Conservation of a Chromosome 8 Inversion and Exon Mutations Confirm Common Gulonolactone Oxidase Gene Evolution Among Primates, Including H. Neanderthalensis.

Ascorbic acid functions as an antioxidant and facilitates other biochemical processes such as collagen triple helix formation, and iron uptake by cells. Animals which endogenously produce ascorbic acid have a functional gulonolactone oxidase gene (GULO); however, humans have a GULO pseudogene (GULOP) and depend on dietary ascorbic acid. In this study, the conservation of GULOP sequences in the primate haplorhini suborder were investigated and compared to the GULO sequences belonging to the primates strepsirrhini suborder. Phylogenetic analysis suggested that the conserved GULOP exons in the haplorhini primates experienced a high rate of mutations following the haplorhini/strepsirrhini divergence. This high mutation rate has decreased during the evolution of the haplorhini primates. Additionally, indels of the haplorhini GULOP sequences were conserved across the suborder. A separate analysis for GULO sequences and well-conserved GULOP sequences focusing on placental mammals identified an in-frame GULO sequence in the Brazilian guinea pig, and a potential GULOP sequence in the pika. Similar to haplorhini primates, the guinea pig and lagomorph species have experienced a high substitution rate when compared to the mammals used in this study. A shared synteny to examine the conservation of local genes near GULO/GULOP identified a conserved inversion around the GULO/GULOP locus between the haplorhini and strepsirrhini primates. Fischer's exact test did not support an association between GULOP and the chromosomal inversion. Mauve alignment showed that the inversion of the length of the syntenic block that the GULO/GULOP genes belonged to was variable. However, there were frequent rearrangements around ~ 2 million base pairs adjacent to GULOP involving the KIF13B and MSRA genes. These data may suggest that genes acquiring deleterious mutations in the coding sequence may respond to these deleterious mutations with rapid substitution rates.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

In Silico Examination of Single Nucleotide Missense Mutations in NHLH2, a Gene Linked to Infertility and Obesity.

Continual advances in our understanding of the human genome have led to exponential increases in known single nucleotide variants. The characterization of each of the variants lags behind. For researchers needing to study a single gene, or multiple genes in a pathway, there must be ways to narrow down pathogenic variants from those that are silent or pose less pathogenicity. In this study, we use the NHLH2 gene which encodes the nescient helix-loop-helix 2 (Nhlh2) transcription factor in a systematic analysis of all missense mutations to date in the gene. The NHLH2 gene was first described in 1992. Knockout mice created in 1997 indicated a role for this protein in body weight control, puberty, and fertility, as well as the motivation for sex and exercise. Only recently have human carriers of NHLH2 missense variants been characterized. Over 300 missense variants for the NHLH2 gene are listed in the NCBI single nucleotide polymorphism database (dbSNP). Using in silico tools, predicted pathogenicity of the variants narrowed the missense variants to 37 which were predicted to affect NHLH2 function. These 37 variants cluster around the basic-helix-loop-helix and DNA binding domains of the transcription factor, and further analysis using in silico tools provided 21 SNV resulting in 22 amino acid changes for future wet lab analysis. The tools used, findings, and predictions for the variants are discussed considering the known function of the NHLH2 transcription factor. Overall use of these in silico tools and analysis of these data contribute to our knowledge of a protein which is both involved in the human genetic syndrome, Prader-Willi syndrome, and in controlling genes involved in body weight control, fertility, puberty, and behavior in the general population, and may provide a systematic methodology for others to characterize variants for their gene of interest.

Inactivating NHLH2 variants cause idiopathic hypogonadotropic hypogonadism and obesity in humans.

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans.

Pro-opiomelanocortin Neurons and the Transcriptional Regulation of Motivated Exercise.

Hypothalamic pro-opiomelanocortin (POMC) neurons are key sensory neurons for energy balance. The basic helix-loop-helix transcription factor NHLH2 is expressed in POMC neurons, and Nhlh2 knockout mice show adult-onset obesity with low exercise behavior. Evidence is presented to explore the hypothesis that NHLH2 transcriptional activity within POMC neurons is crucial for maintaining motivated spontaneous activity and enforced exercise.

A Genetic Basis for Motivated Exercise.

Prior research has demonstrated a genetic basis for motivated exercise, with evidence of a role for nescient helix-loop-helix-2 (NHLH2/Nhlh2). Nhlh2 transcriptionally regulates the monoamine oxidase A (MAO-A) gene. This article examines the evidence for the hypothesis that polymorphisms in NHLH2 or MAO-A contribute to differences in the human motivation for exercise and physical activity. The genetic pathways that link exercise and motivation are discussed.

Non-Coding RNAs in Human Health and Diseases.

Non-coding RNAs (ncRNAs) are, arguably, the enigma of the RNA transcriptome. Even though there are more annotated ncRNAs (25,967) compared to mRNAs (19,827), we know far less about each of the genes that produce ncRNA, especially in terms of their regulation, molecular functions, and interactions. Further, we are only beginning to understand the role of differential regulation or function of ncRNAs caused by genetic and epigenetic perturbations, such as single nucleotide variants (SNV), deletions, insertions, and histone/DNA modifications. The 22 papers in this Special Issue describe the emerging roles of ncRNAs in neurological, cardiovascular, immune, and hepatic systems, to name a few, as well as in diseases such as cancer, Prader-Willi Syndrome, cardiac arrhythmias, and diabetes. As we begin to understand the function and regulation of this class of RNAs, strategies targeting ncRNAs could lead to improved therapeutic interventions for some conditions.

Analysis of SNHG14: A Long Non-Coding RNA Hosting SNORD116, Whose Loss Contributes to Prader-Willi Syndrome Etiology.

The Small Nucleolar Host Gene 14 (SNHG14) is a host gene for small non-coding RNAs, including the SNORD116 small nucleolar C/D box RNA encoding locus. Large deletions of the SNHG14 locus, as well as microdeletions of the SNORD116 locus, lead to the neurodevelopmental genetic disorder Prader-Willi syndrome. This review will focus on the SNHG14 gene, its expression patterns, its role in human cancer, and the possibility that single nucleotide variants within the locus contribute to human phenotypes in the general population. This review will also include new in silico data analyses of the SNHG14 locus and new in situ RNA expression patterns of the Snhg14 RNA in mouse midbrain and hindbrain regions.

Self-perceptions of critical thinking skills in university students are associated with BMI and exercise.

Objective: To assess the role of body mass index (BMI) and exercise levels in self-perception of critical thinking skills. Participants: Three hundred forty-seven students from an upper-division nutrition class over two consecutive years. Methods: A pre/post survey with a 15-week intervention assessed perceived critical thinking skills in a blended classroom. Results: Students gained in perceived critical thinking skills in six areas over the semester. A higher BMI was associated with decreased perception of one's ability to think logically, along with increased perception that memorization was the key to success. Those that exercised reported that they had strong critical thinking skills compared to those that exercised less frequently. Conclusions: A blended classroom approach was effective in increasing multiple areas of perceptions of critical thinking. However, some perceptions of critical thinking are viewed differently for those of different BMIs and exercise frequency. Consequently, designing interventions specifically targeting those with higher BMIs, could work to erase these inequities.

Dietary Conjugated Linoleic Acid Reduces Body Weight and Fat in Snord116m+/p- and Snord116m-/p- Mouse Models of Prader-Willi Syndrome.

Prader-Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix-loop-helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p-) would respond similarly. We found that while Snord116m+/p- mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS.

New gene targets in the study of hypogonadotropic hypogonadism.

The incidence of congenital hypogonadotropic hypogonadism (HH) is approximately 1-10 in 100,000 live births. Known syndromes, such as Kallman syndrome, caused by a mutation in the KAL-1 gene, and other genes listed in the Online Mendelian Inheritance in Man database, account for 2/3 of the cases. The rest of these cases where there is no known genetic cause for HH are termed idiopathic. In this editorial, I describe each of the articles in the Special Issue on Hypogonadotropic Hypogonadism, with a focus on new genes that might be included in future screens of idiopathic patients.

A low-cost, in silico nutritional genomics course-based undergraduate research experience applicable to multiple disciplines.

This article describes the development and assessment of a Nutritional Genomics course, designed to be held in a regular classroom during normal class periods, with few extra costs to the students or the department. The course was run as an upper-level undergraduate and lower-level graduate student course. Student taking the course spent 11 weeks learning and then 4 weeks using various in silico methods to independently characterize genes of interest in the field. During the last 4 weeks of the course, students combined their methods to test a hypothesis they generated about a gene they have not yet studied and completed a final report in the form of a journal article. Two students have published or are in the process of publishing work from their final project. Validated surveys of genetic knowledge given at least 6 months following the course indicated a very high level of genetic knowledge retainment, and favorable attitudes toward genetics testing and medical use of genetics. Finally, self-perceived critical thinking skills were high, and students indicated that they perceived these skills to be gained by their participation in the course. Materials and syllabus provided in the manuscript makes this CURE easily transferrable to other disciplines.

Nescient helix-loop-helix 2 interacts with signal transducer and activator of transcription 3 to regulate transcription of prohormone convertase 1/3.

Mechanisms controlling body weight involve gene regulation through the activation of signal transduction pathways. The Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway is the mechanism primarily used by leptin in the hypothalamus. The transcription factor nescient helix-loop-helix 2 (Nhlh2) is a downstream target of leptin signaling and is expressed in proopiomelanocortin arcuate neurons. Proopiomelanocortin is cleaved by prohormone convertase 1/3 (PC1/3) to produce peptides that regulate the body's response to energy availability. Previous studies show that the PC1/3 promoter contains STAT3 sites mediating leptin-induced PC1/3 expression, and that Nhlh2 is required for hypothalamic PC1/3 expression because Nhlh2 knockout mice have reduced PC1/3 mRNA levels. Studies herein reveal that leptin-induced PC1/3 gene expression is abrogated in N2KO mice, and that in a hypothalamic cell line both STAT3 and Nhlh2 are required for the full transcriptional response of a PC1/3 reporter gene after leptin stimulation. Furthermore, it is shown that Nhlh2 binds to E-box motifs found adjacent to STAT3 sites in the PC1/3 promoter both in vitro and in chromatin immunoprecipitation assays. Finally, two different protein-protein interaction assays confirm the presence of a STAT3:Nhlh2 heterodimer on the PC1/3 promoter. The Nhlh2:STAT3 heterodimer may be an important transcriptional regulator of other hypothalamic genes in the leptin signaling pathway. These data confirm Nhlh2 as an integral element of the Janus kinase/STAT signaling pathway and are the first to demonstrate coordinated control of PC1/3 transcription by Nhlh2 and STAT3 after leptin stimulation.

Nhlh2: a basic helix-loop-helix transcription factor controlling physical activity.

In mice, targeted deletion of the basic helix-loop-helix transcription factor, nescient helix-loop-helix 2 (Nhlh2), leads to adult-onset obesity and reduced physical activity. We propose the novel hypothesis that transcriptional activity by Nhlh2 (NHLH2 in humans) controls either the ability or the motivation for exercise.

Reduced activity without hyperphagia contributes to obesity in Tubby mutant mice.

The Tub gene was originally identified as a spontaneous mutation in C57Bl/6J mice, and associated with adult-onset obesity (Tub MUT mice). Although the original Tub MUT mouse was identified over 15 years ago, there have been few reports on the animal's food intake, body fat percentage or energy expenditure. In this study, we report food intake, body weight from 5-20 weeks, body fat, body temperature and three different measures of physical activity behavior. Tub MUT mice display reduced food intake, uncharacteristic of many obese mouse models, and reduced voluntary wheel running with normal home cage ambulatory behavior. We conclude that motivation for food and exercise is an underlying defect in TUB MUT mice.

Energy balance pathways converging on the Nhlh2 transcription factor.

Multiple regulatory pathways exist to control the expression levels of neuropeptides in response to body weight and energy availability changes. Since many neuropeptides are first synthesized in a pro-neuropeptide form, the availability of processing enzymes in a neuron can control the amount of active mature neuropeptide produced at any given time. In this review, we will focus on the regulation of prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2), as well as downstream neuropeptide genes. Evidence from our laboratory suggests that Nescient helix-loop-helix 2 (Nhlh2) regulates the transcription of PC1 and PC2, possibly in conjunction with the leptin-stimulated transcription factor, STAT3. Furthermore, Nhlh2 itself is a target of leptin and other energy availability signals, with high levels of expression during energy surplus, and low levels of expression in conditions of reduced energy availability such as food deprivation or cold exposure. Overall, coordinate regulation of Nhlh2, PC1, PC2 and downstream hypothalamic neuropeptides such as thyrotropin releasing hormone (TRH) and pro-opiomelanocortin (POMC) does lead to energy balance modulation and ensuing long-term changes in body weight.

Predisposition to arrhythmia and autonomic dysfunction in Nhlh1-deficient mice.

Nhlh1 is a basic helix-loop-helix transcription factor whose expression is restricted to the nervous system and which may play a role in neuronal differentiation. To directly study Nhlh1 function, we generated null mice. Homozygous mutant mice were predisposed to premature, adult-onset, unexpected death. Electrocardiograms revealed decreased total heart rate variability, stress-induced arrhythmia, and impaired baroreceptor sensitivity. This predisposition to arrhythmia is a likely cause of the observed death in the mutant mice. Heterozygosity for the closely related transcription factor Nhlh2 increased the severity of the Nhlh1-null phenotype. No signs of primary cardiac structural or conduction abnormalities could be detected upon necropsy of the null mice. The pattern of altered heart rhythm observed in basal and experimental conditions (stress and pharmacologically induced) suggests that a deficient parasympathetic tone may contribute to the arrhythmia in the Nhlh1-null mouse. The expression of Nhlh1 in the developing brain stem and in the vagal nuclei in the wild-type mouse further supports this hypothesis. The Nhlh1 mutant mouse may thus provide a model to investigate the contribution of the autonomic nervous system to arrhythmogenesis.

Phylogenetic Analysis of the SNORD116 Locus.

The SNORD116 small nucleolar RNA locus (SNORD116@) is contained within the long noncoding RNA host gene SNHG14 on human chromosome 15q11-q13. The SNORD116 locus is a cluster of 28 or more small nucleolar (sno) RNAs; C/D box (SNORDs). Individual RNAs within the cluster are tandem, highly similar sequences, referred to as SNORD116-1, SNORD116-2, etc., with the entire set referred to as SNORD116@. There are also related SNORD116 loci on other chromosomes, and these additional loci are conserved among primates. Inherited chromosomal 15q11-q13 deletions, encompassing the SNORD116@ locus, are causative for the paternally-inherited/maternally-imprinted genetic condition, Prader-Willi syndrome (PWS). Using in silico tools, along with molecular-based and sequenced-based confirmation, phylogenetic analysis of the SNORD116@ locus was performed. The consensus sequence for the SNORD116@ snoRNAs from various species was determined both for all the SNORD116 snoRNAs, as well as those grouped using sequence and location according to a human grouping convention. The implications of these findings are put in perspective for studying SNORD116 in patients with inherited Prader-Willi syndrome, as well as model organisms.

Increased body weight affects academic performance in university students.

For K-12 students, obesity has been linked to student educational achievements. The study objective was to determine whether academic performance in university students is correlated with BMI. Students from two consecutive academic years (Jan-May 2013 and Jan-May 2014) were given an optional class survey in May, as extra credit. Of the 452 students that completed the survey, 204 females and 75 males (N = 279; 73% female and 27% male) consented to participate in the study. The number of correct answers to problem-solving questions (PSQs) and the overall final grade for the class were compared to the calculated BMI using linear regression with a Pearson's R correlation and unpaired t-tests. BMI was significantly negatively correlated with student's final grades (P = 0.001 Pearson's r = - 0.190) and PSQs were positively correlated with final grades (P < 0.001; Pearson's r = 0.357). Our findings show a correlation between healthy body weight and improved academic performance. Further, the data suggest that future research in the area of body weight, diet, and exercise and any correlations of these with academic performance in college students are warranted.

A molecular conundrum involving hypothalamic responses to and roles of long non-coding RNAs following food deprivation.

Long non-coding RNAs (lncRNAs) are one of most poorly understood RNA classes in the mammalian transcriptome. However, they are emerging as important players in transcriptional regulation, especially within the complexity of the nervous system. This review summarizes the known information about lncRNAs, and their roles in endocrine processes, as well as the lesser-known information about lncRNAs in the brain, and in the neuroendocrine hypothalamus. A "call-to-action" is presented for researchers to use archival transcriptome data to characterize differentially expressed lncRNA species within the hypothalamus. In accordance, we analyze for differential-expression of lncRNA between normal mice and mice with a targeted deletion of the nescient helix-loop-helix 2 gene, and between C57Bl/6 and 129Sv/J mice. Finally, strategies and approaches for researchers to analyze their own datasets or those on the NCBI GEO datasets repository are provided, in hopes that future studies will reveal many new roles for lncRNAs in hypothalamic physiological responses, solving this so-called "molecular conundrum" once and for all.