NNAT Gene Research Articles

Understanding the impact of structural modifications at the NNAT gene’s post-translational acetylation site: in silico approach for predicting its drug-interaction role in anorexia nervosa

PurposeAnorexia nervosa (AN) is a neuropsychological public health concern with a socially disabling routine and affects a person’s healthy relationship with food. The role of the NNAT (Neuronatin) gene in AN is well established. The impact of mutation at the protein’s post-translational modification (PTM) site has been exclusively associated with the worsening of the protein’s biochemical dynamics.MethodsTo understand the relationship between genotype and phenotype, it is essential to investigate the appropriate molecular stability of protein required for proper biological functioning. In this regard, we investigated the PTM-acetylation site of the NNAT gene in terms of 19 other specific amino acid probabilities in place of wild type (WT) through various in silico algorithms. Based on the highest pathogenic impact computed through the consensus classifier tool, we generated 3 residue-specific (K59D, P, W) structurally modified 3D models of NNAT. These models were further tested through the AutoDock Vina tool to compute the molecular drug binding affinities and inhibition constant (Ki) of structural variants and WT 3D models.ResultsWith trained in silico machine learning algorithms and consensus classifier; the three structural modifications (K59D, P, W), which were also the most deleterious substitution at the acetylation site of the NNAT gene, showed the highest structural destabilization and decreased molecular flexibility. The validation and quality assessment of the 3D model of these structural modifications and WT were performed. They were further docked with drugs used to manage AN, it was found that the ΔGbind (kcal/mol) values and the inhibition constants (Ki) were relatively lower in structurally modified models as compared to WT.ConclusionWe concluded that any future structural variation(s) at the PTM-acetylation site of the NNAT gene due to possible mutational consequences, will serve as a basis to explore its relationship with the propensity of developing AN.Level of evidenceNo level of evidence—open access bioinformatics research.

Insilico identification of the rare-coding pathogenic mutations and structural modeling of human NNAT gene associated with anorexia nervosa.

Increased susceptibility towards anorexia nervosa (AN) was reported with reduced levels of neuronatin (NNAT) gene. We sought to investigate the most pathogenic rare-coding missense mutations, non-synonymous single-nucleotide polymorphisms (nsSNPs) of NNAT and their potential damaging impact on protein function through transcript level sequence and structure based in silico approaches. Gene sequence, single nucleotide polymorphisms (SNPs) of NNAT was retrieved from public databases and the putative post-translational modification (PTM) sites were analyzed. Distinctive in silico algorithms were recruited for transcript level SNPs analyses and to characterized high-risk rare-coding nsSNPs along with their impact on protein stability function. Ab initio 3D-modeling of wild-type, alternate model prediction for most deleterious nsSNP, validation and recognition of druggable binding pockets were also performed. AN 3D therapeutic compounds that followed rule of drug-likeness were docked with most pathogenic variant of NNAT to estimate the drugs' binding free energies. Conclusively, 10 transcript (201-205)-based nsSNPs from 3 rare-coding missense variants, i.e., rs539681368, rs542858994, rs560845323 out of 840 exonic SNPs were identified. Transcript-based functional impact analyses predicted rs539681368 (C30Y) from NNAT-204 as the high-risk rare-coding pathogenic nsSNP,deviating protein functions. The 3D-modeling analysis of AN drugs' binding energies indicated lowest binding free energy (ΔG) and significant inhibition constant (Ki) with mutant models C30Y. Mutant model (C30Y) exhibiting significant drug binding affinity and the commonest interaction observed at the acetylation site K59. Thus, based on these findings, we concluded that the identified nsSNP may serve as potential targets for various studies, diagnosis and therapeutic interventions. No level of evidence-open access bioinformatics research.

A next generation sequencing gene panel for use in the diagnosis of anorexia nervosa.

The aim of this study was to increase knowledge of genes associated with anorexia nervosa (AN) and their diagnostic offer, using a next generation sequencing (NGS) panel for the identification of genetic variants. The rationale underlying this test is that we first analyze the genes associated with syndromic forms of AN, then genes that were found to carry rare variants in AN patients who had undergone segregation analysis, and finally candidate genes intervening in the same molecular pathways or identified by GWAS or in mouse models. We developed an NGS gene panel and used it to screen 68 Italian AN patients (63 females, 5 males). The panel included 162 genes. Family segregation study was conducted on available relatives of probands who reported significant genetic variants. In our analysis, we found potentially deleterious variants in 2 genes (PDE11A and SLC25A13) associated with syndromic forms of anorexia and predicted deleterious variants in the following 12 genes: CD36, CACNA1C, DRD4, EPHX2, ESR1, GRIN2A, GRIN3B, LRP2, NPY4R, PTGS2, PTPN22 and SGPP2. Furthermore, by Sanger sequencing of the promoter region of NNAT, we confirmed the involvement of this gene in the pathogenesis of AN. Family segregation studies further strengthened the possible causative role of CACNA1C, DRD4, GRIN2A, PTGS2, SGPP2, SLC25A13 and NNAT genes in AN etiology. The major finding of our study is the confirmation of the involvement of the NNAT gene in the pathogenesis of AN; furthermore, this study suggests that NGS-based testing can play an important role in the diagnostic evaluation of AN, excluding syndromic forms and increasing knowledge of the genetic etiology of AN. Level I, experimental study.

172-OR: Perinatal Exposure to a Western Diet Alters Hypothalamic Gene Expression and Predisposes to Diabetes in Adult Male Mice

Cardiometabolic diseases are among the most prevalent diseases worldwide. Maternal epigenetic imprinting affects gene expression in offspring, leading to increased disease susceptibility later in life. We showed previously that perinatal exposure to high fat diet (HFD) leads to obesity, fasting hyperglycemia, glucose intolerance, elevated heart rate and hypertension, exclusively in male mice. Here, we hypothesize that epigenetic maternal programming alters gene expression in the hypothalamus to promote the cardiometabolic disturbances seen in male offspring. To investigate this, C57BL6/J dams were fed a HFD or regular diet (RD) for 1 month and mated with RD-fed males. All offspring were maintained on RD after weaning. At 3 months of age, hypothalami from programmed (HFD-RD) and control (RD-RD) offspring were harvested for mRNA sequencing (Illumina NextSeq). Results show 11 differentially expressed genes (DEG) in HFD-RD males in comparison to RD-RD males. Using iPathway analysis, some of the DEGs were involved in insulin secretion and synaptic vesicle cycle pathways. Among the DEG, Snap25, coding a protein of the exocytosis machinery important for neurotransmitters, and insulin release was upregulated (p= 2.31E-06). Additionally, Nnat gene, involved in appetite suppression and regulation of synaptic plasticity in the hypothalamus, as well as insulin secretion was downregulated (p= 0.04955). MiRNA6236 (p= 2.28E-09) and Rn45s (p= 0.002), whose deficiency in islets is known to be associated with prediabetes in mice, were both downregulated in programmed males. Interestingly, with exception of Nnat gene downregulation (p= 0.001), all the male DEGs were unaltered in HFD-RD females compared to RD-RD females. In conclusion, perinatal exposure to HFD programs sex-specific alterations in gene expression in the hypothalamus, possibly impairing the central metabolic regulation and predisposing to type 2 diabetes. Disclosure M. Elgazzaz: None. E. D. Lazartigues: None. F. Mauvais-jarvis: None. Funding American Diabetes Association (1-19-IBS-291 to E.D.L.); U.S. Department of Veterans Affairs (BX004294)

Identification of differentially methylated regions (DMRs) of neuronatin in mice.

BackgroundNeuronatin (NNAT) is a paternal-inherited imprinted gene, first discovered in the rat neonatal brain, where it plays vital roles for neuronal growth, brain development, and metabolic regulation. The maternal imprint of NNAT has been identified in mice; however, the differentially methylated regions (DMRs) involved in the monoallelic expression of NNAT have not yet been investigated.ResultsIn this study, we confirmed expression of two isoforms of the NNAT (α and β) in the mice brain via quantitative RT-PCR. Additionally, the methylation profile of the CpG island located in the NNAT gene locus was determined in the mice liver, brain, sperm, and the MII oocyte via bisulfite sequencing PCR.ConclusionIn summary, we provide the first evidence for tissue- and gamete-specific methylation patterns of CpG3 that are located on exon 1, to be putative DMR of NNAT in mice.

Necdin and neuronatin genes expression among diploid parthenogenetic, IVF and in vivo derived female sexed embryos during preimplantation development in goat

The present research work was undertaken to compare the expression of necdin (Ndn) and neuronatin (Nnat) genes among diploid parthenogenetic (DIP), female sexed in vivo and female sexed in vitro produced embryonic cell colony. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and activated by ethanol (7%) for 5 min followed by incubation with 2mM 6-dimethyl amino purine (DMAP) for 4 hrs for DIP embryos production. Embryonic cell colony were developed from 8–16 and morula stage DIP, IVF and in vivo derived embryos and these colonies were used for studying the Ndn and Nnat gene expression. There was no expression of Ndn gene in both the stages of DIP embryos, while this gene was expressed almost similarly in IVF and in vivo derived 8–16 cell stages but down regulated significantly in morula of IVF compared to in vivo derived embryonic cell colony. The Nnat gene expression was absent in in vivo derived 8–16 cell and morula stage embryos, while it was expressed in both the stages of DIP and IVF embryos. Further, it was observed that the expression of this gene was significantly lower in DIP embryos of both the stages as compared to IVF one. There was no significant difference of this gene expression between 8–16 cell and morula of DIP but morula of IVF showed significantly higher expression than 8–16 cell stage.

Neuronatin is related to keratinocyte differentiation by up-regulating involucrin

BackgroundNeuronatin (Nnat), which is a neuronal developmental and differentiation molecule, is expressed in the endoplasmic reticulum of non-neuronal cells and is involved in insulin secretion from pancreatic β-cells by plausibly modulating their intracellular calcium concentration. However, the role of Nnat in keratinocyte differentiation remains unclear. ObjectiveTo unveil a possible integration of Nnat in controlling the keratinocyte differentiation markers such as involucrin, cytokeratin1, filaggrin, loricrin and S100A7. MethodsImmunohistological staining was done using psoriasis, chronic eczema, lichen planus and normal skin. Immunofluorescence staining, Western blotting and semi-quantitative real-time PCR were performed for detecting Nnat, involucrin, cytokeratin1, filaggrin, loricrin and S100A7 using human keratinocytes with or without Nnat gene transfection. Small interference RNA was applied to knockdown the Nnat gene expression. ResultsNnat existed in normal human epidermis and cultured keratinocytes. In the hyperplastic epidermis of psoriasis, chronic eczema and lichen planus, over-expression of Nnat was evident along with involucrin and cytokeratin1 expression. Coordinate up-regulation of Nnat and involucrin, but not cytokeratin1, was demonstrated in cultured keratinocytes under differentiation stimuli such as extracellular calcium elevation, exposure to phorbol myristate acetate, and increased cell density. Transfection of small intereference RNA for Nnat decreased the mRNA levels of Nnat and involucrin, but not of cytokeratin1. Furthermore, a gene transfection assay showed increased involucrin expression in the Nnat-transfected keratinocytes than in mock-transfected counterparts, without any appreciable influence on cytokeratin1, filaggrin, loricrin and S100A7 expression. ConclusionThese data indicate that Nnat is related to keratinocyte differentiation by up-regulating involucrin expression.

Memory-related gene expression profile of the male rat hippocampus induced by teeth extraction and occlusal support recovery

ObjectivesThe present study aimed to identify the effect of memory-related genes on male rats tested for spatial memory with either molar teeth extraction or its restoration by occlusal support using experimental dentures. DesignMemory-related genes were detected from hippocampi of male Wistar rats (exposed to teeth extraction with or without dentures, or no extraction (control)) (7-week old) after behavioural testing (via the radial maze task) using a DNA microarray. The time course of the expression of these genes was evaluated by quantitative real-time polymerase chain reaction (PCR) (on 49-week-old rats). ResultsIn preliminary experiments, to determine which memory genes are affected by spatial memory training, DNA microarray analysis revealed that thyrotropin-releasing hormone (Trh) and tenascin XA (Tnxa) were up-regulated and neuronatin (Nnat) and S100a9 were down-regulated after the maze training. The expression of Tnxa, Nnat and S100a9 of 49-week-old rats (during the time course) via quantitative real-time PCR was consistent with the results of microarrays of the preliminary experiment. Expression of Trh that was evaluated by quantitative real-time PCR did not agree with the results for this gene from the microarray for all groups. Therefore, expression of Trh may have increased in only young, trained rats. The expression of S100a9 prior to the maze task was down-regulated in only the extraction group. ConclusionThese results demonstrated that Trh, Tnxa and Nnat genes were affected according to the degree of memory in male rats. This study also indicated that S100a9 is a memory-related gene, which is affected by the presence of occlusal support.

Selective Methylation of CpGs at Regulatory Binding Sites Controls NNAT Expression in Wilms Tumors

Aberrant expression of imprinted genes, such as those coding for the insulin-like growth factor 2 (IGF2) and neuronatin (NNAT), is a characteristic of a variety of embryonic neoplasms, including Wilms tumor (WT). In case of IGF2, it is generally accepted that loss of imprinting in a differentially methylated region of the IGF2/H19 locus results in biallelic expression and, thus, upregulation of the gene. In this study we examined methylation pattern at potential regulatory elements of the paternally expressed NNAT gene in a cohort of WT patients in order to further characterize the molecular mechanism causing overexpression of this regulatory gene. We demonstrate that transcriptional upregulation of NNAT in WT is grossly independent of the bladder cancer-associated protein (BLCAP) gene, an imprinted gene within the imprinted domain of the NNAT locus. However, expression of the BLCAP transcript isoform v2a formerly known to be selectively expressed from the paternal allele in brain was associated with high expression of NNAT. This contrasts the situation we found at the IGF2/H19 locus, which shows high overexpression of IGF2 and inversely correlated expression of the H19 gene in WT. An analysis of DNA methylation in two potential regulatory regions of the NNAT locus by pyrosequencing revealed significant hypomethylation of the tumors compared to normal kidney tissue. Interestingly, the difference in DNA methylation was highest at CpGs that were observed within three putative binding sites of the CCCTC-binding factor CTCF. Most importantly, hypomethylation of both NNAT regulatory regions is significantly associated with the upregulation of NNAT expression and the BLCAP_v2a transcript. Our data indicate that the methylation status of a not-yet-described regulatory element within the NNAT locus that contains four potential CTCF binding sites determines the expression level of NNAT and the nearby located BLCAP_v2a transcript, thereby suggesting a functional role in the aberrant upregulation of NNAT in WT.

The Imprinted Gene Neuronatin Is Regulated by Metabolic Status and Associated With Obesity

Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.

Neuronatin: A New Inflammation Gene Expressed on the Aortic Endothelium of Diabetic Mice

OBJECTIVE—Identification of arterial genes and pathways altered in obesity and diabetes.RESEARCH DESIGN AND METHODS—Aortic gene expression profiles of obese and diabetic db/db, high-fat diet–fed C57BL/6J, and control mice were obtained using mouse Affymetrix arrays. Neuronatin (Nnat) was selected for further analysis. To determine the function of Nnat, a recombinant adenovirus (Ad-Nnat) was used to overexpress the Nnat gene in primary endothelial cells and in the mouse aorta in vivo.RESULTS—Nnat, a gene of unknown vascular function, was upregulated in the aortas of db/db and high-fat diet–fed mice. Nnat gene expression was increased in db/db mouse aorta endothelial cells. Nnat protein was localized to aortic endothelium and was selectively increased in the endothelium of db/db mice. Infection of primary human aortic endothelial cells (HAECs) with Ad-Nnat increased expression of a panel of nuclear factor-κB (NF-κB)-regulated genes, including inflammatory cytokines, chemokines, and cell adhesion molecules. Infection of mouse carotid arteries in vivo with the Ad-Nnat increased expression of vascular cell adhesion molecule 1 protein. Nnat activation of NF-κB and inflammatory gene expression in HAECs was mediated through pathways distinct from tumor necrosis factor-α. Nnat expression stimulated p38, Jun NH2-terminal kinase, extracellular signal–related kinase, and AKT kinase phosphorylation. Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF-κB–induced gene expression.CONCLUSIONS—Nnat expression is increased in endothelial cells of obese and diabetic mouse blood vessels. The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases.

NNAT and DIRAS3 genes are paternally expressed in pigs

Although expression and epigenetic differences of imprinted genes have been extensively characterised in man and the mouse, little is known on livestock species. In this study, the polymorphism-based approach was used to detect the imprinting status of NNAT and DIRAS3 genes in five heterozygous pigs (based on SNP) of Large White and Meishan F1 hybrids. The results show that both genes were paternally expressed in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). In addition, the NNAT gene had two transcripts in all tested tissues, which is consistent with its counterpart in man and cattle.

Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia.

Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.