ResearchPad - genetics-and-development-(including-gene-regulation) https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[MON-721 Crude Protein of Pyropia Yezoensis Protects Against Tumor Necrosis Factor-á-Induced Myotube Atrophy by Regulating the Mitogen-Activated Protein Kinase and Nuclear Factor-Kappab Signaling Pathways in C2C12 Myotubes]]> https://www.researchpad.co/article/elastic_article_8812 Proinflammatory cytokines induce ubiquitin-proteasome-dependent proteolysis by activating intracellular factors in skeletal muscle, leading to muscle atrophy. Therefore, we investigated the protective effect of Pyropia yezoensis crude protein (PYCP) on tumor necrosis factor (TNF)-α-induced muscle atrophy in vitro. Mouse skeletal muscle C2C12 myotubes were treated for 48 h with TNF-α (20 ng/mL) in the presence or absence of PYCP (25, 50, and 100 μg/mL). PYCP at concentrations up to 100 μg/mL did not affect cell viability. Exposure to TNF-α for 48 h significantly decreased the diameter of myotubes, which was increased by treatment with 25, 50, and 100 μg/mL PYCP. PYCP inhibited TNF-α-induced intracellular reactive oxygen species accumulation in C2C12 myotubes. In addition, PYCP significantly reduced the levels of phosphorylated p38 and JNK. Moreover, by inhibiting the degradation of inhibitor of kappaB-α, PYCP significantly suppressed the TNF-α-induced increased transcriptional activity and nuclear translocation of nuclear factor-kappaB (NF-κB). Furthermore, PYCP inhibited E3-ubiquitin ligases in TNF-α-treated C2C12 myotubes. In conclusion, PYCP ameliorated TNF-α-induced muscle atrophy by inhibiting the mitogen-activated protein kinase-mediated NF-κB pathway, indicating that it has therapeutic potential for related disorders.

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<![CDATA[SAT-713 Novel Estrogenic Gene Regulation Induced by OTC Medications Containing Paraben Preservatives Is Dependent on Concentration that Varies Between Products and Batches]]> https://www.researchpad.co/article/elastic_article_8792 Methylparaben, ethylparaben, and propylparaben are widely used as preservatives in food products, cosmetics, and pharmaceuticals. Parabens have been shown to be weak estrogens and this laboratory has described that extracts of some over the counter (OTC) medications with paraben preservatives can induce estrogen activity in tissue culture-based bioassays. At the same time, this laboratory determined that extracts from OTC medications containing the laxative bisacodyl induce estrogen activity regardless of parabens present and that bisacodyl is estrogenic. The current report describes the use of paraben standards and LC-MS analysis to determine paraben concentrations in extracts from OTC medications (Calcium Carbonate, Bisacodyl, Ibuprofen, Diphenhydramine, and Benzoyl Peroxide) used in previous studies. Also described is the application of the Qiagen RT2 Profiler PCR Array for Human Estrogen Receptor Signalling to determine gene induction profiles in MCF-7 cells treated with methyl, ethyl or proplyparaben, or each of the five pairs of OTC medication extracts (with or without parabens) relative to estradiol treatment. LC-MS analysis of extracts confirmed that five of six OTC medications labeled as paraben-free contain no detectable parabens, while one “paraben free” extract included measurable levels of parabens. At the same time, all of the extracts of OTC medications with paraben ingredients, some of which induce estrogen activity, were found to contain a wide range of paraben concentrations. A threshold range of paraben concentration in OTC medications is required to induce estrogen activity in bioassays. Analysis of paraben concentrations of extracts from different product lots of the same OTC medication identified discrepancies in the amount of paraben between batches. PCR Array profiles of the three paraben standards and the OTC medication extracts share some gene induction characteristics induced by estradiol. At the same time, methyl, ethyl and propylparabens induced unique gene array profiles that are shared by the OTC medication extracts containing parabens. The extracts of OTC medications containing bisacodyl stimulated a distinct gene induction profile that has some features of the profiles of estradiol and paraben treatment. This study highlights both the capacity for paraben preservatives in OTC medications to induce novel estrogen activity (gene induction) and the importance of determining the paraben concentration in OTC medications to determine estrogen potential. While the capacity for OTC medications containing parabens or other estrogenic substances to induce estrogen activity in individuals using the medications is unclear, consumers may want to know the potential for estrogen activity in these products.

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<![CDATA[SUN-710 Custom Panel to Diagnosis Genetic Endocrine Disorders in a Tertiary Academic Hospital]]> https://www.researchpad.co/article/elastic_article_8748 Next-generation sequencing (NGS) has been transforming the endocrine diagnostic methodology allowing the genetic testing to assume an exploratory role rather than only a confirmatory one. This is possible due to lower costs and increased yield of information. A way to further increase efficiency and sensitivity for variant detection is the use of a sequencing custom panel selecting specific genes for screening. In endocrine disorders, the complex and intricate genotype-phenotype relations and occurrence of diverse comorbidities made the diagnosis challenging. Our aim is to analyze the efficiency of a multigenic panel for molecular diagnosis of endocrine disorders in patients assisted in a tertiary academic hospital, as well as to train academic and medical faculties in the use of molecular tools. Genomic DNA from 282 patients was extracted from blood sample using standard procedures. Sanger method was previously used to screen some candidate genes in half of the patients. The custom panel was designed with 651 genes using the SureDesign tool (Agilent technologies), either associated with the phenotype (OMIM) or candidate genes that englobes developmental (DD), metabolic (MD), and adrenal (AD) disorders. Libraries were prepared with SureSelectXT Target Enrichment kit (Agilent Technologies). The enriched DNA libraries were sequenced in NextSeq 500 (Illumina) with High Output V2 kit (2 x 150 bp). The raw data was aligned to hg19 with BWA-MEM, variant calling was performed using FreeBayes and annotated with ANNOVAR. Filtering took into consideration the rarity (≤1%) of variants in population databases and those in exonic or splice site regions. Variants found were then classified according ACMG/AMP criteria. The categories of Pathogenic (P) and Likely Pathogenic (LP) were considered for molecular diagnosis, while variants of uncertain significance (VUS) were only reported. The average result of 3 runs was: 159Kmm2 of cluster density, 76.5 % of Q30 and 76.6 Gb of data were generated. The mean coverage depth of the targeted regions in panel sequencing data was 237x (SD±110x), with at least 96.3% of the sequenced bases being covered more than 20-fold. Out of the 282 patients, we identified 65 LP/P variants (23%), 22 VUS (8%) and 195 remained undiagnosed (69%). Considering the solved cases, 54 (19.1%) have DD, 6 (2.1%) have MD and 5 (1.8%) have AD. Taking into account that half of the patients had already been previously screened, the data enable new findings in known genes. The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the big picture of the molecular pathways behind each disorder. This may be particularly helpful considering the higher diagnosis of DD cases. A precise genetic etiology provides improvement in understanding the disease, guides decisions about prevention or treatment, and brings comfort to the affected families.

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<![CDATA[SAT-717 Region-Specific Effects of the Exposome on Brain Monoamine Levels in Female Rats]]> https://www.researchpad.co/article/elastic_article_8726 Prenatal programming with endocrine disrupting chemicals (EDCs), in particular the ubiquitous plasticizers bisphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP), can induce long-lasting behavioral changes in rats. Additionally, changes in estrogen are correlated with the development of mood disorders in women; however, the underlying neurobiological mechanisms are unclear. This study was conducted to determine the cumulative effects of prenatal exposure to EDCs followed by chronic estradiol treatment in adult female rats on monoamine levels in the prefrontal cortex (PFC) and hippocampus (HC). Dams were orally administered saline (control; 10 µL/kg), BPA (B; 5 µg/kg), DEHP (D; 7.5 mg/kg) or a combination of BPA+DEHP (B+D) during days 6 through 21 of pregnancy. Adult female offspring were sham-implanted or implanted with pellets that release 17β-estradiol (E2) for 90 days (20 ng/day; Innovative Research America). The offspring then underwent a battery of behavioral tests at the end of treatment. Brains collected from the offspring were sectioned and the PFC and HC were microdissected and analyzed for levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT), using High-Performance Liquid Chromatography (HPLC). Significant reductions in monoamine levels were observed in the PFC while NE and 5-HT levels were markedly reduced in the HC after prenatal exposure to D or BD. BPA’s effects on monoamines were comparatively modest. E2 exposure increased DA but decreased 5-HT levels in the PFC of control animals. Prenatal exposure to EDCs made the offspring non-responsive to E2. The marked reduction in monoamine levels could have implications for learning and memory.

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<![CDATA[SAT-724 Endocrine Disruption by Phthalate Exposure in the Pediatric Intensive Care Unit]]> https://www.researchpad.co/article/elastic_article_8707 Aim: Pediatric intensive care relies on plastic indwelling medical devices softened by phthalates. Phthalates leach into the circulation and concerns about toxicity were raised. Exceeding a certain threshold of di(2-ethylhexyl)phthalate (DEHP) exposure in the pediatric intensive care unit (PICU) has been associated with an attention deficit 4 years later (1). Moreover, DEHP and its metabolites have endocrine disrupting properties. Critically ill children reveal the non-thyroidal illness syndrome (2) and unexplained relatively low cortisol (3). Whether DEHP exposure in PICU has endocrine disruptive effects is unknown. We investigated whether DEHP exposure in the PICU, exceeding the previously identified “toxic” threshold for attention, is independently associated with thyroid- and HPA-axis alterations upon PICU discharge. Methods: In this preplanned secondary analysis of the PEPaNIC RCT (N=1440) (4), plasma DEHP metabolite concentrations (MEHP, 5OH-MEHP, 5cx-MEPP, 5oxo-MEHP) were quantified for all patients with a last PICU day sample (N=920). Minimal DEHP exposure was defined as the product of the total DEHP metabolite concentrations on the last PICU day and duration of PICU stay, with 0.551 µmol/L.days identified as “toxic” threshold (1). Serum TSH, total T4, total T3 and rT3 concentrations were quantified for patients with an available last day sample (N=913). For patients with a last day plasma sample and who did not receive corticosteroids (N=391), plasma ACTH, total cortisol, albumin and CBG concentrations were quantified and free cortisol calculated. Multivariable linear regression analyses, adjusted for baseline risk factors and for duration of PICU stay, assessed whether exceeding the previously determined threshold of toxic DEHP exposure was independently associated with the hormone levels on the last PICU day. Main results: Median total DEHP metabolite concentration was 0.101 (IQR 0.049 - 0.279) µmol/L on the last PICU day. Minimal DEHP exposure was 0.337 (IQR 0.161 - 0.880) µmol/l.days, and 328 patients (35.7%) exceeded the toxic threshold. Exceeding this threshold was independently associated with lower total T4 (P=0.002), total T3 (P=0.02) and total cortisol (P=0.001), and higher rT3 (P=0.01) concentrations on the last PICU day, but not with TSH, ACTH or free cortisol. Conclusion: Critically ill children had DHEP metabolites in plasma upon PICU discharge and more than a third were exposed to toxic levels. Toxic DEHP exposure was an independent contributor to the severity of the non-thyroidal illness phenotype and to lower cortisol upon PICU discharge. Future research should assess whether such endocrine-disruptive impact of DHEP exposure in the PICU plays a role in the long-term developmental legacy of critical illness in children. 1 Verstraete et al Intensive Care Med 2016 2 Jacobs et al Thyroid 2019 3 Jacobs et al Intensive Care Med 2019 4 Fivez et al N Engl J Med 2016

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<![CDATA[MON-711 Induction of Apolipoprotein A1 Gene Expression by the Rare Sugar Allulose]]> https://www.researchpad.co/article/elastic_article_8671 Apolipoprotein A-I (apo A-I) is the primary protein component of high-density lipoprotein (HDL) and has many well documented properties which promote cardiovascular health. However, clinical trials designed to increase HDL levels by preventing its catabolism have failed in their primary endpoints in decreasing the risk of cardiovascular disease. Alternative strategies to increase de-novo apo A-I production may be more attractive. We recently demonstrated that the rare sugar allulose decreases oxidative stress and endoplasmic reticulum stress in both endothelial cells and hepatocytes. During these studies we demonstrated that allulose also induces apo A-I secretion by HepG2 cells. Apo A-I, albumin, and SP1 levels were measured by Western blot. Apo A-I and glyceraldehyde-3-phosphate (GAPDH) mRNA levels were measured by quantitative real-time polymerase chain reaction. The effect of allulose on apo A-I promoter activity was measured using transient transfection assays with several plasmids containing various segments and mutations in the apo A-I gene promoter. Apo A-I protein and mRNA levels in cells treated with allulose increased more than two-fold in a dose-dependent manner. These changes were due to the ability of allulose to induce apo A-I gene promoter activity. Using a series of deletion constructs, an allulose-response element was identified in the apo A-I gene promoter which was previously shown to confer induction of apo A-I gene expression by insulin and epidermal growth factor (EGF), the insulin response core element (IRCE). Mutation of the IRCE decreased the ability of allulose and insulin to induce apo A-I promoter activity. Allulose treatment also increased expression of the transcription factor SP1, which had been shown previously be essential for the effects of insulin and EGF on apo A-I promoter activity. In conclusion, allulose increased apo A-I gene expression in HepG2 hepatocytes. This effect was mediated by the IRCE in the apo A-I gene promoter and the transcription factor SP1. The rare sugar allulose may have novel anti-atherogenic properties, in part, by increasing HDL levels.

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<![CDATA[MON-708 Characterizing DNA Methylation Signatures in Adipose Tissue from Metabolic Impaired Asymptomatic Individuals]]> https://www.researchpad.co/article/elastic_article_8669 Obesity remains as a global epidemic characterized by progressive metabolic dysregulation in glucose homeostasis. Along with a genetic association in the development of T2D, epigenetic regulation has been suggested as a significant contributor in altered gene expression. Recent studies have described DNA methylation changes in insulin-sensitive tissues involved in T2D pathogenesis, however epigenetic dynamics on early stages to metabolic alterations is still unclear.

We investigated potential DNA methylation signatures in 34 asymptomatic individuals from the GEMM family study. We compared differentially methylated CpG sites (DMC: B value>0 and delta Beta >|10%|; Infinium EPIC array) from subcutaneous adipose tissue (SCAT) in different groups of individuals according to BMI (kg/m2) and HbA1c (%) levels as follow: Group A Control (C): n=9, 22.0±1.9 kg/m2, 4.8±0.3%; Group B Overweight (OW) with normal HbA1c: n=6, 27.8±1.6 kg/m2, 5±0.2%; Group C Obese (OB) with normal HbA1c: n=6, 34.6±4.2 kg/m2, 5.2±0.2%; Group D Prediabetes (PD): n=7, 31.1±5.7 kg/m2, 5.9±0.2% and Group E T2D: n=6, 30.6±7.3 kg/m2, 7.2±0.9%.

We found 43 overlapping genes with shared pathways in all groups, mainly those related to metabolism and adipogenesis. We also documented particular altered methylated genes, in each group (OW: 386, OB:1005, PD:76 and T2D:189). Pathway enrichment analysis in OB and T2D was mainly related to glucose metabolism, while in OW and PD was NOTCH signaling. All groups displayed a consistent hypermethylation in RARA, ESR1 and NCOR2, well known genes involved in lipid metabolism. Additionally, we describe for the first time, a progression toward hypomethylation in ARHGAP15 and MTAP, related with an impaired metabolic status. Otherwise, analysis of overlapping CpG sites revealed a consistently hypermethylated state in OW (86.42%), OB (86.48%) and PD (51.72%), in contrast with the hypomethylation state (56.3%) observed in the T2D group, previously observed elsewhere (1).

In conclusion, comparison of methylation in SCAT obtained from OW, OB, PD and T2D individuals, display potential pathways and DMC signatures specific in each group. Common novel overlapping genes in global DNA methylation profiles of SCAT, were also observed.

Reference: (1) Barajas-Olmos et al., BMC Med Genet. 2018 Feb 21;19(1):1-8.

Nothing to Disclose: FE, FB, AM, EH, GEMM, ER, RB, LO.

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<![CDATA[SAT-715 Bisphenol-A Alters Pancreatic B-Cell Proliferation and Mass in an Estrogen Receptor Beta-Dependent Manner]]> https://www.researchpad.co/article/elastic_article_8659 Bisphenol-A (BPA) is one of the highest volume chemicals produced worldwide. It is used as the base compound in the manufacture of polycarbonate plastics, epoxies and resins. Humans are consistently exposed to BPA and consistently it has been detected in the majority of individuals examined. Experimental research in animals, as well as human epidemiological studies, converge to conclude that BPA is a risk factor for the development of type 2 diabetes. In previous studies we have demonstrated that the exposure to BPA during embryonic development promote an increment of pancreatic β-cell mass. This was correlated with increased β-cell division and altered global gene expression in pancreatic β-cells. The aim of this work was to determinate whether ERβ was involved in the in the β-cell mass and proliferation increment observed in male mice offspring. ERβ+/- pregnant mice were treated with vehicle or BPA (10 μg/kg/day) from day 9 to 16 of gestation. Offspring pancreatic β-cell mass was measured at postnatal day 0 (P0) and 30 (P30). For ex vivo experiments Wild-type (WT) and ERβ-/- neonates as well as adult male and female mice were used. For in vitro, single islets cells were cultured for 48 h in the presence of 10 μmol/L BrdU, and vehicle, BPA (1, 10, 100 nM) or the specific ERβ agonist WAY200070 (1, 10, 100 nM). β-cell proliferation rate was quantified as the percentage of BrdU-positive pancreatic β-cells. In vivo exposure to BPA during pregnancy promoted an increment of pancreatic β-cell mass and proliferation in WT mice at P30 which was absent in ERβ -/- mice. In order to explore if these changes were related to a direct action of BPA on pancreatic β-cell division we performed a series of ex vivo experiments. Augmented β-cell proliferation rate was observed in BPA-exposed β-cells isolated from both adult male and female WT animals in comparison to controls. The increment was significant at all BPA doses tested. The effect was imitated by the selective ERβ agonist, WAY200070, and was abolished in cells from ERβ-/- mice. We also explored the effects of BPA in pancreatic β-cells from neonates and found an increment in BPA-exposed cells compared to controls, although the difference was only significant at the dose of 1 nM. A similar effect was observed in neonate cells treated with WAY200070 (10 nM). The effects on β-cell replication were abolished in cells from ERβ-/- neonate mice treated either with BPA or WAY200070. Our findings suggest that BPA modulate pancreatic β-cell growth and mass in an ERβ-dependent manner. This could have important implications for metabolic programming of T2DM. Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) grants BPU2017-86579-R (AN) and BFU2016-77125-R (IQ); Generalitat Valenciana PROMETEO II/2015/016 (AN). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

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<![CDATA[SUN-719 The Impact of FOXA3 on Testicular Steroidogenesis]]> https://www.researchpad.co/article/elastic_article_8656 The Forkhead box(Fox) transcription factors are evolutionarily conserved in organisms and regulate diverse biological processes during development as well as adult life. Among the Fox family, FoxA subfamily members Foxa1-3 have been termed `pioneer’ transcription factors as they bind both nucleosome-bound and nucleosome-free DNA targets with the same recognition site. Foxa3 is the only member of FoxA subfamily that is expressed in both male and female gonads. In the testis, Foxa3 is expressed in spermatids and interstitial Leydig cells. We focused our study to elucidate the role of FOXA3 in Leydig cells and its impact on testicular steroidogenesis. Expression of FOXA3 dramatically decreased in mouse Leydig cells during testicular development. In addition, the time-dependent expression of FOXA3 showed an opposite pattern to that of steroidogenic genes in cAMP-induced primary Leydig cells. Meanwhile, Nur77 is among the prime regulators of steroidogenesis in the testicular Leydig cells. Overexpression of FOXA3 in MA-10 cells (mouse Leydig tumor cell line) repressed the cAMP-induced Nur77 promoter activity, which further resulted in the reduced activity of Nur77-target steroidogenic gene promoters (StAR, CYP17A1and 3β-HSD). Similar to above results, the expression of Nur77 and its target genes,StAR, 3β-HSD and CYP11A1, were repressed by adenovirus-mediated overexpression of FOXA3 in mouse primary Leydig cells, although the expression of CYP17A1, another steroidogenic gene, was differentially affected. These results suggest that FOXA3 locally regulates the expression of steroidogenic genes through Nur77 during testicular development.

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<![CDATA[MON-725 Transcriptome Profiling in Postnatal Pituitary Gland Identifies Cell Type-Driven Sex-Specific Changes]]> https://www.researchpad.co/article/elastic_article_8581 The pituitary gland is integral to the regulation of growth, metabolism, puberty, reproduction, and stress responses. Previously, we found that many genes associated with age-at-menarche in genome-wide association studies (GWAS) displayed increasingly sex-biased expression across the pubertal transition in the mouse pituitary. However, whether this trend exists beyond puberty-related genes was not known. In addition, the regulatory mechanisms underlying these gene expression changes remained to be explored. To answer these questions, we profiled the transcriptome, including microRNAs, of mouse pituitary in both sexes across pubertal transition in an unbiased manner and leveraged a recently published pituitary single cell transcriptome to explore cellular composition changes. We found that the most dynamic temporal changes in both mRNA and miRNA expression occur prior to puberty, underscoring a role for regulation of early pituitary postnatal development. We also observed ~900 genes displaying sex-biased expression patterns, arising during early development and becoming increasingly biased across puberty, including known sex-biased genes such as Fshb and Lhb. However, sex differences in miRNA expression are less pronounced, only 13 miRNAs were found to be sex-biased, suggesting lesser contribution of miRNAs to sex-biased gene expression relative to other forms of regulation. To assess whether pituitary cellular composition could underlie changes in gene expression across pubertal transition, we performed single cell deconvolution of our bulk pituitary gland gene expression. Interestingly, we found that sex differences in cell proportions were estimated to emerge across puberty: a greater proportion of lactotropes was found among females, and greater proportions of gonadotropes and somatotropes were found among males. We observed sex-biased expression patterns of marker genes for these cell types, including Prl, Fshb, and Gh. This finding suggests that cell proportion differences between sexes likely contribute to whole pituitary transcriptome changes we observed, however, to what extent remains to be studied. Together our study indicates that miRNAs play a substantial role in regulation of pituitary postnatal development but that differences in cellular composition may contribute more robustly to sex-biased gene expression.

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<![CDATA[SUN-712 Familial 46, XY Complete Female External Sex Development with a Non-Mosaic Inherited SRY Gene Variation]]> https://www.researchpad.co/article/elastic_article_8569 Context: SRY, an architectural transcription factor containing a SOX-related high-mobility group (HMG) box, initiates testis formation in the mammalian bipotential gonadal ridge. Inherited human SRY mutations can be associated with differences in sexual differentiation (DSD) with variable phenotypes in a family.

Objective: To describe the clinical, histopathological and molecular features of a novel inherited SRY allele (pMet64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt.

Design, Setting, Participants and Outcome Measurements: Following DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated.

Results: Breast development in P1 and P2was Tanner II and IV, respectively. Müllerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadoblastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancersEnh13 and TESCO.

Conclusion: The partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the “the father-daughter paradox”).Sex steroids biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

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<![CDATA[SAT-LB132 3-Generation Study of Metabolic Disruption by Pregnancy Serum PFASs: Associations With Abdominal and Whole-Body Obesity in Granddaughters in a 60-Year Follow-Up of the Child Health and Development Studies Cohort]]> https://www.researchpad.co/article/elastic_article_8558 Introduction. We previously found a 3.6-fold increased risk of breast cancer in daughters associated with high maternal (F0) early postpartum serum EtFOSAA combined with high F0 cholesterol (https://doi.org/10.1016/j.reprotox.2019.06.012). Here we test the hypothesis that F0 early postpartum EtFOSAA, in combination with F0 serum cholesterol predicts abdominal obesity (waist circumference > 88cm) and/or whole-body overweight or obesity (body mass index > 25 kg/m2) in daughters (F1) at age 30 and granddaughters (F2) at age 20. Methods. We measured F1 and F2 weight, height, waist circumference and blood pressure when F1 were an average age of 50 years and adult F2 were an average age of 20 years (N=213 triads). F1 also reported their weight at age 30, near the mean age of their pregnancies with their daughters (F2) to allow control for obesity during F2 gestation. EtFOSAA, PFOS, and cholesterol were assayed in archived early postpartum F0 serum samples collected within 3 days of delivery. Results. F0 cholesterol significantly (p<0.05) modified the association of F0 EtFOSAA with self-reported obesity at age 30 in F1 and measured abdominal and whole-body obesity, and blood pressure at age 20 in F2. Association patterns were similar for all outcomes: F0 EtFOSAA was associated with high metabolic risk when F0 serum cholesterol was low (Quartile 1): e.g. 20-year-old F2 had an estimated 2.3 fold increase in the joint risk of abdominal and whole-body obesity over the inter-quartile range of F0 ETFOSAA, 95% Confidence Interval= 1.1, 4.8. F0 EtFOSAA associations with F2 metabolic risk were independent of F0 race, early pregnancy overweight (BMI >25 kg/m2), and serum PFOS. F1 obesity at age 30 did not mediate F0 EtFOSAA associations with F2 outcomes, but additionally predicted high metabolic F2 risk. Conclusions. Findings support the hypothesis that in utero exposure to EtFOSAA impacts metabolic risk factors in female F2 exposed as germline and also independently via promotion of overweight in F1 (their mothers) during F2 gestation.

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<![CDATA[OR24-07 Fetal Sex Impacts First Trimester Maternal-Fetal Communication in Humans]]> https://www.researchpad.co/article/elastic_article_8549 The placenta serves as a regulator of fetal growth throughout pregnancy. Signaling at the maternal-fetal interface is critical during placentation and lays the groundwork for placenta function, affecting pregnancy outcomes. Fetal growth is impacted by fetal sex, with males larger than females, and maternal gestational diabetes and obesity independently increase the risk of macrosomia in male fetuses only. We previously demonstrated differentially expressed genes (DEGs) among sexes involves ancient canonical pathways and metabolic functions in placenta tissue. As these are likely impacted by signaling at the maternal-fetal interface, our aim here was to identify sex differences in signaling at the maternal-fetal interface and among individual cell types within the placenta to explain these differences. RNA-sequencing of first trimester placenta and maternal decidua as well as single cell RNA-sequencing in first trimester placenta was performed in ongoing pregnancies. We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. From these, 35 of 115 receptors and/or ligand genes were also found to be upstream regulators of pathways critical in sexually dimorphic placentation which may define regulation. Single cell analysis identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen presenting cells, and endothelial cells), and all had sexually dimorphic genes. Among individual cell types, ligands from the CC-family of cytokines were most highly representative in females, with their corresponding receptors present on the maternal surface. Furthermore, upstream regulator analysis of sexually dimorphic genes demonstrated TGFβ1 and estradiol to significantly affect all cell types. Dihydrotestosterone, which is produced by the male fetus, was an upstream regulator that was most significant for the trophoblast population. In addition, gene ontology enrichment analysis identified distinctive enriched functions between male and female trophoblasts, with cytokine mediated signaling pathways most representative. MUC15 and NOTUM were the most highly expressed sexually dimorphic autosomal genes found in distinct cell types of the trophoblast population, cell types critical for placentation and nutrient exchange. Thus, differences in hormone and immune signaling pathways may account for differential gene expression and differences in trophoblast function during placentation, which may in turn explain developmental differences, including fetal size, well-being, and overall outcomes.

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<![CDATA[OR24-04 Ovarian Follicle Survival Is Determined by Follicle-Stimulating Hormone Receptor (FSHR) and Estrogen Receptor (GPER) Heteromers]]> https://www.researchpad.co/article/elastic_article_7191 Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression. These concepts are challenged in vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclears and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH/FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers at the cell surface. The role of FSHR/GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. GPER/FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in vivo for inhibiting cAMP-linked apoptosis. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR/GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.

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<![CDATA[MON-722 Cross-Species Glucocorticoid-Sensitive Posterior Dentate Gyrus Gene Network: Developing a Polygenic Score Associated to Susceptibility to Depression After Early Life Adversity Exposure in Humans]]> https://www.researchpad.co/article/elastic_article_6954 Exposure to stress during the life-course has consistently been associated with neuropsychological disorders, but the precise role of stress released glucocorticoids remains unclear in this context. We aimed at using hippocampal gene expression data from macaques to identify clusters of genes sensible to glucocorticoid exposure and create a biologically relevant polygenic score to investigate emotional disorders in a child and adult humans exposed to early adversity. RNA-sequencing data from the posterior dentate gyrus (pDG) of adult Macaca fascicularis females treated with Betamethasone (glucocorticoid) or saline injections for 8 consecutive days were analyzed from two cohorts: Singapore (reference) and Vietnam (replication) with N=12/each. Weighted gene co-expression network analysis (WGCNA) was used to identify clusters (modules) of co-expressed genes associated with betamethasone. In Singaporean animals, genes were clustered in 52 modules, in which 5 were associated with betamethasone. Two modules were preserved in a replication dataset (Vietnam) and in data from female rats treated with corticosterone for 6 weeks, being the black module (557 genes, P=0.01, r=0.7) the one having the highest correlation with glucocorticoid exposure. Gene ontology analysis (FDR<0.05, Metacore®) revealed that this module is associated with transcription processes. The SNPs derived from genes within the module were used to calculate an expression-based polygenic risk score (ePRS) in the human samples, weighing each SNP by the slope of the association between genotype and gene expression (GTex). Linear regression analysis showed a significant interaction between ePRS and early adversity on the Dominique - major depressive disorder domain (β=1304; P=0.003; N=65) in girls aged 6 years (MAVAN), in which a higher ePRS was associated with more symptoms as the adversity scores increases (simple slope analysis,P=0.004). A comparable interaction between the ePRS and postnatal adversity was also observed in adult women (UK Biobank), in which there was an increased risk for early depression onset (β= -424.3, P=0.04; N=13899). In the adult cohort, whole brain gray matter volume was also associated with differences in the expression of the genes that composed the ePRS-black network (main ePRS effect, β=1865776, P=0.03, N=10902). Glucocorticoid exposure affects a specific group of genes in pDG of adult female macaques and rats, influencing transcriptional processes. Variations in the expression of this gene network sensible to glucocorticoids were associated with susceptibility for the development of depression in girls and adult women exposed to early life adversity. These show the importance of glucocorticoids on the development of depressive symptoms. The gene network affected by glucocorticoids can guide future pharmacological or mechanistic studies in other samples or species.

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<![CDATA[MON-709 Single-Cell RNA-Sequencing Deciphers POMC Neuron Destiny]]> https://www.researchpad.co/article/elastic_article_6880 The hypothalamus is one of the critical brain nodes regulating body weight and energy homeostasis. Within this node, Pomc neurons sense nutrient and hormonal signals to release melanocortin peptides that induce satiety, whereas AgRP/NPY neurons exert opposite effects by releasing AGRP that promotes feeding. Immature neurons in the hypothalamic ventricular zone start to express Pomc at E10.5, reach a maximum number at E14.5 and then decrease to stabilize at E18.5. However, it remains elusive how Pomc expressing precursors adopt their final cell fates. Therefore, the goal of this study was to decipher the temporal sequence of transcription factor (TF) expression leading to the terminal differentiation of POMC neurons. Red fluorescent cells collected from dissociated hypothalami of Pomc-tDimer-dsRed mice at six critical developmental time points - E11.5, E13.5, E15.5, E17.5, P5 and P12- were FACS sorted for the 10X genomics scRNA-seq pipeline. Unsupervised cell clustering identified 11 distinct clusters based on their transcriptional profiles. Eight of the clusters were highly-enriched for neuronal signature genes and were further characterized based on their transcript levels for Pomc (high, medium or low) and other distinct feature genes. Cells in the Pomchigh cluster expressed genes identified previously to modulate Pomc expression, including Isl1, Nkx2-1, and Tbx3, together with several novel candidate TFs. Unexpectedly, Nr5a1, the ventromedial hypothalamic nucleus marker gene encoding SF1, was highly expressed in the Pomchigh cluster at early stages. One of the Pomclow clusters highly expressed Otp, Agrp, Npy, Sst and Calcr while a second was highly enriched with Tac2, Kiss1, Pdyn, Prlr, Ar and Esr1 transcripts. All the clusters showed direct correlations of embryonic stage with the expression of progressively more mature markers of differentiation, thereby extending previous reports of these clusters based on single time points. Moreover, our results uncovered five novel Pomc neuron clusters with unique patterns of TF gene expression. For comparison of these data to the adult hypothalamus, we performed a TRAP-Seq study using PomcCreERT,Rosa26eGFP-L10a mice. Prdm12 and Tbx3 were among the most highly differentially expressed TFs in the POMC neuron affinity purified translatome. Similarly, Cited1, Npy2r, and Asb4 were highly expressed in both the Pomchigh cluster and the TRAP-Seq derived POMC translatome. This comprehensive molecular characterization of POMC cells during development sheds new light on the molecular diversification of early POMC neuron precursors and provides a valuable resource for elucidating the regulatory mechanisms defining POMC neuron subgroups in the hypothalamus.

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<![CDATA[MON-712 Restoration of Growth and Fertility in Zebrafish (Danio Rerio) Model with PROP1 Knockout Generated by CRISPR/Cas9 Genomic Editing]]> https://www.researchpad.co/article/elastic_article_6801 Introduction: Hypopituitarism is defined as the deficiency of one or more pituitary hormones and can occur due to pathogenic allelic variants in transcription factors involved in pituitary development. PROP1 gene is responsible for progenitor cell migration from the marginal zone to the anterior lobe, and its terminal differentiation into corticotropes and gonadotropes cell lines besides somatotropes, lactotropes and thyrotropes due to POU1F1 (also known as PIT1) activation. In humans, mutations in the PROP1 gene are the most common cause of congenital hypopituitarism with GH, TSH, LH/FSH, and progressive ACTH deficiencies. A dwarf phenotype with short stature, pituitary hormone deficiency, and infertility has been described in humans and Ames mice lineage harboring mutations in the PROP1/Prop1 gene. Another valuable animal model used in basic research is the zebrafish (Danio rerio) due to a high homology in neuroendocrine functioning. To test the potential of this model, in our previous study, a 32bp insertion carrying a stop codon was directed into the second exon of prop1 with CRISPR/Cas9, establishing a homozygous mutant strain (prop1mut). Objective: To characterize the phenotype and expression patterns of transcription factors and hormones in the zebrafish prop1mut lineage. Methods: prop1, pit1, and gh1 mRNA levels were analyzed during embryonic development at 24 and 72 hours post-fertilization (hpf). RNA from 30 pooled embryos was extracted using DirectZol RNA Miniprep. cDNA was synthesized from 1ug of total RNA using High-Capacity cDNA Reverse Transcription Kit and qPCR was performed using SYBR Green PCR Master Mix. Gene expression was normalized to ef1a and the prop1mut group was compared with the control wild type group (WT). Animals were kept in the tanks at a density of 15 animals/liter and images were acquired at 13 and 20 days post fertilization (dpf) after brief anesthetization using a stereomicroscope and measured in ImageJ software to determine the larval standard length from nose to the end of the spinal cord. Results: At 24 and 72hpf, prop1mut embryos expressed the altered prop1 mRNA at similar levels to the prop1 expression observed in WT. Lower pit1 expression in prop1mut embryos was observed at both periods (p<0.01). Albeit in low levels, similar gh1 expression was observed in both lineages at 24hpf, and prop1mut embryos presented lower gh1 expression at 72hpf (p<0.001). prop1mut larvae presented a significant decrease in size at 13dpf (p<0.001) but not at 20dpf. Conclusion: In this study, the prop1mut zebrafish model exhibited a dwarf phenotype during larval development associated with diminished pit1 and gh1 expression during the embryonic stage. Additionally, in the juvenile stage, the development rate in prop1mut animals was restored, presenting similar standard lengths observed in WT animals.

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<![CDATA[SAT-728 The Regulation of Tumor Suppressor Genes P53, BRCA-2, and Cell Cycle Protein p21 by Bisphenol S (BPS) in MCF-7 and T47-D Breast Cancer Cells]]> https://www.researchpad.co/article/elastic_article_6729 Bisphenol A (BPA) is considered to be an endocrine disrupting chemical (EDC), which mimics endogenous hormones and is linked to various cancers. Bisphenol S (BPS) is a BPA analogue, often used in plastics. BPS can leach into food and drink products, exposing humans to these chemicals. Evidence suggests BPS is also an EDC with similar endocrine disrupting effects. Despite hopes for a safer alternative, research has shown BPS possesses estrogenic activity due to its structural similarities with its analogue BPA. Previously we have shown the effects of BPS on estrogen receptor-alpha (ERα) and BRCA-1 in both MCF-7 and T-47D breast cancer cells. The wild-type p53 and BRCA-2 work to prevent cancer by monitoring and repairing DNA damage; however, in breast cancer patients these genes are often mutated. Mutated p53 will induce the cell cycle protein p21 to act as an oncogenic transcription factor. In the present study, we have examined the effects of BPS, alone and in combination with hormones and anti-hormones, on p53, BRCA-2, and p21 in both MCF-7 and T-47D cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay, and RT-qPCR analyses. Western blot studies revealed alterations in the expression of p53, BRCA-2, and p21 related with varying concentrations of BPS (4-20 µM). In comparison to the control, p53 expression increased (65-95%) in the presence of BPS in both MCF-7 and T-47D cells. In addition, BRCA-2 expression revealed a similar increase in both cell lines when treated with BPS. However, p21 expression decreased (approximately 50%) with increasing concentrations in both cell lines. For further evaluation, an optimal concentration of 8 µM BPS was then used in combination with various hormones and anti-hormones. Compared to the control, BPS and E2 were up regulated in a similar fashion to p53. A similar trend in the effects on BRCA-2 expression was depicted in T-47D and MCF-7 cells. However, in p21, BPS and E2 were down regulated in both MCF-7 and T-47D breast cancer cells. In order to determine the influence of BPS on the growth of breast cancer cells, image cytometric analysis with propidium iodide staining was utilized to quantify alterations in T-47D and MCF-7 cell numbers and viability. Upon treatment of BPS concentrations (4-20 µM), an increase in cellular proliferation (12-60% increase) occurred in both cell lines. These cellular proliferative effects of BPS and E2 were sensitive to combination treatments with anti-estrogens. Confocal microscopy was utilized to examine the cytolocalization of p53 upon exposure to BPS alone and in combination with hormones and anti-hormones. The results from this study will yield a greater understanding of the molecular regulation of BPS action via the p53, BRCA-2, and p21 signaling pathways linked with breast cancer.

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<![CDATA[SUN-711 Association of Single Nucleotide Polymorphisms of CYP11B2, CYP11B1 and CYP17A1 with Primary Aldosteronism in a Multi-Ethnic Malaysian Cohort]]> https://www.researchpad.co/article/elastic_article_6719 Primary aldosteronism (PA), also known as Conn’s syndrome, is a common curable cause of hypertension. Family studies of essential hypertensive patients suggest that heritable genetic factors play a role in blood pressure regulation1. Interestingly, single nucleotide polymorphisms (SNP) in genes encoding enzymes involved with adrenal steroidogenesis, CYP11B2, CYP11B1 and CYP17A1, associate with increased risk of hypertension2. Therefore, we analysed whether selected SNPs in these genes are associated with PA. We performed an association study using genotype imputation for selected SNPs of the steroidogenic enzyme genes CYP11B2 (rs4546, rs1799998, rs13268025), CYP11B1 (rs6410, rs149845727), and CYP17A1 (rs1004467, rs138009835, rs2150927) from a pilot genome wide association study of Malaysian PA patients and healthy controls which was merged with the Singapore Genome Variation Project (SGVP) population dataset3. Genotype imputation for minor and major alleles was validated using PCR sequencing (n>10 for each genotype). Further, one SNP from each steroidogenic enzyme (CYP11B2:rs1799998, CYP11B1:rs6410 and CYP17A1:rs1004467) was validated using commercial TaqMan genotyping assays on the ABI 7000 Sequence Detection System which was performed on 149 PA patients and 78 non-hypertensive healthy individuals. Case-control genetic association analysis was performed at http://www.oege.org/software/orcalc.html and the association between genotypes and phenotypes was done using the independent-samples Kruskal-Wallis test on SPSS (version 25). The Minor Allele Frequencies (MAFs) for rs1004467, rs6410 and rs1799998 were similar to East Asian populations but differed significantly different from European, African, American and South Asian populations (rs1004467 MAF: C=0.258/298, rs6410 MAF: A=0.265/298, rs1799998 MAF: C=0.225/298). In Chinese patients matched by gender, heterozygotes for rs6410 had significantly increased risk of PA compared to common homozygotes (OR: 3.15, 95% CI: 1.01–9.8, p=0.04). Across patients of different ethnicity, the distribution of aldosterone levels was significantly different (p=0.039). In summary, only SNP rs6410 in Chinese patients matched by gender showed association with PA in our South East Asian cohort. More functional experiments need to be done to find out whether this is causal for PA or whether the SNP is in linkage disequilibrium with the actual functional causative SNPs. Once the functional SNP is known, identification of these germline variants in asymptomatic family members would allow early screening of PA to be offered and potentially provide novel drug targets to treat the disease.

References: 1Timberlake et al., Curr Opin Nephrol Hypertens. 2001 Jan;10(1):71-9. 2MacKenzie et al., Int J Mol Sci. 2017 Mar 7;18(3). pii: E579. 3Teo et al., Genome Res. 2009 Nov;19(11):2154-62.

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<![CDATA[SUN-723 CDH2 Gene Analysis in a Cohort of Patients with Congenital Hypopituitarism]]> https://www.researchpad.co/article/elastic_article_6687 Introduction: Hypopituitarism is defined as a deficiency of one or more pituitary hormones. Pathogenic allelic variants in genes implicated in pituitary development were associated in 15% of the patients with congenital hypopituitarism (CH). To improve the molecular diagnosis we performed whole exome sequencing of ten patients born from consanguineous parents with CH. One patient with GH, TSH, ACTH and LH/FSH deficiencies presented an allelic variant c.865G>A, p.V289I in CDH2 gene (exon 7) in homozygous state that was absent in populational databanks. CDH2 produces an N-cadherin protein implicated in cellular adhesion and is responsible for epithelial-mesenchymal transition during pituitary development and differentiation. Aim: To analyze the CDH2 gene in a cohort of unrelated patients with CH. Methods: We selected 143 patients with CH from a single Brazilian center. Genomic DNA, extracted by salting out technique, was submitted to PCR amplification of 15 coding regions, except CG rich exon 1, of the CDH2 gene followed by the Sanger sequencing. Rare allelic variant frequency (MAF<1%) was searched in the populational data bank (ExAC, gnomAD, ABraom). Bioinformatic sites (Human Splicing Finder, Polyphen2, Mutation Taster and Mutation assessor) were used to look for deleterious effects. Results: Three allelic variants were found in this cohort. The allelic variant CDH2 (c.865G>A, p.V289I) was found in heterozygous state in a male patient with short stature diagnosed with GH and TSH deficiencies at the age of 11 that evolved with LH/FSH and ACTH deficiencies. Family segregation showed 3 among 11 normal siblings heterozygous carriers. This variant is rare, in heterozygous state, in populational data bank and it was predicted as deleterious or possibly harmful. The allelic variant c.1202C> A (p.A401D), in exon 9, was found in heterozygous state in a female patient with isolated GH deficiency and intellectual disability. The variant was absent in the databases and predicted as deleterious or disease-causing. The variant was absent in the mother and stepsister and the father was not available for testing. The c.1430_1431delCCinsTG allelic variant (p.P477L) was found in heterozygous state in a patient with septo-optic dysplasia, GH, TSH and ACTH deficiencies. It was absent in the databases and was predicted as deleterious or disease causing. The Human Splicing Finder predicted exonic splicing enhancer breakdown leading to the loss of 93 nucleotides. Normal mother is heterozygous carrier suggesting incomplete penetrance. Conclusion: Heterozygous variants in CDH2 were found in 2% of a cohort of Brazilian patients with congenital hypopituitarism and none in homozygous or compound heterozygous state. Further CDH2 analyses in unrelated patients from different ethnic backgrounds are needed to establish the role CDH2 variants in the etiology of congenital hypopituitarism.

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