Estrogen receptor signaling and targets: Bones, breasts and brain (Review).

Estrogens are involved in a number of physiological functions, including in the development of the brain, growth, reproduction and metabolism. The biological actions of estrogens are achieved by binding to estrogen receptors (ERs) in numerous types of tissues. ERα and ERß belong to the nuclear receptor superfamily and the G­protein coupled ER1 (GPER1) is a membrane receptor. The primary biologically active estrogen, 17ß­estradiol demonstrates a high affinity for ERs. Mechanistically, estrogens bind to the ERs in the nucleus, and the complex then dimerize and bind to estrogen response elements (EREs) located in the promoter regions of the target genes. This is referred to as the genomic mechanism of ERs' function. Furthermore, ERs can also act through kinases and other molecular interactions leading to specific gene expression and functions, referred to as the non­genomic mechanism. While ERα and ERß exert their functions via both genomic and non­genomic pathways, GPER1 exerts its function primarily via the non­genomic pathways. Any aberrations in ER signaling can lead to one of a number of diseases such as disorders of growth and puberty, fertility and reproduction abnormalities, cancer, metabolic diseases or osteoporosis. In the present review, a focus is placed on three target tissues of estrogens, namely the bones, the breasts and the brain, as paradigms of the multiple facets of the ERs. The increasing prevalence of breast cancer, particularly hormone receptor­positive breast cancer, is a challenge for the development of novel antihormonal therapies other than tamoxifen and aromatase inhibitors, to minimize toxicity from the long treatment regimens in patients with breast cancer. A complete understanding of the mechanism of action of ERs in bones may highlight options for novel targeted treatments for osteoporosis. Likewise, the aging of the brain and related diseases, such as dementia and depression, are associated with a lack of estrogen, particularly in women following menopause. Furthermore, gender dysphoria, a discordance between experienced gender and biological sex, is commonly hypothesized to emerge due to discrepancies in cerebral and genital sexual differentiation. The exact role of ERs in gender dysphoria requires further research.

Obesogens in Adolescence: Challenging Aspects and Prevention Strategies.

Childhood obesity has become a global epidemic, with significant increases in prevalence over recent decades. While excessive calorie consumption and physical inactivity are known factors, emerging research highlights the role of endocrine-disrupting chemicals (EDCs), particularly obesogens, in obesity's pathogenesis. This review explores the historical context of the environmental obesogens hypothesis, their sources, mechanism of action, impact on prenatal and postnatal development, and epigenetics. Additionally, it discusses the long-term consequences of childhood obesity and proposes prevention strategies that will mitigate negative health effects. Obesogens were found to disrupt hormonal balance and metabolic processes through various mechanisms such as altering gene expression, hormonal interference, and inflammation. Especially significant was exposure during critical windows of development, which correlates with an increased risk of obesity in childhood or adolescence. Long-term effects of childhood obesity include chronic health conditions and psychosocial issues. A comprehensive approach is necessary to address childhood obesity encompassing genetic, environmental, and lifestyle factors. Prevention strategies should focus on reducing obesogen exposure, promoting healthy lifestyles, and implementing regulatory policies. Future research should investigate obesogens-diet interactions, microbiome impacts, and combined obesogens effects. Long-term human studies are also crucial for validating findings from animal models and allowing for informed decision-making to combat the obesity pandemic.

Genetic diagnosis of endocrine disorders in Cyprus through the Cyprus Institute of Neurology and Genetics: an ENDO-ERN Reference Center.

The report covers the current and past activities of the department Molecular Genetics-Function and Therapy (MGFT) at the Cyprus Institute of Neurology and Genetics (CING), an affiliated Reference Center for the European Reference Network on Rare Endocrine Conditions (Endo-ERN).The presented data is the outcome of > 15 years long standing collaboration between MGFT and endocrine specialists from the local government hospitals and the private sector. Up-to-date > 2000 genetic tests have been performed for the diagnosis of inherited rare endocrine disorders. The major clinical entities included Congenital Adrenal Hyperplasia (CAH) due to pathogenic variants in CYP21A2 gene and Multiple Endocrine Neoplasia (MEN) type 2 due to pathogenic variants in the RET proto-oncogene. Other rare and novel pathogenic variants in ANOS1, WDR11, FGFR1, RNF216, and CHD7 genes were also found in patients with Congenital Hypogonadotropic Hypogonadism. Interestingly, a few patients with Disorders of Sexual Differentiation (DSD) shared rare pathogenic variants in the SRD5A2, HSD17B3 and HSD3B2 while patients with Glucose and Insulin Homeostasis carried theirs in GCK and HNF1A genes. Lastly, MGFT over the last few years has established an esteemed diagnostic and research program on premature puberty with emphasis on the implication of MKRN3 gene on the onset of the disease and the identification of other prognosis biomarkers.As an Endo-ERN member MGFT department belongs to this large European network and holds the same humanistic ideals which aim toward the improvements of health care for patients with rare endocrine conditions in respect to improved and faster diagnosis.

The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes.

Objective: The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). Methods: 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Results: Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). Conclusion: The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.

Reduced serum concentrations of biomarkers reflecting Leydig and Sertoli cell function in male patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a recessive condition that affects the adrenal glands. Despite life-long replacement therapy with glucocorticoids and mineralocorticoids, adult patients with CAH often experience impaired gonadal function. In pubertal boys and in men with CAH, circulating testosterone is produced by the adrenal glands as well as the testicular, steroidogenic cells. In this European two-center study, we evaluated the function of Leydig and Sertoli cells in 61 boys and men with CAH, primarily due to 21-hydroxylase deficiency. Despite conventional hormone replacement therapy, our results indicated a significant reduction in serum concentrations of both Leydig cell-derived hormones (i.e. insulin-like factor 3 (INSL3) and testosterone) and Sertoli cell-derived hormones (i.e. inhibin B and anti-Müllerian hormone) in adult males with CAH. Serum concentrations of INSL3 were particularly reduced in those with testicular adrenal rest tumors. To our knowledge, this is the first study to evaluate circulating INSL3 as a candidate biomarker to monitor Leydig cell function in patients with CAH.

Stress, Thyroid Dysregulation, and Thyroid Cancer in Children and Adolescents: Proposed Impending Mechanisms.

Stress is a potential catalyst for thyroid dysregulation through cross-communication of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid (HPT) axes. Stress and stressors exposure motivates molecular mechanisms affecting compound feedback loops of the HPT axis. While there is evidence of connection between stress and thyroid dysregulation, the question whether this connection is implicated in the development of thyroid cancer (TC) remains unanswered. In view of the rising incidence of TC in both adults and children alongside the increasing stress in our modern society, there is a need to understand possible interrelations between stress, thyroid dysregulation, and TC. Prolonged glucocorticoid secretion due to stress interferes with immune system response by altering the cytokines, inducing low-grade chronic inflammation, and suppressing function of immune-protective cells. Chronic inflammation is a risk factor linked to TC. The role of autoimmunity has been a matter of controversy. However, there is epidemiological connection between autoimmune thyroid disease (AITD) and TC; patients with AITD show increased incidence in papillary thyroid carcinoma (PTC), and those with TC show a high prevalence of intrathyroidal lymphocyte infiltration and thyroid autoantibodies. Timing and duration-dependent exposure to specific endocrine disrupting chemicals (EDCs) has an impact on thyroid development, function, and proliferation, leading to thyroid disease and potentially cancer. Thyroid hormone imbalance, chronic inflammation, and EDCs are potential risk factors for oxidative stress. Oxygen free radicals are capable of causing DNA damage via stimulation of the mitogen-activating protein kinase or phosphatidylinositol-3-kinase and/or nuclear factor kB pathways, resulting in TC-associated gene mutations such as RET/PTC, AKAP9-BRAF, NTRK1, RAASF, PIK3CA, and PTEN. Stressful events during the critical periods of prenatal and early life can influence neuroendocrine regulation and induce epigenetic changes. Aberrant methylation of tumor suppressor genes such as P16INK4A, RASSF, and PTEN is associated with PTC; histone H3 acetylation is shown to be higher in TC, and thyroid-specific noncoding RNAs are downregulated in PTC. This review focuses on the above proposed mechanisms that potentially lead to thyroid tumorigenesis with the aim to help in the development of novel prognostic and therapeutic strategies for TC.

Methylation status of hypothalamic Mkrn3 promoter across puberty.

Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting region associated with Prader-Willi syndrome. Imprinted MKRN3 is expressed in hypothalamic regions essential for the onset of puberty and mutations in the gene have been found in patients with central precocious puberty. The pubertal process is largely controlled by epigenetic mechanisms that include, among other things, DNA methylation at CpG dinucleotides of puberty-related genes. In the present study, we investigated the methylation status of the Mkrn3 promoter in the hypothalamus of the female mouse before, during and after puberty. Initially, we mapped the 32 CpG dinucleotides in the promoter, the 5'UTR and the first 50 nucleotides of the coding region of the Mkrn3 gene. Moreover, we identified a short CpG island region (CpG islet) located within the promoter. Methylation analysis using bisulfite sequencing revealed that CpG dinucleotides were methylated regardless of developmental stage, with the lowest levels of methylation being found within the CpG islet region. In addition, the CpG islet region showed significantly lower methylation levels at the pre-pubertal stage when compared with the pubertal or post-pubertal stage. Finally, in silico analysis of transcription factor binding sites on the Mkrn3 CpG islet identified the recruitment of 29 transcriptional regulators of which 14 were transcriptional repressors. Our findings demonstrate the characterization and differential methylation of the CpG dinucleotides located in the Mkrn3 promoter that could influence the transcriptional activity in pre-pubertal compared to pubertal or post-pubertal period. Further studies are needed to clarify the possible mechanisms and effects of differential methylation of the Mkrn3 promoter.

Molecular modelling of novel ADCY3 variant predicts a molecular target for tackling obesity.

Severe early­onset obesity is mainly attributed to single gene variations of the hypothalamic leptin­melanocortin system, which is critical for controlling the balance between appetite and energy expenditure. Adenylate cyclase 3 (ADCY3), a transmembrane enzyme localized in primary neuronal cilia, is a key genetic candidate, which appears to have an essential role in regulating body weight. The present study aimed to identify ADCY3 genetic variants in severely obese young patients of Greek­Cypriot origin by genomic sequencing. Apart from previously reported variants, the novel and probably pathogenic variant c.349T>A, causing a p.Leu117Met substitution within one of the two pseudo­symmetric halves of the transmembrane part of the protein, was reported. Molecular modelling analysis used to delineate bonding interactions within the mutated protein structure strongly suggested a change in interactive forces and energy levels affecting the pseudo­twofold symmetry of the transmembrane domain of the protein and probably its catalytic function. These results support the involvement of ADCY3 in the pathology of the disease and point towards the requirement of defining protein function and evaluating the clinical significance of the detected variants.

Pathogenic and Low-Frequency Variants in Children With Central Precocious Puberty.

Background: Central precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP. Methods: Fifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed. Results: Three previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP. Conclusion: The results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP.

Microcalcifications without a thyroid nodule as the sole sign of papillary thyroid carcinoma.

SUMMARY: Our objective is to demonstrate the importance of considering microcalcifications even without evidence of nodules as a potential sign of malignancy. Current guidelines, such as those of the British Thyroid Association, acknowledge the clinical significance of microcalcifications only when found within nodules. In this case, they are considered a suspicious feature, classifying the nodules as U5 (i.e. high risk) where fine-needle aspiration biopsy (FNAB) is warranted, following the high likelihood of cancer in these nodules. In addition, there is a dearth of evidence of ultrasound scan (USS) detection of microcalcifications in the thyroid gland outside of nodules, along with their associated clinical implications. Yet, this clinical manifestation is not so infrequent considering that we do encounter patients in the clinic showing these findings upon ultrasound examination. Three patients who presented to our clinic with thyroid-related symptoms were shown to have areas of microcalcifications without a nodule upon sonographic evaluation of their thyroid gland. These incidentally detected hyperechoic foci were later confirmed to correspond to areas of papillary thyroid carcinoma (PTC) on histopathological examination of resected tissue following thyroidectomy. Four more cases were identified with sonographic evidence of microcalcifications without nodules and given their clinical and other sonographic characteristics were managed with active surveillance instead. LEARNING POINTS: Echogenic foci known as microcalcifications may be visible without apparent association to nodular structures. Microcalcifications without nodules may not be an infrequent finding. Microcalcifications are frequently indicative of malignancy within the thyroid gland even without a clearly delineated nodule. Empirically, the usual guidelines for the management of thyroid nodules can be applied to the management of microcalcifications not confined to a nodule, but such a finding per se should be classified as a 'high-risk' sign.