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Context: The endocrine function of human fetal adrenals (HFA) is activated already during first trimester, but changes in adrenal steroidogenesis during fetal life are not well characterized. Objective: This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from 1st and 2nd trimester. Design and Setting: Male and female HFA samples from gestational week (GW) 8-19 were examined, including a total of 101 samples from 83 fetuses. Main Outcome Measure(s): Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS. Results: Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2 and SULT2A1 was significantly higher in 2nd trimester compared with 1st trimester (P

Over 90% of the human genome expresses many different forms of non-protein-coding (nc) RNAs in addition to mRNAs. They are categorized into short and long ncRNAs. Although their overall functions and biological importance are still under investigation, they appear to be essential for supporting complex human activities mainly by regulating genome transcription and/or protein translation. It is known that some short RNAs are identified in several body fluids of humans, including serum, urine and saliva. Biological and/or pathological roles of serum micro (mi) RNAs have recently been reported, whereas little is known about other short non-coding (snc) RNAs circulating in serum. Here we present a comprehensive analysis on the short RNAs present in mouse serum using the RNA-sequencing by employing two endocrine organs (adrenal gland and ovary) and liver as controls. We found that serum has numerous short RNA reads and most of them (~90%) are unable to be mapped to the reference genome. Among the mapped reads, short RNAs expressed from the intergenic area (intergenic sncRNAs) represent the majority (97.7%) of short RNAs appeared in mouse serum. These intergenic sncRNAs are mapped to the intergenic area located closely to the nearby protein-coding genes (~20 kb distance), indicating their potential regulatory functions on the transcription of these coding genes. Most of the serum intergenic sncRNAs are distinct from those identified in the three control organs, whereas the intergenic sncRNAs found in the control organs highly overlap with each other. Among known sncRNAs, fragments of ribosomal (r) RNAs (72.0%), transfer (t) RNAs (18.1%), piwi-interacting (pi) RNAs (4.01%) and micro (mi) RNAs (3.96%) are most abundant, whereas small nuclear (sn) RNAs (1.46%) and small nucleolar (sno) RNAs (0.44%) are found as minor fractions. Very few short RNAs derived from mRNAs or long non-coding RNAs are also identified in mouse serum. Interestingly, many of the mRNA fragments identified in serum are derived from the genes associated with the transmembrane signaling pathways in the Ingenuity® Pathway Analysis, suggesting easy liberation of these mRNA fragments from respective cells possibly due to their local translation as reported in neuronal cells. Taken together, our findings indicate that sncRNAs, particularly intergenic sncRNAs, rRNA fragments and tRNAs, are abundant in mouse serum. These results suggest in humans that they may have important biological actions and/or potential clinical implications as valuable clinical markers/therapeutic targets in addition to miRNAs.

Background: The Megalencephaly Capillary Malformation syndrome (MCAP) is a genetic overgrowth disorder characterized by brain overgrowth, polymicrogyria, vascular malformations, and segmental somatic overgrowth secondary to activating mutations in the PI3K-AKT-mTOR pathway (PIK3CA). Congenital macrosomia and postnatal asymmetric overgrowth is typical in this condition, however both growth failure and hypoglycemia have been reported in a significant fraction of affected children raising the concern for growth hormone deficiency (GHD). Methods: We performed an observational multi-institution study of children with MCAP and GHD. Medical records were abstracted including growth trajectories, laboratory data, and treatment response to growth hormone (GH), if applicable. Results: Eight participants (4 female, 4 male) were diagnosed with GHD at mean age of 3.2 years. All had very low or undetectable insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3) concentrations. Four underwent GH stimulation testing and all had insufficient responses with a median peak GH of 3.1 ng/ml (range 1.1-7.3). Growth patterns revealed a drastic decline in height z-scores within the first year of life from greater than 2.0 at birth to less than -2.0 z-score by 12 months of age. Growth velocity was in the low-normal range between 1-4 years of age therefore height z-scores were stable but remained low. Four of the 8 had hypoglycemia with fasting. Central hypothyroidism was diagnosed in one and adrenal insufficiency in another. Brain MRI reports were available in 7 children and all had a structurally normal pituitary gland. Three children were treated with GH; one became inconsolable after one week of treatment and it was discontinued. The other two children have continued on GH with excellent responses in linear growth velocity. Conclusion: Profound GHD is a notable feature of MCAP, a condition with gain-of-function mutations promoting cell growth and proliferation. Therefore, children with MCAP and either hypoglycemia and/or postnatal growth failure should be evaluated for GHD. The universally low IGF-BP3 levels in this cohort were striking given this is an unusual finding even in GHD. Additional research is needed into the mechanisms underlying this phenomenon as well as whether treatment with GH is effective and safe in this condition.

The 18-kDa translocator protein TSPO is a conserved ubiquitous high affinity cholesterol binding protein localized in the outer mitochondrial membrane. It plays a critical role in the segregation and transport of free cholesterol (FC) from the outer to the inner mitochondrial membrane in steroidogenic cells. Numerous studies have shown the presence of TSPO in liver. Imaging studies in humans, using specific TSPO ligands, showed that TSPO is highly expressed and accurately mirrors the histological picture of non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The function of TSPO in simple steatosis and NASH is unknown. Wild type Huh7 (WT) is a well-differentiated human hepatocyte-derived carcinoma cell line, which contains TSPO in mitochondria. In the present study, we generated a stable TSPO knockdown (TSPO KD) in the Huh7 cell line. To mimic hepatic steatosis in vitro, we treated WT and TSPO KD cells with either acetylated LDL (AcLDL), to deliver cholesterol into the cells, or AcLDL plus 58035, an Acetyl-CoA Acetyltransferase (ACAT) and thus cholesterol esterification inhibitor, to induce the accumulation of FC. AcLDL treatment significantly increased FC levels in WT cells; this effect was more pronounced in TSPO KD cells. In contrast, treatment of the cells with AcLDL and 58035 resulted in reduced FC levels likely due to the damage induced by FC accumulation that led to reduced cell proliferation. Considering that TSPO binds FC with high affinity, we investigated the underlying mechanism/link between TSPO KD and FC accumulation by examining the conversion between FC and cholesterol ester (CE) upon AcLDL treatment. The results obtained showed that knocking down TSPO expression reduced ACAT2 (transforming FC to CE) levels, but not cholesterol ester hydrolase (CEH) converting CE to FC, expression. TSPO was found to directly interact with ACAT2. Under these conditions, the accumulation of FC led to the appearance of lipid droplets (LD) of increased size in TSPO KD compared to WT cells, suggesting that TSPO plays a role in LD formation and/or dynamics in liver cells. GRP78, IRE1A, and PERK endoplasmic reticulum stress markers were also elevated in TSPO KD cells following AcLDL treatment while the mitochondrial membrane potential decreased compared to WT cells. Moreover, increased numbers of lysosomes and autophagosomes were observed in TSPO KD cells, an effect further potentiated by the treatment with AcLDL plus 58035. This finding was validated by the up regulation of the LAMP1 and LC3B, lysosome and autophagic structure markers, respectively. Taken together, these data suggest that under cholesterol treatment, TSPO loss would trigger ER stress, mitochondrial dysfunction, and lipid droplet accumulation via FC accumulation, and ultimately cause intracellular protein and organelle degradation, hallmarks of hepatic steatosis.

Central nervous system including brain and retina is composed of appropriate number and subtypes of neurons derived from neural progenitor cells. However, our understanding of the proliferation and differentiation of neural progenitor cells is limited. COUP-TFI and -TFII genes, encoding two nuclear receptors, are associated with neurodevelopmental disease and heart defects respectively. Seven-up, the homologous gene of COUP-TF gene in Drosophila, is expressed and required in photoreceptor cell precursors. We observed that along dorso-ventral axis, COUP-TFI and -TFII genes are differentially expressed in mouse retinal stem cells. Minor defects in the eye are generated in either COUP-TFI or -TFII single gene knockout mouse with RXCre. Interestingly, loss of both genes in the retinal stem cells leads to failed differentiation of photoreceptors and thinner retina. The reduced bipolar cells, amacrine cells, horizontal cells, and ganglionic cells are also displayed in the double mutant retina. In addition, the development of Muller cells is repressed accompanying with fibrosis. Mechanistic studies reveal that COUP-TF genes may regulate the differentiation of photoreceptors through Crx and Nrl genes, and modulate the proliferation of retinal stem cells through P27 and P57 genes. Thus, our study shed new light on the understanding of evolutionarily conserved function of COUP-TF genes in the differentiation of photoreceptor cells.

Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome, associated with tumours such as pleuropulmonary blastoma (PPB), ovarian Sertoli-Leydig cell tumours (SLCT), multinodular goiter (MNG), cystic nephroma (CN), embryonal rhabdomyosarcoma (ERMS) and others. Germline mutations in DICER1 cause an alteration in miRNAs processing, deregulating target oncogenes and leading to elevated risk of tumorigenesis. In most reported cases, there is a heterozygous germline mutation and a somatic second hit mutation. Aim: Analyze the presence of germline and somatic alterations in the DICER1 gene in 6 patients with paediatric tumours associated with DICER1 spectrum. Methods: Automated sequencing of DICER1 gene from gDNA extracted from blood and formalin-fixed, paraffin-embedded tumour tissues of affected subjects and relatives. Cases: 5 girls (P1-P5) and 1 boy (P6), (P1: bilateral SLCT, P2 and P3: SLCT and MNG, P4: MNG and uterine myosarcoma, P5: vaginal rhabdomyosarcoma, and P6: CN). Chronological age at diagnostic was 5, 12, 15, 16, 13 y 2, respectively. Results: We detected 5 novel heterozygous mutations: p.Trp1098* in P1, p.Phe351fs*1 in P2, p.Arg1596Glyfs*24 in P4, c.3269+1G>A in P5, p.Asp244Glyfs*27 in P6, and one previously described heterozygous deletion p.Asp1437Metfs*16 in P3.These mutations (P1-P4 and P6) predict the presence of a truncated protein losing its essential domains for the enzymatic activity and, in one P5 an alteration in the splicing process. The tissue analysis revealed p.Asp1709Glu in MNG and p.Glu1705Lys in SLCT in P2, and p.Glu1705Lys in MNG in P3,previously described as hotspot somatic mutations. Conclusions: We report five novel germinal mutations in DICER1 gene and two somatic hotspot mutations in MNG and SLCT tissue samples. These findings confirm that a second hit event is involved in the mechanism of MNG and SLCT development. Molecular analysis of DICER1 gene allows to perform genetic counselling about familial recurrence risk.

Introduction: Type 4 familial partial lipodystrophic syndrome (FPLD4) is an autosomal dominant disease due to frameshift variants of PLIN1 gene. This gene encodes perilipin 1, a protein playing a key role in the structure of the adipocyte lipid droplet and in the regulation of lipolysis. Only 6 independent families with FPLD4 have been described. The aim of this work was to identify the major signs of the disease in the largest cohort of FPLD4 patients reported to date. Patients and Methods: Clinical and biological phenotype of 11 new patients with a family history of lipodystrophic syndrome and bearing a frameshift variant of PLIN1 gene, were investigated after written informed consent. Histological examination, and perilipin 1 protein expression analyses by western blot on whole cell extracts were performed on biopsies of adipose tissue in 2 patients. Results: We identified 3 heterozygous frameshift variants of PLIN1, segregating with the disease in 11 patients from 5 independent families. Our team had already described the p.Val398Glyfs*167 variant, and 2 new variants p.Gly362Glyfs*63 and p.Pro403Argfs*164 were identified. In all patients next generation sequencing (NGS) excluded any other molecular defect known to be responsible for lipodystrophy. On the studied adipose tissue samples, the PLIN1 variant leads to an abnormal longer form of perilipin 1 with fat fibrosis. The main signs of FPLD4 associate a partial, but also sometimes generalized lipoatrophy of post-pubertal occurrence and a phenotype of variable severity including muscular hypertrophy, acromegaloid facies, hirsutism, and metabolic complications (hypertriglyceridemia, insulin-resistance, diabetes, liver steatosis). Lipoatrophy might be missing or difficult to identify in men and youth. Leptin level was low or inappropriate to BMI, which was below 30 but not always very low. Conclusion: The clinical phenotype of FPLD4 is quite homogeneous, but the clinical diagnosis requires a careful clinical examination and/or an expert advice. An early genetic diagnosis is recommended to confirm the disease and prevent metabolic complications.

Background: Turner syndrome (TS) is a disorder characterized by complete or partial absence of the second sexual chromosome and the most commonly found abnormalities are short stature and gonadal failure. Most TS patients need estrogen replacement but individual outcomes on uterine volume, breast development and vertebral bone density are highly variable. To our knowledge, the impact of polymorphisms in estrogen receptor 1 (ESR1) over the variability in the development of secondary sexual characteristics and in adult height has not yet been evaluated in TS patients. Objective: To investigate the association among five ESR1 polymorphisms (rs543650, rs1038304, rs2046210, rs2234693 and rs9340799) and breast development, uterine volume, vertebral bone density, FSH and LH levels and adult height in TS patients. Methods: We selected 80 TS patients receiving adult estrogen dose for a minimum period of one year. Patients who had spontaneous complete pubertal development or who have Y chromosome material in karyotype were excluded from analysis. They were submitted to clinical and laboratory evaluation, pelvic ultrasound, bone densitometry and genotyping of the five chosen ESR1 polymorphisms through real time PCR. These polymorphisms were selected through active search in Pubmed and in GWAS catalogs, based on the following criteria: recurrence in studies, being Tag-single nucleotide polymorphisms (Tag-SNPs) or having a minor allele frequency (MAF) which allowed the present study with the available sample. Results: 61% of patients had 45,X karyotype and their mean target height was 158.9cm. 82% of them were treated with rhGH for 5 years on average and they reached a mean adult height of 149cm. Their average age of puberty onset was 14.4 years old and 10% of them had spontaneous thelarche. Their mean uterine volume was 30.1cc but ranged from 5.9 to 83cc. 91% attained adult breast development (Tanner 5) and 93% had normal vertebral bone mass after correction for height (by using the online calculator from University of Washington: courses.washington.edu/bonephys). Only 15% had FSH and/or LH lower than 10 IU/L in two or more measurements. Considering the ESR1 polymorphisms, all of them were in Hardy-Weinberg equilibrium. Statistical analysis with multiple linear regression showed that none of the studied polymorphisms was able to predict any of the above estrogen therapy outcomes in an isolated manner. Conclusion: We could not find a relationship among the studied ESR1 polymorphisms and estrogen therapy outcomes in patients with TS. However, the high variability in estrogen replacement results suggests that genetic variants play an important role. Other efforts to identify polymorphisms associated to estrogen outcomes may allow for individualization of treatment in TS.

Background: Skeletal dysplasia is a frequent cause of congenital disproportionate short stature, and genetic defects of cartilage extracellular matrix (ECM) affect growth plate structure and functions. However, precise diagnosis remains difficult due to high heterogeneity of phenotypes. Objective: We sought to identify phenotype spectrum and genetic causes for Chinese patients with skeletal related short stature. Patients and Methods: 86 short stature patients clinically diagnosed or suspected of skeletal dysplasia from 2013 to 2018 were included, and clinical information were systematically collected. Next generation sequencing (NGS) was informed and conducted in all these patients, and pathogenicity of candidate genes were analyzed based on clinical data. Results: 45 of the 86 patients had confirmed mutations, and 46.7% of them (21 to 45) were identified with heterozygous mutations relevant of cartilage extracellular matrix. 19 variants of 5 genes were identified including 9 of COL2A1, 1 of COL9A1, 4 of COL10A1, 4 of COMP and 3 of FBN1. The age at diagnosis was 9.47±5.46 years, and the height SDS was -4.78±1.65. The phenotypic spectrum ranged from proportionate short stature with mild upper limb deformity to typical, severe disproportionate manifestations. Deformities of limbs and vertebral anomalies were more frequently encountered presentations. Patients with COL2A1 mutations were more likely to show facial features, pectus carinatum, scoliosis and epiphyseal dysplasia while those with COL10A1 variants mainly presented genu varum and scoliosis. 5 probands (2 COL10A1, 3 COL2A1) were maternal inherited and 12 of 14 affected family members were female. Individuals with COMP variants had the severest height loss (medium height, -6.24 SDS; range, -7.56 to -5.97) and showed typical short-limb dwarfism, irregular epiphyses and dysfunction of joints in early ages. None of the patients with FBN1 mutations had significant facial features or spinal deformity, but bowing of limbs, brachydactyly and decreased joint mobility were visible. 4 of 20 individuals underwent rhGH therapy but had limited effects. Facial dysmorphism was a significant feature to exclude patients with FGFR3 mutations, while limb and spine deformities were frequently encountered in both ECM and FGFR3 patients. Conclusion: NGS is an efficient tool to identify genetic cause of skeletal dysplasia due to its clinical and molecular heterogeneity, and variants of genes encoding cartilage extracellular matrix are the most common cause. Sources of Research Support: The National Key Research and Development Program of China” (No. 2016YFC0901501) and CAMS Innovation Fund for Medical Science (CAMS-2016-I2M-1-002). Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Context: Guidelines has systematically suggested genetic testing for a wide spectrum of phenotypes in order to confirm or exclude surely the diagnosis of multiple endocrine neoplasia type 1 (MEN1). However, the probability of find a germline MEN1 mutation has showed extremely variable (5-95%) when different phenotypes are considered. The prompt recognition of potential clinical predictors could anticipate the result of the genetic testing in the different clinical scenarios. Objective: To investigate if specific phenotypic features may predict the presence of germline MEN1 mutations in index-cases with clinical diagnosis of MEN1 syndrome. Methods and Patients: 110 MEN1 index cases were included: amplicons based on long-range PCR covering the full MEN1 open reading frame of 94 MEN1 index cases (49 familial, 45 sporadic) were sequenced by targeted-Next Generation Sequencing (tNGS) MiSeq Illumina platform while Sanger Sequencing (SS) was approached to the other 16 MEN1 index cases (2 familial, 14 sporadic). MLPA assay was supported to 31 MEN1-negative patients by tNGS/SS whose DNA samples were available. Results: 68 index cases (62%) had germline MEN1 mutations. With the tNGS/SS/MLPA protocol, the mutation detectability rate was higher in familial MEN1 (90%, 46/51 vs. 37%; 22/59; p, < 0.05). In comparison with MEN1-positive patients, MEN1-negative cases were older (53±12 vs. 39±13; p, < 0.05) and most of them had no more than two MEN1-related tumors (76%, 32/42 vs. 10%, 7/68; p, < 0.05). Considering the subset of 59 patients with negative familial history, the 22 MEN1-positive patients revealed clinical predictors for detection of MEN1 mutation when they were compared with 37 truly non-MEN1 mutation carriers. Thus, sporadic MEN1-positive patients had higher significantly prevalence of the following predictors. 1) ≥ 3 tumors, 86% (19/22) versus 11% (4/37); 2) PETs, 86% (19/22) versus 22% (8/37); 3) which were multifocal, 100% versus 1/8 (9%); 4) with frequent synchronic association of functioning and NF/PETs, 47% (9/19) versus 0% and; 5) high frequency of malignancies, 58% (11/19) versus 5% (2/37). Indeed, there was confirmation of these predictors comparing all 68 MEN1-positive carriers against 42 MEN1-negative cases. Conclusions: The strategy adopted to support the diagnosis of MEN1 based on analysis of clinical predictors and genetic testing for tNGS/SS-MLPA underscored that the positive familial history, occurrence of three or more MEN1 tumors, presence of PETs, especially multifocal PETs, and malignancies are strong predictors to detection of MEN1 mutations.

Obesity and type 2 diabetes in adult life are influenced by the intrauterine environment. The fetus adapts to the environment to which it was exposed, leading potentially to resistance to similar exposures experienced after birth. Studies in both mice and humans suggest that exposure of pregnant females to nutrient deprivation may lead their offspring to develop obesity, insulin resistance and ultimately cardiovascular disease in their adulthood. As the cytoprotective transcription factor Nrf2 (Nfe2l2) is known to be activated upon oxidative and electrophilic stress and has an emerging role in metabolism and specifically in hepatic gluconeogenesis and lipogenesis, we hypothesized that it should be important in the adaptation of the embryos to the metabolic stress induced by maternal caloric restriction. To this end, pregnant C57BL6 mice underwent calorie restriction (CR) by providing them with 50% of the necessary calories per body weight (assessed in pilot experiments) from gestation day 10 to gestation day 16. This CR scheme guarantees the delivery of viable pups that weigh 50% less than the pups whose mothers were fed ad libitum standard chow diet. This CR experiment was performed using either wild-type (WT) or Nrf2 knockout (Nrf2KO) parents. Pregnant females of both genotypes fed ad libitum were used as control. The pregnant females were sacrificed on gestational day 16 and embryos were extracted. Embryonic liver was used for DNA extraction for sex determination by PCR and for RNA preparation to be used for quantitative real-time PCR (qPCR). Statistical significance was set at p

Purpose: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We reported that angiotensin II could change the DNA methylation status around transcription factor binding sites and a transcription start site (TSS) and activate expression of the aldosterone synthase gene (CYP11B2). In order to clarify the effect of potassium on the methylation status of CYP11B2 H295R cells were treated with high concentration of potassium and the methylation status of CYP11B2 was examined. Methods: H295R cells were treated with 16mM potassium for 7 days and observed the dynamic changes in DNA methylation patterns of the CYP11B2 promoter for 28 days. The gene expression of CYP11B2 was measured by real time quantitative PCR. Isolated DNAs from H295R cells were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions. Non-treated cells served as a control. Human CYP11B2 ELISA, chromatin accessibility by real-time PCR (CHART-PCR), DNA methylation and demethylation activity assay were done as previously reported. Results: DNA demethylation at CpG1 (Ad1) was detected within 2 days after stimulation. DNA at CpG2 (Ad5) and CpG3 and DNA around a transcription start site (CpG-1/-2) were demethylated at day 4 in cells treated with potassium. CYP11B2 mRNA levels in cells treated with potassium were significantly higher than those in non-treated cells at days 7 and 9. Similar changes were observed in the levels of CYP11B2 protein. CHART-PCR revealed that potassium treatment increased chromatin accessibility around Ad1 at days 2, 4, 7, 9 and 11, with accessibility increasing from 67% to 70%. Potassium treatment significantly reduced DNA methylation activity at days 7 and 9. Demethylation activity was not affected by potassium. Conclusion: Our data indicated that binding of transcription factors initiates chromatin relaxation and transcription, which are followed by DNA demethylation around transcription factor binding sites and a TSS in the CYP11B2 promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of CYP11B2 expression from an inactive to an active state.

Background: Multiple Endocrine Neoplasia Syndrome Type 2 (MEN2) is an inherited endocrine disorder characterized by the development of pheochromocytoma, medullary thyroid carcinoma (MTC) and parathyroid neoplasia. MEN2 is autosomal dominant and occurs from activating missense variants in the RET proto-oncogene. The estimated prevalence of MEN2 is thought to be 1 per 30,000 in the general population, and although rare, the identification of MEN2 can aid with early screening and prevention for patients and their families. Aim: The aim of our study was to investigate the prevalence and clinical manifestations of pathogenic RET variants in the multi-ethnic BioMe Biobank. Methods: We used exome sequencing and electronic health record (EHR) data from BioMe Biobank participants, who are recruited from primary care and specialty clinics across a single U.S. Health System. We evaluated exome sequence data from 30,000 BioMe participants for RET variants designated as pathogenic for MEN2 according to the clinical genetics database ClinVar. We reviewed EHR data for carriers of pathogenic RET variants. Results: In the BioMe Biobank, 5 ClinVar pathogenic variants were identified in the RET gene. Fifteen participants were heterozygous carriers of these variants; 5 were of self-reported African ancestry, 5 of Hispanic/Latino ancestry, 2 of European ancestry, and 3 of other or unknown ancestry. The age range of carriers was 23-78 years, and 60% were female. The estimated prevalence of pathogenic RET variant carriers in BioMe was ~1 in 2000. Based on EHRs, only 3 carriers (20%) were identified from usual clinical care as having the diagnosis of MEN2A. All 3 carriers with diagnosed MEN2A had the 10:43114500:T:C variant (rs75076352) and had a history of MTC and pheochromocytoma. One of the 3 carriers had a history of elevated parathyroid hormone levels and renal calculi without hypercalcemia. Two of the 3 carriers had a documented family history of MEN2A. Of the 12 pathogenic RET variant carriers without known MEN2, 9 (75%) were diagnosed with hypertension. 7 (58%) carried the 10:43119548:G:A variant (rs79658334). Of these 7 carriers, one had a thyroid nodule, three had autoimmune thyroid disease, one had an adrenal cortical adenoma with primary hyperaldosteronism and hypokalemic periodic paralysis, and two had no known endocrine conditions. Conclusions: These results from the BioMe Biobank reveal a higher prevalence of pathogenic RET variants than previously recognized. Carriers of pathogenic RET variants demonstrate highly variable expressivity for traits for which earlier diagnoses might improve outcomes; these findings have implications for cascade testing of family members. Genomic screening for pathogenic RET variants will likely improve our understanding of MEN2 on a more molecular basis.

The maintenance of homeostasis is fundamental for the efficient execution of all biological processes. A series of adaptive responses, pertinent to all scales of biological organization, have evolved to assist cells, tissues, organs and indeed individuals to attain homeostasis in response to stimuli that tend to abolish it. At the biochemical level a well appreciated such response is the unfolded protein response (UPR) that is triggered during stress of the endoplasmic reticulum (ER), a component of the integrated stress response. Common stimuli that may inflict ER stress are special diets, especially rich in calories and/or lipids, hypoxia, and even aging. While virtually all cells in every tissue and species are capable of inflicting the UPR during stress of the ER, little is known with regards to how this response operates across species, in genetically diverse individuals, or even different tissues of the same individual. By studying ER stress in cultured cells and tissues of genetically diverse animals of the genus Peromyscuswe started addressing these questions and explored their biological ramifications. We discovered that despite the fact that high variation exists between individuals in the expression levels of various UPR components (BiP, GRP94, calnexin, ATF4 and CHOP, a stunningly high degree of coordination is maintained between the expression of different chaperones, with some animals exhibiting uniformly high and others exhibiting moderate or lower response. Noteworthy, by comparing the UPR profile in tissues from a panel of genetic diverse P. leucopus we found that different tissues have their own resilience to stress. In example, despite the inherent variation, brain displayed the tightest coordination between the levels of expression of different chaperones that implies the lowest resilience which in turn is consistent with elevated demand of the tissue for increased homeostatic performance. In these studies, lungs exhibited moderate and the liver the lowest robustness in the degree of coordination in the chaperones examined which in turn may reflect moderate or high resilience for the corresponding tissue respectively. This is in line with each tissue’s ability to be in direct contact and to process exogenously inflicted, naturally occurring stimuli such as the breathing air or the food respectively. How factors such as aging or special diet affect the degree of coordination will be discussed. These results underscore the significance of coordination - as opposed to mere expression levels - and imply that the ability to adapt at different environments and indeed the susceptibility to disease may constitute reflections of the inherent resilience of different tissues in maintaining coordination of gene expression.

Menopause is associated with significant adverse metabolic changes and an increased cardiovascular risk profile. Though well described, the underlying molecular mechanisms for these changes remain unclear. The liver is both mediator and target of metabolic derangement. In this study we used an unbiased RNA-sequencing approach to discover differently expressed genes in the oophorectomized mouse liver as well as response to treatment with 17-β-estradiol (E2). 5 groups of 9-week-old C57BL/6J female mice (10 per group) were subjected to ovariectomy (OVX) or sham operation and left untreated for 6 weeks to allow for development of metabolic changes. At this point 2 groups were sacrificed and tissues harvested. The rest of the mice were treated with E2 or vehicle for 6 weeks and then sacrificed. RNA-sequencing was performed on liver samples 6 weeks post surgery. Differential gene expression was assessed using DESeq2 and changes in gene sets using Gene set enrichment analyses (GSEA). Quantitative Real Time PCR was used to validate select differently expressed genes, as well as to assess changes with E2 treatment at 12 weeks post surgery. We found a total of 103 genes (35 up and 68 downregulated) to be differently expressed (fold change ≥ 2, adjusted P ≤ 0.05, max count ≥ 30) between OVX and SHAM at 6 weeks post surgery. GSEA showed significant upregulation (FDR < 0.01) in gene sets for: fatty acid metabolism (normalized enrichment score = 5.13), oxidative phosphorylation (3.91), myc targets (3.81), peroxisome (2.94), adipogenesis (2.82), androgen response (2.56), UV response up (2.49) and TNF-α signaling via NF-Kβ (2.17). Significant downregulated gene sets were: mitotic spindle (-2.88) and G2M checkpoint (-2.4). Specific differently expressed genes, known to be related to metabolic pathways, were validated using real-time PCR and analyzed at 6- and 12-weeks post-surgery with or without E2 treatment. Surprisingly, ENHO, encodes for Adropin, a secreted peptide and known regulator of fat metabolism, associated with cardiovascular health, was found to be significantly decreased at 6 weeks (fold change -2.1, adjusted p = 7.2e-9) and 12 weeks after OVX (-1.42, p=0.008), though it wasn’t changed following treatment with E2. Importantly, reduced ENHO expression was found in cardiomyocytes 12 weeks post-surgery (relative mRNA expression for OVX vs SHAM = 0.44, P=0.03). Our results show that ovariectomy induces transcriptional changes in liver expression of gene sets related to metabolism and inflammation. The role of ENHO encoding for adropin as a biomarker and therapeutic target in menopause-associated metabolic derangement remains to be investigated.

Background: Next-generation sequencing (NGS) consists of massive parallel processing libraries where millions of reads are generated in a single sequencing run. To ensure efficient sequencing, the amount of DNA pool libraries must be measured precisely because it influences the density of the clusters formation. There are different quantification methods aiming to obtain high density clusters with Q30>80%, avoiding the negative effects of overclustering. Our aim was to compare the density of the clusters in exome libraries quantified by two different methodologies qPCR and MiSeq Nano V2 method. Exome libraries were prepared using SureSelect Human All Exons kits (Agilent Technology) and sequenced on Illumina HiSeq2500 platform (Illumina) using the V4 high output sequencing kit. The pools were prepared in equimolar concentrations using qPCR (KAPA Library Quantification Kit), to achieve final pool library concentration of 10 nM. Three groups were defined: the original 10 nM group (n=16); a qPCR group (n=16) in which the pool concentrations were corrected by qPCR and the MiSeq Nano V2 group (n=12) where pool concentrations were corrected by clustering data of MiSeq Nano in a direct linear proportion. The statistics analysis was performed by Kruskal-Wallis followed by Dunn test. Cluster density values are reported as the absolute distance from the optimal density (1000 k/mm2 of flow cell) specified for V4 kit. Results: The 10 nM group showed a median of 191, iqr (interquartile range) = 48-273, the qPCR group the median was 80, iqr = 30-135 and the MiSeq Nano V2 group the median was 46, iqr = 13-78. Therefore, the MiSeq NanoV2 method was significantly better than the other groups at either generating closer distances from the target cluster density value (Kruskal-Wallis test, p

BACKGROUND: Studies have identified the intrauterine environment as a regulating factor of the methylation profiles of DNA of newborns. In Mexicans a SLC16A11 risk haplotypes is associated with type 2 diabetes mellitus. It is in our interest to evaluate SLC16A11 expression from the earliest stages of human development in the cells of umbilical cord blood of newborn offspring of mothers with and without gestational diabetes mellitus (GDM), and thus identify indirectly epigenetic modifications due to maternal hyperglycemic status. To date is scarce this kind of studies. OBJECTIVE: To determine the SLC16A11 gene expression, in the cells of umbilical cord blood of newborn offspring of mothers with or without GDM. MATERIAL AND METHODS: Pregnant patients submitting to cesarean, with or without GDM, were invited to participate in at Gyneco-obstetric hospital of IMSS, Mexico City. RNA of peripheral blood cells (PBC) from mothers with or without GDM, and from the umbilical cord blood cells of their newborn offspring, were extracted. Briefly, to evaluate differences in SLC16A11 gene expression, cDNA was generated by using retro-transcriptase reaction and then subjected to end point PCR with specific oligonucleotides. Variables were analyzed with t-Student test using the SPSS program. Values of p < 0.05 were considered statistically significant. RESULTS: Mean age of mothers was 30 ± 4.25 years. GDM mothers were heavier (32.90 ± 3.7 vs 30.12 ± 3.4 kg/m2, p=0.032), but there were no differences in glycemic control (HbA1c)(5.4 ± 0.6 vs 5.2 ± 0.6, p=0.333). Somatometric measurements of offspring showed no significant differences (weight: 3013.33 ± 675.54 vs 3135.71g ± 740.59, p=0.626 and length: 48.50 ± 3.03 vs 49.17cm ± 1.56, p=0.368). Analysis SLC16A11 gene expression did not showed differences in mothers, (p=0.059), and in offspring (p=0.315). Because there is a SLC16A11 gene expression, this could suggest that the SLC16A11 gene promoter could not be methylated. CONCLUSIONS: No modifications in SLC16A11 gene expression were present in GDM, which suggests that GDM does not modify the molecular mechanism in SLC16A11 RNA expression.

Background: Reduced muscle and bone-mass and quality as well as impaired energy homeostasis are seen in both aging and the metabolic syndrome (MetS). We previously showed that exercise and Angiotensin1-7 (A1-7) ameliorate this burden in young rats, but their effect vis-a-vis aging is unknown. Aims: To assess the effects of exercise under normal and high-fat diet (HFD) with or without A1-7 on bone architecture and muscle quality as well as on musculoskeletal and fat epigenetic signatures in aged mice. Methods: 19-months-old-mice receiving HFD or normal-chow (NC) with or without 3-months exercise (treadmill, 6 days/week, 20 min/day or A1-7 (0.6 mcg/kg/d). Bone was evaluated by microCT. Global quantification of the epigenetic modification 5-methyl-or hydroxyl-methyl cytosine (5mC; 5hmc) by LC-MS/MS in white/brown adipose tissue (WAT; BAT) and Gastrocnemius muscle. Results: Old, sedentary mice were metabolically "starved" with low serum glucose and triglycerides. (A) Neither exercise nor high fat diet elicited sustained changes in body weight in old mice. B) Exercise per se rehabilitated glucose, triglycerides levels and muscle quality (enhanced muscle-fiber sectional area), accompanied by reduction in muscle 5mC and in BAT 5hmc (p

DNA methylation has been recognized as a putative regulatory mechanism for CYP11B2 in primary hyperaldosteronism. We aimed to investigate the DNA methylation and the expression of a panel of genes encoding several enzymes involved in steroidogenesis in adrenocortical benign tumors. We collected a total of 60 adrenocortical tissues, including 9 non-functioning adrenal adenomas, 9 adenomas associated with autonomous cortisol secretion, 17 adenomas associated with Cushing’s syndrome, 13 Conn’s adenomas and 12 tissues derived from adrenal gland adjacent to the Conn’s adenomas. Non-functioning tumors and autonomous cortisol secretion were defined according to cortisol levels after 1 mg dexamethasone suppression test ≤ or > 50 nmol/L, respectively. The DNA methylation level of CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DHCR24, HSD3B1, HSD3B2, NR5A1, STAR, and TSPO was evaluated by quantitative Bisulfite Next Generation Sequencing (bisulfite-NGS). Bioinformatic analysis was performed in a GalaxyProject environment and processed by BSPAT (Bisulfite Sequencing Pattern Analysis Tool). Spearman correlation coefficients were calculated using IBM SPSS 21 (IBM). CYP11B1, CYP11B2, CYP17, CYP21, STAR and β-actin gene expressions were examined by quantitative Real-Time PCR using a Sybr Green Assay kit (Thermo Fisher Scientific). The equation 2−ΔΔCt was used to calculate the fold changes in gene expression between the categories of samples. CYP11B2 was significantly hypomethylated in Conn’s adenoma, when compared to other adrenal tissues (P

Background: Congenital GH deficiency (GHD) can be isolated (IGHD) or combined with other pituitary hormone deficiencies (CPHD). The identification of mutations has clinical implications for the management of patients and genetic counseling. Objective: to conduct a prospective molecular-genetic analysis in genes associated with IGHD or CPHD. Method: 119 patients (65 males) with IGHD (n= 27) or CPHD (n= 92) were studied using target gene approach. Targeted regions (involving 26 genes associated with GHD) were captured using Agilent Sure Select technology. Sequencing was performed with Illumina NextSeq. All variants were absent or extremely rare in public databases and were classified according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP). Results: Eight patients (6.7%) had variants classified as pathogenic (n = 7) or likely pathogenic (n = 4). Two loss of function mutations were found in homozygous state in GHRHR receptor [c.57+1G>A];[c.57+1G>A] and [c.820_821insC];[c.820_821insC]. Two nonsense variants were in heterozygous state in GLI2 [c.1681G>T] and OTX2 [c.319C>T] and two heterozygous missense variants were identified in TGIF1 [c.260A>T] and GHSR [c.545T>C] - the last variant was present in two different individuals. A compound heterozygous variant in PROP1 [c.301_302del];[ c.109+1G>A] was previously published with a cohort of PROP1 Brazilian patients. Conclusion: The panel established the diagnosis of 8 patients and the patients with negative results are candidates for whole exome sequencing.

Background: APECED syndrome is a rare monogenic disease caused by homozygous mutation of AIRE gene. It classically presents with chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency with an early onset in childhood. Non-endocrine manifestations as ectodermic dystrophy, asplenism and pneumonitis are also observed but their incidence remains unknown and their mechanisms not well understood. APECED has been poorly reported in France although it is widely described in several European countries. The aim of this study was to report on rare manifestations of APECED syndrome in a French cohort. Patients and methods: We performed a multicentric prospective observational study in France in order to collect clinical, biological, immunological and genetic data, after written informed consent in the frame of a PHRC (Hospital Project of Clinical Research #1927). Bronchiolitis, splenic atrophy and ocular manifestations were systematically investigated. Results We enrolled 25 patients between 2009 and 2016. Clinically, the median age at diagnosis was 12 while the median age of the first manifestation was 6, so there was diagnostic delay. The median number of manifestations was seven. 76 % of patients presented with the classical triad. Reduction of lung function was observed in 62% of patients, asplenism in 26%, and ocular manifestation in 33%. Genetically, eleven mutations of the AIRE gene were identified, two of which never previously reported: an intronic variation c.653-70G>A (intron 5) in a patient with hypoparathyroidism as unique manifestation , and c.1066del (p.Arg356GlyfsX22) (exon 9) in a patient from Guadeloupe with composite heterozygous mutations (c.967_979del13; exon 8). The most common AIRE mutation was the mutation R257X. Median age was 26 and sex ratio was 1.1. Biologically, 100% of tested sera were positive for anti-IFNα-antibodies, 15/18 for anti-IL-22, and 13/18 for anti-IL-17F antibodies. Conclusion: This series shows a high genotypic and phenotypic variability of APECED in France, that could be explained by different ethnic origins. The systematic screening for non-classic manifestations shows a more frequent occurrence than in other series. Systematic screening of asplenism and bronchiolitis could be a useful strategy to make an earlier diagnosis, to prevent infections by vaccination and to treat earlier pulmonary involvement. Lastly, antibodies against Th17 cytokines appear as good soluble markers for diagnosis of non-classical presentation of the syndrome.

Circular RNAs (circRNAs) are a recently identified class of non-coding RNAs participating in multiple biological process and tumor progress. However, the expression of circRNAs in parathyroid carcinoma (PCa) and parathyroid adenoma (PAd) remains unknown. The expression profile of circRNAs was assessed by microarray in tissues of 6 parathyroid carcinomas, 6 adenomas and 4 normal parathyroid glands. A circRNA-mRNA network was constructed to explore the possible significance of circRNAs in the parathyroid carcinoma. Moremore, the expression of circRNAs and corresponding mRNAs were validated in a cohort of parathyroid neoplasms by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Compared to the normal parathyroid, the differentially expressed circRNAs were as many as 5310 in PCa and 1055 in PAd. Among these differentially expressed circRNAs, the expression of four circRNAs, including hsa_ circRNA_0035563 (p=0.006), hsa_circRNA_0017545 (p=0.009), hsa_circRNA_0001687 (p=0.005) and hsa_circRNA_0075005 (p=0.001), and four mRNAs (MYC, FSCN1, ANXA2 and AKR1C3) was then confirmed by RT-qPCR. Consequently, hsa_circ_0075005 was the most promising diagnostic marker to distinguish PCa from PAd, with the AUC value of 0.770 (p=0.001) via receiver operator characteristic curve analysis. Furthermore, high expression level of hsa_circ_0035563 was found to be related with the CDC73 mutations (p=0.022), as well as the disease recurrence in the patients with PCa (p=0.042). In conclusion, this is the first study to reveal the expression profile of circRNAs in parathyroid neoplasms. The hsa_circ_0075005 and MYC mRNA may be used as novel diagnostic markers for PCa. High expression level of hsa_circ_0035563 was found to be related with the CDC73 mutations and disease recurrence in the patients with PCa, but the detailed molecular mechanisms of their pathogenic contribution are still in need of further investigation.

Skeletal muscle lipid metabolism significantly modulates systemic metabolic status; unsurprisingly, aberrant skeletal muscle nutrient handling is closely associated with metabolic diseases. The mechanisms underlying skeletal muscle regulation of lipid metabolism remain to be fully elucidated. Previous studies have shown that mice deficient in nuclear receptor PPARδ suffer from deranged skeletal muscle lipid handling; a similar phenotype is observed in mice deficient in the transcription factor Krüppel-like factor 15 (KLF15). Preliminary data support the existence of KLF15-PPARδ cooperativity in regulating skeletal muscle lipid metabolism, and thus we sought to 1) define the metabolic role of skeletal muscle specific KLF15; and 2) elucidate the molecular basis of KLF15-PPARδ interaction and impact on skeletal muscle lipid metabolism. We generated a skeletal muscle specific KLF15 knockout (K15-SKO) mouse and characterized the metabolic phenotype of this animal. K15-SKO mice have increased body weight and fat mass, elevated circulating free-fatty acids and triglyceride, insulin insensitivity, and glucose intolerance compared to controls. Importantly, K15-SKO mice demonstrated decreased skeletal muscle expression of a number of lipid flux genes, many of which are targets of PPARδ. To determine the necessity of KLF15 for PPARδ-mediated gene expression, we depleted KLF15 in C2C12 cells and looked at a number of PPARδ targets. The expression levels of Fatp1, Cpt1b, and Scl25a20 were attenuated - this response was unchanged in the presence or absence of GW501516 (GW), a PPARδ agonist. We used Seahorse cell metabolism analyzer to assess fatty acid oxidation and observed that knockdown of KLF15 reduces GW induction of palmitate oxygen consumption rates. We conducted co-transfection studies and determined that KLF15 and PPARδ act synergistically on the Fatp1 promoter. Co-immunoprecipitation studies confirmed physical interaction between KLF15 and PPARδ. Finally, control and K15-SKO mice were gavaged with GW for 10 days, and K15-SKO animals demonstrated attenuated induction of a number of PPARδ targets in skeletal muscle. Taken together, these data suggest that skeletal muscle specific KLF15 is critical in the regulation of lipid handling and necessary for optimal PPARδ-mediated regulation of skeletal muscle lipid metabolism. References: 1. Manickam, R., Wahli, W. Roles of peroxisome proliferator-activated receptor β/δ in skeletal muscle physiology. Biochimie136 (2017). 2. Haldar, S.M., et al. Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation. Proc Natl Acad Sci U S A109 (2012). 3. Tanaka, T., et al. Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci U S A100 (2003). Funding sources: NCATS, T32; NHLBI, T32; NIDDK, R01 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Background: Short stature is a common pediatric disorder, affecting 3% of the population; however, the identification of its cause is limited by its largely variable clinical presentation and genetic heterogeneity. Identification of novel genetic causes of short stature is of great importance to customize the therapeutic approach of this entity. The especial AT-rich sequence binding protein1 gene (SATB1) encodes a chromatin organizer with important roles in cell growth and immunosuppression. SATB1 is highly expressed in the pituitary gland, where it regulates the differentiation of PIT1 positive cells. Indeed, the conditional knockout of Satb1 in the mouse pituitary decreases growth hormone expression, leading to reduced growth. Aim: To investigate the putative role of SATB1 in growth in a cohort of individuals with short stature and in a zebrafish knockout (KO) model. Methods: We obtained germline DNA samples from a cohort of 287 Brazilian patients diagnosed with short stature (height A was identified in 3/33 (9.1%) of SGA patients, but only in 2/143 (1.4%) of ISS patients. No CNVs were observed. In the zebrafish model, satb1 knockout led to significantly smaller size at 3 wpf, compared with heterozygous and wild type controls (median standard length: 6.6, 7.4, and 7.8 cm, and mean weight: 2.7, 4.3, and 4.6 mg, respectively, P

INTRODUCTION: Single nucleotide variants (SNV) are the most common type of genetic variation among humans. The high-throughput sequencing methods increase the number of identified variants in the human genome but functional studies for disease-associated variants is laborious and time consuming. Different computational methods have been developed to predict SNP pathogenicity and phenotypic effect. They are based on different variant parameters, such as sequence homology, proteins structure and evolutionary conservation. OBJECTIVE/METHODS: To evaluate the performance of the 10 widely used pathogenicity prediction tools available in the internet in relation with functional studies: Fathmn, Mutation Acessor, Phanter and Sift sites (based on evolutionary information) and Mutation Taster, Polyphen-2, Align GVGD, CAAD, Provean and SNP-and-Go sites (based in a combination of protein structural, functional parameters and evolutionary information). We analyzed 40 described pathogenic mutation in 4 different genes associated with disorder of sex development (DSD): 17β-hydroxysteroid dehydrogenase (HSD17B3), Steroidogenic factor 1 (SF-1/NR5A1), Androgen receptor (AR) and Luteinizing hormone/chorionic gonadotropin receptor (LHCGR). All mutations are already published and functional studies showed loss of function proteins. To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF >0.01) benign SNVs described in the same 4 DSD. The quality of the predictions was analyzed by five parameters: accuracy, precision, sensitivity, specificity and Matthews correlation coefficient (MCC). RESULTS: No method was in full accordance with the functional study. The best accuracy was observed in the Polyphen-2 and SNP-and-Go programs (0.81 and 0.82); the best precision was observed in Mutation Accessor and SNP-and-Go (0.96 and 0.84). The best specificity was observed for Mutation Accessor and SNP-and-GO (0.96 and 0.83). Five programs (Phanter, Sift, Mutation Taster, Polyphen-2 and CAAD) showed sensitivity > 0.80, but only SNP-and-GO program had specificity of 0.83. Performance ranged from poor (Align GVGD - MCC 0.13) to reasonably good (SNP-and-GO - MCC 0.63). CONCLUSION: Computational algorithms are important tools for SNV analysis but their correlation with functional studies is extremely variable. The overall best performing methods among the 10 prediction tools was SNP-and-GO, with accuracy reaching 0.82.

Diabetes and obesity are metabolic indicators of an energy imbalance in the melanocortin system. Agouti related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulate energy through the release of its neuropeptide- AgRP, along with Neuropeptide Y and GABA. AgRP acts on melanocortin 4 receptor (MC4-R) by stimulating appetite and decreasing energy expenditure. Overexpression of AgRP is known to contribute to the development of diabetes and obesity. Expression of the Agrp gene is regulated by weight loss, presumably via leptin. We found that the Ensembl database specifies that for the mouse Agrp gene there are three AgRP transcripts which differ in their 5’ untranslated regions and share the same coding exons. To determine if the AgRP mRNA isoforms are relevant for weight control, we collected tissue samples from adult mouse hypothalamus, and designed a quantitative PCR assay for each of the three AgRP mRNA isoforms. After establishing the presence of each isoform in the hypothalamus, we used these assays to determine the influence of fasting on the expression levels of the three different AgRP transcripts. We also sought to determine whether the sequences upstream of each of the three isoforms could function as promoters to drive gene expression. To determine if each upstream region can act as a promoter, we cloned the upstream regions into a reporter plasmid expressing FLAG-tagged red fluorescent protein. Each promoter construct was transfected into a neuronal cell line established from NPY/AgRP-GFP neurons. We found that all three AgRP mRNA isoforms are expressed in the adult mouse hypothalamus. While fasting caused an induction of the total AgRP mRNA (fed=1.00, fasted= 6.29; p