The p.Cys1281Tyr variant in the hinge module/flap region of thyroglobulin causes intracellular transport disorder and congenital hypothyroidism.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) variants causes very low serum TG levels with normal or enlarged thyroid glands, depending on the severity of the defect, and with autosomal recessive inheritance. The purpose of this study was to functionally characterize p.Cys1281Tyr variant in the TG gene in order to increase our knowledge of the molecular mechanisms associated with CH. In order to find evidence that support the hypothesis that the p.Cys1281Tyr variant would affect the TG folding were performed amino acid prediction, 3D modeling and transient expression analysis in HEK293T cells. 18 of the 21″in silico" algorithms predict a deleterious effect of the p.Cys1281Tyr variant. The full-length 3D model p.Cys1281Tyr TG showed disulfide bond cleavage between the cysteines at positions 1249 and 1281 and rearrangement of the TG structure, while transient expression analysis indicated that p.Cys1281Tyr causes retention of the protein inside the cell. Consequently, these results show that this pathogenic variant makes it impossible for TG to fulfill its function in the biosynthesis process of thyroid hormones, causing CH. In conclusion, our results confirm the pathophysiological importance of misfolding of TG as a consequence of p.Cys1281Tyr variant located in the hinge module/flap region of TG.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review.

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

Mutational screening of the TPO and DUOX2 genes in Argentinian children with congenital hypothyroidism due to thyroid dyshormonogenesis.

PURPOSE: Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the preventable causes of both cognitive and motor deficits. We present a genetic and bioinformatics investigation of rational clinical design in 17 Argentine patients suspected of CH due to thyroid dyshormonogenesis (TDH). METHODS: Next-Generation Sequencing approach was used to identify variants in Thyroid Peroxidase (TPO) and Dual Oxidase 2 (DUOX2) genes. A custom panel targeting 7 genes associated with TDH [(TPO), Iodothyrosine Deiodinase I (IYD), Solute Carrier Family 26 Member 4 (SLC26A4), Thyroglobulin (TG), DUOX2, Dual Oxidase Maturation Factor 2 (DUOXA2), Solute Carrier Family 5 Member 5 (SLC5A5)] and 4 associated with thyroid dysembryogenesis [PAX8, FOXE1, NKX2-1, Thyroid Stimulating Hormone Receptor (TSHR)] has been designed. Additionally, bioinformatic analysis and structural modeling were carried out to predict the disease-causing potential variants. RESULTS: Four novel variants have been identified, two in TPO: c.2749-2 A > C and c.2752_2753delAG, [p.Ser918Cysfs*62] and two variants in DUOX2 gene: c.425 C > G [p.Pro142Arg] and c.2695delC [p.Gln899Serfs*21]. Eighteen identified TPO, DUOX2 and IYD variants were previously described. We identified potentially pahogenic biallelic variants in TPO and DUOX2 in 7 and 2 patients, respectively. We also detected a potentially pathogenic monoallelic variant in TPO and DUOX2 in 7 and 1 patients respectively. CONCLUSIONS: 22 variants have been identified associated with TDH. All described novel mutations occur in domains important for protein structure and function, predicting the TDH phenotype.

Curating the gnomAD database: Report of novel variants in the thyrogobulin gene using in silico bioinformatics algorithms.

Thyroglobulin (TG) is a large glycosylated protein of 2767 amino acids, secreted by the thyrocytes into the follicular lumen. It plays an essential role in the process of thyroid hormone synthesis. TG gene variants lead to permanent congenital hypothyroidism. In the present work, we report a detailed population and bioinformatic prediction analyses of the TG variants indexed in the Genome Aggregation Database (gnomAD). The results showed a clear predominance of nonsense variants in the European (Finnish), European (Non-Finnish) and Ashkenazi Jewish ethnic groups, whereas the splice site variants predominate in South Asian and African/African-American populations. In total, 282 novel TG variants were described (47 missense involving the wild-type cysteine residues, 177 missense located in the ChEL domain and 58 splice site variants) which were not reported in the literature and that would have deleterious effects in prediction programs. In the gnomAD population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:320. In conclusion, we provide an updated and curated reference source for the diagnosis of thyroid disease, mainly to congenital hypothyroidism due to TG deficiency. The identification and characterization of TG variants is undoubtedly a valuable approach to study the TG structure/function relations and an important tool for clinical diagnosis and genetic counseling.

Molecular analysis of thyroglobulin mutations found in patients with goiter and hypothyroidism.

Thyroid dyshormonogenesis due to thyroglobulin (TG) gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. Up to now, one hundred seventeen deleterious mutations in the TG gene have been identified and characterized. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report eight patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and image evaluation. Sequencing of DNA, genotyping, as well as bioinformatics analysis were performed. Molecular analyses revealed three novel inactivating TG mutations: c.5560G>T [p.E1835*], c.7084G>C [p.A2343P] and c.7093T>C [p.W2346R], and four previously reported mutations: c.378C>A [p.Y107*], c.886C>T [p.R277*], c.1351C>T [p.R432*] and c.7007G>A [p.R2317Q]. Two patients carried homozygous mutations (p.R277*/p.R277*, p.W2346R/p.W2346R), four were compound heterozygous mutations (p.Y107*/p.R277* (two unrelated patients), p.R432*/p.A2343P, p.Y107*/p.R2317Q) and two siblings from another family had a single p.E1835* mutated allele. Additionally, we include the analysis of 48 patients from 31 unrelated families with TG mutations identified in our present and previous studies. Our observation shows that mutations in both TG alleles were found in 27 families (9 as homozygote and 18 as heterozygote compound), whereas in the remaining four families only one mutated allele was detected. The majority of the detected mutations occur in exons 4, 7, 38 and 40. 28 different mutations were identified, 33 of the 96 TG alleles encoded the change p.R277*. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of the predicted TG misfolding and therefore thyroid hormone formation as a consequence of truncated TG proteins and/or missense mutations located within its ACHE-like domain.

Glutamate neurotransmission is affected in prenatally stressed offspring.

Previous studies from our laboratory have shown that male adult offspring of stressed mothers exhibited higher levels of ionotropic and metabotropic glutamate receptors than control rats. These offspring also showed long-lasting astroglial hypertrophy and a reduced dendritic arborization with synaptic loss. Since metabolism of glutamate is dependent on interactions between neurons and surrounding astroglia, our results suggest that glutamate neurotransmitter pathways might be impaired in the brain of prenatally stressed rats. To study the effect of prenatal stress on the metabolism and neurotransmitter function of glutamate, pregnant rats were subjected to restrain stress during the last week of gestation. Brains of the adult offspring were used to assess glutamate metabolism, uptake and release as well as expression of glutamate receptors and transporters. While glutamate metabolism was not affected it was found that prenatal stress (PS) changed the expression of the transporters, thus, producing a higher level of vesicular vGluT-1 in the frontal cortex (FCx) and elevated levels of GLT1 protein and messenger RNA in the hippocampus (HPC) of adult male PS offspring. We also observed increased uptake capacity for glutamate in the FCx of PS male offspring while no such changes were observed in the HPC. The results show that changes mediated by PS on the adult glutamatergic system are brain region specific. Overall, PS produces long-term changes in the glutamatergic system modulating the expression of glutamate transporters and altering synaptic transmission of the adult brain.

Prenatal restraint stress decreases the expression of alpha-7 nicotinic receptor in the brain of adult rat offspring.

Prenatal stress (PS) strongly impacts fetal brain development and function in adulthood. In normal aging and Alzheimer's disease, there is hypothalamic-pituitary-adrenal axis dysfunction and loss of cholinergic neurons and neuronal nicotinic acetylcholine receptors (nAChRs). This study investigated whether prenatal restraint stress affects nAChR expression in the brain of adult offspring. For PS, pregnant dams were placed in a plastic restrainer for 45 min, three times daily during the last week of pregnancy; controls were undisturbed. Male offspring were analyzed at postnatal day (PND) 60 (n = 4 rats per group). Western blot (WB) and fluorescence microscopy showed that PS decreased α7-AChR subunit expression (∼50%) in the frontal cortex in the adult offspring. PS decreased significantly the number of α7-AChR-expressing cells in the medial prefrontal cortex (by ∼25%) and in the sensory-motor cortex (by ∼20%) without affecting the total cell number in those areas. No alterations were found in the hippocampus by quantitative polymerase chain reaction (qPCR), or WB analysis, but a detailed fluorescence microscopy analysis showed that PS affected α7-AChR mainly in the CA3 and dentate gyrus subfields: PS decreased α7-AChR subunit expression by ∼25 and ∼30%, respectively. Importantly, PS decreased the number of α7-AChR-expressing cells and the total cell number (by ∼15 and 20%, respectively) in the dentate gyrus. PS differently affected α4-AChR: PS impaired its mRNA expression in the frontal cortex (by ∼50%), without affecting protein levels. These results demonstrate that disturbances during gestation produce long-term alterations in the expression pattern of α7-AChR in rat brain.

Intrastriatal 6-OHDA lesion differentially affects dopaminergic neurons in the ventral tegmental area of prenatally stressed rats.

Exposure to a variety of stressful events during the last week of pregnancy in rats interferes with the correct progeny development, which in turn leads to delays in motor development, impaired adaptation to stressful conditions, altered sexual behaviour, learning deficits, neuronal development and brain morphology. Many of these alterations have been attributed to changes in dopamine (DA) neurotransmission and occur primarily in the mesolimbic system. We found that prenatally stressed offspring showed higher levels of cells expressing tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and that these cells were more susceptible to a neurochemical insult with 6-hydroxy-DA (6-OHDA) in adulthood. Moreover, prenatally stressed rats presented differences in terms of the number and asymmetry of neuronal nitric oxide synthase-expressing cells in the VTA and nucleus accumbens, respectively. Similar to the results described for TH-expressing cells, the nitrergic systems were differentially regulated after 6-OHDA lesion in control and prenatally stressed rats. These results indicated that prenatal stress affects the dopaminergic and nitrergic systems in the mesolimbic pathway. In addition, we propose that the mesolimbic areas are more susceptible than the motor areas to a neurochemical insult during adult life.

Prenatal stress changes the glycoprotein GPM6A gene expression and induces epigenetic changes in rat offspring brain.

Prenatal stress (PS) exerts strong impact on fetal brain development and on adult offspring brain functions. Previous work demonstrated that chronic stress alters the mRNA expression of GPM6A, a neuronal glycoprotein involved in filopodium extension. In this work, we analyzed the effect of PS on gpm6a expression and the epigenetic mechanisms involved. Pregnant Wistar rats received restraint stress during the last week of gestation. Male offspring were sacrificed on postnatal days 28 and 60. Hippocampus and prefrontal cortex samples were analyzed for gene expression (qPCR for mRNAs and microRNAs), methylation status (bisulfite conversion) and protein levels. Hippocampal neurons in culture were used to analyze microRNA overexpression effects. Prenatal stress induced changes in gpm6a levels in both tissues and at both ages analyzed, indicating a persistent effect. Two CpG islands in the gpm6a gene were identified. Variations in the methylation pattern at three specific CpGs were found in hippocampus, but not in PFC samples from PS offspring. microRNAs predicted to target gpm6a were identified in silico. qPCR measurements showed that PS modified the expression of several microRNAs in both tissues, being microRNA-133b the most significantly altered. Further studies overexpressing this microRNA in neuronal cultures showed a reduction in gmp6a mRNA and protein level. Moreover filopodium density was also reduced, suggesting that GPM6A function was affected. Gestational stress affected gpm6a gene expression in offspring likely through changes in methylation status and in posttranscriptional regulation by microRNAs. Thus, our findings propose gpm6a as a novel target for epigenetic regulation during prenatal stress.

Age-dependent effects of prenatal stress on the corticolimbic dopaminergic system development in the rat male offspring.

We have previously demonstrated that prenatal stress (PS) exerts an impairment of midbrain dopaminergic (DA) system metabolism especially after puberty, suggesting a particular sensitivity of DA development to variations in gonadal hormonal peaks. Furthermore we demonstrated that PS alters the long term androgens profile of the rat male offspring from prepubertal to adult stages. In this work we evaluated the sexual hormones activational effects on the DA system by analysing PS effects on the dopaminergic D2-like (D2R) and on the gonadal hormones receptor levels on cortical and hippocampal areas of prepubertal and adult male offspring. We further evaluated the dendritic arborization in the same areas by quantifying MAP2 immunoexpresion. Our results show that PS affected oestrogen receptor alpha (ERα) expression: mRNA er1s and ERα protein levels were decreased on prefrontal cortex and hippocampus of adult offspring. Moreover, PS reduced D2R protein levels in hippocampus of prepubertal rats. Morphological studies revealed that prepubertal PS rats presented decreased MAP2 immunoexpression in both areas suggesting that PS reduces the number of dendritic arborizations. Our findings suggest that PS exerts long-term effects on the DA system by altering the normal connectivity in the areas, and by modulating the expression of D2R and ERα in an age-related pattern. Since the developing forebrain DA system was shown to be influenced by androgen exposure, and PS was shown to disrupt perinatal testosterone surges, our results suggest that prenatal insults might be affecting the organizational role of androgens and differentially modulating their activational role on the DA development.

Prenatal maternal restraint stress exposure alters the reproductive hormone profile and testis development of the rat male offspring.

Several studies have demonstrated that the presence of stressors during pregnancy induces adverse effects on the neuroendocrine system of the offspring later in life. In the present work, we investigated the effects of early programming on the male reproductive system, employing a prenatal stress (PS) paradigm. This study found that when pregnant dams were placed in a plastic restrainer three times a day during the last week of pregnancy, the offspring showed reduced anogenital distance and delayed testicular descent. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels were decreased at postnatal day (PND) 28 and testosterone was decreased at PND 75. Increased testosterone plus dihydrotestosterone (T + DHT) concentrations correlated with increased testicular 5α Reductase-1 (5αR-1) mRNA expression at PND 28. Moreover, PS accelerated spermatogenesis at PND 35 and 60, and increased mean seminiferous tubule diameter in pubertal offspring and reduced Leydig cell number was observed at PND 35 and 60. PS offspring had increased androgen receptor (AR) mRNA level at PND 28, and at PND 35 had increased the numbers of Sertoli cells immunopositive for AR. Overall, the results confirm that stress during gestation can induce long-term effects on the male offspring reproductive system. Of particular interest is the pre-pubertal imbalance of circulating hormones that probably trigger accelerated testicular development, followed by an increase in total androgens and a decrease in testosterone concentration during adulthood. Exposure to an unfavourable intrauterine environment might prepare for harsh external conditions by triggering early puberty, increasing reproductive potential.

Gestational restraint stress and the developing dopaminergic system: an overview.

Prenatal stress exerts a strong impact on fetal brain development in rats impairing adaptation to stressful conditions, subsequent vulnerability to anxiety, altered sexual function, and enhanced propensity to self-administer drugs. Most of these alterations have been attributed to changes in the neurotransmitter dopamine (DA). In humans; dysfunction of dopaminergic system is associated with development of several neurological disorders, such as Parkinson disease, schizophrenia, attention-deficit hyperactivity disorder, and depression. Evidences provided by animal research, as well as retrospective studies in humans, pointed out that exposure to adverse events in early life can alter adult behaviors and neurochemical indicators of midbrain DA activity, suggesting that the development of the DA system is sensitive to disruption by exposure to early stressors. The purpose of this article is to provide a general overview of published studies and our own study related to the effect of prenatal insults on the development of DA metabolism and biology, focusing mainly in articles involving prenatal-restraint stress protocols in rats. We will also attempt to make a correlation between theses alterations and DA-related pathological processes in humans.