinogens that play a role in TC, such as eating habits, living in volcanic areas, and xenobiotic elements. Among eating habits, iodine intake represents one of the more discussed elements, because its deficiency is associated with follicular thyroid carcinomas (FTCs), while its progressive increment seems to be responsible for PTC. The gas, ash, and lava emissions of volcanoes are composed of various toxic compounds that pollute ground water, vegetables, and animals, contaminating humans via the food chain. Finally, the risk of developing PTC has also been associated with exposure of the population to xenobiotics in the environment or in the home. Their carcinogenic effects are probably caused by their accumulation, but additional studies are necessary to better understand the mechanisms of action.

High Sensitive Long Period Fiber Grating Biosensor for Cancer Biomarker Detection

We report an innovative fiber optic biosensor based on Long Period Gratings (LPGs) working in reflection configuration for real time monitoring of human Thyroglobulin (Tg), a protein marker of differentiated thyroid cancer. A standard LPG is first transformed in a practical probe working in reflection mode, and then it is coated with a single layer of atactic polystyrene (aPS) in order to increase its surrounding refractive index sensitivity and to provide, at the same time, the desired interfacial properties for a stable anti-Tg antibody. The functionalized reflection-type LPG biosensor clearly demonstrates the effectiveness and sensitivity of the developed biosensing platform, allowing the real time and label-free detection of Tg in the needle washouts of fine-needle aspiration biopsies, at concentrations useful for pre- and post-operative assessment of the biomarker levels. Analyte recognition and capture were confirmed with a parallel on fiber ELISA-like assay using, in pilot tests, the biotinylated protein and HRP-labeled streptavidin for its detection. Dose-dependent experiments showed that the detection is linearly dependent on concentration within the range between 0 and 4 ng/mL, while antibody saturation occurs for higher protein levels.