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We report the construction and analysis of a new reference genome assembly for Rattus norvegicus , the laboratory rat, a widely used experimental animal model organism. The assembly has been adopted as the rat reference assembly by the Genome Reference Consortium and is named GRCr8. The assembly has employed 40× Pacific Biosciences (PacBio) HiFi sequencing coverage and scaffolding using optical mapping and Hi-C. We used genomic DNA from a male BN/NHsdMcwi (BN) rat of the same strain and from the same colony as the prior reference assembly, mRatBN7.2. The assembly is at chromosome level with 98.7% of the sequence assigned to chromosomes. All chromosomes have increased in size compared with the prior assembly and k -mer analysis indicates that the subject animal is fully inbred and that the genome is represented as a single haploid assembly. Notable increases are observed in Chromosomes 3, 11, and 12 in the prospective rDNA regions. In addition, Chr Y has increased threefold in size and is more consistent with the rat karyotype than previous assemblies. Several other chromosomes have grown by the incorporation of sizable discrete new blocks. These contain highly repetitive sequences and encode numerous previously unannotated genes. In addition, centromeric sequences are incorporated in most chromosomes. Genome annotation has been performed by NCBI RefSeq, which confirms improvement in assembly quality and adds more than 1100 new protein coding genes. PacBio Iso-Seq data have been acquired from multiple tissues of the subject animal and are released concurrently with the new assembly to aid further analyses., (© 2024 Li et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: The NIH All of Us Research Program aims to advance personalized medicine by not only linking patient records, surveys, and genomic data but also engaging with participants, particularly from groups traditionally underrepresented in biomedical research (UBR). This study details how the dialogue between scientists and community members, including many from communities of color, shaped local research priorities., Materials and Methods: We recruited area quantitative, basic, and clinical scientists as well as community members from our Community and Participant Advisory Boards with a predetermined interest in All of Us research as members of a Special Interest Group (SIG). An expert community engagement scientist facilitated 6 SIG meetings over the year, explicitly fostering openness and flexibility during conversations. We qualitatively analyzed discussions using a social movement framework tailored for community-based participatory research (CBPR) mobilization., Results: The SIG evolved through CBPR stages of emergence, coalescence, momentum, and maintenance/integration. Researchers prioritized community needs above personal academic interests while community members kept discussions focused on tangible return of value to communities. One key outcome includes SIG-driven shifts in programmatic and research priorities of the All of Us Research Program in Southeastern Wisconsin. One major challenge was building equitable conversations that balanced scientific rigor and community understanding., Discussion: Our approach allowed for a rich dialogue to emerge. Points of connection and disconnection between community members and scientists offered important guidance for emerging areas of genomic inquiry., Conclusion: Our study presents a robust foundation for future efforts to engage diverse communities in CBPR, particularly on healthcare concerns affecting UBR communities., (Published by Oxford University Press on behalf of the American Medical Informatics Association 2024.)

Background: While intermittent fasting leads to weight loss and improved glucose metabolism, food insecurity, the insufficient access to food for a healthy life, is associated with obesity and adverse cardiometabolic health, especially in women. We aimed to characterize the effects of intermittently restricted feeding on energy balance and glucose tolerance in female mice., Methods: Female C57BL/6J mice were fed a high-fat, high-sucrose diet and intermittently food restricted to 60% of control littermates' ad libitum intake, starting at weaning and until week 19. Restricted mice were subsequently allowed ad libitum access to the same diet. Body composition and energy balance were measured at weeks 18.5, 19, 30, and 40. At week 42, mice underwent an intraperitoneal glucose tolerance test and plasma appetitive hormones measurements after nutrient gavage., Results: During the food restriction phase, restricted mice accrued lower weight and fat mass than controls despite periodic ad libitum food access. Reintroduction of continuous ad libitum food caused increased food intake during the light phase and increased body mass in restricted mice. Minor differences in body composition-adjusted energy expenditure between groups were observed at week 40. At week 42, glucose tolerance was impaired in restricted mice compared to controls, and trends toward lower levels of postprandial anorexigenic hormones glucagon-like peptide-1 and pancreatic polypeptide were observed., Conclusion: Our findings suggest that repeated intermittent food restriction leads to changes in eating behavior that predispose to glucose intolerance when food is freely available. Future studies are needed to elucidate the specific mechanisms underlying these changes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Gene pathways and gene-regulatory networks are used to describe the causal relationship between genes, based on biological experiments. However, many genes are still to be studied to define novel pathways. To address this, a gene-clustering algorithm has been used to group correlated genes together, based on the similarity of their gene expression level. The existing methods cluster genes based on only one type of omics data, which ignores the information from other types. A large sample size is required to achieve an accurate clustering structure for thousands of genes, which can be challenging due to the cost of multi-omics data. Meta-analysis has been used to aggregate the data from multiple studies and improve the analysis results. We propose a computationally efficient meta-analytic gene-clustering algorithm that combines multi-omics datasets from multiple studies, using the fixed effects linear models and a modified weighted correlation network analysis framework. The simulation study shows that the proposed method outperforms existing single omic-based clustering approaches when multi-omics data and/or multiple studies are available. A real data example demonstrates that our meta-analytic method outperforms single-study based methods.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A major focus of academia, industry, and global governmental agencies is to develop and apply artificial intelligence and other advanced analytical tools to transform health care delivery. The American Heart Association supports the creation of tools and services that would further the science and practice of precision medicine by enabling more precise approaches to cardiovascular and stroke research, prevention, and care of individuals and populations. Nevertheless, several challenges exist, and few artificial intelligence tools have been shown to improve cardiovascular and stroke care sufficiently to be widely adopted. This scientific statement outlines the current state of the art on the use of artificial intelligence algorithms and data science in the diagnosis, classification, and treatment of cardiovascular disease. It also sets out to advance this mission, focusing on how digital tools and, in particular, artificial intelligence may provide clinical and mechanistic insights, address bias in clinical studies, and facilitate education and implementation science to improve cardiovascular and stroke outcomes. Last, a key objective of this scientific statement is to further the field by identifying best practices, gaps, and challenges for interested stakeholders.

Objective: Food insecurity (FI) is associated with poor metabolic health. It is assumed that energy intake and diet quality underlie this association. We tested the hypothesis that dietary factors (quantity and quality) mediate the association of FI with excess weight, waist circumference and glycemic control [glycohemoglobin (A1C)]., Methods: A mediation analysis was performed on data from the National Health And Nutrition Examination Survey using FI as an independent variable; body mass index (BMI), waist circumference, and A1C as metabolic outcome variables and total energy intake, macronutrients, and diet quality measured by the Healthy Eating Index-2015 (HEI-2015) as potential mediators., Results: Despite a greater prevalence of obesity in participants experiencing FI, daily reported energy intake was similar in food-secure and -insecure subjects. In adjusted analyses of the overall cohort, none of the examined dietary factors mediated associations between FI and metabolic outcomes. In race-stratified analyses, total sugar consumption was a partial mediator of BMI in non-Hispanic Whites, while diet quality measures (HEI-2015 total score and added sugar subscore) were partial mediators of waist circumference and BMI, respectively, for those in the "other" ethnic group., Conclusion: Dietary factors are not the main factors underlying the association of FI with metabolic health. Future studies should investigate whether other social determinants of health commonly present in the context of FI play a role in this association., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Agouti-related peptide (AgRP/ Agrp ) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp -expressing cells ( Klf4 Agrp -KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C , ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp . We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity. NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

Metabolic diseases are a host of complex conditions, including obesity, diabetes mellitus, and metabolic syndrome. Endocrine control systems (eg, adrenals, thyroid, gonads) are causally linked to metabolic health outcomes. N/NIH Heterogeneous Stock (HS) rats are a genetically heterogeneous outbred population developed for genetic studies of complex traits. Genetic mapping studies in adult HS rats identified loci associated with cardiometabolic risks, such as glucose intolerance, insulin resistance, and increased body mass index. This study determined underappreciated metabolic health traits and the associated endocrine glands within available substrains of the HS rat founders. We hypothesize that the genetic diversity of the HS rat founder strains causes a range of endocrine health conditions contributing to the diversity of cardiometabolic disease risks. ACI/EurMcwi, BN/NHsdMcwi, BUF/MnaMcwi, F344/StmMcwi, M520/NRrrcMcwi, and WKY/NCrl rats of both sexes were studied from birth until 13 weeks of age. Birth weight was recorded, body weight was measured weekly, metabolic characteristics were assessed, and blood and tissues were collected. Our data show wide variation in endocrine traits and metabolic health states in ACI, BN, BUF, F344, M520, and WKY rat strains. This is the first report to compare birth weight, resting metabolic rate, endocrine gland weight, hypothalamic-pituitary-thyroid axis hormones, and brown adipose tissue weight in these rat strains. Importantly, this work unveils new potential for the HS rat population to model early life adversity and adrenal and thyroid pathophysiology. The HS population likely inherited risk alleles for these strain-specific traits, making the HS rat a powerful model to investigate interventions on endocrine and metabolic health., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Rare diseases individually affect relatively few people, but as a group they impact considerable numbers of people. The Rat Genome Database (https://rgd.mcw.edu) is a knowledgebase that offers resources for rare disease research. This includes disease definitions, genes, quantitative trail loci (QTLs), genetic variants, annotations to published literature, links to external resources, and more. One important resource is identifying relevant cell lines and rat strains that serve as models for disease research. Diseases, genes, and strains have report pages with consolidated data, and links to analysis tools. Utilizing these globally accessible resources for rare disease research, potentiating discovery of mechanisms and new treatments, can point researchers toward solutions to alleviate the suffering of those afflicted with these diseases., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Environmental bisphenol compounds like bisphenol F (BPF) are endocrine-disrupting chemicals (EDCs) affecting adipose and classical endocrine systems. Genetic factors that influence EDC exposure outcomes are poorly understood and are unaccounted variables that may contribute to the large range of reported outcomes in the human population. We previously demonstrated that BPF exposure increased body growth and adiposity in male N/NIH heterogeneous stock (HS) rats, a genetically heterogeneous outbred population. We hypothesize that the founder strains of the HS rat exhibit EDC effects that were strain- and sex-dependent. Weanling littermate pairs of male and female ACI, BN, BUF, F344, M520, and WKY rats randomly received either vehicle (0.1% EtOH) or 1.125 mg BPF/l in 0.1% EtOH for 10 weeks in drinking water. Body weight and fluid intake were measured weekly, metabolic parameters were assessed, and blood and tissues were collected. BPF increased thyroid weight in ACI males, thymus and kidney weight in BUF females, adrenal weight in WKY males, and possibly increased pituitary weight in BN males. BUF females also developed a disruption in activity and metabolic rate with BPF exposure. These sex- and strain-specific exposure outcomes illustrate that HS rat founders possess diverse bisphenol-exposure risk alleles and suggest that BPF exposure may intensify inherent organ system dysfunction existing in the HS rat founders. We propose that the HS rat will be an invaluable model for dissecting gene EDC interactions on health., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The laboratory rat, Rattus norvegicus, is an important model of human health and disease, and experimental findings in the rat have relevance to human physiology and disease. The Rat Genome Database (RGD, https://rgd.mcw.edu) is a model organism database that provides access to a wide variety of curated rat data including disease associations, phenotypes, pathways, molecular functions, biological processes, cellular components, and chemical interactions for genes, quantitative trait loci, and strains. We present an overview of the database followed by specific examples that can be used to gain experience in employing RGD to explore the wealth of functional data available for the rat and other species. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Navigating the Rat Genome Database (RGD) home page Basic Protocol 2: Using the RGD search functions Basic Protocol 3: Searching for quantitative trait loci Basic Protocol 4: Using the RGD genome browser (JBrowse) to find phenotypic annotations Basic Protocol 5: Using OntoMate to find gene-disease data Basic Protocol 6: Using MOET to find gene-ontology enrichment Basic Protocol 7: Using OLGA to generate gene lists for analysis Basic Protocol 8: Using the GA tool to analyze ontology annotations for genes Basic Protocol 9: Using the RGD InterViewer tool to find protein interaction data Basic Protocol 10: Using the RGD Variant Visualizer tool to find genetic variant data Basic Protocol 11: Using the RGD Disease Portals to find disease, phenotype, and other information Basic Protocol 12: Using the RGD Phenotypes & Models Portal to find qualitative and quantitative phenotype data and other rat strain-related information Basic Protocol 13: Using the RGD Pathway Portal to find disease and phenotype data via molecular pathways., (© 2023 Wiley Periodicals LLC.)

A common preclinical model of hypertension characterized by low circulating renin is the "deoxycorticosterone acetate (DOCA)-salt" model, which influences blood pressure and metabolism through mechanisms involving the angiotensin II type 1 receptor (AT 1 R) in the brain. More specifically, AT 1 R within Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) has been implicated in selected effects of DOCA-salt. In addition, microglia have been implicated in the cerebrovascular effects of DOCA-salt and angiotensin II. To characterize DOCA-salt effects upon the transcriptomes of individual cell types within the ARC, we used single-nucleus RNA sequencing (snRNAseq) to examine this region from male C57BL/6J mice that underwent sham or DOCA-salt treatment. Thirty-two unique primary cell type clusters were identified. Sub-clustering of neuropeptide-related clusters resulted in identification of three distinct AgRP subclusters. DOCA-salt treatment caused subtype-specific changes in gene expression patterns associated with AT 1 R and G protein signaling, neurotransmitter uptake, synapse functions, and hormone secretion. In addition, two primary cell type clusters were identified as resting versus activated microglia, and multiple distinct subtypes of activated microglia were suggested by sub-cluster analysis. While DOCA-salt had no overall effect on total microglial density within the ARC, DOCA-salt appeared to cause a redistribution of the relative abundance of activated microglia subtypes. These data provide novel insights into cell-specific molecular changes occurring within the ARC during DOCA-salt treatment, and prompt increased investigation of the physiological and pathophysiological significance of distinct subtypes of neuronal and glial cell types., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wagner, Deng, Claflin, Ritter, Cui, Nakagawa, Sigmund, Morselli, Grobe and Kwitek.)

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The Rat Genome Database (RGD, https://rgd.mcw.edu) has evolved from simply a resource for rat genetic markers, maps, and genes, by adding multiple genomic data types and extensive disease and phenotype annotations and developing tools to effectively mine, analyze, and visualize the available data, to empower investigators in their hypothesis-driven research. Leveraging its robust and flexible infrastructure, RGD has added data for human and eight other model organisms (mouse, 13-lined ground squirrel, chinchilla, naked mole-rat, dog, pig, African green monkey/vervet, and bonobo) besides rat to enhance its translational aspect. This article presents an overview of the database with the most recent additions to RGD's genome, variant, and quantitative phenotype data. We also briefly introduce Virtual Comparative Map (VCMap), an updated tool that explores synteny between species as an improvement to RGD's suite of tools, followed by a discussion regarding the refinements to the existing PhenoMiner tool that assists researchers in finding and comparing quantitative data across rat strains. Collectively, RGD focuses on providing a continuously improving, consistent, and high-quality data resource for researchers while advancing data reproducibility and fulfilling Findable, Accessible, Interoperable, and Reusable (FAIR) data principles., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The COVID-19 pandemic stemmed a parallel upsurge in the scientific literature about SARS-CoV-2 infection and its health burden. The Rat Genome Database (RGD) created a COVID-19 Disease Portal to leverage information from the scientific literature. In the COVID-19 Portal, gene-disease associations are established by manual curation of PubMed literature. The portal contains data for nine ontologies related to COVID-19, an embedded enrichment analysis tool, as well as links to a toolkit. Using these information and tools, we performed analyses on the curated COVID-19 disease genes. As expected, Disease Ontology enrichment analysis showed that the COVID-19 gene set is highly enriched with coronavirus infectious disease and related diseases. However, other less related diseases were also highly enriched, such as liver and rheumatic diseases. Using the comparison heatmap tool, we found nearly 60 percent of the COVID-19 genes were associated with nervous system disease and 40 percent were associated with gastrointestinal disease. Our analysis confirms the role of the immune system in COVID-19 pathogenesis as shown by substantial enrichment of immune system related Gene Ontology terms. The information in RGD's COVID-19 disease portal can generate new hypotheses to potentiate novel therapies and prevention of acute and long-term complications of COVID-19.

Uncovering genotype-phenotype relationships is a fundamental challenge in genomics. Gene prioritization is an important step for this endeavor to make a short manageable list from a list of thousands of genes coming from high-throughput studies. Network propagation methods are promising and state of the art methods for gene prioritization based on the premise that functionally related genes tend to be close to each other in the biological networks. Recently, we introduced PhenoGeneRanker, a network-propagation algorithm for multiplex heterogeneous networks. PhenoGeneRanker allows multi-layer gene and phenotype networks. It also calculates empirical p values of gene and phenotype ranks using random stratified sampling of seeds of genes and phenotypes based on their connectivity degree in the network. In this study, we introduce the PhenoGeneRanker Bioconductor package and its application to multi-omics rat genome datasets to rank hypertension disease-related genes and strains. We showed that PhenoGeneRanker performed better to rank hypertension disease-related genes using multiplex gene networks than aggregated gene networks. We also showed that PhenoGeneRanker performed better to rank hypertension disease-related strains using multiplex phenotype network than single or aggregated phenotype networks. We performed a rigorous hyperparameter analysis and, finally showed that Gene Ontology (GO) enrichment of statistically significant top-ranked genes resulted in hypertension disease-related GO terms.

Central obesity is genetically complex, and its exponential increase in the last decades have made it a critical public health issue. The Lyon Hypertensive (LH) rat is a well-characterized hypertensive model that also exhibits spontaneous and profound differences in body weight and adiposity, relative to its metabolically healthy control, the Lyon Normotensive (LN) rat. The mechanisms underlying the body weight differences between these strains are not well-understood, thus a congenic model (LH 17 LNa) was developed where a portion of the proximal arm of LN chromosome 17 is introgressed on the LH genomic background to assess the contribution of LN alleles on obesity features. Male and female LH 17 LNa rats were studied, but male congenics did not significantly differ from LH in this study. Female LH 17 LNa rats exhibited decreases in total body growth, as well as major alterations to their body composition and adiposity. The LH 17 LNa female rats also showed decreases in metabolic rate, and a reduction in food intake. The increased adiposity in the female LH 17 LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia. Sequencing of the parental strains identified a novel frameshift mutation in the candidate gene Ercc6l2 , which is involved in transcription-coupled DNA repair, and is implicated in premature aging. The discovery of the significance of Ercc6l2 in the context of female-specific adipocyte biology could represent a novel role of DNA repair failure syndromes in obesity pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clark, Wagner, Holl, Reho, Tutaj, Smith, Dwinell, Grobe and Kwitek.)

Hypertension characterized by low circulating renin activity accounts for roughly 25%-30% of primary hypertension in humans and can be modeled experimentally via deoxycorticosterone acetate (DOCA)-salt treatment. In this model, phenotypes develop in progressive phases, although the timelines and relative contributions of various mechanisms to phenotype development can be distinct between laboratories. To explore interactions among environmental influences such as diet formulation and dietary sodium (Na) content on phenotype development in the DOCA-salt paradigm, we examined an array of cardiometabolic endpoints in young adult male C57BL/6J mice during sham or DOCA-salt treatments when mice were maintained on several common, commercially available laboratory rodent "chow" diets including PicoLab 5L0D (0.39% Na), Envigo 7913 (0.31% Na), Envigo 2920x (0.15% Na), or a customized version of Envigo 2920x (0.4% Na). Energy balance (weight gain, food intake, digestive efficiency, and energy efficiency), fluid and electrolyte homeostasis (fluid intake, Na intake, fecal Na content, hydration, and fluid compartmentalization), renal functions (urine production rate, glomerular filtration rate, urine Na excretion, renal expression of renin, vasopressin receptors, aquaporin-2 and relationships among markers of vasopressin release, aquaporin-2 shedding, and urine osmolality), and blood pressure, all exhibited changes that were subject to interactions between diet and DOCA-salt. Interestingly, some of these phenotypes, including blood pressure and hydration, were dependent on nonsodium dietary components, as Na-matched diets resulted in distinct phenotype development. These findings provide a broad and robust illustration of an environment × treatment interaction that impacts the use and interpretation of a common rodent model of low-renin hypertension.

Biological interpretation of a large amount of gene or protein data is complex. Ontology analysis tools are imperative in finding functional similarities through overrepresentation or enrichment of terms associated with the input gene or protein lists. However, most tools are limited by their ability to do ontology-specific and species-limited analyses. Furthermore, some enrichment tools are not updated frequently with recent information from databases, thus giving users inaccurate, outdated or uninformative data. Here, we present MOET or the Multi-Ontology Enrichment Tool (v.1 released in April 2019 and v.2 released in May 2021), an ontology analysis tool leveraging data that the Rat Genome Database (RGD) integrated from in-house expert curation and external databases including the National Center for Biotechnology Information (NCBI), Mouse Genome Informatics (MGI), The Kyoto Encyclopedia of Genes and Genomes (KEGG), The Gene Ontology Resource, UniProt-GOA, and others. Given a gene or protein list, MOET analysis identifies significantly overrepresented ontology terms using a hypergeometric test and provides nominal and Bonferroni corrected P-values and odds ratios for the overrepresented terms. The results are shown as a downloadable list of terms with and without Bonferroni correction, and a graph of the P-values and number of annotated genes for each term in the list. MOET can be accessed freely from https://rgd.mcw.edu/rgdweb/enrichment/start.html., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Model organism research is essential for discovering the mechanisms of human diseases by defining biologically meaningful gene to disease relationships. The Rat Genome Database (RGD, ( https://rgd.mcw.edu )) is a cross-species knowledgebase and the premier online resource for rat genetic and physiologic data. This rich resource is enhanced by the inclusion and integration of comparative data for human and mouse, as well as other human disease models including chinchilla, dog, bonobo, pig, 13-lined ground squirrel, green monkey, and naked mole-rat. Functional information has been added to records via the assignment of annotations based on sequence similarity to human, rat, and mouse genes. RGD has also imported well-supported cross-species data from external resources. To enable use of these data, RGD has developed a robust infrastructure of standardized ontologies, data formats, and disease- and species-centric portals, complemented with a suite of innovative tools for discovery and analysis. Using examples of single-gene and polygenic human diseases, we illustrate how data from multiple species can help to identify or confirm a gene as involved in a disease and to identify model organisms that can be studied to understand the pathophysiology of a gene or pathway. The ultimate aim of this report is to demonstrate the utility of RGD not only as the core resource for the rat research community but also as a source of bioinformatic tools to support a wider audience, empowering the search for appropriate models for human afflictions., (© 2021. The Author(s).)

Cardiovascular disease represents the leading cause of death in the United States, and metabolic diseases such as obesity represent the primary impediment to improving cardiovascular health. Rodent (mouse and rat) models are widely used to model cardiometabolic disease, and as a result, there is increasing interest in the development of accurate and precise methodologies with sufficiently high resolution to dissect mechanisms controlling cardiometabolic physiology in these small organisms. Further, there is great utility in the development of centralized core facilities furnished with high-throughput equipment configurations and staffed with professional content experts to guide investigators and ensure the rigor and reproducibility of experimental endeavors. Here, we outline the array of specialized equipment and approaches that are employed within the Comprehensive Rodent Metabolic Phenotyping Core (CRMPC) and our collaborating laboratories within the Departments of Physiology, Pediatrics, Microbiology & Immunology, and Biomedical Engineering at the Medical College of Wisconsin (MCW), for the detailed mechanistic dissection of cardiometabolic function in mice and rats. We highlight selected methods for the analysis of body composition and fluid compartmentalization, electrolyte accumulation and flux, energy accumulation and flux, physical activity, ingestive behaviors, ventilatory function, blood pressure, heart rate, autonomic function, and assessment and manipulation of the gut microbiota. Further, we include discussion of the advantages and disadvantages of these approaches for their use with rodent models, and considerations for experimental designs using these methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reho, Nakagawa, Mouradian, Grobe, Saravia, Burnett, Kwitek, Kirby, Segar, Hodges, Sigmund and Grobe.)

Metabolic syndrome (MetS) is a highly heritable disease and a major public health burden worldwide. MetS diagnosis criteria are met by the simultaneous presence of any three of the following: high triglycerides, low HDL/high LDL cholesterol, insulin resistance, hypertension, and central obesity. These diseases act synergistically in people suffering from MetS and dramatically increase risk of morbidity and mortality due to stroke and cardiovascular disease, as well as certain cancers. Each of these component features is itself a complex disease, as is MetS. As a genetically complex disease, genetic risk factors for MetS are numerous, but not very powerful individually, often requiring specific environmental stressors for the disease to manifest. When taken together, all sequence variants that contribute to MetS disease risk explain only a fraction of the heritable variance, suggesting additional, novel loci have yet to be discovered. In this article, we will give a brief overview on the genetic concepts needed to interpret genome-wide association studies (GWAS) and quantitative trait locus (QTL) data, summarize the state of the field of MetS physiological genomics, and to introduce tools and resources that can be used by the physiologist to integrate genomics into their own research on MetS and any of its component features. There is a wealth of phenotypic and molecular data in animal models and humans that can be leveraged as outlined in this article. Integrating these multi-omic QTL data for complex diseases such as MetS provides a means to unravel the pathways and mechanisms leading to complex disease and promise for novel treatments. © 2022 American Physiological Society. Compr Physiol 12:1-40, 2022., (Copyright © 2022 American Physiological Society. All rights reserved.)

Bisphenol F (BPF) is increasingly substituting bisphenol A in manufacturing polycarbonates and consumer products. The cardiometabolic effects of BPF in either humans or model organisms are not clear, and no studies to date have investigated the role of genetic background on susceptibility to BPF-induced cardiometabolic traits. The primary goal of this project was to determine if BPF exposure influences growth and adiposity in male N:NIH heterogeneous stock (HS) rats, a genetically heterogeneous population. Littermate pairs of male HS rats were randomly exposed to either vehicle (0.1% ethanol) or 1.125 µg/ml BPF in 0.1% ethanol for 5 weeks in drinking water starting at 3 weeks-of-age. Water consumption and body weight was measured weekly, body composition was determined using nuclear magnetic resonance, urine and feces were collected in metabolic cages, and blood and tissues were collected at the end of the study. BPF-exposed rats showed significantly increased body growth and abdominal adiposity, risk factors for cardiometabolic disease. Urine output was increased in BPF-exposed rats, driving a trend in increased creatinine clearance. We also report the first relationship between a bisphenol metabolizing enzyme and a bisphenol-induced phenotype. Preliminary heritability estimates of significant phenotypes suggest that BPF exposure may alter trait variation. These findings support BPF exposure as a cardiometabolic disease risk factor and indicate that the HS rat will be a useful model for dissecting gene by BPF interactions on metabolic health., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We present a genome assembly from an individual male Rattus norvegicus (the Norway rat; Chordata; Mammalia; Rodentia; Muridae). The genome sequence is 2.44 gigabases in span. The majority of the assembly is scaffolded into 20 chromosomal pseudomolecules, with both X and Y sex chromosomes assembled. This genome assembly, mRatBN7.2, represents the new reference genome for R. norvegicus and has been adopted by the Genome Reference Consortium., Competing Interests: Competing interests: J. Threlfall was an employee of F1000Research up until January 2021., (Copyright: © 2021 Howe K et al.)

Obesity represents the single greatest ongoing roadblock to improving cardiovascular health. Prolonged obesity is associated with fundamental changes in the integrative control of energy balance, including the development of selective leptin resistance, which is thought to contribute to obesity-associated hypertension, and adaptation of resting metabolic rate (RMR) when excess weight is reduced. Leptin and the melanocortin system within the hypothalamus contribute to the control of both energy balance and blood pressure. While the development of drugs to stimulate RMR and thereby reverse obesity through activation of the melanocortin system has been pursued, most of the resulting compounds simultaneously cause hypertension. Evidence supports the concept that although feeding behaviors, RMR, and blood pressure are controlled through mechanisms that utilize similar molecular mediators, these mechanisms exist in anatomically dissociable networks. New evidence supports a major change in molecular signaling within AgRP (Agouti-related peptide) neurons of the arcuate nucleus of the hypothalamus during prolonged obesity and the existence of multiple distinct subtypes of AgRP neurons that individually contribute to control of feeding, RMR, or blood pressure. Finally, ongoing work by our laboratory and others support a unique role for AT 1 (angiotensin II type 1 receptor) within one specific subtype of AgRP neuron for the control of RMR. We propose that understanding the unique biology of the AT 1 -expressing, RMR-controlling subtype of AgRP neurons will help to resolve the selective dysfunctions in RMR control that develop during prolonged obesity and potentially point toward novel druggable antiobesity targets that will not simultaneously cause hypertension.

Background: The clinical diagnosis of genetic renal diseases may be limited by the overlapping spectrum of manifestations between diseases or by the advancement of disease where clues to the original process are absent. The objective of this study was to determine whether genetic testing informs diagnosis and facilitates management of kidney disease patients., Methods: We developed a comprehensive genetic testing panel (KidneySeq) to evaluate patients with various phenotypes including cystic diseases, congenital anomalies of the kidney and urinary tract (CAKUT), tubulointerstitial diseases, transport disorders and glomerular diseases. We evaluated this panel in 127 consecutive patients ranging in age from newborns to 81 years who had samples sent in for genetic testing., Results: The performance of the sequencing pipeline for single-nucleotide variants was validated using CEPH (Centre de'Etude du Polymorphism) controls and for indels using Genome-in-a-Bottle. To test the reliability of the copy number variant (CNV) analysis, positive samples were re-sequenced and analyzed. For patient samples, a multidisciplinary review board interpreted genetic results in the context of clinical data. A genetic diagnosis was made in 54 (43%) patients and ranged from 54% for CAKUT, 53% for ciliopathies/tubulointerstitial diseases, 45% for transport disorders to 33% for glomerulopathies. Pathogenic and likely pathogenic variants included 46% missense, 11% nonsense, 6% splice site variants, 23% insertion-deletions and 14% CNVs. In 13 cases, the genetic result changed the clinical diagnosis., Conclusion: Broad genetic testing should be considered in the evaluation of renal patients as it complements other tests and provides insight into the underlying disease and its management., (© The Author(s) 2019. Published by Oxford University Press on behalf of ERA-EDTA.)

Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2 BN /Mcwi; consomic S2 B ) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[ rs13453786-rs66377062 ]/Aek; S2 B a) and middle segment (SS.BN-[ rs106982173-rs65057186 ]/Aek; S2 B b) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2 B a rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase ( Enpep ) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 ( Hs2st1 ) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1 , associated with increased vascular inflammation under HSD.

Complex diseases such as hypertension, cancer, and diabetes cause nearly 70% of the deaths in the U.S. and involve multiple genes and their interactions with environmental factors. Therefore, identification of genetic factors to understand and decrease the morbidity and mortality from complex diseases is an important and challenging task. With the generation of an unprecedented amount of multi-omics datasets, network-based methods have become popular to represent the multilayered complex molecular interactions. Particularly node embeddings, the low-dimensional representations of nodes in a network are utilized for gene function prediction. Integrated network analysis of multi-omics data alleviates the issues related to missing data and lack of context-specific datasets. Most of the node embedding methods, however, are unable to integrate multiple types of datasets from genes and phenotypes. To address this limitation, we developed a node embedding algorithm called Node Embeddings of Complex networks (NECo) that can utilize multilayered heterogeneous networks of genes and phenotypes. We evaluated the performance of NECo using genotypic and phenotypic datasets from rat ( Rattus norvegicus ) disease models to classify hypertension disease-related genes. Our method significantly outperformed the state-of-the-art node embedding methods, with AUC of 94.97% compared 85.98% in the second-best performer, and predicted genes not previously implicated in hypertension.

Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide ( Agrp ) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.

Cascade-specific termination of G protein signaling is catalyzed by the RGS (regulator of G protein signaling) family members, including RGS2 . Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated by these hormones. Mutations in RGS2 are associated with human hypertension and increased risk of developing preeclampsia and its sequelae. RGS family members are known to influence maternal vascular function, but the role of RGS2 within the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the development of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared with clinically matched controls and RGS2 is known to be a CREB-responsive gene, RGS2 mRNA was reduced in placentas from pregnancies affected by preeclampsia. Experimentally reducing Rgs2 expression within the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in pregnant wild-type C57BL/6J mice. Stimulation of RGS2 transcription within immortalized human HTR8/SVneo trophoblasts by cAMP/CREB signaling was discovered to be dependent on the activity of histone deacetylase activity, and more specifically, HDAC9 (histone deacetylase-9), and HDAC9 expression was reduced in placentas from human pregnancies affected by preeclampsia. We conclude that reduced expression of RGS2 within the placenta may mechanistically contribute to preeclampsia. More generally, this work identifies RGS2 as an HDAC9 -dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.

Formed in late 1999, the Rat Genome Database (RGD, https://rgd.mcw.edu) will be 20 in 2020, the Year of the Rat. Because the laboratory rat, Rattus norvegicus, has been used as a model for complex human diseases such as cardiovascular disease, diabetes, cancer, neurological disorders and arthritis, among others, for >150 years, RGD has always been disease-focused and committed to providing data and tools for researchers doing comparative genomics and translational studies. At its inception, before the sequencing of the rat genome, RGD started with only a few data types localized on genetic and radiation hybrid (RH) maps and offered only a few tools for querying and consolidating that data. Since that time, RGD has expanded to include a wealth of structured and standardized genetic, genomic, phenotypic, and disease-related data for eight species, and a suite of innovative tools for querying, analyzing and visualizing this data. This article provides an overview of recent substantial additions and improvements to RGD's data and tools that can assist researchers in finding and utilizing the data they need, whether their goal is to develop new precision models of disease or to more fully explore emerging details within a system or across multiple systems., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Metabolic syndrome is a complex disorder that comprises several other complex disorders, including obesity, hypertension, dyslipidemia, and diabetes. There are several rat models that encompass component features of MetS. Some models are inbred strains selected for one or more traits underlying MetS; others are population models with genetic risk for MetS traits, are induced by environmental stressors such as diet, are spontaneous monogenic mutant models, or are congenic strains derived from a combination of these models. Together they can be studied to identify the genetic and physiological underpinnings of MetS to identify candidate genes or mechanisms for study in human MetS subjects.

Previously, we reported that cervical vagal nerve stimulation (VNS) increases blood glucose levels and inhibits insulin secretion in anesthetized rats through afferent signaling. Since afferent signaling is also thought to mediate the therapeutic effects of VNS in patients with therapy-refractory epilepsy and major depression, the question arises if patients treated with VNS develop impaired glucose tolerance. Thus, we hypothesized that cervical VNS impairs glucose tolerance in conscious rats. Rats (n = 7) were instrumented with telemetric blood pressure sensors and right- or left-sided cervical vagal nerve stimulators (3 V, 5 Hz, 1 msec pulse duration, 1 h on 1 h off). Glucose tolerance tests (GTTs, 1.5 g dextrose/kg BW, i.p.) were performed after overnight fasting with the stimulators on or off (sham stimulation) in randomized order separated by 3-4 days. Overnight VNS did not alter mean levels of blood pressure or heart rate, but increased fasted blood glucose levels (140 ± 13 mg/dL vs. 109 ± 8 mg/dL, P < 0.05). The area under the blood glucose concentration curves of the GTTs was larger during VNS than sham stimulation (3499 ± 211 mg/dL*h vs. 1810 ± 234 mg/dL*h, P < 0.05). One hour into the GTTs, the serum insulin concentrations had decreased during VNS (-0.57 ± 0.25 ng/mL, P < 0.05) and increased during sham stimulation (+0.71 ± 0.15 ng/mL, P < 0.05) compared to the fasted baseline levels. These results demonstrate that chronic cervical VNS elevates fasted blood glucose levels and impairs glucose tolerance likely through inhibition of glucose-induced insulin release in conscious rats. It remains to be determined if patients treated with VNS are at greater risk of developing glucose intolerance and type 2 diabetes., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: The genomes of laboratory rat strains are characterised by a mosaic haplotype structure caused by their unique breeding history. These mosaic haplotypes have been recently mapped by extensive sequencing of key strains. Comparison of genomic variation between two closely related rat strains with different phenotypes has been proposed as an effective strategy for the discovery of candidate strain-specific regions involved in phenotypic differences. We developed a method to prioritise strain-specific haplotypes by integrating genomic variation and genomic regulatory data predicted to be involved in specific phenotypes. Specifically, we aimed to identify genomic regions associated with Metabolic Syndrome (MetS), a disorder of energy utilization and storage affecting several organ systems., Results: We compared two Lyon rat strains, Lyon Hypertensive (LH) which is susceptible to MetS, and Lyon Low pressure (LL), which is susceptible to obesity as an intermediate MetS phenotype, with a third strain (Lyon Normotensive, LN) that is resistant to both MetS and obesity. Applying a novel metric, we ranked the identified strain-specific haplotypes using evolutionary conservation of the occupancy three liver-specific transcription factors (HNF4A, CEBPA, and FOXA1) in five rodents including rat. Consideration of regulatory information effectively identified regions with liver-associated genes and rat orthologues of human GWAS variants related to obesity and metabolic traits. We attempted to find possible causative variants and compared them with the candidate genes proposed by previous studies. In strain-specific regions with conserved regulation, we found a significant enrichment for published evidence to obesity-one of the metabolic symptoms shown by the Lyon strains-amongst the genes assigned to promoters with strain-specific variation., Conclusions: Our results show that the use of functional regulatory conservation is a potentially effective approach to select strain-specific genomic regions associated with phenotypic differences among Lyon rats and could be extended to other systems.

Hypertension is a major risk factor for cardiovascular disease, Type 2 diabetes, and end organ failure, and is often found concomitant with disorders characteristic of the Metabolic Syndrome (MetS), including obesity, dyslipidemia, and insulin resistance. While the associated features often occur together, the pathway(s) or mechanism(s) linking hypertension in MetS are not well understood. Previous work determined that genetic variation on rat chromosome 17 (RNO17) contributes to several MetS-defining traits (including hypertension, obesity, and dyslipidemia) in the Lyon Hypertensive (LH) rat, a genetically determined MetS model. We hypothesized that at least some of the traits on RNO17 are controlled by a single gene with pleiotropic effects. To address this hypothesis, consomic and congenic strains were developed, whereby a defined fragment of RNO17 from the LH rat was substituted with the control Lyon Normotensive (LN) rat, and MetS phenotypes were measured in the resultant progeny. Compared to LH rats, LH-17LN consomic rats have significantly reduced body weight, blood pressure, and lipid profiles. A congenic strain (LH-17LNc), with a substituted fragment at the distal end of RNO17 (17q12.3; 74-97 Mb; rn4 assembly), showed differences from the LH rat in blood pressure and serum total cholesterol and triglycerides. Interestingly, there was no difference in body weight between the LH-17LNc and the parental LH rat. These data indicate that blood pressure and serum lipids are regulated by a gene(s) in the distal congenic interval, and could be due to pleiotropy. The data also indicate that body weight is not determined by the same gene(s) at this locus. Interestingly, only two small haplotypes spanning a total of approximately 0.5 Mb differ between the LH and LN genomes in the congenic interval. Genes in these haplotypes are strong candidate genes for causing dyslipidemia in the LH rat. Overall, MetS, even in a simplified genetic model such as the LH-17LN rat, is likely due to both independent and pleiotropic gene effects.

Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs., (© 2016 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of American Society of Transplant Surgeons.)

Background: The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon hypertensive and Lyon normotensive rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, an F2 intercross between Lyon hypertensive and Lyon normotensive was comprehensively studied., Methods and Results: Multidimensional data were obtained including genotypes of 1536 single-nucleotide polymorphisms, 23 physiological traits, and >150 billion nucleotides of RNA-seq reads from the livers of F2 intercross offspring and parental rats. Phenotypic and expression quantitative trait loci (eQTL) were mapped. Application of systems biology methods identified 17 candidate MetS genes. Several putative causal cis-eQTL were identified corresponding with phenotypic QTL loci. We found an eQTL hotspot on rat chromosome 17 that is causally associated with multiple MetS-related traits and found RGD1562963, a gene regulated in cis by this eQTL hotspot, as the most likely eQTL driver gene directly affected by genetic variation between Lyon hypertensive and Lyon normotensive rats., Conclusions: Our study sheds light on the intricate pathogenesis of MetS and demonstrates that systems biology with high-throughput sequencing is a powerful method to study the pathogenesis of complex genetic diseases., (© 2015 American Heart Association, Inc.)

Background: The metabolic syndrome (MetS), a complex disorder involving hypertension, obesity, dyslipidemia and insulin resistance, is a major risk factor for heart disease, stroke, and diabetes. The Lyon Hypertensive (LH), Lyon Normotensive (LN) and Lyon Low-pressure (LL) rats are inbred strains simultaneously derived from a common outbred Sprague Dawley colony by selection for high, normal, and low blood pressure, respectively. Further studies found that LH is a MetS susceptible strain, while LN is resistant and LL has an intermediate phenotype. Whole genome sequencing determined that, while the strains are phenotypically divergent, they are nearly 98% similar at the nucleotide level. Using the sequence of the three strains, we applied an approach that harnesses the distribution of Observed Strain Differences (OSD), or nucleotide diversity, to distinguish genomic regions of identity-by-descent (IBD) from those with divergent ancestry between the three strains. This information was then used to fine-map QTL identified in a cross between LH and LN rats in order to identify candidate genes causing the phenotypes., Results: We identified haplotypes that, in total, contain at least 95% of the identifiable polymorphisms between the Lyon strains that are likely of differing ancestral origin. By intersecting the identified haplotype blocks with Quantitative Trait Loci (QTL) previously identified in a cross between LH and LN strains, the candidate QTL regions have been narrowed by 78%. Because the genome sequence has been determined, we were further able to identify putative functional variants in genes that are candidates for causing the QTL., Conclusions: Whole genome sequence analysis between the LH, LN, and LL strains identified the haplotype structure of these three strains and identified candidate genes with sequence variants predicted to affect gene function. This approach, merged with additional integrative genetics approaches, will likely lead to novel mechanisms underlying complex disease and provide new drug targets and therapies.

The angiotensinogen gene is genetically linked with hypertension, but the mechanistic basis for association of sequence variants in the promoter and coding region of the gene remains unclear. An E-box at position -20 has been hypothesized to control the level of angiotensinogen expression, but its mechanistic importance for angiotensinogen expression in human tissues is uncertain. We developed an allele-specific polymerase chain reaction-based assay to distinguish between angiotensinogen mRNA derived from variants at the -20 position (rs5050) in the angiotensinogen promoter in adipose tissues obtained during surgery. The assay takes advantage of linkage disequilibrium between the rs5050 (located in the promoter) and rs4762 (located in the coding region) single nucleotide polymorphisms. This strategy allowed us to assess the level of allele-specific expression in A-20C heterozygous subjects comparing the relative proportion of each allele with the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that angiotensinogen mRNA derived from the -20C allele is expressed significantly higher than that derived from the -20A allele in subcutaneous adipose tissue, and increased expression correlates with enriched chromatin binding of upstream stimulatory factor-2 to the -20C E-box compared with -20A. This may be depot selective because we were unable to detect these differences in omental adipose. This provides the first data directly comparing expression of angiotensinogen mRNA and differential transcription factor binding derived from 2 variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.

We characterize a novel otoferlin mutation discovered in a sibling pair diagnosed with auditory neuropathy spectrum disorder and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the otoferlin splice donor site of exon 28 (IVS28 + 1G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (0.78 ms) compared to subjects with sensorineural hearing loss (average: adults 0.71 ms, children 0.85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered., (© 2013 S. Karger AG, Basel.)

Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin resistant, and hyperleptinemic but also develop a dysregulated immune system. Using marker-assisted breeding to introgress the Koletsky rat leptin receptor mutant (lepr-/lepr-), we developed a novel congenic BBDR.(lepr-/lepr-) rat line to study the development of obesity and type 2 diabetes (T2D) in the BioBreeding (BB) diabetes-resistant (DR) rat. While heterozygous lepr (-/+) or homozygous (+/+) BBDR rats remained lean and metabolically normal, at 3 wk of age all BBDR.(lepr-/lepr-) rats were obese without hyperglycemia. Between 45 and 70 days of age, male but not female obese rats developed T2D. We had previously developed congenic BBDR.(Gimap5-/Gimap5-) rats, which carry an autosomal frameshift mutation in the Gimap5 gene linked to lymphopenia and spontaneous development of type 1 diabetes (T1D) without sex differences. Because the autoimmune-mediated destruction of pancreatic islet beta-cells may be affected not only by obesity but also by the absence of leptin receptor signaling, we next generated BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) double congenic rats carrying the mutation for Gimap5 and T1D as well as the Lepr mutation for obesity and T2D. The hyperleptinemia rescued end-stage islets in BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats and induced an increase in islet size in both sexes, while T1D development was delayed and reduced only in females. These results demonstrate that obesity and T2D induced by introgression of the Koletsky leptin receptor mutation in the BBDR rat result in islet expansion associated with protection from T1D in female but not male BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats. BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats should prove valuable to study interactions between lack of leptin receptor signaling, obesity, and sex-specific T2D and T1D.

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

To dissect the genetic architecture controlling blood pressure (BP) regulation in the spontaneously hypertensive rat (SHR) we derived congenic rat strains for four previously mapped BP quantitative trait loci (QTLs) in chromosomes 2, 4, and 16. Target chromosomal regions from the Brown Norway rat (BN) averaging 13-29 cM were introgressed by marker-assisted breeding onto the SHR genome in 12 or 13 generations. Under normal salt intake, QTLs on chromosomes 2a, 2c, and 4 were associated with significant changes in systolic BP (13, 20, and 15 mmHg, respectively), whereas the QTL on chromosome 16 had no measurable effect. On high salt intake (1% NaCl in drinking water for 2 wk), the chromosome 16 QTL had a marked impact on SBP, as did the QTLs on chromosome 2a and 2c (18, 17, and 19 mmHg, respectively), but not the QTL on chromosome 4. Thus these four QTLs affected BP phenotypes differently: 1) in the presence of high salt intake (chromosome 16), 2) only associated with normal salt intake (chromosome 4), and 3) regardless of salt intake (chromosome 2c and 2a). Moreover, salt sensitivity was abrogated in congenics SHR.BN2a and SHR.BN16. Finally, we provide evidence for the influence of genetic background on the expression of the mapped QTLs individually or as a group. Collectively, these data reveal previously unsuspected nuances of the physiological roles of each of the four mapped BP QTLs in the SHR under basal and/or salt loading conditions unforeseen by the analysis of the F2 cross.

Positional cloning of lymphopenia (lyp) in the BB rat revealed a frameshift mutation in Gimap5, a member of at least seven related GTPase Immune Associated Protein genes located on rat chromosome 4q24. Our aim was to clone and sequence the cDNA of the BB diabetes prone (DP) and diabetes resistant (DR) alleles of all seven Gimap genes in the congenic DR.lyp rat line with 2 Mb of BB DP DNA introgressed onto the DR genetic background. All (100%) DR.(lyp/lyp) rats are lymphopenic and develop type 1 diabetes (T1D) by 84 days of age while DR.(+/+) rats remain T1D and lyp resistant. Among the seven Gimap genes, the Gimap5 frameshift mutation, a mutant allele that produces no protein, had the greatest impact on lymphopenia in the DR.(lyp/lyp) rat. Gimap4 and Gimap1 each had one amino acid substitution of unlikely significance for lymphopenia. Quantitative RT-PCR analysis showed a reduction in expression of all seven Gimap genes in DR.(lyp/lyp) spleen and mesenteric lymph nodes when compared to DR.(+/+). Only four; Gimap1, Gimap4, Gimap5, and Gimap9 were reduced in thymus. Our data substantiates the Gimap5 frameshift mutation as the primary defect with only limited contributions to lymphopenia from the remaining Gimap genes.